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Temperature Effects on Ovary Swelling in Sweet Pepper

Permanent Link: http://ufdc.ufl.edu/UFE0041206/00001

Material Information

Title: Temperature Effects on Ovary Swelling in Sweet Pepper Physiology and Anatomy
Physical Description: 1 online resource (176 p.)
Language: english
Creator: Cruz-Huerta, Nicacio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: anatomy, capsicum, carbohydrates, ovary, pepper, photosynthesis, temperature
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: TEMPERATURE EFFECTS ON OVARY SWELLING IN SWEET PEPPER: PHYSIOLOGY AND ANATOMY. In bell peppers (Capsicum annuum L.), low night temperature and high source:sink ratios during the preanthesis stage cause swollen ovaries, i.e., abnormally enlarged ovaries, resulting in low quality fruits. The physiological reasons for this ovary response are still unclear, but excess carbohydrate accumulation in ovaries has been implicated. In this research, it was found that low night temperature (LNT, 12degreeC) induced ovary swelling in three types of sweet peppers (cherry, elongated, and blocky bell), with the greatest response occurring in bell peppers. Three to four weeks of continuous LNT were required for maximum response, which coincides with the timing required for flower bud initiation in pepper. This suggests that flowers must be exposed to LNT soon after initiation in order for this response to occur. A second set of experiments showed that both LNT and fruit removal (i.e. increasing the source:sink ratio) increased the incidence of swollen ovaries in sweet pepper. Fruiting plants under LNT and non-fruiting plants under optimum night temperature (20degreeC) produced an intermediate-sized ovary, suggesting that night temperature of 20degreeC combined with high source:sink ratio or night temperature of 12degreeC combined with low source:sink ratio can partially overcome the detrimental effects of low night temperature or high source:sink ratio on ovary swelling in pepper. Both LNT and fruit removal decreased net carbon exchange rate without affecting total plant dry weight. This suggests that excess availability of current photosynthate (via maintaining similar carbon exchange rates and reducing plant growth) is not the mechanism that results in the increase in swollen ovaries observed under both LNT and high source:sink conditions. Ovary carbohydrate analysis and anatomical analysis revealed marked differences between swollen ovaries produced under LNT vs swollen ovaries produced under high source:sink ratios. Low night temperature increased floral ovary reducing sugar and starch concentration, while high source:sink ratios had no effect on ovary carbohydrate concentrations. Ovaries developed under LNT had thicker ovary walls and greater transverse area, with only slight increases in cell size and number. In contrast, ovaries developed under high source:sink ratios increased floral ovary size mainly through increased cell size. Finally, both LNT and high source:sink ratio increased ovary size through mechanisms that appear to be different.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nicacio Cruz-Huerta.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Darnell, Rebecca L.
Local: Co-adviser: Williamson, Jeffrey G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041206:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041206/00001

Material Information

Title: Temperature Effects on Ovary Swelling in Sweet Pepper Physiology and Anatomy
Physical Description: 1 online resource (176 p.)
Language: english
Creator: Cruz-Huerta, Nicacio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: anatomy, capsicum, carbohydrates, ovary, pepper, photosynthesis, temperature
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: TEMPERATURE EFFECTS ON OVARY SWELLING IN SWEET PEPPER: PHYSIOLOGY AND ANATOMY. In bell peppers (Capsicum annuum L.), low night temperature and high source:sink ratios during the preanthesis stage cause swollen ovaries, i.e., abnormally enlarged ovaries, resulting in low quality fruits. The physiological reasons for this ovary response are still unclear, but excess carbohydrate accumulation in ovaries has been implicated. In this research, it was found that low night temperature (LNT, 12degreeC) induced ovary swelling in three types of sweet peppers (cherry, elongated, and blocky bell), with the greatest response occurring in bell peppers. Three to four weeks of continuous LNT were required for maximum response, which coincides with the timing required for flower bud initiation in pepper. This suggests that flowers must be exposed to LNT soon after initiation in order for this response to occur. A second set of experiments showed that both LNT and fruit removal (i.e. increasing the source:sink ratio) increased the incidence of swollen ovaries in sweet pepper. Fruiting plants under LNT and non-fruiting plants under optimum night temperature (20degreeC) produced an intermediate-sized ovary, suggesting that night temperature of 20degreeC combined with high source:sink ratio or night temperature of 12degreeC combined with low source:sink ratio can partially overcome the detrimental effects of low night temperature or high source:sink ratio on ovary swelling in pepper. Both LNT and fruit removal decreased net carbon exchange rate without affecting total plant dry weight. This suggests that excess availability of current photosynthate (via maintaining similar carbon exchange rates and reducing plant growth) is not the mechanism that results in the increase in swollen ovaries observed under both LNT and high source:sink conditions. Ovary carbohydrate analysis and anatomical analysis revealed marked differences between swollen ovaries produced under LNT vs swollen ovaries produced under high source:sink ratios. Low night temperature increased floral ovary reducing sugar and starch concentration, while high source:sink ratios had no effect on ovary carbohydrate concentrations. Ovaries developed under LNT had thicker ovary walls and greater transverse area, with only slight increases in cell size and number. In contrast, ovaries developed under high source:sink ratios increased floral ovary size mainly through increased cell size. Finally, both LNT and high source:sink ratio increased ovary size through mechanisms that appear to be different.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nicacio Cruz-Huerta.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Darnell, Rebecca L.
Local: Co-adviser: Williamson, Jeffrey G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041206:00001


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TEMPERATURE EFFECTS ON OVARY SWELLING IN SWEET PEPPER:
PHYSIOLOGY AND ANATOMY




















By

NICACIO CRUZ-HUERTA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010

































2010 Nicacio Cruz-Huerta





















To my wife Miriam and my daughter Diana, with love









ACKNOWLEDGMENTS

Many people and institutions have been involved and made possible my studies. I

am expressing here my gratitude to them.

My gratitude and appreciation go to my advisor Dr. Rebecca L. Darnell, for her

continuous counseling, guidance, encouragement, help, and patience throughout my

entire graduate program, and for the financial support. Without it, it would have been

impossible to reach the final goal. I also thank Drs. Jeff Williamson, Karen Koch, Ken

Boote and Don Huber, for their assistance in this project. What I have learned from

each of them will make me a better professor and researcher.

I thank Steven Hiss for all the good advice and help during the experimental and

laboratory phases. I thank Valerie Jones, for her help in the carbohydrate analysis. I

thank Elio Jovicich, Paul Miller, and Mike McCaffery for all the good advice on how to

deal with growth chambers and pepper pests and diseases under Florida conditions,

and lab work. I also thank Drs. Tim Martin, Joseph Vu, Gloria Moore, Paul Lyrene,

Charles Guy, Byung-Ho Kang, and Mr. Dale, and Mrs. Kelley for letting me use their

laboratories and equipment during my research and/or helping with their expertise. I

extend my gratitude to all Horticultural Sciences Department staff, students and faculty

that in one way or another enriched my work and life during my doctoral program.

I thank the institutions that financially supported me during my studies: the

Mexican Council for Science and Technology (CONACYT), Colegio de Postgraduados,

Horticultural Sciences Department of the University of Florida, and the Florida-Mexico

Institute.









I am grateful to my parents, Arturo and Reina, my brothers and sisters and to the

Cruz-Diaz and Carrillo-Moreno families, for giving me the encouragement and their

continuous support.

I specially thank Paola, Oscar, Sonya, Jack, Patty, Omar, Mireya, Julio, Nancy,

Jose, Rebecca, Tim, Desire, Nadia, and their families for their friendship, help, and

support when my family needed it the most. I thank Mexicans in Gainesville and all our

new friends in Gainesville.

Finally, my deepest gratitude goes to Miriam and Diana, for making the hardest

moments easier, and for being with me in this journey.









TABLE OF CONTENTS

page

A C KNO W LEDG M ENTS ......... ............... ............................................. ...............

LIS T O F TA B LE S ............. .. ..... ......................................................... ...... ........ 8

LIST O F FIG URES........................................... ............... 10

A BST RA CT ............... ... ..... ......................................................... ...... 13

CHAPTER

1 INTR O D U CT IO N ............................................................................................ 15

2 LITERATURE REVIEW ................... ........................... 18

General Description of the Plant ........................ ....................... 18
Fruit Anatomy/Morphology .................................................................. .................... 19
Fruit Growth and Development ................................. ..... 20
Flower Development .............. ........................... 21
Flower Abscission and Fruit Set .............. ................ ............... 21
Fruit Growth............................................. ............... 23
Carbon Economy in the Fruit .......................................................... 28
Respiratory Losses.............................. ............... 28
Carbon Gain .................................................................................................... 29
Fruit photosynthesis ......................................... .... ... .. ..... 29
Imported carbon ....................... ......... ...... ............... 31
Abnormal Flower and Fruit Development ............... ........................... 39

3 LOW NIGHT TEMPERATURE INCREASES OVARY SIZE IN SWEET PEPPER.. 45

Introduction ........................... ............... 45
Material and Methods ........... ......... .... ....... ......... 48
Plant Material and Growing Conditions .............. ......... .... ............ 48
E x p e rim e nt 1 ..................................................................... 5 0
Experiment 2 ...................................................................... ......... ................... 51
S tatistica l A na lysis ........................................................................ ......... 52
Results ............................. .................................... 53
E x p e rim e nt 1 ..................................................................... 5 3
Experiment 2 ...................................................................... ......... ................... 56
D is c u s s io n ................................................................................................. 5 8
C o n c lu s io n s ............................................................................................... 6 4






6









4 LOW NIGHT TEMPERATURE AFFECTS CARBON EXCHANGE RATES OF
S W E ET P E P P E R LEA V E S ............................................................ ... ................. 76

Introduction ............... ...... ............................................................ ...... 76
M material and M methods ......... ........................... ... ............ ....................... 80
Plant Material and Growing Conditions ..................................... .............. 80
Experim ent 1 .................... ................. .................................. 81
Experim ent 2 ............. .... .......................................... ... ............ 82
Data Analysis ................ ......... .......... ......... 83
Results ............................. .................................... 83
E x p e rim e nt 1 ..................................................................... 8 3
Experiment 2 ...................................................................... ......... ................... 86
D is c u s s io n ................................................................................................. 8 8
C o n c lu s io n s ............................................................................................... 9 1

5 BELL PEPPER OVARY SWELLING DUE TO LOW NIGHT TEMPERATURE
AND SOURCE SINK RATIO: OVARY WALL ANATOMY AND
CARBOHYDRATES...................................... 105

Intro d uctio n ............. ......... .. ................................. ................... ............... 10 5
Materials and Methods......................................... 107
P lant M material .................................................................................. 107
Experimental Description...................... ............... 108
Anatomy ......... ..... .... ...... .... ......... ......... ........... 109
Carbohydrates ................ ......... ......... ............... 111
Data Analysis ................. ....................... ......... 113
R e su lts ........... .... ....................................................... ............................... 1 14
Ovary Size ................................................. 114
Anatomy ............. ............. ................... 116
Carbohydrates ................ ......... ......... ............... 119
D discussion ............... .................................................................................... 120
Conclusions .................... .... ......... ......................... 127

6 SUMMARY .......................... ................... .................... 148

APPENDIX

ADDITIONAL TABLES AND FIGURES .................................. ....... 151

REFERENCES ............................... ............... 159

BIOGRAPHICAL SKETCH ........................ .................. 176









LIST OF TABLES


Table page

3-1 Relationship between days after treatment and harvest interval defined for
statistical analysis in two groups of sweet pepper cultivars.............................. 65

3-2 Number of flowers harvested at anthesis and number of nodes bearing
flowers in six cultivars of sweet pepper grown at 22/20C or 22/12C
day/night temperatures. ................ ....... ....... .......... .............. 65

3-3 Main effect of night temperature during the preanthesis stage on ovary
characteristics of sweet pepper flowers measured at anthesis........................... 66

3-4 Main effect of cultivar on characteristics of ovaries at flower anthesis in sweet
pepper. .............................................................................. ......... 66

3-5 Main effect of harvest interval on ovary characteristics of six cultivars of
sw e e t p e p p e r ................ ........................................................... 6 7

3-6 Interaction of night temperature and cultivar on ovary characteristics of sweet
pepper flowers harvested at anthesis during the duration of the experiment or
only during the last two harvest intervals ............ ....................................... 68

3-7 Coefficients of conversion to estimate ovary volume at anthesis stage as a
function of its fresh weight in six cultivars of sweet pepper grown under two
night temperatures ..................................... ........... 68

3-8 Main effect of night temperature during the preanthesis stage on ovary
characteristics of sweet pepper flowers at anthesis................... ............ 69

3-9 Main effect of cultivar on ovary characteristics of sweet pepper flowers at
anthesis. .................................................... ................. ....... ......... 69

3-10 Main effect of node on ovary characteristics of sweet pepper flowers at
anthesis. ................ ......... ..................... ......... ........... 70

3-11 Interaction of night temperature and cultivar on ovary characteristics of sweet
pepper flowers harvested at anthesis ................................. ............. .......... 70

3-12 P values of t-test comparison between night temperatures of 12 and 20C
within each cultivar and node for ovary fresh weight (FW), diameter (Diam.),
and length ..................... .............. ................. ...... ......... 71

4-1 Effects of night temperature and cultivar on fresh weight of flower and flower
parts, ovary diameter and ovary length in sweet pepper ................................ 93









4-2 P-values for effects of night temperature (Temp), cultivar (CV), and days
after treatment (DAT) and their interactions on leaf gas exchange
param eters. ................................................................... ......... 93

4-3 Main effects of night temperature and cultivar on mean carbon exchange
rate, stomatal conductance and intercellular C02 in sweet pepper leaves
measured six hours after lights were on. ................. .... ....................... ...... 94

4-4 Main effects of night temperature and cultivar on daily net photosynthesis,
daily night respiration, and calculated daily total C02 gain in sweet pepper
leaves. .................. ....... .. ..................... ......... ................. 94

4-5 Night temperature and cultivar effects on vegetative growth of sweet pepper
at the end of the 45-day experiment period ............. ...... .................. 95

4-6 Main effects of night temperature and presence/absence of fruits on fresh
weight of the flower, flower parts, ovary diameter, and ovary length in
'Legionnaire' bell pepper. ................................... ...... .................. 95

4-7 P-values for effects of night temperature (Temp), presence/absence of fruits
(Fruits), and days after treatment (DAT) and their interactions on leaf gas
exchange parameters in 'Legionnaire' bell pepper ................. .................. 96

4-8 Main effects of night temperature and presence/absence of fruits on carbon
exchange rate, stomatal conductance and intercellular C02 in 'Legionnaire'
bell pepper leaves. ....................... .. ............... ................................ 96

4-9 Night temperature and presence/absence of fruit effects on growth of
'Legionnaire' bell pepper at the end of the 45-day experiment period ............... 97

5-1 Dehydration and resin embedding steps for 'Legionnaire' bell pepper ovaries
previously fixed in FAA containing 50% ethanol............... ........................ 128

5-2 Main effects of night temperature and presence/absence of fruits on the fresh
weight, diameter, and length of the ovary in 'Legionnaire' bell pepper flowers
harvested at anthesis ....................... ................ .............. .............. 128

5-3 Main effect of days after treatment on the fresh weight, diameter, and length
of the ovary in 'Legionnaire' bell pepper flowers harvested at anthesis ........... 129

5-4 Main effects of night temperature and presence/absence of fruits on soluble
sugar and starch concentration in ovaries (ovary wall and placenta) of
'Legionnaire' bell pepper flowers harvested at anthesisz............................... 129

5-5 Main effects of night temperature and presence/absence of fruits on soluble
sugar and starch content in ovaries (ovary wall and placenta) of 'Legionnaire'
bell pepper flowers harvested at anthesis. ....... ...... ..... .................. 130









LIST OF FIGURES


Figure page

2-1 Schematic drawing of pepper branching and flowering sequence ................ 43

2-2 Schematic diagram of a pepper plant ......... ...................... ................ .......... 44

2-3 Pepper fruit and its parts. ............ ........ ............. ........ ............... 44

3-1 Schematic drawing of pepper branching and flowering sequence ................ 72

3-2 Schematic drawing of a sweet pepper plant pruned to two main axes with
lateral branches............... ........ ...................... .. ...... ......... 73

3-3 Ovary fresh weight of sweet pepper flowers at anthesis developed under
22/12C or 22/20C day/night temperatures for discrete harvest intervals......... 74

3-4 Ovary fresh weight of sweet pepper flowers harvested at anthesis from the
second to the seventh nodes above the first flowering node in plants grown
at day/night temperatures of 22/12C or 22/20C................ ... ............ 75

4-1 Effect of days after treatment began on net carbon exchange rate and
stomatal conductance, and intercellular CO2 of sweet pepper leaves .............. 98

4-2 Effect of night temperature and cultivar on net carbon exchange rate (CER)
of sweet pepper leaves over time ........... ......... .................. ..... .. 99

4-3 Effects of night temperature and cultivar on stomatal conductance (gs) of
sweet pepper leaves over time................................................ .................. 100

4-4 Effect of night temperature, cultivar, and days after treatments began on leaf
respiration in sweet pepper plants during the day and night ............................ 101

4-5 Effect of days after treatment began on net carbon exchange rate, stomatal
conductance, and intercellular CO2 of 'Legionnaire' bell pepper leaves......... 102

4-6 Effect of night temperature and days after treatment began on net carbon
exchange rate, stomatal conductance, and intercellular CO2 of 'Legionnaire'
be ll pepper leaves. ...................... .. .......... .................................. .... 103

4-7 Effect of presence or absence of developing fruits and days after treatment
began on net carbon exchange rate and stomatal conductance of
'Legionnaire' bell pepper leaves. .............. ........ ....... .. ............. 104

5-1 Schematic representation of harvesting times for anatomical analysis in
relation to fruit growth in the fruiting plants ............... ........ .............. 131









5-2 Transverse cross section of the ovary wall of ovaries at flower anthesis in
'Leg ionna ire' be ll pepper. ............... ... .................................... ............... 13 1

5-3 Relationship between the measured and the calculated area of a transverse
section in the ovary of 'Legionnaire' bell pepper ................. ....... ........... 132

5-4 Interaction of night temperature and presence/absence of fruits on ovary
characteristics of flowers harvested at anthesis in 'Legionnaire' bell pepper.... 133

5-5 Effect of night temperature and days after treatment on ovary fresh weight of
flowers at anthesis in 'Legionnaire' bell pepper. .................... ..... ............ 134

5-6 Effect of presence/absence of fruits and days after treatment on ovary size of
flowers harvested at anthesis in 'Legionnaire' bell pepper ............................ 135

5-7 Effect of night temperature on ovary size and ovary wall thickness and area
in 'Legionnaire' bell pepper flowers at anthesis. ............. ....... ............ 136

5-8 Effect of night temperature on ovary wall cell size and cell number in
'Legionnaire' bell pepper flowers harvested at anthesis. ................................ 137

5-9 Median cross section of ovaries in 'Legionnaire' bell pepper flowers
developed under two night temperatures. ........ ... .................................... 138

5-10 Effect of presence or absence of fruits on ovary size and ovary wall thickness
and area in 'Legionnaire' bell pepper flowers harvested at anthesis. ............... 139

5-11 Effect of the presence or absence of developing fruits on ovary wall cell size
and cell number in 'Legionnaire' bell pepper flowers harvested at anthesis..... 140

5-12 Median cross section of ovaries in 'Legionnaire' bell pepper flowers
developed in fruiting and non-fruiting plants. ............... ............. ............... 141

5-13 Effect of harvest time on the ovary size and ovary wall thickness and area in
'Legionnaire' bell pepper flowers harvested at anthesis. ................................ 142

5-14 Effect of harvest time on ovary wall cell size and cell number in 'Legionnaire'
bell pepper flowers harvested at anthesis. ................................................. 143

5-15 Interaction of night temperature and presence/absence of fruits on ovary size
and ovary wall thickness and area in 'Legionnaire' bell pepper flowers
harvested at anthesis. ............. ..... ............. ......... .... ......... ............... 144

5-16 Interaction of night temperature and presence/absence of fruits on ovary wall
cell size and cell number in 'Legionnaire' bell pepper flowers harvested at
a n th e s is ...................... .. .. ......... .. .. .. ..... .................................. 14 5









5-17 Effect of harvest time and presence/absence of developing fruits on ovary
size and ovary wall thickness and area in 'Legionnaire' bell pepper flowers
harvested at anthesis. ............. ..... ............. ......... .... .......... ............... 146

5-18 Effect of harvest time and presence/absence of developing fruits on ovary
wall cell size and cell number in 'Legionnaire' bell pepper flowers harvested
at anthesis. ............................ ...................... .... ......... 147









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

TEMPERATURE EFFECTS ON OVARY SWELLING IN SWEET PEPPER:
PHYSIOLOGY AND ANATOMY

By

Nicacio Cruz-Huerta

August 2010

Chair: Rebecca L. Darnell
Cochair: Jeffrey G. Williamson
Major: Horticultural Science

In bell peppers (Capsicum annuum L.), low night temperature and high source:sink

ratios during the preanthesis stage cause swollen ovaries, i.e., abnormally enlarged

ovaries, resulting in low quality fruits. The physiological reasons for this ovary response

are still unclear, but excess carbohydrate accumulation in ovaries has been implicated.

In this research, it was found that low night temperature (LNT, 12C) induced ovary

swelling in three types of sweet peppers (cherry, elongated, and blocky bell), with the

greatest response occurring in bell peppers. Three to four weeks of continuous LNT

were required for maximum response, which coincides with the timing required for

flower bud initiation in pepper. This suggests that flowers must be exposed to LNT soon

after initiation in order for this response to occur. A second set of experiments showed

that both LNT and fruit removal (i.e. increasing the source:sink ratio) increased the

incidence of swollen ovaries in sweet pepper. Fruiting plants under LNT and non-fruiting

plants under optimum night temperature (200C) produced an intermediate-sized ovary,

suggesting that night temperature of 200C combined with high source:sink ratio or night

temperature of 120C combined with low source:sink ratio can partially overcome the









detrimental effects of low night temperature or high source:sink ratio on ovary swelling

in pepper. Both LNT and fruit removal decreased net carbon exchange rate without

affecting total plant dry weight. This suggests that excess availability of current

photosynthate (via maintaining similar carbon exchange rates and reducing plant

growth) is not the mechanism that results in the increase in swollen ovaries observed

under both LNT and high source:sink conditions. Ovary carbohydrate analysis and

anatomical analysis revealed marked differences between swollen ovaries produced

under LNT vs swollen ovaries produced under high source:sink ratios. Low night

temperature increased floral ovary reducing sugar and starch concentration, while high

source:sink ratios had no effect on ovary carbohydrate concentrations. Ovaries

developed under LNT had thicker ovary walls and greater transverse area, with only

slight increases in cell size and number. In contrast, ovaries developed under high

source:sink ratios increased floral ovary size mainly through increased cell size. Finally,

both LNT and high source:sink ratio increased ovary size through mechanisms that

appear to be different.









CHAPTER 1
INTRODUCTION

Pepper (Capsicum annuum L.) is an important cultivated species worldwide. World

production is led by China, Mexico, Turkey, Indonesia, Spain and the U.S. The U.S.

ranks sixth and produces 3.5% of the world's sweet and pungent (chile) peppers (FAO,

2007). In 2007, harvested area in the U.S. was 21,974 ha for bell pepper and 9,996 ha

for chile pepper, accounting for about 7.5% of the total harvested area of vegetable

crops (USDA Economic Research Service, 2008). After California, Florida is the

second largest pepper producing state (USDA Economic Research Service, 2008). In

2007, harvested area in Florida was 7,365 ha of bell peppers (USDA National

Agricultural Statistics Services, 2008), and -1200 ha of hot peppers (Li et al., 2006).

Peppers also have a cultural importance in countries like Mexico because of their

variety of uses. In addition to the traditional use of peppers in the cuisine of several

cultures, peppers are also important as a source of natural pigments (paprika pepper),

capsaicin (hot peppers, used in pharmaceutical industries and food preparation) (Surh

and Lee, 1995; Duarte et al., 2004), antioxidants (colored bell peppers) (Ishikawa, 2003;

Mateos et al., 2003; Sun et al., 2007) and as ornamental plants (Stummel and Bosland,

2006). Bosland (1999) described pepper's importance based on three aspects: historic,

general use (fresh vegetable, condiment, ornamental), and medicinal use.

Pepper fruit development depends on proper flower development, successful

pollination, fertilization and fruit set, suitable fruit growth, and ripening in pigmented

varieties. Therefore, favorable conditions for the proper growth and development of the

sexual organs are a requirement to ensure high fruit set, yield and quality (Testa et al.,

2002). Adverse environmental conditions, such as extremes in temperature, light,









and/or water availability negatively affect early flower development, fruit set and fruit

growth. These effects can include altered pollen and/or ovary development and flower

bud and flower abortion (Rylski and Spigelman, 1982; Polowick and Sawhney, 1985;

Rylski, 1985; Aloni et al., 1991a; Mercado et al., 1997b; Mercado et al., 1997c; Aloni et

al., 1999).

Fruit size and uniform shape are two important characteristics that determine fruit

quality. These characteristics are determined by a number of factors, including

assimilate accumulation in the developing flower/fruit and flower/fruit cell number and

cell size. In turn, assimilate accumulation and cell number/size are determined by

several factors, including temperature and source:sink ratios.

Previous research has shown that pepper grown under cool night temperatures

develop a high percentage of malformed fruits that are not marketable. Low

temperatures induce flower deformation, including swollen flowers and ovaries (i.e.,

flowers and ovaries that are larger than the ones developed under optimum

temperature), resulting in fruit malformation (Mercado et al., 1997c; Aloni et al., 1999).

Swollen ovaries also develop under high source:sink ratios in pepper (Aloni et al.,

1999). In both cases (i.e. cool night temperatures and high source:sink ratios), swollen

flower buds are reported to have increased reducing sugars and starch concentration

compared to normal (i.e. not swollen) flower buds (Aloni et al., 1999). Although several

studies have been done, the relation between assimilate availability and ovary swelling

in pepper remains unclear. Furthermore, the anatomical basis for ovary swelling and

deformation under these conditions is unknown.









The central hypothesis of this study is that both low night temperature and high

source:sink ratio increase ovary size and therefore ovary swelling via effects on

assimilate availability/accumulation and/or cell size/number. The objectives of this

research were to determine the effects of night temperatures and source:sink ratios on:

1) ovary size in sweet pepper cultivars that differ in fruit size and shape; 2) net carbon

exchange rates, growth, and ovary swelling in two cultivars of pepper; 3) ovary soluble

sugar and starch concentration in bell pepper ovaries; and 4) cell number and cell size

of bell pepper floral ovaries at anthesis.









CHAPTER 2
LITERATURE REVIEW

General Description of the Plant

Pepper is the common name for several species from the genus Capsicum. There

are five botanical species that are commercially cultivated. Four of these species (C.

frutescens, C. chinense, C. baccatum, and C. pubescens) play a role in local economies

but are not widely cultivated. C. annuum, which includes the long-fruited bell varieties, is

the most widely cultivated around the world, both in temperate and tropical areas

(Bosland, 1996; Wien, 1997).

Bell pepper plants are herbaceous and either annual or perennial in their native

habitat. Their shoot architecture is determined by their sympodial growth habit (Elitzur et

al., 2009). Plant height varies from 50 to 90 cm under field conditions, but under

greenhouse conditions, plant height may vary from 0.7 to 2.5 m. (Vilmorin-Diaz, 1977;

Nuez-Viials et al., 1996).The main stem bears 8 to 15 leaves forming a helix around the

stem, with a 2/5 phyllotaxy, and terminates in a flower (Child, 1979). The two or three

nodes below the terminal flower in the main stem bear sympodial shoots (Elitzur et al.,

2009) or first lateral branches (Child, 1979). A sympodial shoot is composed of a leaf

and the internode connected to the previous node. The sympodial shoot terminates in a

flower, and contains a bud at the leaf axil that develops two opposite leaves. The two

opposite leaves and the terminal flower from the preceding shoot compose a sympodial

unit (Figure 2-1A). As the two leaves expand, the corresponding stem internodes

elongate and 'carry up' the leaves, which are finally located above the preceding flower

(Figure 2-1B). Again, shoots end in a terminal flower, new leaves develop from the leaf

axil, and internodes elongate (Figure 2-2A). After the first branching point, every new









leaf axil below the floral terminus of a sympodial shoot develops two opposite new

leaves. The cycle of development repeats itself and could, in theory, continue for an

indefinite period of time. (Elitzur et al., 2009).However, differential growth between the

two leaves developing from the same axilar bud may result in asymetric sympodial

development, i.e., one sympodial shoot has a smaller leaf and a shorter internode

compared to the other shoot (Figure 2-2B). The suppression of one sympodial shoot

that leads to the suppression of subsequent sympodial shoots may occur at any time.

Such suppression is especially common in sympodia 3 and greater, and results in a

great variability of final pepper plant architecture (Steer and Pearson, 1976; Child, 1979;

Elitzur et al., 2009). Because of the flowering pattern, leaf and flower number ratio is

close to one (Heuvelink and Marcelis, 1996).

Fruit Anatomy/Morphology

The pepper fruit is a berry composed of three or four carpels (Steer and Pearson,

1976). The main parts are the peduncle, calyx, pericarp, placenta, and seeds (Figure

2-3).

The peduncle is comprised of the epidermis (rectangular-square or oblong cells),

hypodermis (one to three layers of cells that are partially lignified), cortex parenchymaa

cells with irregular form), vascular tissue and pith (in large fruits). The peduncle has

internal and external phloem tissue, and in the outer side of the external phloem tissue,

phloem fibers occur singly, in pairs, or in small groups separated by parenchyma tissue.

There is a ring of xylem tissue, broken by radial xylem rays (Parry, 1969; Rylski, 1986).

The calyx is the connection between the peduncle and the pericarp and placenta

itself (Rylski, 1986). The pericarp represents more than 80% of the fruit's fresh weight

and is the edible part. The pericarp is comprised of the epidermis, the hypodermis, the









mesocarp (with chloroplasts), the inner mesocarp which includes fibrovascular bundles

(xylem and phloem tissues), and the endocarp. Between the mesocarp and endocarp

there are cells called "giant cells", responsible for the blisters seen on the inner surface

(Parry, 1969; Rylski, 1986). Pericarp thickness appears to be related to the number of

cell layers in the ovary wall (Munting, 1974; Ali and Kelly, 1992).

Fruit Growth and Development

Fruit production is the final phase of a continuous physiological process that

begins with successful pollination and fertilization of flowers and ends with fruit

maturation and ripening. Fertilization is usually required for successful fruit set and

development, otherwise the flower senesces and no fruit is formed (Gillaspy et al.,

1993; Testa et al., 2002). In some cases, however, parthenocarpic fruits may develop

due to genetic or environmental factors (e.g. extreme temperatures, low relative

humidity) or by application of plant growth regulators to the flower (Fos and Nuez, 1996;

Fos et al., 2000; Heuvelink and Korner, 2001; Sato et al., 2001; Gorguet et al., 2005). In

tomato, an increased level of auxins and gibberellins in the ovary can substitute for

pollination and trigger fruit development, therefore those hormones are considered as

the key elements in parthenocarpic fruit development (Gorguet et al., 2005).

Fruit shape, fruit size, pericarp thickness, and carpel number are important

characteristics used to differentiate pepper types and varieties. In sweet pepper, fruit

shape, fruit size and uniformity of carpels are the main determinants of fruit quality

(Aloni et al., 1999). Fruit development comprises several stages, including flower and

early ovary development, fruit abscission/fruit set, and fruit growth.









Flower Development

Flower initiation in bell pepper starts when the main stem reaches ~the sixth leaf

(Choi and Gerber, 1992). At day/night temperatures of 28/15C, it takes approximately

30 days from the sixth leaf to anthesis of the first flower (Cruz-Huerta, 2001). There is

no specific information about the duration of cell division and cell enlargement in sweet

pepper ovary. The ovary in a bell pepper flower comprises the ovary wall, placental

tissue, and the ovules, which will develop into seeds after fertilization. Based on

morphological observations, Munting (1974) suggested that most cell division in the

ovary takes places before anthesis, similar to some other crops. In cucumber (Cucumis

sativus L), for example, approximately 70% of cell division takes place before anthesis

(Marcelis and Baan-Hofman-Eijer, 1993) and in tomato (Lycopersicon esculentum Mill.)

fruit, cell division ends about two weeks after anthesis (Mapelli et al., 1978).

Flower Abscission and Fruit Set

Flower bud and flower abscission is a serious problem in sweet pepper, especially

in the large-fruited varieties. Consequences of flower abscission vary from delayed fruit

production to low fruit yield depending on when the stress occurs (Wien, 1997). Factors

that trigger flower abscission are high temperature (Bakker, 1989b; Turner and Wien,

1994a), low light intensity (Turner and Wien, 1994b, a), water stress (Wien, 1997), low

relative humidity (Bakker, 1989a), developing fruits on the plant (Marcelis and Baan-

Hofman-Eijer, 1997; Marcelis et al., 2004), and biotic factors like pests and diseases

(Johnstone et al., 2005).

Of these factors, the most common cause of flower bud and flower abscission

appears to be high temperature (> 21 C during the night or > 32C during the day)

(Rylski and Spigelman, 1982; Aloni et al., 1991b), as occurs in tomato (Sato et al.,









2001; Gorguet et al., 2005). In general, optimum temperature for fruit set in sweet

pepper ranges from 22 to 26 oC during the day and from 15 to 18 oC during the night

(Rylski and Spigelman, 1982; Bakker, 1989b; Bhatt and Srinivasa-Rao, 1993b; Turner

and Wien, 1994a). Combinations of high temperature with other stress factors increase

abscission. For instance, high air temperature combined with low soil moisture

(Cochran, 1936 cited by Wien, 1997) or low light intensity (Turner and Wien, 1994b)

increased flower bud and flower abscission in pepper plants compared to high

temperatures alone. Low night temperatures, on the other hand, cause low pollen

fertility and swollen ovaries resulting in production of small, deformed and/or

parthenocarpic fruits (Rylski and Spigelman, 1982; Polowick and Sawhney, 1985;

Pressman et al., 1998a; Aloni et al., 1999). Detailed description of the effects of low

night temperature on flower and fruit development is given in the section "Abnormal

Flower and Fruit Development" on page 39.

Factors related to flower bud and flower abortion in pepper can be grouped into

two main categories. The first group comprises those factors, such as extreme

temperatures (Pressman et al., 1998a; Aloni et al., 2001) and low relative humidity

(Bakker, 1989a), that impair pollination. Pollination and resultant seed number are

important for fruit set. Well-pollinated fruits grown under optimal conditions have

between 150-300 seeds (Aloni et al., 1999), although Marcelis and Baan-Hofman-Eijer

(1997) found that 50-100 seeds/fruit was sufficient for maximal fruit set. Furthermore,

they found that fruit set decreased as seed number fell below 50 seed per fruit. The

second group comprises those factors that reduce carbohydrate availability. High

temperature (Turner and Wien, 1994a), water stress (Aloni et al., 1991a), low light









intensity (Turner and Wien, 1994b; Aloni et al., 1996), some pests or diseases

(Johnstone et al., 2005), and developing fruits on the plant (Marcelis and Baan-Hofman-

Eijer, 1997; Marcelis et al., 2004) may reduce photosynthesis and/or sugar availability in

pepper plant. Pepper cultivars that exhibit high flower abscission generally have a lower

capacity to accumulate sugars and starch in their flowers compared to cultivars that

exhibit low abscission (Aloni et al., 1996). Marcelis et al. (2004) reported that when

source strength was decreased in pepper by shading, high plant density, or leaf

pruning, the rate of flower bud abortion increased linearly. Similar results have been

reported in other species. For example, in maize (Zea mays L), water deficits inhibit

photosynthesis, and the decrease in photosynthate flux to the developing organs

appears to trigger abortion (Boyer and Westgate, 2004).

Under optimal growing conditions, the petals and ovary of pepper contain the

highest amount of starch and sucrose per organ compared with the rest of flower

structures, and the highest absolute and relative activity of sucrose synthase (Aloni et

al., 1996). Although a continuous flux of sucrose into the developing flower is required

for successful fruit set (Aloni et al., 1997), conditions that result in excess sugar

accumulation in the flower bud may cause swollen ovaries (see "Abnormal Flower and

Fruit Development" on page 41) that develop into abnormal fruits.

Fruit Growth

The fruit growth period in sweet pepper depends on the cultivar and the

temperature. Fruit reached the mature stage approximately 65 days after anthesis

(DAA) in 'Mazurka' (Marcelis and Baan-Hofman-Eijer, 1995b) and 'Ariane' (Cruz-Huerta,

2001), but this period was 75 days in 'Domino' (Tadesse et al., 2002) and more than

100 days in 'California' (Pretel et al., 1995). Temperature also affects the fruit growth









period. The growth period of pepper fruit 'Delphin' decreased as daily mean

temperatures increased, regardless of the day/night temperature amplitude (Bakker,

1989b). In tomato, Walker and Ho (1977) found that carbon import was inhibited by fruit

cooling (5 C) and enhanced by fruit warming (35 C) compared with controls (25 C).

Both fresh and dry matter accumulation in pepper follow a single sigmoid curve,

but the pattern differs among cultivars (Munting, 1974; Nielsen et al., 1991; Marcelis

and Baan-Hofman-Eijer, 1995b). In 'Mazurka' (Marcelis and Baan-Hofman-Eijer,

1995b), fruit fresh weight reached its maximum value between 40 and 45 DAA (stage of

marketable green); but the fruit continued gaining dry weight until 60-65 DAA (stage of

marketable red); 'Domino' (Tadesse et al., 2002) required 56 days and 77 days, while

'California' (Pretel et al., 1995) required 80 and 100 days to reach its maximum fresh

and dry weight, respectively.

The absolute growth rate in 'Mazurka' fruit followed a bell-shaped curve (Marcelis

and Baan-Hofman-Eijer, 1995b). In this cultivar, the maximum rate of fresh (>6 g d-1)

and dry (>0.4 g d-1) weight gain occurred about the fourth week after anthesis. However,

in 'Domino' there was no period of peak growth because fruit fresh weight and volume

were predominantly linear until 8 weeks after anthesis (Tadesse et al., 2002). Hall

(1977) reported the maximum rate of dry weight gain (0.2 g d-1) occurred approximately

40 DAA, while the maximum relative growth rate (RGR, g DW g1 d1) occurred during

the first days after anthesis. Based on the data reported by Marcelis and Baan-Hofman-

Eijer (1995b), the maximum RGR in pepper fruit takes place in the first three days after

anthesis, and Nielsen et al. (1991) found the maximum RGR during the first 11 days

after anthesis.









Growth pre- and post-anthesis determines fruit shape and size in pepper. Fruit

shape is defined primarily in the preanthesis stages of flower development, as a result

of genetic and environmental control (Munting, 1974; Perin et al., 2002; Weiss et al.,

2005). In bell pepper, as well as spherical and ovoid pepper cultivars, fruit shape is

defined before pollination occurs, by the pattern and rate of cell division, most of which

occurs before bloom (Munting, 1974; Aloni et al., 1999; Meijer and Murray, 2001).

However, in long-fruited pepper genotypes, fruit shape is defined by both cell division

(pre and postanthesis) and cell elongation (postanthesis) (Munting, 1974).

The shape of the ovary at anthesis may not be related to the final fruit shape

(Munting, 1974; Wien, 1997). One parameter to measure the shape is the

length:diameter ratio (LDR). In bell peppers, the LDR in the ovary is about 0.9 and it

increases to about 1.1 at fruit maturity. In long-fruited genotypes, however, the ratio may

change from about 1.0 to 2.2 or even as high as about 9 in mature fruits (Munting, 1974;

Wien, 1997). During the first two weeks after anthesis, relatively higher growth rates

were observed in the base of the ovary of long and ovoid pepper fruits compared to the

growth rates in the tip. This basal growth was attributed to cell division and elongation

(Munting, 1974). Ultimately, final fruit shape and regularity is the result of processes that

take place preanthesis (locule number) and postanthesis (uniformity of the locules in

each fruit) (Rylski, 1986; Ali and Kelly, 1993; Wien, 1997; Aloni et al., 1999; Perin et al.,

2002).

As with shape, final fruit size and weight (fresh and dry) are determined mainly by

cell number at anthesis and cell elongation during anthesis and postanthesis (Munting,

1974; Coombe, 1976; Rylski, 1986; Ali and Kelly, 1993; Bertin et al., 2002). Smaller









contributions to weight are due to cell division after anthesis and increases in cell solute

concentration (Coombe, 1976).

Fruit cell number and cell size are influenced by several factors, including water

supply (Ho et al., 1987) and assimilate supply (Bertin et al., 2002). Cell expansion and

therefore cell size is influenced by water relations (Ho et al., 1987). Mild water deficit

(Ho et al., 1987) or high vapor pressure deficit (vpd) conditions (Leonardi et al., 2000),

both of which decrease water import during cell elongation, may reduce final fruit size

and fresh weight. Sugar import; however, may not be affected, resulting in smaller fruit

with increased soluble solids.

Assimilate supply also influences fruit size by affecting source sink relations.

Flowers and the small fruits just after anthesis represent a small sink (Archbold et al.,

1982; Aloni et al., 1991b). In young pepper plants bearing one small fruit, one flower,

and one flower bud, for example, the flower bud imports up to 3.6 mg of sucrose per

day, which represents 3.2% of the exported sucrose, while the flower imports 3.7% and

the fruit 45.9% of those carbohydrates. However, in fruitless plants of pepper, about

18.3% of the exported sucrose is allocated to flowers and flower buds (Aloni et al.,

1991b).

Developing fruits, however, rapidly become an important sink. In sweet pepper,

when fruit growth rate is close to its maximum, fruit may represent up to 90% of the dry

matter accumulation in the plant for a short period of time (= 4 days) and more than

80% for a period of 20 days (Hall, 1977). Over the entire development period, fruit

represents between 30 and 65% of the dry matter accumulation (Nielsen and Veierskov,

1988; Bhatt and Srinivasa-Rao, 1993b; Cruz-Huerta etal., 2005). Similar values have









been found for tomato (Ho, 1984). Photosynthate accumulation in the fruit depends on

sink strength rather than on the capacity of the peduncle to transport assimilates (Zhang

et al., 2005). Small tomato fruits (= 20% of their maximum volume) imported carbon at

an absolute rate nearly twice that of the larger fruits (80-90% of its maximum volume)

even though the peduncle's phloem area in the smaller fruits was also smaller (Walker

and Ho, 1977). Walker and Ho (1977) concluded that in tomato fruit, sink strength

depends more on sink activity than on sink size, especially when the fruit is small.

Presence of fruits also modifies dry matter distribution in the plant. Rapidly growing

fruits decrease growth rate of all other organs (Hall, 1977).

Ali and Kelly (1992) found that defruiting the first and second nodes in pepper

plants increased the number of cell tiers in the ovary wall, the final fruit weight, and

pericarp thickness of the fruit in the third node, but had no effect on the fruit length or

diameter. However, when the sink competition was alleviated in the flower bud stage,

fruit length, diameter, weight and pericarp thickness were increased. Assuming the final

cell size was similar in the fruits of all treatments, these findings suggest that decreased

competition during the entire period of ovary development increased both periclinal and

anticlinal cell divisions in the ovary wall; however, decreased competition in the later

stages of growth increased only anticlinal cell division. It remains unknown whether in

pepper, cell number is more important than cell size in defining fruit size under

carbohydrate limiting and non-limiting conditions.

Position of the fruit on the plant also influences final fruit size. The first fruits, which

are borne in the lower part of the plant, are usually larger, and fruit size decreases as

they are formed in the upper levels (Khah and Passam, 1992; Cruz-Huerta, 2001).









However, this influence seems to be related to fruit cell number (Bertin et al., 2002).

Within a tomato truss, proximal fruits are bigger and already contain more cells at

anthesis than distal ones (Bangerth and Ho, 1984). Bertin et al. (2002) found that the

tomato fruit cell number was similar in fruits within the same truss under low sink

competition conditions. However, when competition increased, cell number declined

more in the distal than in proximal fruits.

Even though the pepper fruit is the main sink in the plant, its capacity to

metabolize assimilates, and therefore to grow, seems to be affected by its previous

assimilate supply (Koning and Marcelis, 1998). When source-limited fruits were

changed to non-limiting source conditions, it took 2 to 3 weeks before fruit growth rate

was as fast as that from fruits growing continuously under non-limiting conditions.

Carbon Economy in the Fruit

The assimilate supply to fruit is determined primarily by carbohydrate allocation, as

carbon is one of the most important components of the fruit's dry matter. Fruit carbon

accumulation is the balance between carbon loss (via fruit respiration) and carbon gain

(via fruit photosynthesis and imported carbon).

Respiratory Losses

Respiration rates per unit weight are higher in young fruits of sweet pepper

compared with older fruits (Pretel et al., 1995; Tadesse et al., 2002), especially during

the first three weeks of growth. This is similar to other fruits such as tomato (Xu et al.,

1997), cucumber (Marcelis and Baan-Hofman-Eijer, 1995c) and blueberry (Birkhold et

al., 1992). In some fruits, there may be a second peak in respiration when maximum

fresh weight or maturation approaches. For example, in 'Domino' bell pepper (Tadesse

et al., 2002), C02 production increased when fruit reached the maximum fresh weight;









however, in 'California' bell pepper (Pretel et al., 1995) C02 liberation remained low until

fruit was red. As an indirect way to measure respiration, Villavicencio et al. (2001)

measured CO2 concentration inside the 'Camelot' pepper fruit and found that the

concentration increased from 33 to 66 mg L-1 from the mature green stage to the red

stage, and then decreased.

Respiration losses in the fruits may represent an important proportion of the total

carbon requirement. For instance, in cucumber, respiration losses accounted for 13 to

15% of the total cumulative carbon requirement, irrespective of temperature and

number of fruits competing for assimilates (Marcelis and Baan-Hofman-Eijer, 1995c),

and in tomato, such losses are 25% (Tanaka et al., 1974a). In blueberry, respiration

losses were 37% of the total carbon requirement (Birkhold et al., 1992). There is no

specific information for pepper, but the respiratory costs might be lower than they are in

tomato and cucumber because of carbon fixation in pepper fruit (Steer and Pearson,

1976) and the possibility of CO2 refixation (Bower et al., 2000).

Carbon Gain

Fruit photosynthesis

Fruit carbon fixation or refixation has been reported for several species,

particularly in the early stages of fruit development. In general, carbon fixation in fruit is

10 to 100 times lower than that of the respective leaves (Blanke and Lenz, 1989; Bower

et al., 2000) and comprises only a small percentage of the total carbon requirement for

fruit development. Steer and Pearson (1976) calculated that pepper fruits were able to

fix up to 13% of the required carbon. Tomato fruit photosynthesis contributions ranged

from 10 to 15% (Tanaka et al., 1974a; Hetherington et al., 1998), in cucumber the range

was from 1 to 5% (Marcelis and Baan-Hofman-Eijer, 1995a), and in blueberry about









15% (Birkhold et al., 1992). Blueberry fruit photosynthesis supplied 50% of the carbon

required during the first 10 days after flowering and 85% during the 5 days after petal

fall (Birkhold et al., 1992). Some small fruits even exhibit net C02 fixation under light

conditions. Xu et al. (1997) found that small tomato fruits were net photosynthesizers

under photosynthetic photon flux (PPF) greater than 300 pmol m-2 s-1. Birkhold et al.

(1992) found that blueberry fruits exhibited net photosynthesis from petal fall through

fruit color break. In Cinnamomum camphora, C02 refixation accounted for 22.9% of the

carbon balance of the fruit (Ogawa and Takano, 1997). In addition, results of Smillie et

al. (1999) suggested that the photosynthetic activity found in the calyx, green shoulder,

pericarp and locular parenchyma of tomato fruits plays a significant role in providing

carbon assimilates to the fruit.

Fruit photosynthesis differs from leaf photosynthesis in several aspects. Pepper

fruit enzymatic activity of ribulose bisphosphate carboxylase oxygenase (Rubisco) on

a fresh weight basis is only one fifteenth of the activity found in leaves (Steer and

Pearson, 1976). Chloroplasts in the outer fruit layers are typically sun chloroplasts but

evolve to shade chloroplasts in the inner tissues as light penetration decreases (Blanke

and Lenz, 1989). Stomata in the fruit, if present, are 10 to 100 times fewer in number

than in the respective leaves (Blanke and Lenz, 1989). In pepper fruits no stomata were

found (Blanke and Holthe, 1997).

Blanke and Lenz (1989) and Blanke (1998) proposed that fruit photosynthesis is

mainly intended to refix C02 from mitochondrial respiration of predominantly imported

carbon. Gas exchange through the fruit epidermis in fruits like apple and pepper is low,

which favors higher C02 concentrations in the center of the fruit, since seeds are an









important source of respired C02 (Blanke and Lenz, 1989; Blanke and Holthe, 1997;

Villavicencio et al., 2001). Part of this C02 may be refixed by phosphoenolpyruvate

carboxylase (PEPC) and possibly phosphoenolpyruvate carboxykinase (PEPCk) by

3-carboxylation and accumulation of malic acid in the vacuole (Blanke and Lenz, 1989);

however, no diurnal variation in the pH has been measured. Pepper fruits remain green

during most of the growing period. In addition, the large cavity and relatively thin

pericarp may favor a higher light intensity throughout the tissue, increasing

photosynthetic activity and possibly the capacity to refix more carbon than other fruits.

Imported carbon

Carbon imported by the fruit comes from either current photosynthesis or

previously stored carbohydrates. There is no specific research in pepper addressing

translocation of previously stored carbon to the fruit. In non-fruiting plants of pepper, the

stem becomes the main sink, but there is no evidence that it stores soluble sugars or

starch that are later used by the fruit (Hall and Milthorpe, 1978). In tomato, the stem is

also a major sink for assimilates and it functions as a storage organ. However, there is

little evidence to suggest that these assimilates are used in fruit production, except

when the plant is in the final stages of its growth cycle, or if premature leaf loss occurs

(Hocking and Steer, 1994).

By far, the most important source of imported carbon to developing fruit comes

from current photosynthesis. Although leaves supply the majority of photosynthate for

fruit development (Steer and Pearson, 1976), green stems and petioles may also fix

C02. Xu et al. (Xu et al., 1997) found that net photosynthesis in petioles of young

tomato leaves (5th and 10th node from the apex) was positive when PPF was higher

than 100 pmol m-2 s-1, but in petioles from older leaves (15 and 18th nodes) net CO2









exchange was close to 0 even at higher PPF. In contrast, there was no net

photosynthesis in the stem, but the higher the irradiance, the lower the rates of net CO2

efflux regardless of stem age, indicating some carbon fixation was occurring.

Leaf and plant photosynthesis are affected by a variety of factors including leaf

area, leaf age, presence of fruits, light intensity, and temperature.

a) Leaf area. Pepper plants can compensate photosynthetically for certain

amounts of defoliation. Defoliation of up to 50% in young pepper plants increased net

photosynthesis in the remaining leaves such that final fruit fresh and dry matter were not

affected (Bhatt and Srinivasa-Rao, 1993a). In tomato, partial leaf removal increased net

assimilation rate of the remaining leaves, so fruit growth was not affected (Tanaka and

Fujita, 1974).

b) Leaf age. Photosynthetic rates vary with leaf age. Very young leaves have a

negative carbon exchange rate and may represent the major sink for photosynthates

manufactured within them (Tanaka et al., 1974b). In pepper 'California Wonder',

maximum photosynthetic rate occurs when leaf area is ~ 10 cm2 (Steer, 1971), the time

at which stomatal density is highest (Steer, 1972). The photosynthetic rate decreases

slightly until the leaf reaches its maximum size, and then remains steady for a period of

time (Steer, 1971). Hall (1977) reported that the photosynthetic rate of pepper leaves

that expand during or immediately after anthesis in fruiting plants remains steady

throughout fruit growth. Older leaves generally exhibit lower photosynthetic rates

compared to younger leaves (Steer and Pearson, 1976). In tomato, Bolanos and Hsiao

(1991) reported that maximum leaf photosynthesis was reached near the time of full

expansion and declined steadily thereafter. Also in tomato, Xu et al. (1997) found that









net photosynthesis of the 10th, 15th and 18th leaves from the apex were only 50, 21

and 7% of that of the 5th leaf, and suggested that leaves below the 18th node can be

eliminated to improve air circulation and reduce humidity within the crop without

affecting C02 fixation. Photosaturation may also differ with leaf age, where old leaves

photosaturate at lower light intensities. In addition, the surplus of photosynthates from

young, upper leaves may depress the photosynthetic activity of old leaves (Tanaka et

al., 1974a).

c) Developing fruits. Developing fruits may affect plant photosynthesis by

decreasing leaf area and/or increasing leaf photosynthetic rates. Compared with non-

fruiting plants, plants with developing fruits often have reduced total leaf area (due to

fewer and smaller leaves) and increased life span of functional leaves (Hall, 1977;

Klaring et al., 1996). Decreases in leaf area of fruiting pepper plants compared to non-

fruiting plants were significant as soon as 2 weeks after anthesis (Hall, 1977; Bhatt and

Srinivasa-Rao, 1989). By 80 DAA (2 weeks after maturation of first fruit), functional leaf

area in fruiting plants was about 50% that of non-fruiting plants. However, in non-fruiting

plants, leaf senescence and abscission rates were seven-fold or more greater than in

fruiting plants (Hall, 1977). This phenomenon is not seen in all pepper cultivars. Bhatt

and Srinivasa-Rao (1997) reported no differences in total leaf area in bell pepper with

different fruit loads.

Developing fruits may also affect photosynthesis by increasing leaf CO2

assimilation rates or by causing a partial recovery of the leaf photosynthesis rate in

older leaves (Hall and Brady, 1977). Leaf C02 assimilation rates may increase up to

65% in fruiting plants compared to non-fruiting plants (Hall and Brady, 1977; Nilwik,









1980; Bhatt and Srinivasa-Rao, 1989), or in the same plants before and after fruit set

and development (Cruz-Huerta et al., 2005). In addition, removing pepper fruits during

rapid growth reduced the rate of C02 assimilation by up to 30% in the leaves close to

the fruits, mainly due to changes in both the leaf and the intracellular resistance (Hall

and Milthorpe, 1978). However, removal of the first two flowers or fruits in pepper plants

had no consistent effect on the photosynthetic rates of fully expanded and exposed

leaves (Bhatt and Srinivasa-Rao, 1997). Bertin et al. (2001) reported that decreasing

sink competition during tomato fruit cell elongation decreased fruit dry matter content,

suggesting that reduced sink strength inhibited leaf photosynthetic rates.

d) Light. Light is one of the most important factors in plant productivity

(Papadopoulos and Pararajasingham, 1997). It is generally accepted that most C3

species photosaturate between 500 and 1000 PPF (full sun light intensity is ~2000 PPF)

(Taiz and Zeiger, 2006); however, there is great variation, even within species. For

example, Xu et al. (1997) found that young tomato leaves reached their maximum

photosynthetic rate between 750 and 800 pmol m-2 s-1 while older leaves saturated at

about 300 pmol m-2 s-1. In pepper, E-Jaimez et al. (2005) reported that light saturation in

bell pepper grown in a greenhouse occurred ~ 1500 pmol m-2 s-1 PPF. Decreasing light

intensity from 1800 to 1000 pmol m-2 s-1 PPF decreased photosynthesis ~20% on sunny

days; however photosynthesis decreased ~40% on cloudy days due to higher stomatal

resistance (E-Jaimez et al., 2005).

Photoperiod also affects plant productivity. Some Capsicum annuum genotypes

have the ability to acclimate to supplementary light, even to continuous light, by

increasing chlorophyll content (Murage and Masuda, 1997), carboxylation capacity,









electron transport capacity, and the light saturation point (Dorais et al., 1995). Demers

et al. (1998) reported that continuous light increased plant growth (fresh and dry basis)

and yield for the first five weeks in pepper compared to a 14 h photoperiod; however,

after five weeks, growth under continuous light decreased,. The authors suggested that

the negative effects of continuous light on plant growth were due to a limitation in

photosynthate export, as evidenced by high leaf starch content and leaf deformation.

However, Dorais et al. (1996), reported that after 15 weeks of growing bell pepper under

extended photoperiod (18 h) or continuous lighting, fruit yield increased compared with

plants grown under 8 or 12 h photoperiods, indicating increased photosynthate

allocation to fruits.

e) Temperature. Temperatures above or below the optimal range for

photosynthesis reduce net photosynthesis and alter biomass partitioning, favoring

vegetative over reproductive growth (Ahmed et al., 1993; Wang et al., 1997; Diczbalis

and Menzel, 1998). Temperature effects on photosynthesis depend on several factors,

including the plant organ exposed (root vs shoot), the time of the day (day vs night), and

the duration of the event.

Some species tolerate root temperatures up to 35C without decreasing

photosynthetic rates (Du and Tachibana, 1994; Nada et al., 2003); however, root

temperatures above 38C generally result in severe decreases in photosynthesis (Du

and Tachibana, 1994; Diczbalis and Menzel, 1998; Nada et al., 2003). The reduction in

photosynthesis is initially caused by stomatal closure triggered by water stress (He et

al., 2001) or abscisic acid (ABA) accumulation (Nada et al., 2003). Other physiological

disorders, including impaired activation of Rubisco and decreased chlorophyll content,









also play an important role under prolonged high root temperatures (Du and Tachibana,

1994; Nada et al., 2003).

Variable root temperatures (from 23C to 40C) decreased photosynthesis in

lettuce (He et al., 2001) and dry matter accumulation in pepper (Dodd et al., 2000),

However, Gosselin and Trudel (1986) reported that maximum dry weight in pepper was

found at root temperatures of 24C, while maximum photosynthesis occurred at root

temperatures of 36C. In tropical rambutan (Nephelium lappaceum), maximum

photosynthetic rates occurred at 28C root temperature, and photosynthesis decreased

significantly at lower (15C) or higher (38C) root temperatures (Diczbalis and Menzel,

1998).

Excessively high shoot temperatures decrease photosynthesis and increase

respiration, reducing carbohydrate availability (Taiz and Zeiger, 2006). Decreases in

photosynthetic rates in C3 plants are often attributed to stomatal closure (Taiz and

Zeiger, 2006), followed by the inactivation of Rubisco and/or denaturation of Rubisco

activase, which facilitates the carbamylation and maintenance of Rubisco activity

(Hendrickson et al., 2008, and references there in).

Low temperatures also reduce shoot photosynthesis. This effect is more serious in

chilling-sensitive species, such as cucumber, tomato, and sweet pepper (Li et al.,

2003b). Studies in cucumber, sweet pepper (Li et al., 2003b), and Arabidopsis (Zhang

and Scheller, 2004) showed that low temperature (4C) combined with low light intensity

(100-150 PPF) damaged PSI without damaging PSII. However, cold temperatures and

high irradiances damaged PSII (Liu et al., 2001). Pepper plants that were cold-hardened









(5 d at 14C and 250 PPF) were more tolerant to chilling stress, but tolerance was only

expressed by a faster recovery (Liu et al., 2001).

Night shoot temperature also affects photosynthesis. Night temperatures between

15 and 25 C decrease photosynthesis in grapevine due to a delay in stomatal opening

the next morning, although stomatal conductance recovers as temperatures increase

during the day (Hendrickson et al., 2004a; Hendrickson et al., 2004b). Low night

temperatures, especially below 10C, may also decrease photosynthesis by decreasing

ribulose-1,5-bisphosphate (RuBP) regeneration and Pi availability for recycling

(Hendrickson et al., 2004a; Hendrickson et al., 2004b), reducing activity of PSII (Sundar

and Reddy, 2000; Bertamini et al., 2005), and decreasing leaf chlorophyll content

(Sundar and Reddy, 2000; Bertamini et al., 2005). Reduction in Rubisco amount and

activity, as well as other photosynthetic/carbohydrate metabolizing enzymes, such as

fructose bisphosphatase (FBPase), and sucrose phosphate synthase, may also occur in

response to low night temperature (Sundar and Reddy, 2000; Ramalho et al., 2003;

Hendrickson et al., 2004a; Hendrickson et al., 2004b; Bertamini et al., 2005), However,

species/cultivars tolerant to low night temperatures increase the activity of Rubisco and

other photosynthetic enzymes, including phosphoenolpyruvate carboxylase, NADP-

malate dehydrogenase, NADP-malic enzyme, and stromal FBPase (Du et al., 1999).

In several tropical crops, including sugar cane, bean, soybean, coffee, maize, and

peanut (Wolfe, 1991; Ying et al., 2000, and references there in; Ramalho et al., 2003;

Wang et al., 2004), one cold night may decrease photosynthetic rates 5 to 80%. Sweet

pepper seems to be very sensitive to low night temperatures. Bhatt and Srinivasa-Rao

(1993b) reported that decreasing night temperatures from 22 to 17C decreased net









C02 assimilation rates up to 30%. During maize grain-filling, one night of exposure to

4C decreased the carbon exchange rate 14 to 30%, depending on the genotype (Ying

et al., 2000). Greater reductions were found when maize plants were exposed to two or

three cold nights at 4C. Maintaining plants in the dark at 15 to 180C for an hour before

light exposure reduced the negative effect of cold night temperature on maize

photosynthesis (Ying et al., 2002). The reduction in maize leaf photosynthesis after cold

exposure was linearly related to the incident PPF level the next day, but fluorescence

and C02 exchange readings suggest that the reduction in photosynthesis is not

associated with photoinhibition (Ying et al., 2002). Similarly, Hendrickson et al. (2004a;

2004b) found that chilling-induced reduction in grapevine photosynthesis was not due to

photoinhibition. Initial inhibition appeared to be related to stomatal closure, followed by a

biochemical restriction due to limitation of RuBP regeneration and sucrose and starch

synthesis limitation (end-product limitation) (Hendrickson et al., 2004a; 2004b).

Some species have the ability to acclimate to low temperatures. In such cases,

exposure to low temperatures for long period (weeks) markedly increased the maximum

activities of Rubisco, stromal and cytosolic fructose-1,6-bisphosphatase, and sucrose-

phosphate synthase, and increased RuBP regeneration and carboxylation in leaves of

several species, leading to significant increases in whole plant photosynthetic capacity

(Holaday et al., 1992; Hurry et al., 1994; Du et al., 1999; Yamori et al., 2005). Although

net photosynthesis in Parthenium argentatum initially decreased when night

temperature was decreased from 20C to 15C, photosynthetic rates began to recover

after 30 days of low night temperatures, reaching values equal to or higher than initial

values (Sundar and Reddy, 2000). In cotton, Singh et al. (2005) reported that after 30









days of growing plants under decreasing night temperatures from 16 to 11 C (at day

temperatures of 28C), C02 assimilation, stomatal conductance, carboxylation capacity,

electron transport capacity, and triose-phosphate utilization capacity were similar

between leaves developed under low night temperature and leaves developed on

control plants (28/24C day/night). Mercado et al. (1997a) reported that sweet pepper

plants grown under low night temperatures (14 C) showed improved chilling resistance

upon exposure to night temperatures of 6 C compared with plants grown under higher

night temperatures (20 C).

Abnormal Flower and Fruit Development

In pepper, as in most horticultural crops, yield of high quality fruit is as important

as total yield. Fruit weight, size, shape, firmness, color, and disease/pest incidence are

used to evaluate pepper fruit quality (Aloni et al., 1999; Navarro et al., 2002; Kissinger

et al., 2005; Lim et al., 2007). Both final fruit size and shape in pepper are directly

affected by early flower and fruit development (Ali and Kelly, 1992; Aloni et al., 1999).

Early development, in turn, is very sensitive to several factors, including ambient

temperature and carbohydrate availability in the plant.

High temperatures induce flower abortion and impair pollen fertility in several

crops, including tomato (Bertin, 1995; Sato et al., 2001; Sato et al., 2002), pepper

(Polowick and Sawhney, 1985; Turner and Wien, 1994a; Aloni et al., 2001), and

Brassica napus (Young et al., 2004). In tomato and pepper, high temperature stress

also reduces pollen grain number and germination (Aloni et al., 2001; Sato et al., 2004;

Sato and Peet, 2005). In pepper, additional morphological changes are associated with

high temperatures. Day/night temperatures of 28/23C decreased ovary diameter and

increased style length compared to a day/night temperatures of 23/180C (Polowick and









Sawhney, 1985). High temperature after anthesis also reduces fruit and seed growth

and seed quality in chili pepper (Pagamas and Nawata, 2008).

Low temperatures also induce abnormal flower and fruit development in bell

pepper. In winter, cooler temperatures and shorter day lengths increase the percentage

of small, flattened parthenocarpic fruits. Pollen fertility is the main reason for this

condition, but female organs (Polowick and Sawhney, 1985; Pressman et al., 1998a;

Shaked et al., 2004) and other parts of the flower (Aloni et al., 1999) are also affected.

Night temperatures of 15C or less (day temperature of 20 to 25C) increase ovary

diameter in pepper without increasing locule number, creating "swollen" ovaries and

malformed fruit (Polowick and Sawhney, 1985; Rylski, 1985; Bakker, 1989b; Aloni et al.,

1999; Shaked et al., 2004). Low night temperatures also produce abnormalities in the

petal and stamen, and reduce style length. The longer the duration of low night

temperatures, the higher the percentage of swollen flowers and ovaries (Polowick and

Sawhney, 1985; Aloni et al., 1999). Aloni et al. (1999) found that 15 days at night

temperatures of 12 or 180C resulted in 21 and 7% swollen ovaries, respectively. By 49

days, these percentages increased to 78 and 14%, respectively. Cell volume in swollen

ovaries is greater than that in normal ovaries, but cell number remains the same

(Pressman et al., 1998b) suggesting that swollen ovaries develop after cell division

ends.

Although low night air temperatures result in swollen pepper ovaries and

malformed fruit, low night root temperatures do not appear to have the same effect.

Pepper plants grown at night air temperatures of 12C produced low fertility pollen and

small fruits at root night temperatures of either 120 or 20C (Mercado et al., 1997c; Aloni









et al., 1999). These findings suggest that flower deformation and ovary swelling are

favored by low air temperature during the night, regardless of root temperature.

Very high day temperature may reduce negative effects that low night

temperatures have on final fruit shape and size in pepper. Pressman et al. (2006) found

that increasing the day temperature to ~ 35C compensated for some of the deleterious

effects of low night temperature on vegetative and reproductive development,

particularly on stamen and pollen development. However, no information on the effects

of these high day temperatures on ovary development was reported.

Excess carbohydrate availability during early flowering/fruiting also results in

abnormal flower and fruit development in pepper. Increased source/sink ratio by fruit

removal increases the proportion of swollen ovaries (27 mm diameter) compared with

fruiting plants (Aloni et al., 1999). Fresh weight of swollen flowers that developed in

defruited plants was three to four times that of the flowers on fruiting plants (Aloni et al.,

1999). In addition, the percentage of swollen flowers was inversely related to the

number of growing fruits on the plant, and directly proportional to the concentration

(fresh weight basis) of reducing sugars and starch in the flowers developed in those

plants (Aloni et al., 1999). Flowers that develop on defruited plants accumulated four-

fold more 14C02 (600 vs 150 pg d-1) and fresh weight (180 vs 60 mg) than flowers that

develop on fruiting plants (Aloni et al., 1991b; Aloni et al., 1999). These data suggest

that excess assimilates are transported to flower buds on defruited plants, resulting in

ovary swelling and deformation (Aloni et al., 1999).

In summary, flower and ovary swelling in pepper is favored by exposing the shoot

to low night temperature or by removing actively growing fruits. However, the









physiological changes in the plant and flower buds that induce such swelling and

deformation are still not clear.




















Figure 2-1. Schematic drawing of pepper branching and flowering sequence. A)
Meristematic stage. B) Well developed branch. A sympodial shoot bears a
leaf (L1, blue), and terminates in a flower (F2). From the leaf axil, two leaves
(L2) develop. The flower and the two leaves developed from the same
sympodial shoot compose a sympodial unit (F2 and L2, green). L2 leaves are
'pushed up' by internode elongation, so that the leaves appear above the
preceding flower. The shoot terminates in a flower, which is part of the
subsequent sympodial unit. From each of the L2 axils, two new leaves (L3,
red) develop. Each color represents sympodial units of a different order
(Drawing A based on micrograph from Elitzur et al., 2009).











SU2


Figure 2-2. Schematic diagram of a pepper plant. A) Pepper plant has a main stem with
cotyledonary (C) and true leaves (L, yellow). The main stem ends in a
terminal flower (FTF, first terminal flower, brown circle). The terminal flower
and the first two leaves above it (brown) originate from the main stem and
form the main stem sympodial unit. Each new sympodial unit is also
composed of two leaves and a flower (circle), forming sympodial unit 1 (SU),
2 (SU2), and so forth. B) Asymmetric growth of the sympodial components
results in different plant architecture.


Figure 2-3. Pepper fruit and its parts (Modified from Rylski, 1986).









CHAPTER 3
LOW NIGHT TEMPERATURE INCREASES OVARY SIZE IN SWEET PEPPER

Introduction

Pepper (Capsicum annuum L) is an important vegetable crop that belongs to the

Solanaceae family, and whose edible part is botanically a fruit. Historically, peppers

have been grown in the tropics as well as in temperate climates (Eshbaugh, 1993). Hot

peppers are consumed fresh, dried or processed, mostly as spice, or as a source of

food colorants or capsaicin. Sweet peppers, a genetic recessive non-pungent form, are

an important green vegetable crop worldwide, especially in temperate regions

(Eshbaugh, 1993; Bosland, 1996). There is large variability in sweet pepper fruit size

and shape, ranging from cherry (spherical, ~2.5 to 3.5 cm in diameter), to elongated

(banana or cubanelle), to bell (blocky) peppers, which are the most important from an

economic standpoint (Bosland, 1996; Bosland, 1999). In the U.S., per capital

consumption of bell peppers increased from 1 kg in 1970 to ~3 kg in the late 1990s and

has remained steady during the last 10 years (USDA Economic Research Service,

2008).

Pepper plants develop a main stem that bears leaves on the first 8 to 15 nodes

and terminates in a flower. The two or three nodes preceding the terminal flower bear

sympodial shoots (first lateral branches) (Child, 1979; Elitzur et al., 2009). Each

sympodial shoot has a leaf, and terminates in a flower. Two new leaves emerge from

the leaf axil below the floral terminus, and are "pushed" above the terminal flower by

elongation of the new internode (Figure 3-1). From every sympodial shoot, a new

sympodial unit is formed, consisting of the two sympodial shoots, the flower in the

vertex of the shoots (i.e. the terminal flower from the preceding shoot), and the single









leaf at the distal end of each sympodial shoot (i.e. two leaves, due to the presence of

two sympodial shots). This cycle of development repeats itself and could, in theory,

continue for an indefinite period of time (Elitzur et al., 2009). Thus, flowering and fruiting

in pepper is continuous as long as plants are growing.

Fruit shape, size and regularity are important quality components in sweet

peppers. Final fruit shape is the result of genetic and environmental factors (Perin et al.,

2002; Weiss et al., 2005), and it is defined in the early stages of fruit development

(Munting, 1974). In bell, spherical, and ovoid fruited pepper varieties, shape definition

takes place before pollination occurs (Munting, 1974; Aloni et al., 1999; Meijer and

Murray, 2001). However, in long-fruited varieties, fruit shape is defined during both the

preanthesis and postanthesis stages (Munting, 1974).In all genotypes, pollination and

fertilization are key factors in attaining the ideal size, shape and uniformity (Mercado et

al., 1997c). Under optimal growth conditions, bell pepper fruit requires 150-300 seeds to

develop the uniform blocky shape (Aloni et al., 1999). After fertilization and fruit set in

bell pepper, changes in assimilate supply can change the growth rate and therefore the

size, but do not affect fruit shape (Aloni et al., 1999).

Sweet peppers, as with other fresh fruits and vegetables, are a commodity in

demand all year. In order to fill that market need, sweet pepper production during the

winter has been developed in areas with warm or mild winters or even in cold winters

under greenhouse conditions. However, when grown in cold winters under greenhouse

conditions, bell peppers can develop up to 60% of flattened and deformed fruits, and

thus, unmarketable (Rylski, 1973; Ali and Kelly, 1993; Aloni et al., 1999).









Several reports have previously addressed the effect of low night temperature

(LNT) on flower and fruit development and malformation in sweet pepper. However,

they have mainly focused on pollen quality (Mercado et al., 1997b; Pressman et al.,

1998a; Shaked et al., 2004), pollination (Mercado et al., 1997c; Pressman et al.,

1998a), number of seeds (Rylski, 1973; Kato, 1989), and fruit quality (Rylski, 1973;

Kato, 1989; Pressman et al., 2006). Only a few studies have focused on flower and

ovary malformation and swelling, and these studies have been limited to effects in bell

pepper (Pressman et al., 1998b; Aloni et al., 1999). Flower deformations due to LNT

include abnormal and curled petals that do not fully expand, shorter stamens with low

pollen content and low germinability, and larger ovaries with shorter styles compared

with flowers developed under intermediate or high temperatures (Polowick and

Sawhney, 1985; Rylski, 1985; Mercado et al., 1997b; Pressman et al., 1998a; Aloni et

al., 1999; Shaked et al., 2004). The ovary enlargement observed under cool

temperatures is referred to as ovary swelling (Pressman et al., 1998b; Aloni et al.,

1999). Ovaries developed under LNT are impaired in fruit development, even when

pollinated with viable pollen, and result in smaller, irregularly shaped fruit compared with

fruit developed under higher night temperatures (Pressman et al., 1998a).

In addition to LNT, high source:sink ratio can also result in swollen ovaries in bell

pepper (Aloni et al., 1999). High source:sink ratio can be caused by harvesting all the

fruits or by a massive flower drop. When bell pepper plants bearing several fruits in

different stages were artificially defruited, 90% of the flowers that reached anthesis 15

days after defruiting were swollen, compared to 20% in the non-defruited control.

Although partial defoliation of completely defruited plants did not reduce ovary swelling,









some reduction in ovary swelling was observed in subsequently formed flowers when

two developing fruits were allowed to remain on the plant (Aloni et al., 1999).

Although the effects of LNT and high source:sink ratios on ovary swelling in bell

peppers are documented, LNT and high source:sink ratio effects in other types of sweet

peppers are unknown. In addition, in pepper cultivars with blocky or spherical fruit,

ovary shape and cell number are determined in the preanthesis stages; however, in

long-fruited cultivars, cell number and ovary shape are modified in the postanthesis

stages (Munting, 1974). Thus, LNT and/or source sink ratio might affect ovary shape

differently depending on the final fruit shape. Therefore, the hypothesis tested in this

work is that low night temperature increases ovary size and causes ovary swelling in a

range of pepper cultivars differing in final fruit shape and size, but low source:sink ratio

can counteract the effect of LNT on ovary swelling. The objectives were to determine

the effects of LNT and two source sink ratios on ovary size in different sweet pepper

cultivars and to determine the parts of the ovary that are most affected by LNT.

Material and Methods

Plant Material and Growing Conditions

Seedlings of six (Experiment 1) or four (Experiment 2) sweet pepper varieties were

germinated using a mix of commercial potting media (SunGro Metro-Mix Ag-Lite Mix1)

and perlite (4:1 v:v) in 40-cell flats (60 cm3 each cell) in 1.4 m2 growth chambers (E15

Conviron, Winnipeg, Canada). Chamber temperature was set to 22/200C (day/night)

and PPF was maintained between 450-500 pmol m-2 s-1 above the canopy with a 14-




1 Composition: Canadian Sphagnum peat moss (55-65%), horticultural grade vermiculite, dolomite
limestone, starter nutrient charge, and wetting agents (www.sungro.com/products.php, 12/17/2008).









hour photoperiod. Light was provided by fluorescent and incandescent lights. There was

no control of CO2 concentration or relative humidity.

Plants were watered with tap water until the beginning of expansion of the first true

leaf (4 weeks after seeding), when fertigation began. The fertigation protocol differed

with the experiment and is described below. Pests were controlled by using a

combination of biological control (wasps for aphids) and chemical control. Insecticidal

soap (Safer@ Brand Insect Killing Soap, Safer Brand, www.saferbrand.com,

12/17/2008), dicofol (Dicofol 4E, Dicofol 42%, Makhteshim Agan of North America, NY,

USA), or abamectin (Agri-Mek 0.15EC, abamectin 2%, Syngenta Crop Protection,

Greensboro, NC, USA) were sprayed as needed at one-half the lowest recommended

dosage until seedlings were transplanted to containers.

Seedlings at about the sixth-leaf stage (54 days after sowing) were transplanted

into 1.5 L containers using the same media mix. Plants were continuously pruned once

branching began. At the first branching point, two shoots were allowed to grow, forming

the main stem sympodial unit. For the subsequent sympodial units in Experiment 1, the

strongest shoot was allowed to grow (contributing to the main axis of the plant) and the

other shoot was allowed to develop one sympodial unit (forming a lateral branch) before

pruning (Figure 3-2A). In Experiment 2, one-half of the plants were pruned as in Figure

3-2A and the other half were pruned so that the less strong shoot was allowed to

develop only the flower of the next sympodial unit before pruning (Figure 3-2B).

Sympodial units formed on the main stem sympodial shoots were numbered SU1

(sympodial unit 1), which in turn bore SU2. Subsequent sympodial units were numbered

3, 4, ..., n (see Elitzur et al., 2009). For the purpose of this study, nodes above the first









branching point were also numbered according to their sympodial unit in order to

describe each flower's position, i.e., node 1 (corresponding to SU1), node 2, node 3,

etc. Flowers from the main stem and node 1 (i.e. SU1) were removed at the flower bud

stage.

Experiment 1

This experiment was designed to determine the response of six cultivars of sweet

pepper to low night temperature. Four blocky bell-type pepper cultivars ('Ariane',

'Aristotle' with X3R, 'Brigadier', 'Legionnaire'), one elongated-fruit cultivar ('Banana

Supreme', length:diameter ratio ~4), and one cherry-type cultivar ('Red Cherry Sweet',

rounded, 2.5-3.7 cm diameter) were exposed to day temperatures of 220C and one of

two night temperatures: 200 (high night temperature, HNT) or 120C (low night

temperature, LNT). Several cultivars of bell pepper cultivars allowed to test differences

among bell pepper cultivars, since previous work on ovary swelling as a response to

night temperature had been done mostly in 'Mazurka' cultivar (Pressman et al., 1998a;

Pressman et al., 1998b; Aloni et al., 1999). Plants were fertigated starting at about 6

weeks after seeding with 20N:6.7P:16.7K water soluble fertilizer (Scotts-Sierra

Horticultural Products Company, Marysville, OH, www.scottsprohort.com). Application

rates were ~1 mg N per plant per day before transplanting (54 days after sowing) and

from 15 to 50 mg N per plant per day after transplanting, depending on plant age.

Starting at 7 weeks after transplanting, plants were fertigated as needed with the

following nutrient solution (mM): 3.4 Ca(NO3)2, 1.8 KNO3, 1.6 KH2PO4, 0.3 KCI, 2.7

MgSO4, and (pM): 50.2 Fe-EDTA, 3.1 CuSO4, 14.6 MnSO4, 64.8 H3BO3, 4.6 ZnSO4, 0.6

Na2MoO4 (modified from Cruz-Huerta et al., 2005; and Jovicich, 2007). Plants were

watered in the morning to increase root-zone temperature in the LNT treatment (Ying et









al., 2002) so that day temperature was similar in the root and shoot in both LNT and

HNT. Three plants per cultivar were randomly distributed in each growth chamber.

Night temperature treatments began just before anthesis of flowers in node 3

('Ariane' and 'Aristotle'), 4 ('Brigadier', 'Legionnaire' and 'Banana Supreme') or 5 ('Red

Cherry Sweet'). Once treatments started, all the flowers at anthesis were continuously

harvested during a 39-day period in 'Brigadier', 'Legionnaire', 'Banana Supreme' and

'Red Cherry Sweet' and a 64-day period in 'Ariane' and 'Aristotle'. The flower harvest

period was prolonged in these latter two cultivars due to a delay in their growth and

development after transplanting. Plants did not bear any fruit before and during the

experiment. Flower harvests occurred every 2-3 days during the first 50 days and every

day during the last 13 days. A minimum of 66 flowers per night temperature and cultivar

combination were harvested. The flower position in the plant (i.e., node number above

first terminal flower and whether it was in the main axis or in a lateral branch) was

recorded. Ovary fresh weight, volume (measured by water displacement), diameter and

length were recorded. Ovaries were cut in half and ovary wall thickness was also

measured.

Experiment 2

Based on the results from the first experiment, four cultivars, 'Ariane',

'Legionnaire', 'Banana Supreme', and 'Red Cherry Sweet', were selected for

Experiment 2. The criteria used to select the cultivars were 1) contrasting responses to

LNT in bell peppers ('Ariane' and 'Legionnaire'), and 2) contrasting fruit shape and size

(bell, elongated and cherry-fruited varieties). Two night temperatures regimes, high

(HNT, 20C) and low (LNT, 120C), and two levels of source supply (high and low) were

studied. Source supply was modified either by leaving the complete sympodial unit on









every lateral shoot (Figure 3-2A), or removing the leaves and leaving only the flower

(Figure 3-2B). Therefore, starting in sympodial unit 2, high source supply treatment had

twice as many leaves as the low source supply treatment did., i.e., 8 vs. 4 leaves per

sympodial unit, as shown in Figure 3-2.

Each growth chamber was divided into 3 blocks (based on variation in light

intensity within the growth chamber observed in Experiment 1) and within each block, 8

plants were randomly placed (4 cultivars and 2 levels of source supply), with one plant

per treatment using a completely randomized block design. Treatments began just

before anthesis of flowers on the second sympodial unit (node 2). The first terminal

flower and flowers from node 1 were removed.

Flowers were harvested every day at anthesis for up to 45 days, from the second

to the seventh nodes above the first terminal flower. As in Experiment 1, plants did not

bear any fruit. The position of every harvested flower in the plant was recorded. Ovary

fresh weight (FW), volume, diameter, and length were recorded. Ovary wall thickness

and FW were also measured, as was placenta FW.

Statistical Analysis

Data from Experiment 1 were grouped into five harvest intervals (Table 3-1). For

'Brigadier', 'Legionnaire', 'Banana Supreme', and 'Red Cherry Sweet', harvest intervals

were 8 days, and for 'Ariane' and 'Aristotle', harvest intervals were 13 days, since

development was delayed in these two cultivars. Data were analyzed as a mixed model

with repeated measures using cultivar, night temperature and harvest period as main

factors, axis (i.e. main axis or lateral branch) nested within each plant as the repeated

measures subject, and days after anthesis as the covariate.









Experiment 2 originally included three main factors (4 cultivars, 2 temperature

regimes, 2 levels of source supply). Additionally, node number (number of nodes

counted above the first flowering node) and the axis within the plant were included in

the model during the analysis. After the initial statistical analysis, it was found that the

source supply did not significantly affect the parameters evaluated, either as main or

interactive effects. Thus, this factor was not considered in the final analysis.

Data from experiment 2 were also analyzed as a mixed model with repeated

measures, using temperature and cultivar as the factors of the main model, and the

node number and axis (main axis, lateral branch) of the harvested flower as

components of the sub-model. The subject for the repeated measures was an individual

plant.

Data were analyzed using SAS (SAS Institute Inc., 2008), and mean separation

was done using Tukey-Kramer (multiple LSmeans) or t-test (two means). Multiple

LSMeans letter grouping was done using a SAS macro developed by Saxton (1998).

Results

Experiment 1

Approximately 30 flowers per plant reached anthesis and were harvested during

the experiment, with no significant difference in the number of flowers among cultivars

(Table 3-2). Low night temperature (LNT) decreased the number of flowers compared

with high night temperature (HNT) (27 vs 33 flowers, respectively). Within each cultivar,

temperature reduced the number of flowers only in 'Ariane' and 'Legionnaire' (P<0.04).

The decrease in flowers harvested at LNT compared with HNT was accompanied by a

concomitant decrease in number of nodes under LNT (Table 3-2).









All ovary parameters measured were affected by temperature, cultivar, and

harvest interval as main effects. Interaction of temperature with cultivar and harvest

interval was significant in most cases, except for ovary wall thickness and fresh

weight:volume ratio (Table A-1, Appendix). For fresh weight and volume, all interactions

were significant, whereas for the rest of the variables, one or more interactions were

non-significant. The covariate days after anthesis (DAA) and the repeating subject

component (branch within each plant) were also significant for most of the cases.

Ovary FW, volume, diameter, length, and ovary wall thickness were greater in

flowers developed under LNT compared with flowers developed under HNT (Table 3-3).

However, shape was not affected, as indicated by the length:diameter ratio.

There were significant differences among cultivars in ovary size (Table 3-4).

Blocky varieties (bell peppers 'Ariane', 'Aristotle', 'Brigadier', and 'Legionnaire')

developed larger ovaries than the cherry ('Red Sweet Cherry') or elongated ('Banana

Supreme') cultivars. Within the bell pepper types, 'Ariane' and 'Aristotle' developed

smaller ovaries than 'Legionnaire', possibly due to the delayed growth. Differences in

ovary volume and diameter were similar to those in ovary FW; however, length in some

blocky cultivars ('Aristotle' and 'Brigadier') was less than in the non-blocky cultivars

('Banana Supreme' and 'Red Cherry Sweet'), resulting in significant differences in the

length:diameter ratio (0.70 vs 1.1, respectively). Ovary wall thickness was greater in

cherry and elongated-fruited cultivars compared with bell-fruited cultivars.

Ovary fresh weight and volume of flowers harvested at anthesis remained

constant during the first two harvest intervals (i.e., 16 DAT 'Brigadier', 'Legionnaire',

'Banana Supreme' and 'Red Cherry Sweet' and 26 days in 'Ariane' and 'Aristotle', see









Table 3-1 for further details) (Table 3-5), which generally corresponded to the first and

second nodes harvested (Figure A-1, Appendix). Subsequently, ovary FW increased

significantly at a rate of ~6 mg per harvest interval, and ovary volume increased at ~7

mm3 per interval. Similar patterns were observed with diameter and length. Ovary wall

thickness, however, decreased initially, then increased, but did not reach the initial

values. Ovary shape at anthesis (as indicated by the length:diameter ratio) remained

constant during the first four harvest intervals, but was more elongated in the final

interval.

There was a significant interaction between temperature and cultivar on ovary

fresh weight, volume, diameter, length and length:diameter ratio (Table 3-6). When

comparing the average effect of the night temperature treatment on ovary

characteristics, significant differences were detected only in 'Aristotle' and 'Legionnaire'.

However, when only the flowers harvested during the last two harvest intervals of the

experiment (harvest intervals IV and V) are considered, significant differences were

found in 'Aristotle', 'Legionnaire', 'Brigadier', and 'Banana Supreme' (Table 3-6).

The relationship between ovary fresh weight and volume was linear (volume =

b-FW) for every combination of cultivar and temperature. In general, the slopes (b)

ranged from 1.04 to 1.09 in bell peppers, slightly less than 1.00 in 'Red Cherry Sweet',

with intermediate values for 'Banana Supreme' (Table 3-7). Thus, 'Red Sweet Cherry'

ovaries were denser than ovaries of bell pepper cultivars.

There was a significant three-way interaction (temperature x cultivar x harvest

interval) on ovary characteristics (Table A-1). There was no effect of night temperature

during the first three weeks of treatment (i.e., harvest intervals I and II for 'Aristotle' and









'Ariane' and I to Ill for 'Brigadier', 'Legionnaire', 'Banana Supreme', and Red Cherry

Sweet) in any cultivar (Figure 3-3). Subsequently, however, fresh weight of ovaries from

flowers at anthesis in three out of four bell pepper varieties ('Aristotle', 'Brigadier', and

'Legionnaire'), and in the long-fruited variety ('Banana Supreme') increased under LNT

compared with HNT. However, in this experiment, ovary FW of 'Ariane' (bell), and 'Red

Cherry Sweet' (cherry), did not significantly increase under LNT compared with HNT. In

general, three to four weeks of LNT were required to significantly increase ovary fresh

weight compared to HNT treated plants. In four out of six cultivars, ovary FW did not

change under HNT throughout the harvest intervals.

Experiment 2

Ovary characteristics of 'Ariane', 'Banana Supreme', 'Legionnaire', and 'Red

Cherry Sweet' were dependent on night temperature, cultivar, and their interaction

(Table A-2 in Appendix), but were not dependent on number of leaves at the node (i.e.

potential source supply differences), as indicated earlier.

Fresh weight of ovary, placenta and ovary wall were significantly greater in flowers

developed under LNT compared with those developed under HNT (Table 3-8). Ovary

diameter and length were 18% and 15% greater, respectively, under LNT compared

with HNT, while ovary wall thickness was 10% greater in ovaries developed under LNT.

Length:diameter and ovary wall fresh weight:total fresh weight ratios were similar in

both LNT and HNT.

The ovaries at anthesis were larger in the bell pepper types ('Ariane' and

'Legionnaire') than in the banana or cherry types (Table 3-9). In addition, there were

differences in the percent of the total ovary FW that was contributed by the ovary wall.

In 'Ariane' and 'Legionnaire', the ovary wall FW represented ~ 50% of the total FW,









while in 'Banana Supreme' and 'Red Cherry Sweet', the ovary wall represented ~65% of

the total FW. There was also a significant correlation between the ovary fresh weight

and ovary wall thickness for each genotype (r2 > 0.63 for all four genotypes; P<0.01).

In general, flowers from the lowest level nodes had the smallest ovaries, with

ovary FW increasing steadily from 35 mg in node 2 to 56 mg in node 7 (Table 3-10),

representing ~ a 60% increase. The ovary wall and placenta also increased by ~60%,

while the diameter and length increased by 18% and 15%, respectively, as node level

increased.

Interactions between night temperature and cultivar were significant for ovary,

ovary wall and placenta FW, and for ovary diameter, but not for ovary length (Table A-2

in Appendix). Over the course of the experiment, plants grown under LNT produced

flowers with greater ovary and ovary wall FW compared to plants grown under HNT.

The increase in ovary FW under LNT ranged from 50% in 'Banana Supreme' to 76% in

'Legionnaire' (Table 3-11). Similar results were obtained in ovary wall FW, where

increases ranged from 47% ('Red Cherry Sweet') to 85% ('Legionnaire'). However, LNT

increased placenta FW only for bell peppers ('Ariane' and 'Legionnaire'), not for the

banana or cherry cultivars. The ovaries from plants grown under LNT increased in both

diameter and length, except for the long-fruited cultivar, which increased only in length.

When data from nodes 5 to 7 only are considered, the increase in ovary, ovary wall, and

placenta FW under LNT is even more dramatic, resulting in greater ovary swelling under

LNT compared with HNT.

Although ovary size increased as node number increased (Table 3-10), there were

differences among cultivars and temperature treatments in this response (Figure 3-4).









Ovary size increased with node number in all cultivars under LNT, while ovary size

increased with node number under HNT only in 'Legionnaire'. 'Ariane' and 'Legionnaire'

(bell type) responded faster to night temperature treatments than 'Banana Supreme'

and 'Red Cherry Sweet'. In 'Legionnaire', for example, LNT significantly increased the

ovary size compared with HNT starting at node 2, which was harvested 8 days after the

beginning of treatment (DAT) (Table A-3, Appendix). In contrast, in 'Red Cherry Sweet',

significant differences between temperature treatments were not found until node 5 (20

DAT). In 'Ariane', differences in ovary FW due to night temperature treatments began by

node 4 (21 DAT, Table A-3, Appendix). However, maximum ovary swelling was reached

after node 5 for all cultivars (20 DAT in 'Red Cherry Sweet' to 31 DAT in 'Legionnaire').

Although ovary FW increased more than diameter and length at LNT compared

with HNT within each cultivar (Table 3-11), both diameter and length responded as

quickly as FW to night temperature treatments (Table 3-12). In fact, in 'Banana

Supreme' and 'Red Cherry Sweet', significant differences in length were found earlier

(i.e. node 4) than were differences in fresh weight and diameter (i.e. node 5).

Discussion

Most of the studies involving LNT on ovary and fruit development have been on

large-fruited pepper cultivars, including bell-type (Rylski, 1973; Polowick and Sawhney,

1985; Bhatt and Srinivasa-Rao, 1993b; Mercado et al., 1997b; Mercado et al., 1997c;

Pressman et al., 1998a; Pressman et al., 1998b; Aloni et al., 1999; Shaked et al., 2004).

Limited work has been done on small fruited sweet pepper cultivars (Kato, 1989), other

types of peppers such as cayenne and jalapeno (Shaked et al., 2004), or other species

of Capsicum (Mercado et al., 1997c), and these studies focused on pollen development

and fertility, but not on the ovary development.









In the present work, low night temperature increased ovary diameter and fresh

weight in bell pepper flowers, confirming previous results (Polowick and Sawhney,

1985; Pressman et al., 1998b; Aloni et al., 1999). Final ovary diameter of the bell

pepper cultivars used in the present experiments ranged from 5.7 to 6.8 mm under LNT,

and from 4.3 to 6.4 mm under HNT, and these values are within the ranges reported

previously (Pressman et al., 1998b; Aloni et al., 1999; Shaked et al., 2004). Ovary

swelling occurred in all bell, long-fruited and cherry pepper types under LNT. A previous

study (Shaked et al., 2004) reported that night temperatures of 10C increased ovary

diameter of sweet bell and hot long-fruited cayenne pepper flowers at anthesis

compared with ovaries developed at night temperatures of 20C; however, there was no

such effect of LNT on ovary diameter of jalapeno peppers. Our results indicate that

under LNT conditions, the increase in ovary fresh weight is proportionately greater than

the increase in ovary diameter or length. For example, in Experiment 2, there was a

61% increase in ovary FW and only a 15 to 18% increase in ovary diameter and length

under LNT compared with HNT. Most previous studies have measured night

temperature effects on ovary diameter, not fresh weight or length (Polowick and

Sawhney, 1985; Aloni et al., 1999). Our results suggest that LNT effects on ovary

swelling in peppers, and subsequent effects on fruit malformation, may be better

predicted by looking at effects on ovary FW in bell peppers. However, in long-fruited

cultivars such as 'Banana Supreme', ovary length may also be a good indicator of ovary

swelling, as length increased more rapidly than did FW or volume in response to LNT

compared with HNT.









Analysis of continuous harvests over time as a main effect, i.e. harvest intervals in

Experiment 1 and nodes in Experiment 2, revealed that flowers harvested at the

beginning of the experiments had smaller ovaries than the flowers harvested at the end

of the experiments (Table 3-5, Table 3-10). However, when data were analyzed by

cultivar and temperature treatment, there were no significant differences in ovary size

among harvest intervals (Figure 3-3) or nodes (Figure 3-4) in ovaries developed under

HNT, except for 'Ariane' and 'Aristotle' in Experiment 1 and 'Legionnaire' in Experiment

2. Thus, the increase in ovary size over time (harvest intervals or nodes), as observed

in the present work, is primarily due to the effect of LNT. In Experiment 1, 'Ariane' and

'Aristotle' were the two cultivars that suffered delayed growth when transplanted. It is

possible that the stress of transplanting reduced the carbohydrate availability in both

cultivars for a period of time after transplant, resulting in smaller flowers. Carvalho and

Heuvelink (2001) reported that increasing assimilate availability increased flower size in

chrysanthemum. In pepper, plants bearing growing fruits developed ovaries with thinner

ovary wall compared to the ovaries from plants without fruits, and the authors suggested

that assimilate availability limited ovary wall thickness in fruiting plants (Ali and Kelly,

1992). Although ovary size was not recorded by Ali and Kelly (1992), the high

correlation between ovary wall thickness and ovary fresh weight in our work (r2 = 0.63 to

0.70, P<0.001; data not shown) suggests a similar limitation in assimilate availability

may have occurred for 'Ariane' and 'Aristotle' in Experiment 1.

In general, sweet pepper cultivars developing under LNT required from three to

four weeks to develop swollen ovaries. In the main stem of bell pepper, floral initiation

occurs between the sixth and the seventh leaf and is independent of temperature (Choi









and Gerber, 1992). At day/night temperatures of 28/15C, it takes ~ 4 weeks from the

sixth leaf to anthesis of the first flower (Cruz-Huerta, 2001). This suggests that flower

buds must be exposed to LNT within the first week after flower bud initiation in order to

develop a large percentage of swollen ovaries. The exception to this was observed in

'Legionnaire' bell pepper in Experiment 2, in which significant increases in ovary size

occurred after ~ one week of LNT compared with HNT. Even so, ~30 days of LNT were

required for maximum increases in ovary size to be manifested in 'Legionnaire'.

Although a critical period for LNT exposure was revealed in our work (i.e. within

the first week after flower bud initiation), the duration of LNT exposure required for

development of swollen ovaries was not examined, as plants were exposed to low or

high night temperatures continuously throughout the experiments. It is clear; however,

that the percentage of flowers that exhibit ovary swelling increases as the duration of

the LNT exposure increases. This has been demonstrated previously in bell pepper

(Aloni et al., 1999) and reflects the continuous floral initiation nature of pepper.

Previous work on ovary swelling in pepper focused only on the whole ovary. Our

data show that the ovary wall FW: total ovary FW ratio varied among types of cultivars,

averaging ~65% for the non-bell pepper cultivars ('Banana Supreme' and 'Red Cherry

Sweet') and ~50% for the bell pepper cultivars ('Ariane' and 'Legionnaire'). However,

LNT did not change the ratios, indicating that the increase in ovary FW is proportionally

similar for ovary wall. The placenta FW, however, was affected by night temperature in

bell and cherry-type peppers, but not in long-fruited. Therefore, increased ovary FW in

bell and cherry-type peppers caused by LNT was due to increased ovary wall and

placenta FW, and in 'Banana Supreme', was mainly due to ovary wall increase.









Although LNT treatments increased ovary size in both experiments, there were

differences between experiments, as indicated above. Ovaries in Experiment 1 were

larger than in Experiment 2, regardless of cultivar or temperature treatments.

Differences in ovary size between experiments may in part be explained by harvest

frequency (every 3 days in Experiment 1 vs every day in Experiment 2), and in part by

plant density (13 and 17 plants/m2, for Experiments 1 and 2 respectively). Previous

studies have reported that higher plant densities produce smaller fruits (Cebula, 1995;

Cruz-Huerta et al., 2009) and that smaller fruits are correlated with smaller ovaries (Ali

and Kelly, 1992).Therefore it is likely that flowers developed at higher plant densities

had smaller ovaries. Relative response to LNT was also different between Experiments

1 and 2. In Experiment 1, ovary FW and volume were ~10 to 12% greater when

developed under LNT compared with those developed under HNT (Table 3-3) and

~20% greater when only the last two harvest intervals were considered (Table 3-6). In

Experiment 2, FW of ovaries developed under LNT were ~60% greater than that of

ovaries developed under HNT (Table 3-8) or greater when data from nodes 5 to 7 within

each cultivar were considered (Table 3-11). The smaller difference in ovary FW

between LNT and HNT in Experiment 1 may have been due to longer exposure of

ovaries to HNT before harvest, resulting in faster growth, as has been reported by

Rylski (1972). As indicated, ovaries in Experiment 1 were harvested every three days,

while those in Experiment 2 were harvested every day. Thus, ovaries in the HNT

treatment in Experiment 1 were exposed to a longer duration of high temperatures, and

likely grew faster than did ovaries under LNT, reducing the differences in ovary FW

between treatments. There was also a markedly slower response to LNT with respect to









increases in ovary FW with time in Experiment 1 compared with Experiment 2,

particularly in 'Legionnaire', 'Red Cherry Sweet', and 'Ariane'. This may again reflect the

larger ovary size and increased time between flower harvests in Experiment 1

compared with Experiment 2.

Although Aloni et al. (1999) reported that a high source:sink ratio also favors ovary

swelling in bell peppers, we did not find that in the present work. The difference in leaf

number we maintained (by pruning) may not have been great enough to result in a

significant difference in carbohydrate supply to ovaries. Alternatively, the high plant

density in Experiment 2 (17 plants/m2) may have resulted in sufficient leaf shading such

that increasing the number of leaves in a sympodial unit from four to eight did not

increase whole plant photosynthesis and therefore carbohydrate supply. It is unknown,

however, if under lower plant densities leaves developed under low night temperature

conditions acclimate and reach similar photosynthetic rates as leaves developed under

high night temperatures, as has been found in other species (Singh et al., 2005).

The most visible response of bell pepper to low night temperatures during the

preanthesis stages is deformed fruits caused by few or no seeds (Rylski, 1986;

Pressman et al., 1998a) and the formation of swollen ovaries (Rylski, 1972; Polowick

and Sawhney, 1985; Pressman et al., 1998b; Aloni et al., 1999; Shaked et al., 2004).

The absence of seed or reduction in seed number is caused primarily by lack of

successful pollination/fertilization (Kato, 1989; Mercado et al., 1997b; Mercado et al.,

1997c; Shaked et al., 2004). Shaked et al. (2004) and Pressman et al. (2006) reported

that LNT during the 4 days prior to anthesis in bell peppers reduced pollen carbohydrate

concentrations, thereby decreasing pollen viability and germinability. This may explain









why in commercial operations even 2 or 3 nights of low temperature causes deformation

of the fruits developed from flowers that were exposed to LNT prior to anthesis (E.

Jovicich, personal communication).

Conclusions

Low night temperature (12C) induced ovary swelling in three types of sweet

pepper (cherry, elongated, and blocky bell), with the greatest response occurring in bell

peppers. Three to four weeks of continuous low night temperature were required for

maximum response. This timing coincides with the time required for flower bud initiation

in pepper and suggests that flowers must be exposed to LNT soon after initiation in

order for this response to occur. However, the minimum duration of exposure to LNT

required to elicit this response is unknown, as the present research maintained LNT

throughout the course of flower development. Decreasing the source supply by leaf

removal did not influence ovary size, regardless of the temperature regime. However,

plant density may have resulted in plant-to-plant shading such that differences in whole

plant photosynthesis (and therefore source:sink ratios) did not occur. Additional work on

night temperature x source:sink ratio is necessary to further test the hypothesis that

both factors interact to affect ovary swelling in sweet pepper.









Table 3-1. Relationship between days after treatment and harvest interval defined for
statistical analysis in two groups of sweet pepper cultivars.
Days after treatment


Harvest interval Group 1 Group 2
I 1-8 1-13
II 9-16 14-26
III 17-24 27-39
IV 25-32 40-52
V 33-40 53-65
z Group 1: 'Brigadier', 'Legionnaire', 'Banana Supreme', and 'Red Cherry Sweet'; Group
2: 'Ariane' and 'Aristotle'.


Table 3-2. Number of flowers harvested at anthesis and number of nodes bearing
flowers in six cultivars of sweet pepper grown at 22/20C or 22/12C day/night
temperatures.
Number of flowers/plantz Number of nodes
Night temperature (oC) Night temperature (oC)
Cultivar 12 20 Pvaluesy Meanx 12 20 Pvalues Mean
'Ariane' 25.0 33.3 0.04 29.2 aW 6.3 8.7 0.01 7.5 ab
'Aristotle' 31.3 36.0 0.29 33.7 a 8.5 9.7 0.16 9.1 a
'Brigadier' 26.7 28.0 0.82 27.3 a 6.8 7.2 0.69 7.0 b
'Legionnaire' 22.0 30.3 0.04 26.2 a 5.5 8.0 <0.01 6.8 b
'Banana Supreme' 30.0 37.3 0.12 33.7 a 7.3 8.8 0.08 8.1 ab
'Red Cherry Sweet' 27.3 34.0 0.13 30.7 a 6.2 8.3 0.01 7.3 b
Meanv 27.1 33.2 <0.01 30.1 6.8 8.4 <0.01 7.6
z All flowers were continuously harvested at anthesis during a 39-day period for
'Brigadier', 'Legionnaire', 'Banana Supreme' and 'Red Cherry Sweet' and a 64-day
period for 'Ariane' and 'Aristotle'.
SP values were generated from the temperature treatment data within each cultivar; n=3
for flower number per plant and 6 for node number.
x Means were averaged for each cultivar across both temperature treatments; n=6 for
flower number per plant and 12 for node number.
W Means with the same letter in the same column are not significantly different (Tukey-
Kramer, P<0.05).
v Means averaged across all cultivars; n=18 for flower number per plant and 36 for node
number.









Table 3-3. Main effect of night temperature during the preanthesis stage on ovary
characteristics of sweet pepper flowers measured at anthesis.
Temperature treatmentsy
Parameterz 22/12C 22/20C P value
Fresh weight (mg) 84x 76 0.008
Volume (mm3) 88 79 0.002
Diameter (mm) 5.5 5.3 0.003
Length (mm) 4.3 4.2 <0.001
Length:diameter ratio 0.82 0.82 0.59
Ovary wall thickness (mm) 0.59 0.57 0.004
z Flowers were continuously harvested at anthesis during a 39-day period for 'Brigadier',
'Legionnaire', 'Banana Supreme' and 'Red Cherry Sweet' and a 64-day period for
'Ariane' and 'Aristotle'.
Y Day/night temperatures.
x Means were averaged for each temperature treatment across all cultivars and harvest
intervals; n= 597 and 487 for 22/20C and 22/12C, respectively.

Table 3-4. Main effect of cultivar on characteristics of ovaries at flower anthesis in sweet
pepper.
Cultivarz Ovary Volume Diameter Length Length: Ovary wall
FW(mg) (mm3) (mm) (mm) Diameter ratio thickness (mm)
'Ariane' 80 cyx 86 c 5.6 c 4.1 c 0.74 b 0.56 b
'Aristotle' 91 bc 96 bc 5.9 bc 3.9 d 0.66 c 0.55 b
'Brigadier' 101 ab 107 ab 6.1 ab 4.2 bc 0.68 c 0.56 b
'Legionnaire' 112 a 117 a 6.4 a 4.4 a 0.69 c 0.56 b
'Banana Supreme' 48 d 49 d 4.1 d 4.5 a 1.08 a 0.61 a
'Red Cherry Sweet' 48 d 46 d 4.1 d 4.4 ab 1.07 a 0.62 a
z Flowers were continuously harvested at anthesis during a 39-day period for 'Brigadier',
'Legionnaire', 'Banana Supreme' and 'Red Cherry Sweet' and a 64-day period for
'Ariane' and 'Aristotle'.
Y Means were averaged for each cultivar across both temperature treatments and all
harvest intervals. For fresh weight, n = 173, 200, 158, 152, 192, and 171 for'Ariane',
'Aristotle', 'Brigadier', 'Legionnaire', 'Banana Supreme', and 'Red Cherry Sweet',
respectively. For the remaining variables, n was equal to or greater than it was for FW.
x Means with the same letter in the same column are not significantly different (Tukey-
Kramer, P<0.05).









Table 3-5. Main effect of harvest interval on ovary characteristics of six cultivars of
sweet pepper.


Harvest Fresh Volume Diameter Length Length:diameter Ovary wall
interval z,y weight (mg) (mm3) (mm) (mm) ratio thickness (mm)
I 70 d xw 74d 5.2c 4.1 b 0.84 a 0.60 a
II 73 d 76 d 5.3 c 4.2 b 0.82 a 0.58 b
III 79 c 82 c 5.3 bc 4.3 ab 0.84 a 0.56 c
IV 86 b 89 b 5.4 b 4.3 a 0.82 a 0.56 c
V 92 a 97 a 5.7 a 4.3 a 0.79 b 0.58 b
z Flowers were continuously harvested at anthesis during a 39-day period for 'Brigadier',
'Legionnaire', 'Banana Supreme' and 'Red Cherry Sweet' and a 64-day period for
'Ariane' and 'Aristotle'.
Y Harvest intervals were either 8 (39 days total) or 13 (64 days total) days, depending on
cultivar (see details on Table 3-1).
x Means were averaged for each harvest interval across cultivar and temperature
treatment; n = 160, 207, 211, 233, and 273 for intervals 1 to 5, respectively. For fresh
weight, volume, FW:volume ratio, and ovary wall thickness in interval 1, n = 122.
W Means with the same letter in the same column are not significantly different (Tukey-
Kramer, P<0.05).









Table 3-6. Interaction of night temperature and cultivar on ovary characteristics of sweet
pepper flowers harvested at anthesis during the duration of the experiment or
only during the last two harvest intervals.
Fresh weight (mg) Volume (mmd) Diameter (mm) Length (mm)
Night temperature (oC)
CultivarzY 12 20 Pvaluex 12 20 Pvalue 12 20 Pvalue 12 20 Pvalue
All harvest intervals W
ARN 75 86 0.10 81 91 0.17 5.4 5.7 0.10 4.1 4.1 0.74
ATS 101 80 0.003 108 84 0.002 6.2 5.6 0.002 4.0 3.8 0.11
BRI 104 97 0.30 112 101 0.14 6.3 6.0 0.14 4.2 4.1 0.48
LEG 119 105 0.05 126 109 0.03 6.6 6.2 0.07 4.5 4.3 0.09
BAS 54 42 0.10 55 42 0.10 4.3 4.0 0.18 4.7 4.2 <0.001
RCS 50 46 0.57 48 44 0.57 4.2 4.0 0.24 4.4 4.4 0.72
Harvest intervals IV and V v
ARN 87 97 0.22 94 103 0.34 5.7 6.0 0.15 4.2 4.2 1.00
ATS 116 79 <0.001 125 83<0.001 6.5 5.5<0.001 4.0 3.8 0.18
BRI 122 97 0.01 132 102 0.004 6.6 6.0 0.01 4.4 4.1 0.03
LEG 133 112 0.02 141 117 0.02 6.8 6.4 0.12 4.6 4.3 0.01
BAS 65 46 0.03 68 46 0.03 4.5 4.1 0.07 5.1 4.2 <0.001
RCS 61 52 0.34 57 50 0.46 4.5 4.2 0.17 4.6 4.4 0.10
z Flowers were continuously harvested at anthesis during a 39-day period for BRI, LEG,
BAS and RCS and a 64-day period for ARN and ATS.
YARN='Ariane'; ATS = 'Aristotle'; BRI ='Brigadier'; LEG = 'Legionnaire'; BAS = 'Banana
Supreme'; RCS = 'Red Cherry Sweet'.
x P values were generated from the temperature treatment data within each cultivar.
W Means were averaged for each cultivar across all harvest intervals; n = 75 to 107,
depending on temperature treatment and cultivar.
v Last two harvest intervals corresponded to 25 to 39 days after treatments began for
BRI, LEG, BAS and RCS or 40 to 64 days after treatments began for ARN and ARS; n =
30 to 64, depending on temperature treatment and cultivar.

Table 3-7. Coefficients of conversion to estimate ovary volume at anthesis stage as a
function of its fresh weight in six cultivars of sweet pepper grown under two
night temperatures. Volume (mm3) = b FW (mg) (R2>0.99).
Temperature treatment (oC)
Cultivar 22/12 22/20 Average
'Ariane' 1.09 1.07 1.08
'Aristotle' 1.07 1.05 1.06
'Brigadier' 1.08 1.05 1.07
'Legionnaire' 1.06 1.04 1.05
'Red Cherry Sweet' 0.97 0.96 0.96
'Banana Supreme' 1.05 1.00 1.03
Average 1.07 1.04 1.06









Table 3-8. Main effect of night temperature during the preanthesis stage on ovary
characteristics of sweet pepper flowers at anthesis.
Night Ovary Ovary Placenta Ovary wall Diameter Length Length: Ovary wall
temperature z FW wall FWFW(mg) FW:total (mm) (mm) diameter thickness
(mg) (mg) FW ratio (mm)
12C 58**',x 30** 27* 0.56* 4.5* 3.8* 0.91 0.51
200C 36 19 17 0.55 3.8 3.3 0.90 0.46
z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7.
Y *, ** Means are significantly different (t-test) at P<0.05 and P<0.01, respectively.
x Means were averaged across four cultivars and six nodes; n = 514 for 120C and 401
for 200C.

Table 3-9. Main effect of cultivar on ovary characteristics of sweet pepper flowers at
anthesis.
Cultivar z, y Ovary Ovary Placenta Ovary wall Diameter Length Length: Ovary wall
FW wall FW FW (mg) FW:total (mm) (mm) diameter thickness
(mg) (mg) FW ratio (mm)
ARN 61 ax, w 28 a 32 a 0.47 c 4.9 a 3.5 b 0.70 c 0.47 b
LEG 62 a 29 a 33 a 0.46 c 4.9 a 3.4 b 0.69 c 0.46 b
BAS 33b 22b 11 b 0.65 a 3.4 b 3.9a 1.15a 0.50 a
RCS 32 b 20 b 12 b 0.63 b 3.3 b 3.5 b 1.06 b 0.52 a
z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7.
YARN ='Ariane'; LEG = 'Legionnaire'; BAS = 'Banana Supreme'; RCS = 'Red Cherry
Sweet'.
x Means were averaged for each cultivar across both temperature treatments and
nodes.
W Means with the same letter in the same column are not significantly different (Tukey-
Kramer, P<0.05); n =229, 220, 201, and 265 for 'Ariane', 'Legionnaire', 'Banana
Supreme', and 'Red Cherry Sweet', respectively.









Table 3-10. Main effect of node on ovary characteristics of sweet pepper flowers at
anthesis.
Nodez' Y Ovary Ovary Placenta Ovary wall Diameter Length Length: Ovary wall
FW wall FW FW (mg) FW:total (mm) (mm) diameter thickness
(mg) (mg) FW ratio (mm)
2 35 exw 18 e 16 d 0.55 bc 3.8 e 3.3 c 0.91 a 0.46 c
3 39 d 22 d 17 d 0.58 a 3.9 d 3.3 c 0.90 a 0.48 bc
4 46 c 24 c 21 c 0.56 ab 4.1 c 3.5 b 0.91 a 0.48 ab
5 51 b 26 b 25 b 0.54 c 4.2 b 3.7 a 0.91 a 0.50 a
6 54 ab 28 ab 26 ab 0.54 c 4.4 ab 3.7 a 0.90 a 0.50 a
7 56 a 29 a 27 a 0.54 bc 4.5 a 3.8 a 0.90 a 0.51 a
z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7.
Y Days after treatment at which nodes were harvested: 2: 2-9; 3: 6-17; 4: 11-26; 5: 16-
31; 6: 22-38; 7: 28-42, depending on the cultivar.
x Means were averaged across all four cultivars and two temperature treatments; n =
179, 154, 154, 161, 148, 119 for nodes 2, 3, 4, 5, 6, and 7, respectively.
W Means with the same letter in the same column are not significantly different (Tukey-
Kramer, P<0.05).

Table 3-11. Interaction of night temperature and cultivar on ovary characteristics of
sweet pepper flowers harvested at anthesis.
Ovary FW (mg) Ovary wall FW Placenta FW Diameter (mm) Length (mm)
(mg) (mg)
Night temperatures (oC)
CVzy 12 20 Pvalue 12 20 Pvalue 12 20 Pvalue 12 20 Pvalue 12 20 Pvalue
Nodes 2-7
ARN 74x 47 <0.001w 35 22 <0.001 39 26 <0.001 5.4 4.5 <0.001 3.7 3.2 <0.001
LEG 79 45 <0.001 37 20 <0.001 42 25 <0.001 5.4 4.3 <0.001 3.6 3.1 <0.001
BAS 39 26 0.04 26 17 0.01 13 9 0.21 3.5 3.2 0.13 4.2 3.5<0.001
RCS 38 25 0.04 23 16 0.03 15 9 0.06 3.6 3.1 0.007 3.8 3.3 <0.001
Nodes 5-7
ARN 88v 52 <0.001 40 22 <0.001 48 29 <0.001 5.8 4.6 <0.001 4.0 3.3 <0.001
LEG 93 49 <0.001 43 21 <0.001 50 28 <0.001 5.8 4.5 <0.001 3.8 3.1 <0.001
BAS 45 27 0.01 30 18 0.01 15 9 0.09 3.7 3.2 0.04 4.5 3.6 <0.001
RCS 48 25<0.001 29 16 <0.001 19 9 0.01 4.0 3.1 <0.001 4.13.3<0.001
z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7.
YCV=cultivars. ARN: 'Ariane', LEG: 'Legionnaire', BAS: 'Banana Supreme', RCS: 'Red
Cherry Sweet'.
x Means were averaged for each cultivar across all nodes harvested; n = 67 to 134,
depending on temperature treatment and cultivar.
W P values were generated from the temperature treatment data within each cultivar.
v Means were averaged for each cultivar across nodes 5, 6 and 7; n = 23 to 68,
depending on temperature treatment and cultivar.









Table 3-12. P values of t-test comparison between night temperatures of 12 and 20C
within each cultivar and node for ovary fresh weight (FW), diameter (Diam.),
and length.
Cultivar z,y
ARN LEG BAS RCS
Node FW Diam. Length FW Diam. Length FW Diam. Length FW Diam. Length
2 0.86x 0.97 0.42 0.04 0.02 0.04 0.49 0.58 0.04 0.76 0.61 0.98
3 0.15 0.04 0.22 <0.001 <0.001 0.03 0.06 0.15 <0.001 0.76 0.97 0.23
4 <0.001 <0.001 <0.001 <0.001 <0.001 0.004 0.10 0.10 0.002 0.23 0.13 0.003
5 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.08 0.08 <0.001 0.004 <0.001 <0.001
6 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.01 0.14 <0.001 <0.001 <0.001 <0.001
7 <0.001 <0.001 0.002 <0.001 <0.001 0.007 0.08 0.51 <0.001 <0.001 <0.001 <0.001
z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7.
YARN ='Ariane'; LEG = 'Legionnaire'; BAS = 'Banana Supreme'; RCS = 'Red Cherry
Sweet'
x In all cases, P values 50.05 mean that ovaries developed at 12C had significantly
greater fresh weight, diameter or length than the ovaries developed at 200C.






















Figure 3-1. Schematic drawing of pepper branching and flowering sequence. A)
Meristematic stage. B) Well developed branch. The sympodial shoot bears a
leaf (L1, blue), and terminates in a flower (F2). From the leaf axil, two leaves
(L2) develop. The flower and the two leaves developed from the same
sympodial shoot compose a sympodial unit (F2 and L2, green). L2 leaves are
'pushed up' by internode elongation, so that the leaves appear above the
preceding flower. The shoot terminates in a flower, which is part of the
subsequent sympodial unit. From each of the leaf 2 axils, two new leaves (L3,
red) develop. Each color represents sympodial units of a different order
(Drawing A based on micrograph from Elitzur et al., 2009).

























Figure 3-2. Schematic drawing of a sweet pepper plant pruned to two main axes with
lateral branches. Two shoots were allowed to grow at the first branching to
form the main-stem sympodial unit (SU, brown). A) Starting in SU1 (blue), the
strongest shoot was allowed to grow and the second shoot was limited to
development of one sympodial unit (one flower and two leaves) by pruning.
This pruning system was used in all the plants in Experiment 1 and half of the
plants in Experiment 2. B) The strongest shoot was allowed to grow and the
second shoot was pruned so that only the flower of the next sympodial unit
remained. This system was used in half of the plants in Experiment 2. In both
A and B, flowers from the main stem and first SU were removed before
treatment started. SU1= blue; SU2 = green; SU3= red, SU4= white.










a***
a,


'Ariane'


ab, a


a--*' a
a


1-13 14-26 27-39 40-52 53-64


a,* a,***
-,-- -------.*


b b -
a
b a


'Aristotle'


130

110

90 -


70
50 -


'Red Cherry Sweet'





a
ab
ab ab .-*--"

a i
a 11


1-8 9-16 17-24 25-32 33-39 1-8 9-16 17-24 25-32 33-39
Days after treatment

Figure 3-3. Ovary fresh weight of sweet pepper flowers at anthesis developed under
22/12C (--,--) or 22/20C (-*-) day/night temperatures for discrete harvest
intervals. Plants did not bear any fruit before and during the experiment.
Means with the same letter in the same temperature treatment and cultivar
are not significantly different (Tukey-Kramer, P: 0.05). *, ** ***: Means
between temperature treatments within a harvest interval for each cultivar are
significantly different at P50.05, P<0.01, and P50.001, respectively. Range of
n values for harvest intervals: 1st: 7-20; 2nd: 11-25; 3rd: 12-23; 4th: 12-29;
5th: 14-35.


be,** ,
C ^..-- --- .
ab' c "-

ab a
ab ab b




1-13 14-26 27-39 40-52 53-64
ab,* -*
'Legionnaire' ab ,--" a,
b b -- -
S a a a
a
a


'Brigadier'


130

110

90

70

50


130

110

90

70

50


'Banana Supreme'


a**
ab,* a,
b ab --*-"
b ------ ---
Sa a


I I I I I











a ***
'Ariane' a, ab,***
ab,*** '
b,*** -,

/
/
/
C,
c

a a a
a a


100 -

90

80

- 70
E
Su

Z 60
t-

'F 50
S40
- 40 -
-D
U- 30

20

10

0
100

90

80 -


ab,**
^**


a,** 4,
-4 a ***


- _-_..a, ab ab
a b ab
b ab ab


'Banana Supreme'


a* a
a,.- -


a
ab a
--*-'


b .


-a a a a
a a
a a


'Red Cherry Sweet'


S* a, *

a a,


bU


a a a a- a


I I I I I


2 3 4 5 6 7 2 3
Node above first branching


4 5 6 7


Figure 3-4. Ovary fresh weight of sweet pepper flowers harvested at anthesis from the
second to the seventh nodes above the first flowering node in plants grown at
day/night temperatures of 22/12C (--,--) or 22/20C (-*-). Plants did not
bear any fruit. Means with the same letter in the same temperature treatment
and cultivar are not significantly different (Tukey-Kramer, P: 0.05). *, **, ***:
Means between temperature treatments within a cultivar and node are
significantly different at P<0.05, P<0.01, and P<0.001, respectively. Range of
n values for the temperature treatments: 22/12oC: 15-23; 22/20oC: 8-24,
excluding 'Banana Supreme' node 2, where n=2. Days after treatment at
which nodes were harvested: 2: 2-9; 3: 6-17; 4: 11-26; 5: 16-31; 6: 22-38; 7:
28-42, depending on the cultivar.


'Legionnaire'

b,,

c,"*** /
C,

d,*


- 70 -
E
60 60
OU--

(D 50

-c 40

U- 30


20

10 -

0 -----


I I I I I


1- -









CHAPTER 4
LOW NIGHT TEMPERATURE AFFECTS CARBON EXCHANGE RATES OF SWEET
PEPPER LEAVES

Introduction

Pepper (Capsicum annuum L) is an herbaceous species that belongs to the family

Solanaceae (Eshbaugh, 1993), and its fruit is one of the most consumed vegetables

worldwide (Mateos et al., 2003). Pepper fruits, especially sweet peppers, are an

excellent source of essential nutrients, especially vitamin C, 3-carotene, calcium and

antioxidant compounds (LopezHernandez et al., 1996; Mateos et al., 2003; Sun et al.,

2007; Mateos et al., 2009). Cultivated species of peppers were domesticated in tropical

and temperate areas in Mexico, Central America and South America (Bolivia), and are

sensitive to low temperatures (Harlan, 1971; Eshbaugh, 1993).

Low temperature is one of the major factors limiting the productivity and

geographical distribution of many plant species (Li et al., 2003a). Low temperature

effects on physiology and growth have been studied in several agronomic and

horticultural crops (Koscielniak, 1993; Gibson and Mullen, 1996; Boese et al., 1997;

Wang et al., 1997; Roussopoulos et al., 1998; Sukhvibal et al., 1999; Venema et al.,

1999a; Adams et al., 2001; Hendrickson et al., 2004a; Hendrickson et al., 2004b; Singh

et al., 2005; Limin and Fowler, 2006), including pepper (Mercado et al., 1997b;

Pressman et al., 1998b; Aloni et al., 1999; Li et al., 2004; Pressman et al., 2006). One

of the more striking effects of low temperature, particularly low night temperature, on

pepper growth is an increase in swollen floral ovaries and subsequent deformed fruit

(Polowick and Sawhney, 1985; Rylski, 1985; Mercado et al., 1997b; Pressman et al.,

1998a; Aloni et al., 1999; Shaked et al., 2004).









Among other physiological effects, low temperatures reduce photosynthetic rates.

Day temperatures of 6-7C may cause rapid stomatal closure in a variety of plant

species (Boese et al., 1997; Wilkinson et al., 2001) and subsequent photoinhibition (Liu

et al., 2001; Li et al., 2003a; Li et al., 2004; Zhang and Scheller, 2004). Photosynthetic

enzyme activity may also be reduced under low temperatures (Kingston-Smith et al.,

1997).

On the other hand, low night temperatures (i.e., below 10-15C) delay the start

and slow the rate of stomatal opening the following morning, as temperatures increase

(Hendrickson et al., 2004a; Hendrickson et al., 2004b). Night temperatures below 15C

may also decrease photosynthesis by decreasing ribulose-1,5-bisphosphate

regeneration and Pi availability for recycling (Hendrickson et al., 2004a; Hendrickson et

al., 2004b), reducing activity of PSII (Sundar and Reddy, 2000; Bertamini et al., 2005),

and decreasing leaf chlorophyll content (Sundar and Reddy, 2000; Bertamini et al.,

2005). Reduction in Rubisco amount and activity, as well as other

photosynthetic/carbohydrate metabolizing enzymes, such as fructose bisphosphatase

and sucrose phosphate synthase, may also occur in response to low night temperature

(Sundar and Reddy, 2000; Ramalho et al., 2003; Hendrickson et al., 2004a;

Hendrickson et al., 2004b; Bertamini et al., 2005). Low night temperature (5C) followed

by high irradiance (1900 PPF) also decreases photosynthesis by increasing

photoinhibition (Bertamini etal., 2006).

Tropical crops, including pepper, are especially sensitive to low night temperature,

and one cold night of 4 to 12C may decrease photosynthetic rates 5 to 80% (Van-de-

Dijk, 1985; Van-de-Dijk and Maris, 1985; Wolfe, 1991; Janssen et al., 1992; Boese et









al., 1997; Ying et al., 2000, and references therein; Li et al., 2003a; Ramalho et al.,

2003; Wang et al., 2004). Bhatt and Srinivasa-Rao (1993b) reported that net CO2

exchange rates in sweet pepper were 6 to 30% higher at night temperatures of 22C

compared with 170C.

Some species have the capacity to acclimate to low temperatures. In such cases,

exposure to low temperatures for several days may markedly increase the maximum

activities of Rubisco, stromal and cytosolic fructose-1,6-bisphosphatase, and sucrose-

phosphate synthase in leaves, RuBP regeneration and carboxylation, and lead to

significant increases in whole plant photosynthetic capacity (Holaday et al., 1992; Hurry

et al., 1994; Du et al., 1999; Yamori et al., 2005). In pepper, plants that were cold

acclimated (5 days at 14C and 250 PPF) exhibited greater photosynthetic rates

compared to non-acclimated plants upon subsequent exposure to low temperatures

(40C, 1200 PPF) (Liu etal., 2001).

Respiration rates are also affected by low temperature, with rates in both the light

and the dark increasing as temperature increases up to about 40C (Lin and Markhart,

1990; Taiz and Zeiger, 2006). In the short term, respiration is highly responsive to

ambient temperatures; however, in the long-term, temperature acclimation can occur.

Tjoelker et al. (1999) reported that respiration in five species of high-temperature

acclimated plants (30/24C day/night) was lower than that of low-temperature

acclimated plants (18/12C day/night) when measured at temperatures between 12 and

300C.

Net carbon exchange rates (CER) and ovary swelling in pepper are affected not

only by temperature, but also by source:sink ratios. Net CER increased in newly









developed leaves of fruiting compared with non-fruiting pepper (Cruz-Huerta et al.,

2005). In bell pepper, fruiting decreased vegetative growth rates compared to non-

fruiting plants (Hall and Brady, 1977; Hall and Milthorpe, 1978; Bhatt and Srinivasa-Rao,

1989), since growing fruits represent up to 90% of sink demand (Hall, 1977). Defruiting,

which rapidly increases the source:sink ratio, increased the incidence of flower

deformation and swollen ovaries in pepper (Aloni et al., 1999).

The incidence of swollen ovaries in bell pepper due to low night temperature or

high source:sink ratio has been correlated with increased ovary carbohydrate

concentration in the flower bud (Aloni et al., 1999). Under low night temperature, pepper

plants exhibit slower growth rates, resulting in decreased shoot dry weight compared

with plants grown under higher night temperatures (Mercado et al., 1997a). In some

species, leaves developed under low night temperature may acclimate to such

conditions, resulting in carbon exchange rates as high as in plants growing under

warmer temperatures (Singh et al., 2005). Therefore, similar photosynthetic rates and

slower growth rates under low night temperatures may cause excess carbohydrate

accumulation in floral ovaries, resulting in swelling and deformed fruits. Similarly,

developing fruits in bell pepper are a strong sink, and defruiting pepper plants increases

starch concentration in stems (Hall and Milthorpe, 1978), which may also result in an

excess carbohydrate supply to floral ovaries. The hypothesis tested in the present

experiments is that sweet pepper leaves developed under low night temperatures

acclimate to such conditions and have similar carbon exchange rates as leaves

developed under high night temperatures. This, combined with reduced growth under

low night temperatures, increases the incidence of swollen or deformed ovaries in









pepper flowers. A second hypothesis tested is that fruiting decreases the incidence of

swollen ovaries, regardless of night temperature. The objectives were to 1) measure net

CER, growth, and ovary swelling in two cultivars of pepper grown under low and high

night temperatures and 2) determine fruiting effects on net CER, growth, and ovary

swelling in plants grown under two night temperatures.

Material and Methods

Plant Material and Growing Conditions

The experiments were carried out using two types of sweet pepper with

contrasting fruit size and fruit shape 'Legionnaire' bell pepper (Experiments 1, 2, and

3) and 'Red Cherry Sweet' cherry pepper (Experiment 1). Seeds were germinated in a

mix of peat moss, vermiculite, and dolomite limestone (SunGro Metro-Mix Ag-Lite Mix2)

and grown in 1.4 m2 growth chambers (Mod E15, Conviron, Winnipeg, Canada).

Chamber temperature was set to 22/200C (day/night), and PPF was maintained

between 450-500 pmol m-2 s-1 above the canopy with a 14-hour photoperiod, as

described in Chapter 3. Seedlings at about the sixth-leaf stage (~ 50 days after sowing)

were transplanted to 1.5 L containers, using the same growing media for germination,

and maintained in the growth chambers throughout the experiments.

Plants were fertigated as needed with the following nutrient solution (mM): 3.4

Ca(N03)2, 1.8 KNO3, 1.6 KH2PO4, 0.3 KCI, 2.7 MgSO4, and (pM): 50.2 Fe-EDTA, 3.1

CuSO4, 14.6 MnSO4, 64.8 H3B03, 4.6 ZnSO4, 0.6 Na2MoO4 (modified from Cruz-Huerta

et al., 2005; and Jovicich, 2007). Pests were controlled as described in Chapter 3.



2 Composition: Canadian Sphagnum peat moss (55-65%), horticultural grade vermiculite, dolomite
limestone, starter nutrient charge, and wetting agents (www.sungro.com/products.php, 12/17/2008).









Plants were pruned to two main axes, with one lateral sympodial unit (one flower and

two leaves) on every node in the main axis, as described in Chapter 3 (Figure 3-2A).

Two experiments were conducted to measure net CER and biomass accumulation

under low (12C) vs high (200C) night temperatures (Experiments 1 and 2) and different

fruit loads (Experiment 2).

Experiment 1

Eighteen seedlings each of 'Legionnaire' and 'Red Cherry Sweet' were selected

when flowers in nodes 3 or 4 were at anthesis (~20 days after transplanting). Growth

chamber temperatures were set to 220C day, with night temperatures of either 200 (high

night temperature, HNT) or 120C (low night temperature, LNT). Each growth chamber

was divided into three blocks to reduce PPF variation and three plants of each cultivar

were randomly placed within each block. All the flowers were removed; therefore plants

did not bear any fruit.

Gas exchange measurements were performed using a portable photosynthesis

system (LI-6400, Licor, Lincoln, NE, USA) on the most recent mature leaf, which was

the fourth or fifth from the apex. Measurements included net CER, stomatal

conductance (gs), intercellular CO2 concentration (Ci), and leaf dark respiration (RD, day

and night). Net CER measurements were performed about six hours after lights were on

and were done every three days during a 36-day period after treatments started, with a

final measurement at 45 days. This period of time ensured that several new leaves in

each axis completely developed under the temperature treatments. Based on data

reported by Choi and Gerber (1992) and Cruz-Huerta (2001), sweet pepper leaves take

four to five weeks from the beginning of formation until full expansion. Respiration

measurements during the day were performed 60 to 90 seconds after net CER









measurements were completed, by covering the top of the assimilation chamber with an

opaque card, and during the night before the dark period ended. Respiration

measurements during the day were performed only twice at the beginning of the

experiment (1 and 3 days after treatment, DAT) since no difference among treatments

was found. Night respiration rates were measured at 13, 27 and 47 DAT. Fresh weight

of flowers and flower parts (ovary, calyx, and petals) at anthesis and ovary diameter and

length were recorded on flowers harvested from 30 to 37 DAT. At the end of the

experiment, leaf area, dry weight per organ, plant height, and number of internodes

were also recorded.

Experiment 2

Twenty-four 'Legionnaire' seedlings were selected ~30 days after transplanting

and flower buds from the main-stem sympodial unit (first bifurcation) and sympodial unit

1 (node 1) were removed. Flowers on nodes 2 and 3 were hand-pollinated, allowing one

fruit per axis (2 fruits per plant) to develop beyond petal fall, the indicator of fruit set.

About ten days after petal fall, when the fruits were rapidly growing (Marcelis and Baan-

Hofman-Eijer, 1995b), night temperature (HNT vs LNT) and source:sink ratio treatments

began. Fruits were either allowed to grow or were removed in order to vary the

source:sink ratio, resulting in a 2 x 2 factorial (two night temperatures and two

source:sink ratios) with six replications.

As in Experiment 1, net CER measurements were performed using a portable LI-

6400 photosynthesis system. Measurements were done every three to four days during

a 41-day period on one to three of the most recently matured leaves per plant.

Photosynthesis was measured about six hours after lights were on. Measurement on

day 19 was not considered in the final analysis since plants were under water stress









when measured. Fresh weight of flowers and flower parts at anthesis and ovary

diameter and length were recorded on flowers harvested from 30 to 36 DAT. Organ dry

weight, plant leaf area, and plant height were recorded at end of the experiment.

Data Analysis

Data from all experiments were analyzed as a completely randomized block

design, with repeated measures, except for the dry weight in Experiments 1 and 2. The

subject for repeated measures was the individual plant. Comparisons performed were

night temperatures, days after beginning of treatment (Experiments 1 and 2), cultivars

(Experiment 1), and source:sink ratio (Experiment 2). Data from the two measurement

dates for day respiration were pooled and analyzed as a 2x2 factorial (night temperature

x cultivars). Night respiration data were analyzed as a factorial (night temperature x

cultivars x measurement dates) with repeated measures over time.

Results

Experiment 1

Low night temperature increased flower, flower parts, and ovary fresh weight and

ovary size, compared to HNT (Table 4-1). Flower fresh weight in the LNT was 32%

greater than in the HNT. As expected, bell pepper had larger flowers and ovaries than

cherry type pepper. The interaction between cultivar and night temperature was

significant for total flower fresh weight, calyx fresh weight, and ovary diameter and

length. After four weeks of night temperature treatments, ovaries of 'Red Cherry Sweet'

exhibited a proportionately greater increase in size under LNT compared with HNT than

did ovaries of 'Legionnaire'. However, we selected 'Legionnaire' for the subsequent

experiments due to the more rapid response of this cultivar to LNT compared to the

slower initial response in 'Red Cherry Sweet' (Chapter 3).









Carbon exchange rate was significantly influenced by the cultivar, DAT, and

interactions between DAT x cultivar and DAT x temperature (Table 4-2). Stomatal

conductance was influenced by DAT and interactions where DAT was a factor, while Ci

was influenced only by DAT and the interaction of DAT x cultivar.

Net CER was significantly higher in 'Red Cherry Sweet' than 'Legionnaire' over the

course of the experiment (Table 4-3); however, night temperature had no effect on net

CER, stomatal conductance, or intercellular CO2.

Regardless of cultivar, CER decreased over time (Figure 4-1A). There were two

periods where CER decreased rapidly; the first occurring 6 to 9 DAT and the second

occurring 24 to 27 DAT. Stomatal conductance followed a similar pattern over time (i.e.

DAT) as net CER, although with more pronounced decreases (Figure 4-1B). The

intercellular CO2 concentration (Ci) also decreased with time. Ci decreased rapidly from

287 to 196 pmol mol-1 during the first 6 DAT. After the initial decrease, Ci increased to

reach a peak at 19 DAT, followed by a second peak at 30 DAT, before finally

decreasing to -220 pmol mol- after 33 DAT (Figure 4-1 C).

Although the main effect of night temperature on net CER was not significant

(Table 4-3); there was a significant interaction of DAT x temperature on net CER (Figure

4-2A). Three days after the beginning of the experiment, plants under LNT had

significantly greater CER than those under HNT. After that, however, plants under HNT

had either similar or significantly greater CER than those under LNT. Differences in net

CER between cultivars were found between 15 and 21 DAT and at the end of the

experiment (between 36 and 45 DAT), with 'Red Cherry Sweet' exhibiting greater CER

than 'Legionnaire' (Figure 4-2B).









The interaction between DAT x night temperature on stomatal conductance

(Figure 4-3A) was similar to that observed for net CER. Stomatal conductance was

higher under LNT compared with HNT the first three days after treatments began, but

were significantly lower between 21 and 33 DAT. Significant differences in stomatal

conductance between cultivars were found only at 15 and 19 DAT (Figure 4-3B). The

initial decrease in gs, which was similar in both cultivars, was followed by a transient

increase, with a greater increase observed in 'Legionnaire' compared with 'Red Cherry

Sweet' (Figure 4-3B). The increase in gs in 'Red Cherry Sweet' correlated with the

increase observed in CER (Figure 4-2B); however, no such correlation was observed in

'Legionnaire'.

Internal CO2 concentrations were significantly higher under LNT compared with

HNT during the first 6 days of treatment and significantly lower from day 19 to day 24

(Table A-4, Appendix), which corresponded with patterns observed in both CER (Figure

4-2A) and gs (Figure 4-3A). Although the interaction between DAT and cultivar was

significant for Ci, differences between cultivars were observed only at 6 DAT, where Ci

in 'Legionnaire' was significantly greater than in 'Red Cherry Sweet' (Table A-4,

Appendix).

During the day, leaf respiration was not affected by night temperature (P=0.71),

but was affected by cultivar (P50.04, Figure 4-4 A, B). No interaction between night

temperature and cultivar was found (P=0.14). During the night, leaf respiration was

significantly reduced by LNT compared to HNT (P50.001). Night leaf respiration was

lower in 'Legionnaire' bell pepper compared to 'Red Cherry Sweet'. Night respiration

was affected by DAT, increasing from 0.67 pmol m2 s-1 at 13 DAT to 0.85 pmol m2 s-1 at









47 DAT (P50.008). There were no significant interactions among night temperature,

cultivar and DAT on night respiration rate (data not shown).

Assuming constant CER during the day and uniform respiration rates during the

night, carbon balance for leaves was calculated (Table 4-4). At the end of the

experiment, sweet pepper plants under LNT gained less carbon during the day, and

although they respired less during the night, LNT plants gained less carbon than did

HNT plants during a 24-h period. There were no significant effects of cultivar or the

interaction of night temperature x cultivar.

LNT reduced leaf number and specific leaf area compared to HNT, but there was

no significant difference in total leaf area (Table 4-5). No significant temperature effects

were found in plant or organ dry weight. Plants growing under LNT, however, were

shorter due to shorter internodes and had smaller main stem diameter compared to

plants growing under HNT. There were significant differences in leaf number, leaf area,

dry weight, and plant height between cultivars, but there were no significant interactions

between night temperature and cultivar.

Experiment 2

Both LNT and reduced sink demand (i.e, non-fruiting plants) increased fresh

weight of the flower, flower parts, and the ovary diameter compared to HNT (Table 4-6).

Ovary length was affected only by night temperature.

Both LNT and the absence of fruit significantly decreased CER and gs in

'Legionnaire' bell pepper (Tables 4-7 and 4-8). Intercellular CO2 concentration was

decreased under LNT, but was not affected by the presence or absence of fruit.









In general, CER, gs, and Ci decreased over time (i.e., DAT). A dramatic decrease

in CER (26%), gs (58%), and Ci (23%) was observed within 6 days after the beginning

of night temperature treatments (Figure 4-5 A, B, and C). After this initial decrease,

there was little statistical difference in CER, gs, or Ci with time, although there was some

variability. Initial values of CER at 3 DAT in Experiment 2 were similar to the initial rates

in Experiment 1 (11-12 pmol m-2 S-1), and to the rates previously reported by Cruz-

Huerta (2005) at fruit set of the first fruit (12-14 pmol m-2 S-1).

Gas exchange parameters were significantly influenced by the interaction between

night temperature and days after treatment (Figure 4-6 A, B, and C). However, CER

was significantly lower under LNT compared with HNT, except for two measurements

dates (22 and 24 DAT) (Figure 4-6A). Stomatal conductance and intercellular CO2

concentration (Figure 4-6B) responses were very similar to that of CER.

There were no significant interactions between night temperature x fruiting or DAT

x fruiting on gas exchange parameters. Initially, net CER was similar between fruiting

treatments and decreased with time until ~6 DAT. By 15 DAT, CER in non-fruiting plants

decreased markedly, resulting in significantly lower CER compared with fruiting plants

throughout the rest of the experiment (Figure 4-7).

LNT reduced the specific leaf area, but not leaf number or total leaf area (Table 4-

9). No significant temperature effects were found on plant or organ dry weight. Plants

growing under LNT were shorter due to fewer nodes compared to plants growing under

HNT.

Fruiting plants had decreased leaf number per plant and greater specific leaf area

compared to the non-fruiting plants, but leaf area was not affected (Table 4-9). Non-









fruiting plants had increased leaf, stem and root DW compared to fruiting plants.

However, due to the obvious differences in fruit DW, total plant DW was not affected by

the presence/absence of fruits. No interactions between night temperature and fruiting

treatment were found for any of the parameters recorded.

Discussion

Low night temperatures (i.e. less than 15C) and/or low sink demand (i.e.,

presence of developing fruits) induced larger flowers and swollen ovaries in our study,

as has been previously reported (Polowick and Sawhney, 1985; Aloni et al., 1999;

Shaked et al., 2004). Previous results (Aloni et al., 1999) showed that ovary swelling in

bell pepper was correlated with increased concentration of reducing sugars and starch

in the flower bud. Since carbohydrate concentration and content ultimately depends on

plant photosynthesis, we quantified leaf carbon exchange rates, dry weight

accumulation, and incidence of ovary swelling in pepper plants grown under low vs high

night temperature and different source:sink ratios.

In the present work, sweet pepper leaves developed under LNT exhibited lower

net CER compared with leaves developed under HNT. These results do not support our

hypothesis that leaves fully developed under LNT conditions acclimate and maintain

photosynthetic rates as high as those from leaves that developed under HNT. Our

results are in contrast to previous reports on tomato (Van-de-Dijk and Maris, 1985),

cotton (Koniger and Winter, 1993; Singh et al.), guayule (Parthenium argentatum Gray)

(Sundar and Reddy, 2000), and common bean (Phaseolus vulgaris L) (Wolfe, 1991;

Wolfe and Kelly, 1992). In these species, leaves that developed under low night

temperatures exhibited CER similar to or greater than CER of leaves developed under

warm night conditions. However, our results are in agreement with work in other tropical









and subtropical plants, where low night temperature (6 to 10C below the control)

decreased CER between 20 and 50% (Wolfe, 1991; Bruggemann et al., 1992; Diczbalis

and Menzel, 1998; Venema et al., 1999b).

In our experiments, CER and gs decreased over time, independently from night

temperature, cultivar, or presence/absence of developing fruits. In general, CER

decreased significantly in the first 6-9 days after treatments began. The decrease in

CER over time was accompanied by decreases in both gs and Ci concentrations,

suggesting stomatal limitation to CER. This is in agreement with work by Hall and

Milthorpe (1978), who reported that decreased leaf CER in bell pepper was correlated

with decreased leaf and intracellular conductance.

Both net CER and night respiration were reduced by LNT compared to HNT. The

lower respiration during the dark period partially compensated for the carbon balance

during a 24-h period. However, considering only the leaf tissue, carbon gain during a

24-h period was higher under HNT compared to LNT. Whole plant respiration was not

calculated since only leaf respiration data were available. However, on a whole plant

basis, it is likely that differences in net carbon gain between LNT and HNT were

negligible since no differences in DW between LNT and HNT were found. Frantz et al.

(2004) reported that when varying night temperature from 17 to 320C in rapidly growing

lettuce, tomato and soybean, respiration rates during the night increased 2 to 4% per

Celsius degree. However, photosynthetic rates remained steady or decreased slightly

as night temperature increased and dry mass accumulation was not affected.

Although LNT decreased photosynthetic rates without decreasing total plant or

organ dry weight, plant height, stem diameter and specific leaf area were reduced









compared with plants grown under HNT. Similarly, Mercado (1997a) reported that cool

day/night temperatures (25/14C) significantly decreased stem height and specific leaf

area of bell pepper compared with plants grown under warm day/night temperatures

(29/20C).

Fruit removal (Expt. 2) decreased net CER compared to no fruit removal,

supporting previous work in pepper (Hall and Milthorpe, 1978) and tomato (Hucklesby

and Blanke, 1992) and other species (Setter et al., 1980; Fujii and Kennedy, 1985;

Schaffer et al., 1987; Gucci et al., 1991; Gucci et al., 1995) indicating that increasing the

source:sink ratio decreased leaf net CER. This decrease was likely due to non-stomatal

rather than stomatal limitations, as fruit removal decreased CER without affecting Ci.

The most likely non-stomatal limitation is end-product inhibition (Layne and Flore, 1995;

Pieters et al., 2001; Sawada et al., 2001). Plants with low sink strength exhibit

decreased sucrose synthesis due to low demand from the rest of the plant (Pieters et

al., 2001). Low sucrose synthesis reduces the recycling of Pi to the chloroplast, ATP

synthesis and RUBP regeneration, and therefore, photosynthesis (Pieters et al., 2001).

Under sink-limited conditions, leaf starch concentration increases and specific leaf area

decreases (Nederhoff et al., 1992), as was found in our work. In addition, plant

manipulations that cause sugars to accumulate can decrease the expression of

photosynthetic genes and upregulate genes for improved C metabolism, increasing,

among others, sucrose metabolism and starch accumulation (Koch, 1996), which also

explain the decrease in CER due to fruit removal.

Although fruit removal decreased net CER overall, initially there was no effect of

the presence or absence of fruit on CER, as CER in both treatments decreased during









the first 6 DAT and significant differences between fruiting treatments were not

manifested until after ~15 DAT. The initial decrease in CER observed in fruiting plants in

our study is in contrast to work by Cruz-Huerta et al. (2005), who reported that CER in

pepper increased 40 to 50% between fruit set and 14 days after fruit set, and by Van-

de-Dijk and Maris (1985), who reported that net CER increased over time in fruiting

tomato plants. In our experiment, two developing fruits may not have resulted in

sufficient sink demand initially to cause the increase in CER observed in previous work,

where fruit load was greater (Bhatt and Srinivasa-Rao, 1997; Cruz-Huerta et al., 2005).

However, once fruit development progressed, differences in CER between fruiting and

non-fruiting plants became more apparent. Additionally, the increase in root, stem, and

leaf DW observed in non-fruiting compared with fruiting plants may have occurred

quickly in response to the defruiting, thus establishing new sinks in the non-fruiting

treatment and partially masking early effects of defruiting on net CER.

Conclusions

We hypothesized that LNT and/or high source:sink ratio would increase the

incidence of swollen ovaries in pepper either through a combination of maintaining net

CER under conditions of reduced growth rates (LNT), or by reducing collective fruit

demand for assimilates (high source:sink ratio). Both of these conditions would

theoretically result in excess assimilate availability and increased incidence of swollen

ovaries. We found that sweet pepper leaves developed under LNT exhibited lower CER

than leaves developed under HNT, and thus were unable to acclimate to night

temperature conditions. Additionally, plant dry weight accumulation was not affected by

night temperature treatments. Thus, although LNT did increase the incidence of swollen

ovaries, the mechanism was not through maintenance of net CER in combination with









reduced growth rate. On the other hand, fruit removal (i.e. increasing the source:sink

ratio) did decrease net CER and increase the incidence of swollen ovaries. This

suggests that excess availability of current photosynthate may not be the mechanism

that results in the increase in swollen ovaries observed under both LNT and high

source:sink conditions.









Table 4-1. Effects of night temperature and cultivar on fresh weight of flower and flower
parts, ovary diameter and ovary length in sweet pepper.
Fresh weight (mg) Ovary Ovary
Flower Ovary Calyx Petals diameter (mm) length (mm)
Night temperature z,y
12C 287*** 84** 61** 134** 5.3** 4.4***
200C 217 57 46 107 4.6 3.7
Cultivarx
'Legionnaire' 295*** 87*** 60*** 140*** 5.6*** 3.9***
'Red Cherry Sweet' 209 55 48 101 4.3 4.2
Night temp x cultivar
120C 'Legionnaire' 323 aW 98 a 65 a 152a 5.8 a 4.1b
120C 'Red Cherry Sweet' 251 b 70 b 58 b 117b 4.7c 4.6a
200C 'Legionnaire' 267 b 76 b 55 b 129b 5.4 b 3.7c
200C 'Red Cherry Sweet' 167 c 39 c 38 c 85c 3.8 d 3.7c
P-values ns *** ns ****
zFlowers at anthesis were harvested from 30-37 days after treatments began.
YMeans were averaged across cultivars, n = 94 for 12C and 103 for 200C.
xMeans were averaged across temperature, n = 101 for 'Legionnaire' and 96 for 'Red
Cherry Sweet'.
WMeans with the same letters were not significantly different (Tukey-Kramer, P50.05),
n=48, 46, 53, and 50 for 120C -'Legionnaire', 120C -'Red Cherry Sweet', 200C -
'Legionnaire', and 200C -'Red Cherry Sweet', respectively. ns= non significant.
Asterisks indicate means are significantly different (t-test) at P<0.05, P<0.01,
P50.001, respectively.

Table 4-2. P-values for effects of night temperature (Temp), cultivar (CV), and days
after treatment (DAT) and their interactions on leaf gas exchange parameters.
Source of Carbon exchange rate Stomatal conductance Intercellular CO2
variations (pmol m-2 S-1) (mol m-2 S-1) (pL L-1)
Temp 0.11 0.06 0.25
CV 0.01 0.99 0.61
Temp x CV 0.64 0.07 0.21
DAT <0.001 <0.001 <0.001
DAT*CV <0.001 <0.001 <0.001
DAT*Temp 0.01 0.05 0.31
DAT*Temp*CV 0.47 0.05 0.44
Block(Temp)y 0.13 0.39 0.13
Plantx 0.03 0.08 0.03
zDegrees of freedom: 1 for Temp, CV, and Temp x CV; 13 for DAT, DAT*CV,
DAT*Temp, and DAT*Temp*CV.
YRandom effect in the model.
xThe individual plant was the subject for the repeated measures component.









Table 4-3. Main effects of night temperature and cultivar on mean carbon exchange
rate, stomatal conductance and intercellular CO2 in sweet pepper leaves
measured six hours after lights were on.
Carbon exchange rate Stomatal conductance Intercellular CO2
(pmol m-2 s-1) (mol m-2 s-1) (pmol mol-1)
Night temperature z
120C 8.3 0.14 238
200C 9.2 0.16 243
Cultivary
'Legionnaire' 8.5* 0.15 240
'Red Cherry Sweet' 9.1 0.15 241
zMeans were averaged across cultivars and days after treatment, n = 252.
YMeans were averaged across temperature and days after treatment, n = 252.
Asterisks indicate means are significantly different (Tukey-Kramer, P50.05).

Table 4-4. Main effects of night temperature and cultivar on daily net photosynthesis,
daily night respiration, and calculated daily total CO2 gain in sweet pepper
leaves.
Carbon budget (mmol CO2 d1 per plant)z
Daily net Daily night Daily net CO2
photosynthesis respiration gain
Night temperature Y
120C 59* 5** 54*
200C 90 10 80
Cultivarx
'Legionnaire' 70 8 62
'Red Cherry Sweet' 79 7 72
zData were calculated for leaf tissue only, using plant total leaf area, net CER during the
day, and night respiration rates at the end of the experiment (45-47 DAT).
YMeans were averaged across cultivars, n = 18.
xMeans were averaged across temperature, n = 18.
'Asterisks indicate means are significantly different (t-test) at P<0.05) or P<0.01,
respectively.









Table 4-5. Night temperature and cultivar effects on vegetative growth
at the end of the 45-day experiment period.


of sweet pepper


Leaf Leaf SLAz DW (g) Plant Stem Node Internode
no. area Leaf Stem Root Total height diam. number length
(cm2) (cm) (mm) (cm)

Night tempy
12C 68 2273 149 15.2 20.0 10.8 49.3 69 12.1 10.7 4.4
20C 74 2612 184 14.2 24.0 10.7 52.5 83 12.9 11.2 5.4
Values 0.03 0.06 0.006 0.13 0.13 0.72 0.31 0.006 0.05 0.16 0.005

Cultivarx
LEG 60 2741 170 16.2 22.4 11.3 52.6 80 13.6 9.1 4.8
RCS 82 2144 163 13.2 21.6 10.2 49.1 72 11.5 12.8 5.0
Values 0.01 0.001 0.13 0.003 0.28 0.001 0.07 0.003 <0.001 <0.001 0.12
zSLA: specific leaf area (cm2 g-).
YMeans were averaged across cultivars, n = 18.
xLEG: 'Legionnaire', RCS: 'Red Cherry Sweet'. Means were averaged across
temperatures, n = 18.

Table 4-6. Main effects of night temperature and presence/absence of fruits on fresh
weight of the flower, flower parts, ovary diameter, and ovary length in
'Legionnaire' bell pepper.
Fresh weight (mg) Ovary Ovary
Flower Ovary Petals Calyx Stamen Ovary diameter length
wall (mm) (mm)
Night temperature,
12C 451** 171** 148* 95* 26** 73** 7.5** 5.2**
20C 352 121 128 71 21 51 6.5 4.6
Fruits
Non-fruiting plants 431** 156** 146* 94** 25** 66* 7.2** 4.9
Fruiting plants 372 136 130 72 22 58 6.8 4.9
zFlowers at anthesis were harvested from 30-36 days after treatments began.
YMeans were averaged across fruiting treatment, n = 53 and 52 for 12C and 200C,
respectively.
xMeans were averaged across temperature, n = 60 and 45 for non-fruiting and fruiting
plants, respectively.
SAsterisks indicate means are significantly different (t-test) at P<0.01 or P<0.001,
respectively.









Table 4-7. P-values for effects of night temperature (Temp), presence/absence of fruits
(Fruits), and days after treatment (DAT) and their interactions on leaf gas
exchange parameters in 'Legionnaire' bell pepper.
Source of variations Carbon exchange rate Stomatal conductance Intercellular CO2
(pmol m-2 s-1) (mol m-2 s-1) (pmol mol-1)
Temp 0.006 <0.001 <0.001
Fruits 0.003 0.02 0.17
Temp*Fruits 0.33 0.26 0.92
DAT <0.001 <0.001 <0.001
DAT*Temp <0.001 <0.001 <0.001
DAT*Fruits 0.12 0.30 0.88
DAT*Temp*Fruits 0.70 0.87 0.63
Block(Temp) 0.20 0.45 0.47
Plant 0.09 0.21 0.40
zDegrees of freedom: 1 for Temp, Fruits, and Temp*Fruits, 11 for DAT, DAT*Temp,
DAT*Fruits, DAT*Temp*Fruits; not available for Block(Temp) (random factor) or Plant
(subject for the repeated measures).

Table 4-8. Main effects of night temperature and presence/absence of fruits on carbon
exchange rate, stomatal conductance and intercellular C02 in 'Legionnaire'
bell pepper leaves.
Carbon exchange Stomatal conductance Intercellular CO2
rate (pmol m-2 s-1) (mol m-2 s-1) (pmol mol-1)


Night temperatures
12C 8.4* 0.10*** 212***
20C 10.2 0.15 238
Fruits
Non-fruiting plants 8.7** 0.11* 222
Fruiting plants 9.9 0.13 227
zMeasurements were done six hours after lights were on and every three to four days
during a 41-day period on one to three of the most recently matured leaves per plant.
YMeans were averaged across fruiting treatment and days after treatment, n = 144.
xZero or two developing fruits per plant. Means were averaged across temperature and
days after treatments, n = 144.
*, **, ***Means are significantly different (t-test) at P<0.05, P<0.01, and P50.001,
respectively.









Table 4-9. Night temperature and presence/absence of fruit effects on growth of
'Legionnaire' bell pepper at the end of the 45-day experiment period.
Leaf Leaf SLAz DW (g) Plant Node Internode
no. area Leaf Stem Root Fruitsy Total height no. length
(cm2) (cm) (cm)
Night tempx
12C 79 3187 106 30.0 35.3 17.4 18.9 108 75 10.5 4.8
20C 83 3549 135 26.6 38.6 17.3 19.1 107 84 12.1 5.0
Values 0.60 0.310.001 0.21 0.29 0.95 0.95 0.93 0.01 0.03 0.30
Fruits
Non-fruitingy 86 3500 114 30.7 41.5 18.6 6.4 105 82.0 11.8 4.9
Fruiting 76 3236 126 25.9 32.4 16.0 31.6 110 76.9 10.8 4.8
Values 0.03 0.12 0.050.0030.0010.002 0.001 0.12 0.01 0.01 0.76
ZSLA: specific leaf area (cm2 g-).
Yln non-fruiting plants, the fruits were harvested at the beginning of the experiment.
xData were averaged across fruiting treatments, n = 12.
WData were averaged across temperatures, n = 12.










14 A 0.3
SA 0 a 290 a
13 *
12 a b 270 bc bc
11 -
b1 0.2 250 cd
10 bc bc c de
c E c 230 de ef
c ce d cde ef ef
0 E o
E 8 E 210 fg
S)d O.l 1 def def de o
7 ef E
Sd 190 g
LU f
0 6 d -
170

4 .. 0.0 150
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Days after treatment

Figure 4-1. Effect of days after treatment began on (A) net carbon exchange rate (CER)
and (B) stomatal conductance (gs), and (C) intercellular C02 (Ci) of sweet
pepper leaves. Gas exchange was measured six hours after lights were on
and over a 45-day period after the beginning of night temperature treatments
in two cultivars. Data were averaged across night temperatures and cultivars.
Means with the same letters were not significantly different (Tukey-Kramer,
P<0.05), n=36.
















**


--~ 'Legionnaire'
- 'Red Cherry Sweet'


0 10 20 30 40 50 0
Days after treatment


10 20 30 40 50


Figure 4-2. Effect of (A) night temperature and (B) cultivar on net carbon exchange rate
(CER) of sweet pepper leaves over time. Gas exchange was measured six
hours into the light period and over a 45-day period after the beginning of
night temperature treatments. *, **, ***Means between temperature
treatments (A) or cultivars (B) within each measurement time were
significantly different at P<0.05, P<0.01, and P<0.001, respectively, n = 18.









0.35


0.30 *
*
v 0.25 ,


O0.20
E I I






0.05
----12C 'Legionnaire'
--620oC ---'Red Cherry Sweet'
0.00 .. .. .
0 10 20 30 40 50 0 10 20 30 40 50
Days after treatment

Figure 4-3. Effects of (A) night temperature and (B) cultivar on stomatal conductance
(gs) of sweet pepper leaves over time. Measurements were done six hours
after lights were on and over a 45-day period after the beginning of night
temperature treatments. *, **, ***Means between temperature treatments (A)
or cultivars (B) within each measurement time were significantly different at
Ps0.05, P<0.01, and P<0.001, respectively, n = 18.


100









2
i
o
E
0
b b
4-


I I I I I II
12 20 12 20 LEG RCS LEG RCS 13 27 47
Night temperature (C) Cultivar DAT
Figure 4-4. Effect of (A) night temperature, (B) cultivar, and (C) days after treatments
began (DAT) on leaf respiration in sweet pepper plants during the day (w) and
night (m). RCS: 'Red Cherry Sweet', LEG: 'Legionnaire'. For day respiration
(A and B), n = 30; for night respiration (A and B), n=54; and for DAT (C), n =
36. *' **Means are significantly different at P50.05 and P50.001, respectively.
In (C), means with the same letters were not significantly different (Tukey-
Kramer, P<0.05).


101









14 A 0.3 B 300 C
13 a a
12 -a275

b b -0.2 -250 b
0 b c b bc d bcd
E9 bc bcb E E bcd b
9 bc d bcb b 225 bcd

7- ".1 -cde e 200 de
L d d
d d de ef
6 e175 f
5
4 0 150
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
Days after treatment

Figure 4-5. Effect of days after treatment began on (A) net carbon exchange rate (CER),
(B) stomatal conductance (gs), and (C) intercellular CO2 (Ci) of 'Legionnaire'
bell pepper leaves. Measurements were done six hours after lights were on,
and over a 41-day period after the beginning of night temperature and fruiting
treatments. Data were averaged across night temperatures and fruiting
treatments. Means with the same letters were not significantly different
(Tukey-Kramer, P<0.05), n = 24.


102









14 A 0.3 300
SA B C
13 .
12 275
1- ** \*

SE **


S0.1 200

6 I
't* 175 -' 1
5 -12C
S-*-20C
4 0.0 150
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
Days after treatment

Figure 4-6. Effect of night temperature and days after treatment began on (A) net
carbon exchange rate (CER), (B) stomatal conductance (gs), and (C)
intercellular CO2 (Ci) of 'Legionnaire' bell pepper leaves. Measurements were
done six hours after lights were on, and over a 41-day period after the
beginning of the treatments. Data were averaged across fruiting treatments. *
**, *** Means between the two night temperature treatments within each
measuring time were significantly different at P50.05, P<0.01, or P<0.001,
respectively, n=12.


103










14 -

13

12

11

10

9

8

7


* *


I I
Il
I,


-; -Fruits
---- + Fruits


0 10 20 30 40 50 0
Days after treatment


10 20 30 40 50


Figure 4-7. Effect of presence or absence of developing fruits and days after treatment
began on (A) net carbon exchange rate (CER) and (B) stomatal conductance
(gs) of 'Legionnaire' bell pepper leaves. Measurements were done six hours
after lights were on, and over a 41-day period after the beginning of the
treatments. Data were averaged across night temperature treatments. *, **
*** Means between the two night temperature treatments within each
measuring time were significantly different at P<0.05, P<0.01, P<0.001,
respectively. For each mean, n = 12.


104


0.2


S .* .









CHAPTER 5
BELL PEPPER OVARY SWELLING DUE TO LOW NIGHT TEMPERATURE AND
SOURCE SINK RATIO: OVARY WALL ANATOMY AND CARBOHYDRATES

Introduction

In pepper (Capsicum annuum L.), as in all horticultural crops, high quality yield is

as important as total yield. Fruit weight, size, shape, pericarp thickness, and carpel

number and regularity are used to evaluate pepper fruit quality parameters and to

differentiate pepper types and varieties (Aloni et al., 1999; Navarro et al., 2002;

Kissinger et al., 2005; Lim et al., 2007). In bell and spherical peppers, both shape and

size are primarily determined at early preanthesis stage, while in elongated fruits, shape

is determined in both pre and post anthesis (Munting, 1974). Early flower development,

in turn, is very sensitive to several factors, including low ambient temperature and

carbohydrate availability (Ali and Kelly, 1992; Tomer et al., 1998; Aloni et al., 1999).

Low night temperatures affect bell pepper production. In winter, lower

temperatures and shorter day lengths increase the percentage of small, flattened and

parthenocarpic fruits. Poor pollination is the main reason for such problems, but

malformation of female organs (Polowick and Sawhney, 1985; Pressman et al., 1998a;

Shaked et al., 2004) and other parts of the flower (Aloni et al., 1999) also play a role. In

tomato, low night temperature decreases ovule number in one or more locules in some

cultivars, causing malformed fruits (Tomer et al., 1998).

Night temperatures of 15C or less increase ovary diameter in pepper without

increasing locule number, creating "swollen" ovaries and malformed fruit (Polowick and

Sawhney, 1985; Rylski, 1985; Bakker, 1989b; Aloni et al., 1999; Shaked et al., 2004).

The longer the duration of low night temperatures, the higher the percentage of swollen

flowers and ovaries (Polowick and Sawhney, 1985; Aloni et al., 1999) or the greater the


105









swelling (Chapter 3). For instance, under night temperature of 12C, the percentage of

swollen flowers, and therefore ovaries, increased from 21% at 15 days after treatment

to 78% at 49 days after treatment, compared with an increase in swollen flowers from 7

to 14% in plants growing at night temperatures of 180C (Aloni et al., 1999). Evidence

indicates that flower deformation, including ovary swelling, is favored by low shoot

temperature during the night, regardless of root temperature (Polowick and Sawhney,

1985; Mercado et al., 1997c; Aloni et al., 1999).

Increased source/sink ratio also increases the proportion of swollen ovaries. Aloni

et al. (1999) compared fruiting with defruited pepper plants and found that defruiting

increased fresh weight of flowers three to four times that of the flowers on fruiting plants,

resulting in swollen ovaries and malformed fruits. In addition, the percentage of swollen

flowers was inversely related to the number of growing fruits on the plant, and directly

proportional to the concentration of reducing sugars and starch in the flowers developed

in those plants (Aloni et al., 1999). Flowers that developed on defruited plants

accumulated more 14C02 (600 vs 150 pg d-1) and fresh weight (180 vs 60 mg) than

flowers that developed on fruiting plants (Aloni et al., 1991b; Aloni et al., 1999). These

data suggest that excess assimilates are transported to flower buds on defruited plants,

resulting in ovary swelling and deformation (Aloni et al., 1999).

Limited work has been done on the anatomical aspects of swollen ovaries in

pepper. Pressman et al. (1998b) reported that swollen ovaries of bell pepper that

developed in response to cool night temperatures had larger cells than normal ovaries,

but cell number was similar, suggesting that swollen ovaries develop after cell division

ends. However, our findings (Chapter 3) suggest that in order to reach the maximum


106









swelling effect, the cool night temperature stimulus needs to start during the first week

after flower initiation, a stage in which active cell division is taking place (Munting,

1974).

Flower and ovary swelling is favored by exposing the shoot to low night

temperature or by increasing the source:sink ratio. However, the interaction between

night temperature and source-sink modification on ovary swelling is unknown. For these

reasons, the hypothesis tested in this study was that high night temperature (HNT)

combined with high source:sink ratio or low night temperature (LNT) combined with low

source:sink ratio can overcome the detrimental effects of LNT or high source: sink ratio

on ovary swelling. The specific objectives of this study were to 1) investigate whether

ovary size is increased by high source:sink ratio under warm night temperature, and

reduced by low source:sink ratio under low night temperature, 2) investigate the

combined effect of night temperature and source:sink ratio on cell number and cell size

of ovary wall at flower anthesis, and 3) determine the ovary wall and placenta content

and concentration of soluble sugars and starch in bell pepper ovaries developed under

two night temperature regimes and two source:sink ratios.

Materials and Methods

Plant Material

'Legionnaire' bell pepper seedlings were grown in a growth chamber (E15

Conviron, Winnipeg, Canada) set to 22C/20C (day/night temperature), 400-500 pmol

m-2 s-1, and a14-h photoperiod. At the sixth-leaf stage, seedlings were transferred to 1.5

L containers using commercial growing media. Plants were trimmed to two axes, as

described in Chapter 3 (Figure 3-2A). Starting in sympodial unit 1, the strongest shoot

was allowed to grow and the second shoot was limited to development of one


107









sympodial unit (one flower and two leaves) by pruning. Flower buds from the main stem

(first terminal flower) and the first node in the two axes (i.e., sympodial unit 1) were

removed.

Experimental Description

A 2 x 2 factorial experiment (two night temperatures regimes and two source:sink

ratios) was carried out twice. Night temperatures were 200C (HNT) and 120C (LNT) with

the day temperature set to 220C. To establish two different source:sink ratios, one fruit

was allowed to set in the second or third node of each of the two axes in all plants. Five

to 12 days after petal fall (depending on the experiment), fruits on the plants in one

treatment were removed, while fruits on the other treatment were allowed to develop to

maturity. All subsequent flowers and small fruits were removed to avoid further fruit set.

These treatments resulted in low source:sink (fruiting plants, +F) or high source:sink

(non-fruiting plants, -F) ratios. This resulted in four treatment combinations (HNT + F,

HNT F, LNT + F, LNT F). The night temperature and source:sink ratio treatments

started five to twelve days after petal fall, when fruits on fruiting plants exhibited

maximum relative growth rate, according to Marcelis et al. (1995b). Each treatment

combination was replicated six times in a completely randomize block design. All open

flowers were removed when the treatments started. Subsequently, all flowers at

anthesis were harvested for sampling, starting the day after treatments began.

Two experiments were performed. Experiment 1 was designed to determine the

contribution of cell number and cell size to ovary size in the four treatment

combinations. Temperature treatments began 5 to 8 days after petal fall, when one half

of the plants were defruited (high source:sink ratio). Flowers from nodes 4 or 5 and the

subsequent nodes were harvested daily for 42 days after treatments started (DAT).


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Diameter, length and fresh weight of the ovary were recorded and ovaries were fixed in

a formalin-acetic acid-ethanol (FAA) mixture for anatomical studies (see below).

Experiment 2 was designed to determine carbohydrate concentration and content

in the ovary wall and placenta in the four treatment combinations. Developing fruits

were between 8 and 12 days after petal fall when the defruiting was performed and the

night temperature treatments started. Flowers from Experiment 2 were harvested at

anthesis, starting in node 5 and 6, and flowers were harvested daily for 38 days. Fresh

weight, diameter, and length of the ovary were recorded.

Anatomy

Ovaries were individually fixed in FAA. Based on the ovary size data (see below),

an anatomical analysis was performed three times: at 1-2 DAT, at 18-19 DAT, and at

38-40 DAT (see Figure 5-1). A common sampling was used at the beginning of the

experiment, since it was the initial control with no treatment effect. For every treatment

combination and harvest time, the mean fresh weight of all flowers harvested was

calculated. Then, four flowers with fresh weights closest to the mean FW for each

treatment and harvest time were selected for anatomical analysis.

Samples were dehydrated and resin embedded. First, samples were transferred

from the FAA to 50% ethanol to rinse the formalin and acetic acid from the FAA. In

order to facilitate tissue infiltration, the bottom and top sections of the ovary were cut,

and only the middle section was processed. Samples were then dehydrated by

gradually increasing ethanol from 50% to 100%, before placing in 100% acetone (see

Table 5-1). Resin embedding steps were done under partial vacuum. Dehydration and

resin embedding were done using a microprocessor-controlled microwave oven

equipped with a ColdSpot temperature control system (PELCO BioWave Laboratory


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Tissue Processing System, Ted Pella, Inc. Redding, CA, USA), to speed the process.

Microwave power output was set to 180 W, and ColdSpot water bed temperature was

24C. Spurr resin3 (cycloaliphatic epoxide resin [ERL 4221, 4.10g], diglycidyl ether of

polypropylene glycol [DER 736, 1.43g], nonenyl succinic anhydride [NSA, 5.90g] [Ted

Pella, Inc. and PELCO International, Redding, CA], and dimethylaminoethanol [DMAE,

0.1g] [Electron Microscopy Sciences, Hartfield, PA]) was used. After resin embedding,

the samples were put in molds. Molds were kept overnight under vacuum to remove air

bubbles. Samples were then polymerized in an oven at 60C for 24 hours.

Following resin embedding, samples were cut in four pieces, and 700 nm slices

were cut from one piece using a cryo-ultramicrotome (Leica Ultracut UCT,

www.leica.com). Slices were stained with an aqueous solution of 1% toluidine Blue O

and 1% sodium borate, and mounted with immersion oil. Slides were observed in an

Olympus BH2-RFCA fluorescence microscope (Olympus, Melville, NY). Images were

taken with a digital camera Q-Imaging Retiga 2000R and Q-Capture Pro Ver 5.1.1.14

for Win (Q-Imaging, Surrey, BC, Canada), and analyzed using ImageJ software

(Rasband, 2010) after calibration. Ovary wall thickness, thickness of each ovary wall

tissue layer (epidermis, hypodermis, mesocarp, and endocarp), number of cell layers in

the ovary wall, and cell size of the epidermis, hypodermis, mesocarp, giant, and

endocarp cells were measured (Figure 5-2). Average cell size in each tissue was

measured by drawing around a set of cells, measuring the area, and determining cell

number with the Cell Counter plug-in (Kurt De Vos. University of Sheffield, Academic

Neurology, England). There were no differences between hypodermis and epidermis



3 http://www.tedpella.com/technote_html/18300-4221 %20TN.pdf; 3/2/2010.


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cell size, therefore for the final analysis, both tissues were analyzed as epidermal

tissues. No information was recorded for the vascular bundles. Transverse section area

of the ovary wall and ovary wall tissue layers was calculated with the circle and the ring

area formula (ring area = n-r(ri r2)2, where ri equals diameter divided by 2, and r2

equals ri minus tissue thickness). The section area was then corrected with a constant

for shape irregularity. To find the constant, 23 ovaries ranging from 23 to 249 mg FW

(3.5 9.1 mm in diameter and 2.7-5.3 mm in length) were photographed using a

binocular microscope. Total area was then measured from monochromatic images

using ImageJ software (Rasband, 2010), and a linear regression (y = b-x, y = measured

area, b = slope, and x = calculated area) between measured and calculated area was

generated to determine the slope (Figure 5-3).

Carbohydrates

Carbohydrates were analyzed in flowers harvested in Experiment 2 during the

fourth week after the beginning of treatment (261 DAT, nodes 8 to 10 above first

terminal flower), when the maximum difference in ovary size was found. Glucose,

fructose, sucrose and starch were analyzed in the ovary wall and placenta of the ovary.

Harvested flowers were maintained on ice while ovary fresh weight, length and

diameter were recorded. The ovary was then separated into ovary wall and placenta

and stored at -800C (Harris. Kendro Laboratory Products, Asheville, NC, USA). Samples

were lyophilized (Virtis Model 10MR-TR, The Virtis Co., Gardiner, NY, USA) for 72

hours. Samples were subsequently stored at -200C in sealed containers until analysis.

For soluble sugar and starch analysis, samples were weighed and ground in liquid

nitrogen. Samples (10-25 mg) were dissolved in 1 mL 80% ethanol, shaken for 20 min

at 200 rpm in an orbital shaker (Fisher Scientific Model 361; U.S.A.), and centrifuged at


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1380 x g for 10 minutes. The supernatant was removed, and the pellet was re-extracted

with ethanol. The supernatants of the two extractions were combined and total volume

measured. The supernatants were cleared with activated charcoal to remove pigments

and centrifuged at high speed (13,250x g, 4 min). Soluble sugar recovery from samples

was above 95%, as determined by a 14C external standard.

Soluble sugars were analyzed with an enzymatic method (modified from Outlaw

Jr. and Tarczynski, 1984). Three enzyme systems (ES) were used: 1) 'ESA' -

hexokinase (E.C. 2.7.1.1, 0.0565 units/pL) and glucose-6-phosphate dehydrogenase

(E.C. 1.1.1.49, 0.0808 units/pL) to measure glucose; 2) 'ESB' -'ESA' and

phosphoglucoisomerase (EC 5.3.1.9, 0.0741 units/pL) to measure glucose and fructose;

and 3) 'ESC' -'ESB' and invertase (EC 3.2.1.26, 0.7712 units/pL) to measure glucose,

fructose and sucrose. The enzymes were diluted in 1% w/v bovine serum albumin

(BSA, Sigma A6003), 1 M HCI, and 40 mM tris(hydroxymethyl) aminomethane (Fisher

Scientific BP152-500, pH: 10-11.5). For each ES, an aliquot of the cleared sample

(30pL), the assay cocktail (1 mL), and the enzyme system (10 pL) were mixed and

incubated at room temperature for 60 to 90 min, before reading absorbance at 339 nm

in a spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan). The assay cocktail

contained the following components: 170 mM N,N-Bis(2-hydroxyethyl)-2-

aminoethanesulfonic acid (BES, Sigma B4554), 65 mM KOH; 15 mM MgCI2, 1.5 mM

EDTA, 2 mM NADP, 5 mM ATP, and 0.015% w/v BSA.

Sample pellets were analyzed for starch using amyloglucosidase (E.C. 3.2.1.3, 50

units/sample) and measuring resultant glucose equivalents. Pellets were boiled in 2 ml

0.2N KOH for 30 minutes, then acidified with 1 ml of 1M acetic acid. After cooling, 1 mL


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of amyloglucosidase solution (50 units/mL in 0.2M calcium acetate buffer, pH 4.5) was

added and samples were incubated for 18 hours at 37C. Volume was recorded and the

digest was centrifuged for 10 min at 1975 x g. An aliquot of the supernatant was

removed and glucose was quantified. Percent recovery, as estimated using a 14C

external standard, was above 95%.

Data Analysis

Ovary size data from the two experiments were pooled and analyzed together

using mixed models and the SAS program, (SAS Institute Inc., 2008). Flower

harvests were grouped into 4-day intervals, resulting in 11 groups for Experiment 1 and

10 groups for Experiment 2. Data from 5-8 DAT was only from Experiment 2 and data

from 41-44 DAT was only from Experiment 1. Night temperature regime, source:sink

ratio (presence/absence of fruits), days after harvest (grouped in 4-day intervals), and

branch (flowers from the main axis or lateral branches) were considered fixed effects.

Blocks within each experiment and the experiments (1 and 2) were considered as

random effects, and every plant within each experiment was the repeated measures

unit. The anatomy data were analyzed in two phases. A preliminary analysis of variance

performed as a 2 x 2 x 2 factorial (2 night temperatures, 2 source:sink ratios, 2 harvest

times [i.e., without considering the control]) indicated that the temperature x harvest

time and the temperature x source:sink ratio x harvest time were not significant (Table

A-6, Appendix). The final analysis was done as a randomized block design, where all

combinations were handled as treatments (i.e., 9 treatments: 23 factorial + control).

Main effects and specific comparisons were performed based on the preliminary data

and by using contrasts. No comparisons were performed for the non significant


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interactions. Carbohydrate data were analyzed as a 2 x 2 factorial using a completely

randomized block design with 6 replications.

Results

Ovary Size

Low night temperature significantly increased ovary fresh weight, diameter, and

length (Table 5-2; Table A-5, Appendix). Fresh weight increased about 25%, while

diameter and length increased 9 and 6%, respectively.

The presence or absence of fruits also affected ovary size parameters. In non-

fruiting plants, ovary fresh weight of flowers at anthesis was 35% greater than those

from fruiting plants. Both ovary diameter and length also increased in non-fruiting

compared with fruiting plants.

Since flowers at anthesis were harvested over 38-42 days after temperature

treatments started, ovaries were grouped into 10 (Exp. 2) or 11 (Exp. 1) 4-day harvest

intervals. Harvest timing significantly influenced ovary size parameters. In general,

ovary FW increased as harvesting intervals proceeded (Table 5-3). Significant

increases in ovary FW were observed beginning at 13-16 DAT and maximum size was

reached at 25-28 DAT. Based on ovary FW, ovaries could be classified into three

groups: 1 to 12 DAT, 13 to 24 DAT, and 25 to 42 DAT. Ovary diameter increased in a

similar pattern as fresh weight, with significant increases observed beginning at 9-12

DAT. Ovary length was less responsive; ovaries harvested from 1 through 20 DAT were

similar in length and shorter than ovaries harvested from 25 through 44 DAT.

There was a significant interaction between night temperature and

presence/absence of fruits on ovary fresh weight (P<0.02), but not on diameter or length

(P<0.08). Ovary fresh weights in non-fruiting plants under LNT (L-) were the largest,


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averaging 149 mg; while fresh weights were smallest in the HNT with fruits (H+),

averaging 90 mg (Figure 5-4). A similar pattern was observed in ovary diameter and

length, although the interaction was not significant.

The interaction between night temperature and harvest interval was significant for

ovary FW (Figure 5-5), but not for diameter (P<0.36) or length (P<0.76) (Table A-5 and

Figure A-2, Appendix). Ovary fresh weight increased ~42 mg (42%) between harvest

intervals 1-4 DAT and 33-36 DAT in plants developed under LNT, while fresh weight

increased ~25 mg (28%) in ovaries developed under HNT during the same intervals.

Ovary size was also affected by the interaction between days after treatment and

fruiting treatments, which was highly significant for the three parameters (Figure 5-6).

Starting at 5-8 DAT, the ovaries developed on non-fruiting plants had greater FW,

diameter and length than the ovaries developed on fruiting plants. On the non-fruiting

plants, ovary FW, diameter, and length increased steadily from 1-4 DAT through 25-28

DAT, then decreased between 33-36 and 41-44 DAT (t-test, P<0.04 for 33-36 DAT vs

37-40 DAT and P<0.003 for 33-36 DAT vs 41-44 DAT). In fact, pair wise comparisons

revealed that ovary FW and diameter were significantly greater as early as 5-8 DAT

compared to 1-4 DAT (t-test, P 50.01). In fruiting plants; however, there was a

significant reduction in ovary FW and length during the first 5 to 6 harvest intervals

compared to the first harvest interval (t-test, P<0.001), while ovary diameter remained

constant. Subsequently, ovary FW, length, and diameter increased, and by 25-28 DAT

(FW and length) or 29-32 DAT (diameter), were significantly larger than observed at

harvest intervals 17-20 or 21-24 DAT (t-test, P<0.001).


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Anatomy

Ovaries used for the anatomical study were a subsample of the total number

harvested, as described above. Ovaries developed under LNT were significantly larger

compared with ovaries developed under HNT (Figure 5-7A), in accordance with data

presented in Table 5-2. LNT also increased the ovary wall thickness and total

transverse area in the ovary mid-section (Figure 5-7B).

Based on total transverse area of the ovary wall, mesocarp tissue accounted for

72% of the area, followed by giant cells (16%), epidermal tissue (8%) and endocarp

(4%). Although there were differences between LNT and HNT in ovary FW, diameter,

and ovary wall transverse area (Figure 5-7), there were no differences in cell size or cell

number in mesocarp or giant cells between the two night temperature treatments

(Figures 5-8 and 5-9). There was also no difference in the number of cell layers in the

mesocarp (22.0 1.1 vs 20.3 0.7 for LNT and HNT, respectively, data not shown).

Epidermal cells were larger under LNT compared to HNT (P < 0.04; 214 vs 190 pm2),

but there were no differences in cell number (Figures 5-8 and 5-9). There was also no

significant effect of night temperature on endocarp cell size or cell number.

Ovary FW and diameter in non-fruiting plants were significantly greater compared

with ovaries developed on fruiting plants (Figure 5-10), as previously shown in Table

5-2. Ovary wall thickness was also greater in ovaries of non-fruiting compared with

ovaries of fruiting plants (564 vs 444 pm), which in combination with the greater

diameter, resulted in increased total transverse area (12.11 vs 7.73 mm2 for non-fruiting

and fruiting, respectively).

Increases in ovary size in non-fruiting compared with fruiting plants were due to

increased cell size in mesocarp, giant cell, and epidermal tissues, which comprised 96%


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of the ovary wall (Figure 5-11A, B, C; Figure 5-12). Cell number was significantly

greater in ovary endocarp tissue of non-fruiting compared with fruiting plants. There was

also a small, but non-significant, increase in cell number in mesocarp (P<0.09) and

epidermal tissues (P<0.07) of ovaries in non-fruiting plants. Non-fruiting plants also had

a greater number of cell layers in the mesocarp compared to the fruiting plants

(23.01.1 vs 19.40.6 for -F and +F plants, respectively).

Ovary FW increased between 18-19 DAT and 38-40 DAT, while ovary diameter

did not increase (Figure 5-13). Ovary wall thickness at 18-19 DAT (550 pm) was greater

than at 38-40 DAT (458 pm), resulting in a significant increase in ovary wall area at 18-

19 DAT compared with the other two harvest times. Greater ovary fresh weight at 38-40

DAT may be due to greater ovary length in that time (4.3 mm) compared to 1-2 DAT

(3.9 mm) and 18-19 DAT (4.1 mm) (P<0.004, data not shown).

Mesocarp and giant cell size was greater in 18-19 DAT than in 38-40 DAT (Figure

5-14A, B). However, mesocarp cell number increased from 17,100 to 22,000 per cross-

section area in the ovary wall between 1-2 DAT and 38-40 DAT (P<0.06). Regression

analysis indicated a significant increase in giant cell number (cell number = 314.30 +

2.63 x DAT; r2 = 0.99; P<0.01) with days after treatment. A similar trend was observed

in mesocarp cell number (cell number = 17165 + 129.4 x DAT; r 2 = 0.95, P<0.10), and

the increase in cell number may account for the increased ovary FW in 38-40 DAT

compared with 18-19 DAT.

There was a significant interaction between night temperature and

presence/absence of fruit on ovary growth and anatomy (Figures 5-15 and 5-16, Table

A-5, Appendix). Flowers developed under LNT in non-fruiting plants developed the


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largest ovaries (Figure 5-15), while fruiting plants under both night temperatures

developed the smallest ovaries. This differs slightly from the average ovary FW

determined using the entire dataset (Figure 5-4), where ovary FW in fruiting plants

under HNT were significantly smaller than ovaries developed under all other conditions.

Although the trend observed in Figure 5-15 is similar to that observed in Figure 5-4, the

difference is likely due to the smaller sample size used in the anatomical studies, as

well as the slight difference in ovary harvesting times between the anatomical study

(Experiment 1) and the pooled data (Experiments 1 and 2) that was used for ovary

growth measurements.

In the mesocarp, cell size was largest in non-fruiting plants under LNT and

smallest in fruiting plants under HNT (Figure 5-16A). There were no differences due to

temperature, but only due to presence or absence of developing fruits. Mesocarp cell

number, however, was greater in non-fruiting plants under LNT than in the rest of the

combinations (P<0.06). The number of mesocarp cell layers followed the same trend,

with greater number of layers in the non-fruiting plants under LNT (25.0 1.6)

compared with the rest of the treatment combinations (19.1+0.6, 20.70.7 and 19.81.2

for L+, F- and F+, respectively). Size of giant cells was affected only under HNT, where

non-fruiting plants had larger giant cells than did fruiting plants (Figure 5-16B). Such

differences in cell size were compensated for in cell number, so that the total area of

giant cells was similar in the ovaries of both fruiting and non-fruiting plants under HNT

(data not shown).

There were also significant interactions between harvest time and

presence/absence of fruit. Ovary FW and diameter increased at both 18-19 DAT and


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38-40 DAT in non-fruiting plants, compared to fruiting plants (Figure 5-17A). The

smallest ovaries were harvested from fruiting plants at 18-19 DAT, in accordance with

what was previously described in Figure 5-6. Although there were no differences in

ovary FW or diameter between non-fruiting plants at 18-19 DAT and 38-40 DAT, ovary

wall thickness was greatest in non-fruiting plants at 18-19 DAT compared with the other

treatments, resulting in greater ovary wall transverse area (Figure 5-17B). Since there

were no differences in ovary length between 18-19 DAT and 38-40 DAT (4.5 and 4.6

mm, respectively, P<0.50) in non-fruiting plants, the similar ovary FW and diameter

between these two harvest times suggests that the placenta in 38-40 DAT ovaries was

larger than those of 18-19-DAT ovaries. In fruiting plants, there were no differences in

diameter, but ovary FW was greater at 38-40 DAT due to a greater length at 38-40 DAT

(4.1 mm) compared to the diameter at 18-19-DAT (3.6 mm, P<0.001).

Differences in ovary wall transverse area are likely due to the increased cell size of

the mesocarp, giant cells and epidermal tissues in non-fruiting plants at 18-20 DAT

compared to the rest of the treatment combinations (Figure 5-18). There were no

differences between harvest times within fruiting and non-fruiting plants for mesocarp

cell number. There was, however, a trend towards increased cell number in mesocarp,

epidermal tissue and endocarp of non-fruiting compared with fruiting plants.

Carbohydrates

Low night temperature (LNT) increased glucose, fructose and starch concentration

and decreased sucrose concentration in the ovary wall compared to HNT (Table 5-4). In

contrast, soluble sugar and starch concentrations in the placenta were not affected by

night temperature. In general, there were no differences in soluble sugar or starch

concentrations in the ovary wall or in placenta between fruiting and non-fruiting plants,


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with the exception of sucrose in the placenta. There were no significant interactions

between night temperature and presence/absence of fruits on carbohydrate

concentrations (Table A-7, Appendix).

Total content of reducing sugars and starch was also greater in the ovary wall of

ovaries developed under LNT compared to ovaries in the HNT (Table 5-5). Sucrose

content; however, was similar, since the increased DW under LNT was compensated by

the decreased sucrose concentration. In the placenta, fructose and starch content was

greater under LNT compared with HNT; however, sucrose and glucose contents were

not affected. Overall, total content of soluble sugars and starch was 74% greater under

LNT than under HNT.

Soluble sugar content was higher in ovary wall and placenta of non-fruiting

compared with fruiting plants (Table 5-5). Starch content was also higher, although the

increased starch in ovary wall of non-fruiting compared with fruiting plants was not

significant. Overall, carbohydrate content was 31% greater in non-fruiting plants

compared with fruiting plants. No significant interaction was found between night

temperature and presence/absence of fruits on carbohydrate content in the ovary wall

or placenta.

Discussion

Ovary size (and therefore the incidence of swollen ovaries) of 'Legionnaire' bell

pepper increased under LNT compared with HNT, and in non-fruiting (high source:sink

ratio), compared to fruiting (low source:sink ratio) plants, confirming previous reports for

bell and other sweet pepper cultivars (Chapter 3; Pressman et al., 1998b; Aloni et al.,

1999; Shaked et al., 2004). In the present study, ovary FW (which is comprised of ovary

wall FW and FW of the inner placental tissues located in the cavity) increased 25% to


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35% under LNT compared to HNT and non-fruiting compared with fruiting treatments,

respectively. Such increases are comparatively smaller than the values reported by

Aloni et al. (1998b; 1999), who reported increases of 100 to 300% in ovary FW of bell

peppers under LNT or non-fruiting conditions. The reasons for the quantitative

differences in effects of night temperature or source:sink ratio on ovary size of bell

pepper between the two studies may be due to differences in cultivar, plant age, or

environmental conditions used.

Ovary size under LNT increased due to a significant increase in ovary wall

thickness and increased transverse area, without significant increases in cell size or cell

number. However, there was a trend towards larger cells in all ovary wall tissues of

floral ovaries developed under LNT compared with HNT, and a trend towards increased

cell number in the mesocarp and endocarp. Taken together, these increases resulted in

significant increases in ovary FW, diameter, length, and ovary wall thickness and

transverse area in floral ovaries developed under LNT compared with HNT. The trend

in increased cell size observed in the present study is supported by results from a

previous study where LNT increased cell size in mesocarp and giant cells in bell pepper

compared with HNT (Pressman et al., 1998b).

In contrast to the effect of night temperature on ovary swelling, the increase in

floral ovary FW in non-fruiting compared with fruiting plants observed in the present

study was clearly due to significant increases in cell size of the mesocarp, giant cells

and epidermal cells. Actively growing fruits are a much stronger sink for assimilates

compared to flowers (Ali and Kelly, 1992; Marcelis and Baan-Hofman-Eijer, 1997), and

the presence of developing fruits likely reduced the source supply available for floral


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development, resulting in decreased cell size and ovary FW, and therefore the

incidence of swollen ovaries compared with non-fruiting plants. Similarly, the observed

decrease in the number of cell layers in floral ovary mesocarp in fruiting compared with

non-fruiting plants is also a response to increased sink demand of fruiting plants, as

observed by Ali and Kelly (1992).

Differences in ovary FW, diameter, and length due to night temperature were

found within the first four days after treatment. Responsiveness of 'Legionnaire' bell

pepper in the two experiments reported here was similar to the previous response

(Chapter 3), where significant differences due to night temperature were found in the

first week. In that experiment, we found that most sweet pepper cultivars required three

to four weeks of LNT for induction of maximum ovary swelling and concluded that

flowers must be exposed to LNT soon after floral initiation begins. 'Legionnaire' bell

pepper appears more sensitive than other sweet pepper types to LNT induced ovary

swelling and does not appear to require exposure during the first week after flower bud

initiation. Further, although Munting (1974) reported that cell division is the main

process occurring during floral development in pepper prior to anthesis, our results

indicate that cell size is most affected in 'Legionnaire' pre-anthesis in response to LNT

or source:sink modification. Thus, there may be genotypic differences among pepper

cultivars in the intervals of cell division and cell enlargement during flower development

due to the different rates of cell division between genotypes, specially elongated vs

blocky or spherical (Munting, 1974).

The combined effects of low night temperature and source:sink ratio on ovary

swelling in sweet pepper has not been studied previously. In the present research, floral


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ovary FW was greatest in non-fruiting plants under LNT and smallest in fruiting plants

under HNT, suggesting that night temperature and source:sink effects on ovary FW are

cumulative. As hypothesized, fruiting plants under LNT and non-fruiting plants under

LNT had intermediate ovary size. The increased ovary FW in non-fruiting plants under

LNT was due primarily to both increased mesocarp cell number and size, with additional

influences from increased epidermal cell size and increased endocarp cell number. On

the other hand, the decreased ovary FW exhibited by fruiting plants under HNT

compared with other treatments was due to decreased size of mesocarp and giant cells.

Although ovary FW and diameter were greater in non-fruiting compared with

fruiting plants throughout the duration of the experiment, the response over time (i.e.,

over DAT) was clearly different between fruiting and non fruiting plants. In fruiting

plants, floral ovary FW decreased during the first 24 days after treatments started, after

which, ovary FW increased. The decrease was likely due to the high sink demand of the

growing collective fruits. During the first third to the first half of the fruit growth period,

bell pepper fruit reaches the maximum absolute growth rate, gaining over 80% of its

final fresh weight and volume, and over 90% of its final diameter and length (Hall, 1977;

Nielsen et al., 1991; Marcelis and Baan-Hofman-Eijer, 1995b). Flower buds and flowers,

on the other hand, represent the weakest sink in bell pepper plant (Turner and Wien,

1994a). The sink demand of growing bell pepper fruits decreases ~ 35 days after

anthesis (Nielsen et al., 1991; Marcelis and Baan-Hofman-Eijer, 1995b), which

coincides with the increase in floral ovary FW observed in the present study.

In non-fruiting plants, on the other hand, floral ovaries increased in size during the

first seven harvest intervals (i.e., 28 days after treatments started) then remained stable,


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before decreasing during the last two harvest periods. Initial increase in ovary size is

likely due to the lack of fruiting and therefore increased source supply to flowers. As

discussed in Chapter 3 and based on previous reports (Choi and Gerber, 1992; Cruz-

Huerta, 2001), the time from flower initiation to anthesis is ~ 4 weeks in bell pepper.

Thus, maximum size of floral ovaries in non-fruiting plants was reached ~28 days after

treatments began, since these were flowers that developed fully under the non-fruiting

conditions. The decrease in floral ovary size in non-fruiting plants at the end of the

experiment may be related to a decreased photosynthetic rate in the non-fruiting

treatment as previously reported in pepper (Chapter 4; Hall and Milthorpe, 1978) and

tomato (Hucklesby and Blanke, 1992).

Anatomical analysis of a subsample of flowers harvested at discrete intervals (i.e.

1-2 DAT, 18-19 DAT, and 38-40 DAT) revealed that ovary wall transverse area and cell

size of mesocarp, giant cells, and epidermis were significantly greater in ovaries

harvested at 18-19 DAT in non-fruiting plants compared with 38-40 DAT in non-fruiting

or either harvest time in fruiting plants. This correlates well with the larger ovary FW at

18-19 DAT non-fruiting compared with either harvest time in fruiting plants. This does

not explain, however, why there was no difference in ovary FW in flowers harvested at

38-40 DAT vs 18-19 DAT in non-fruiting plants. Since there were no differences in ovary

length between 18-19 and 38-40 DAT in non-fruiting plants, the similar ovary FW and

diameter between the harvest times suggests that the placenta at 38-40 DAT ovaries

was larger than those of 18-19 DAT ovaries. This is supported by previous data

showing that placenta FW increases and the ratio of ovary wall FW:total ovary FW

decreases with harvest interval (Chapter 3).


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The increase in the ovary wall transverse area in ovaries harvested at 38-40 DAT

of non-fruiting plants compared with ovary wall transverse area of fruiting plants at both

harvest times may be due to the sum of the non significant increases in cell numbers of

the mesocarp, epidermis, and endocarp. Changes in source:sink ratios may affect fruit

size by regulating cell division as early as petal and sepal differentiation, and before

carpel initiation (Brukhin et al., 2003; Baldet et al., 2006). Our results suggest that

reducing fruit load initially increased ovary wall cell size, but later, as the fruits became

weaker sinks (Marcelis and Baan-Hofman-Eijer, 1995b), the effect on cell size of

initiating flowers was negligible.

The increase in ovary FW in harvest at 38-40 DAT compared with harvest at 18-19

DAT in fruiting plants was not reflected in significant increases in cell size in ovary wall

tissues. However, there was a trend towards increasing cell number in all ovary wall

tissues in fruiting plants at 38-40 DAT compared with 18-19 DAT. This, combined with

the greater ovary length, likely resulted in greater ovary FW at 38-40 DAT compared

with harvested flowers at 18-19 DAT on fruiting plants.

Carbohydrate accumulation was also affected by both night temperature and

source:sink ratio. As with effects on ovary cell size and number; however, the effects of

night temperature and source:sink ratio on carbohydrate accumulation as related to

ovary size differed. Low night temperature and high source:sink ratio both increased

floral ovary size, but the increase in ovary size was associated with increased ovary

carbohydrate concentration only in the low night temperature, not the high source:sink,

treatment. The increased reducing sugar and starch concentration in floral ovaries


125









developed under LNT compared with HNT may be due to decreased night respiration at

the lower temperature, as observed earlier in this work (see Chapter 4).

Non-fruiting conditions, even though they also caused ovary swelling, did not

increase ovary sugar or starch concentrations. This is in contrast to work by Aloni et al.

(1991b; 1999) who found increased carbohydrate concentrations in flower buds of non-

fruiting compared with fruiting bell pepper. In their work, Aloni et al. (1999) used plants

bearing five fruits that resulted in floral ovary weights that were one-third to one-half the

weight of ovaries on non-fruiting plants. In our work, the plants had only two developing

fruits, resulting in the development of floral ovaries with an average 25% decrease in

FW compared with floral ovaries on non-fruiting plants. It may be that under the

conditions of our experiment, the increased source:sink ratio on the non-fruiting plants

was sufficient to cause increase ovary size and therefore carbohydrate content, but not

sufficient to increase storage of carbohydrate and therefore increase concentration.

Our results are in accordance with previous reports that both LNT and high

source:sink ratio increase ovary size, and therefore, ovary swelling. Our hypothesis that

LNT combined with low source:sink ratio or HNT combined with high source:sink ratio

can overcome the detrimental effects of LNT or high source:sink ratio on ovary swelling

is also supported by our results. However, it appears that the mechanisms by which

floral ovary size increases, resulting in increases in swollen ovaries, differs depending

on whether the swollen ovaries occur in response to LNT or in response to excess

source supply. Ovary swelling caused by LNT is associated with an increased

concentration of reducing sugars and starch in the ovary wall, but not in the placental

tissues, as well as with slight increases in both cell size and cell number in the ovary


126









wall. In contrast, ovary swelling caused by high source:sink ratio seems to be most

clearly associated with increased cell size in the mesocarp, giant cells and epidermal

cells, with negligible effects on carbohydrate concentration.

Conclusions

Both fruiting plants under LNT and non-fruiting plants under HNT produced an

intermediate-sized ovary, which supports our hypothesis that HNT combined with high

source:sink ratio or LNT combined with low source:sink ratio can overcome the

detrimental effects of low night temperature or high source:sink ratio on ovary swelling

in pepper. Ovary fresh weight, diameter and length of flowers at anthesis were

increased by both low night temperature (12C) as well as by absence of fruits. The

mechanisms for the increase in ovary size, however, appear to be different. LNT

significantly increased ovary wall thickness and transverse area, with only slight

increases in cell size and number. Low night temperature also increased floral ovary

reducing sugar and starch concentration. Absence of fruits increased floral ovary size

mainly through increased cell size, with no effect on increasing ovary reducing sugar or

starch concentration.


127









Table 5-1. Dehydration and resin embedding steps for 'Legionnaire' bell pepper ovaries
previously fixed in FAA containing 50% ethanol.
Step Product Processing time Infiltration timey
Dehydration Ethanol 50% 45 seconds 1 min.
75% 45 seconds 1 min.
95% 45 seconds 1 min.
100% 45 seconds 1 min.
Acetone 100% 45 seconds
Acetone 100% Overnight


Spurr resin embedding


Resin: Acetone


30:70 3 min. 5 min.
50:50 3 min. 5 min.
70:30 3 min. 5 min.
100:0 3 min. 5 min.
100:0 Overnight
zTime that the samples were processed in a PELCO BioWave Laboratory Tissue
Processing System with ColdSpot (Ted Pella, Inc. Redding, CA, USA) at 180 W and
waterbed at 240C.
YAfter every step, samples were left on the bench top for various times to improve
infiltration.

Table 5-2. Main effects of night temperature and presence/absence of fruits on the fresh
weight, diameter, and length of the ovary in 'Legionnaire' bell pepper flowers
harvested at anthesis
Fresh weight (mg) Diameter (mm) Length (mm)
Night temperatures
12C 128* 6.6* 4.5*
20C 102 6.0 4.3
Fruits x
Non-fruiting plants 132* 6.7* 4.6*
Fruiting plants 98 5.9 4.2
zPooled data of two experiments. Flowers were continuously harvested for 42 days in
Experiment 1 and 38 days in Experiment 2.
YMeans were averaged across fruiting treatments and harvest intervals; n= 597 and 492
for 20C and 12C, respectively.
xMeans were averaged across temperature treatments and harvest intervals; n= 595
and 494 for non-fruiting and fruiting treatments, respectively.
*Asterisks indicate means are significantly different at P<0.001.


128









Table 5-3. Main effect of days after treatment on the fresh weight, diameter, and length
of the ovary in 'Legionnaire' bell pepper flowers harvested at anthesis.
Days after treatment Fresh weight (mg) Diameter (mm) Length (mm)
1-4 97 e x w 5.8 g 4.3 c
5-8 89 e 5.8 fg 3.9 d
9-12 101 de 6.0 ef 4.3 c
13-16 108 cd 6.2 de 4.3 bc
17-20 104 de 6.2 cde 4.3 bc
21-24 118 bc 6.4 bcd 4.5 ab
25-28 128 ab 6.6 ab 4.6 a
29-32 132 a 6.7 a 4.6 a
33-36 130 a 6.6 ab 4.6 a
37-40 129 ab 6.6 ab 4.6 a
41-44 128 ab 6.6 abc 4.6 a
zPooled data of two experiments. Flowers were continuously harvested during 42 days
in Experiment 1 and 38 days in Experiment 2.
YOvaries were grouped at 4-day intervals. Data from 5-8 DAT were from Experiment 2
only; data from 41-44 DAT were from Experiment 1 only.
xMeans were averaged for each 4-day interval across night temperature and fruiting
treatments; n = 136, 42, 155, 106, 93, 87, 96,121,127, 93, and 33 for intervals 1-4 DAT
to 41-44 DAT, respectively.
WMeans with the same letter in the same column are not significantly different (Tukey-
Kramer, P<0.05).

Table 5-4. Main effects of night temperature and presence/absence of fruits on soluble
sugar and starch concentration in ovaries (ovary wall and placenta) of
'Legionnaire' bell pepper flowers harvested at anthesisz.
Ovary wall (pg/g DW) Placenta (pg/g DW)
Gluy Fru Suc Starch Glu Fru Suc Starch
Temperature
12C 31.9** 31.9* 27.9* 56.9* 62.8 72.9 45.7 99.6
20C 26.2 26.3 35.6 44.8 61.1 65.8 50.5 87.4
Fruits
Non-fruiting 29.0 30.1 31.4 46.6 63.4 68.6 44.2* 87.4
Fruiting 29.1 28.2 32.0 55.1 60.5 70.1 52.1 99.6
zFlowers were harvested 24 to 29 days after treatments started.
YGlu: glucose, Fru: fructose, Suc: sucrose.
*, **: Significant differences at P<0.05 and 0.01 respectively.
xMeans were averaged across fruiting treatments, n =12.
WMeans were averaged across temperature treatments, n = 12.


129









Table 5-5. Main effects of night temperature and presence/absence of fruits on soluble
sugar and starch content in ovaries (ovary wall and placenta) of 'Legionnaire'
bell pepper flowers harvested at anthesis.
Ovary wall content (pg/flower) Placenta content (pg/flower)
Gluy Fru Suc Starch Glu Fru Suc Starch
Temperaturez,
12C 540** 533*** 466 986* 976 1098* 658 1489*
20C 314 319 411 552 771 813 626 1071
Fruits w
Non-fruiting 492* 501* 498* 844 1095** 1158** 729* 1494*
Fruiting 362 350 380 694 653 753 554 1066
zFlowers were harvested 24 to 29 days after treatments started.
YGlu: glucose, Fru: fructose, Suc: sucrose.
*, **: Significant differences at P<0.05 and 0.01, respectively.
xMeans were averaged across fruiting treatments, n =12.
WMeans were averaged across temperature treatments, n = 12.


130





















Flower
initiation


-80


60


c40
a(
Petal
20 fall


1-2 DAT


38-40 DAT


-30 -20 -10 0 10 20 30 40 50 60
Time (days)

Figure 5-1. Schematic representation of harvesting times for anatomical analysis in
relation to fruit growth in the fruiting plants. Dry weight; Fresh
weight. Day 0 represent anthesis. Flower initiation occurs four weeks before
anthesis, and petal fall occurs ~3-4 days after anthesis. Treatments started
~6 days after petal fall. Flowers used in the anatomical analysis reached their
anthesis at 1-2, 18-19 or 38-40 days after treatment (DAT).


Figure 5-2. Transverse cross section of the ovary wall of ovaries at flower anthesis in
'Legionnaire' bell pepper. Tissues: A) epidermis and hypodermis, B)
mesocarp, C) giant cells, D) endocarp, E) ovule, F) placenta tissue, G)
vascular bundle.


131









A 80 -
70 -
E 60
E *
M50 y = 1.0952.x
( 40 r2=0.995
30 -
-0Z P-value < 0.001
2 20
S20 n=23
S10 -

lm mm 0 10 20 30 40 50 60 70 80
Calculated area (mm2)

Figure 5-3. Relationship between the measured and the calculated area of a transverse
section in the ovary of 'Legionnaire' bell pepper. A) Cross section area was
calculated with the average diameter (arrow), then the area was measured
from monochromatic images with imageJ (Rasband, 2010). B) Linear
regression between calculated and measured area.


132










160


140


120


1 a


SOvary FW
*Diameter
O Length


- ~ n ,


-8


-7


-6
E
E
-5

-3 -U
CT
-4 -
o

3 (D
E

-2


-1


- 0


L- L+ H- H+ L- L+ H- H+ L- L+ H- H+
Temperature x fruiting treatment combination

Figure 5-4. Interaction of night temperature and presence/absence of fruits on ovary
characteristics of flowers harvested at anthesis in 'Legionnaire' bell pepper.
Night temperature was either 12 (L) or 20C (H), and plants had either 0 (-) or
two developing fruits (+). Data for the two experiments were pooled for
analysis. Flowers were continuously harvested for 42 days in Experiment 1
and 38 days in Experiment 2. Means were averaged for each combination
across harvest intervals; n = 265, 227, 330, and 267 for L-, L+, H- and H+
treatments, respectively. Means with the same letter in the same parameter
are not significantly different (Tukey-Kramer, P<0.05).


133


|-









160 -
150 -12oC *** *
140 -200C **
-/
-130- /
E120 ,.. _
.-C
110 *
100
_-
( 90
U_
80
70

0 ) .


Days after treatment

Figure 5-5. Effect of night temperature and days after treatment on ovary fresh weight of
flowers at anthesis in 'Legionnaire' bell pepper. Flowers were harvested daily
for 42 days in Experiment 1 and for 38 days in Experiment 2; data were
grouped every 4 days. Data for the two experiments were pooled for analysis.
Data from 5-8 DAT was from Exp. 2 only; data from 41-44 DAT was from Exp.
1 only. Means were averaged for each combination across fruiting treatments;
n = 26 to 32 for 1-4 and 41-44 DAT, and 35 to 86 for the remaining intervals.
** ***: Significant differences between temperature regimes within each
harvest interval at P<0.05, 0.01 and 0.001, respectively.


134













- A -Non-fruiting plants
-- Fruiting plants


*** ***
** *

*** *
-s<..


*


*** ,1


160
150 -
o)140 -
130 -
120 -
D 110

U-
-_ 100 -
2 90
O
L_ 80
0"
7.5


E
6.5 -


E 6.0

5.5 -


**,


5.0



E 4.5
E


c3)
---



m 4.0
_J


*** ***
-*

--A--
*** <-


- ~


**


Days after treatment

Figure 5-6. Effect of presence/absence of fruits and days after treatment on ovary size
of flowers harvested at anthesis in 'Legionnaire' bell pepper. A) Ovary fresh
weight; B) diameter; C) length. Flowers were harvested daily for 42 days in
Experiment 1 and 38 days in Experiment 2; data were grouped every 4 days.
Data for the two experiments were pooled for analysis. Data from 5-8 DAT
was from Exp. 2 only; data from 41-44 DAT was from Exp. 1 only. Means
were averaged for each combination across night temperature treatments; n =
22 to31 for intervals 5-8 DAT and 41-44 DAT, and 39 to 90 for the remaining
intervals. *, ** ***: Significant differences within each harvest interval at
Ps0.05, 0.01 and 0.001, respectively.


135


***
-J'- -.*- .


H
*~ /






Y


0.0 1 ....


A


**









MOvary FW
o Diameter
*** i


7

6

5
E
4

3 1
E
2 Q

1


0.6

0.5

E 0.4
E
2 0.3

S0.2
^ .


12 20
Night temperature


12
(oC)


SOvary wall thickness
3 Ovary wall transverse area
** 12

10

8
6 E
E

4

2


20 12 20


Figure 5-7. Effect of night temperature on ovary size and ovary wall thickness and area
in 'Legionnaire' bell pepper flowers at anthesis. A) ovary size; B) ovary wall
thickness and ovary wall transverse area. *, **, ***: Means between
temperature treatments are significantly different at P<0.05, P<0.01, and
Ps0.001, respectively; n = 16.


136


140

-120
O)
E
-100
F 80

c 60

Lu 40


12 20









*Cell size


DCell number


25 6000


400
350
300
E 250
-200
N
S150
a)
0100
50
0

250

200

E150

N 100
a)
o 50

0


5000

E 4000

U 3000

o 2000

1000


20
co

15

10-
E

5

0






0


E
10
0


12 20


12 20


Night temperature


12 20


12 20


Figure 5-8. Effect of night temperature on ovary wall cell size and cell number in
'Legionnaire' bell pepper flowers harvested at anthesis. A) mesocarp; B) giant
cells; C) epidermal tissues; D) endocarp. Means between temperature
treatments are significantly different at P<0.05; n = 16.


137


250

200

E150

N
* 100

50

0


0.5

0.4
co

0.3 0

0.2 I
E

0.1
C)

0.0

2.0

-0



.5-



0.0
1.0





0.0


















































Figure 5-9. Median cross section of ovaries in 'Legionnaire' bell pepper flowers
developed under two night temperatures. A, C, E: low night temperature, B,
D, F: high night temperature; A, B: total ovary wall; C, D: outer ovary wall; E,
F: inner ovary wall; ep, epidermis; hp, hypodermis; me, mesocarp; vb,
vascular bundle; gc, giant cell; en, endocarp; ov, ovule. Note the thicker ovary
wall under LNT compared to HNT but no difference in cell size.




138









H Ovary FW
O Diameter
***


-F +F -F +F


7

6

5
E
4 &

3 (D
E
2

1


0.6

E 0.5
E
S0.4

S0.3

" 0.2


SOvary wall thickness
B
0 Ovary wall transverse area
12

10

8E
E
6M

4<

2

0
-F +F -F +F


Fruiting treatment

Figure 5-10. Effect of presence (+F) or absence (-F) of fruits on ovary size and ovary
wall thickness and area in 'Legionnaire' bell pepper flowers harvested at
anthesis. A) ovary size, B) ovary wall thickness and ovary wall transverse
area. +F: 2 developing fruits per plant. *, **, ***: Means between fruiting
treatments are significantly different at P<0.05, P<0.01, and Ps0.001,
respectively; n = 16.


139


140

0 120
E
. 100
d3 80
S80

LL AnA













450 -
400
E 350
- 300
N
250
0 200
OC
150
100
50
0
C
250 *


E 200

a)
.U 150

0 100

50

0


*Cell size
OCell number


-F +F -F +F


Fruiting treatment


Figure 5-11. Effect of the presence (+F) or absence (-F) of developing fruits on ovary
wall cell size and cell number in 'Legionnaire' bell pepper flowers harvested at
anthesis. A) mesocarp; B) giant cells; C) epidermal tissues; D) endocarp. +F:
2 developing fruits per plant. *, ** ***: Means between fruiting treatments are
significantly different at P<0.05, P<0.01, and P<0.001, respectively; n = 16.


140


7000

6000

E 5000

S4000

3000
O
2000

1000


o
20 CU
0
0
15 .

10 E

5
o


U -
Z)


0
-c
4 w
3

n
2 E


1
i


0.5

o
0.4
C,
0.3 o
-,

0.2
E

0.1
0
C)
0.0

2.5

2.0
U,
1.5 0
t--

1.0
E
0.5 |
C)
0
0.0


250

_200
E
' 150
N

100

50
50


-F +F


-F +F


















































Figure 5-12. Median cross section of ovaries in 'Legionnaire' bell pepper flowers
developed in fruiting and non-fruiting plants. A, C, E: non-fruiting plants; B, D,
F: fruiting plants (2 developing fruits per plant); A, B: total ovary wall; C, D:
outer ovary wall; E, F: inner ovary wall; ep, epidermis; hp, hypodermis; me,
mesocarp; vb, vascular bundle; gc, giant cell; en, endocarp; ov, ovule. Note
the thicker ovary wall and larger cells in the mesocarp and giant cells in the
non-fruiting compared to the fruiting plants.


141









* Ovary FW
o Diameter
a a


120

_100
E
S80
-c
'T 60
3-
S40
LU


B Ovary wall thickness
0 Ovary wall transverse area

a a 12
ab b b 10
b
8
E
E
6

4 <

2

I II 0


Days after treatment


Figure 5-13. Effect of harvest time on the ovary size and ovary wall thickness and area
in 'Legionnaire' bell pepper flowers harvested at anthesis. A) Ovary size, B)
Ovary wall thickness and ovary wall transverse area. Means with the same
letter are not significantly different at Ps0.05 (LSD test); n = 4 for 1-2 DAT, n
= 16 for 18-19 and 38-40 DAT.


142


5-
E E 0.4
4E E

-a
2 0 0.2

1 -0.1

0 0.0


N^ t, P
NW C


t^ N(: R
NV CJ









*Cell size


OCell number


25 7000


U)
20

15
ioni
10-0
E

5


450
400
350
300
250
3200
. 150
100
S50
0
250

200
E
150
N
U)
S100

50
50


q,oj 0
N:s


Days after treatment


Figure 5-14. Effect of harvest time on ovary wall cell size and cell number in
'Legionnaire' bell pepper flowers harvested at anthesis. A) mesocarp; B) giant
cells; C) epidermal tissues; D) endocarp. Means with the same letter are not
significantly different at P<0.05 (LSD test); n = 4 for 1-2 DAT, n = 16 for 18-19
and 38-40 DAT.


143


6000

5 5000
E
S4000

* 3000

o 2000

1000


4c
U)





1=
0)


0
250


0.5

0.4

0.3

0.21
E

0.1
0
0.0
2.0
C-

1.5 m
o
0

1.0
E

0.5

0.0
0.0


200

E150

S100

0 n


t^ N>I R
NW 9p


~3 9S
~s~s


SN( R
Nt ( 9<









SOvary FW
O Diameter
a r


- u.o
E
S0.5
( 0.4
2 0.3
S0.2
0.1
0.0


* Ovary wall thickness
0 Ovary wall transverse area
a
14
12
b b
b 10-"
c E

6
4
2
S m m I 0


L- L+ H-H+ L- L+ H-H+
Temperature x fruiting


L- L+ H-H+
treatment combination


L- L+ H-H+


Figure 5-15. Interaction of night temperature and presence/absence of fruits on ovary
size and ovary wall thickness and area in 'Legionnaire' bell pepper flowers
harvested at anthesis. Night temperature was either 12 (L) or 20C (H), and
plants had either 0 (-) or 2 developing fruits (+). Means with the same letter
are not significantly different at P<0.05 (LSD test); n=8.


144


160
140
120
100
80
60
40
20
0










*Cell size
OCell number


L- L+ H-H+ L- L+ H-H+
Temperature x fruiting


L- L+ H-H+ L- L+ H-H+
treatment combination


Figure 5-16. Interaction of night temperature and presence/absence of fruits on ovary
wall cell size and cell number in 'Legionnaire' bell pepper flowers harvested at
anthesis. A) mesocarp, B) giant cells, C) epidermal tissues, D) endocarp.
Night temperature was either 12 (L) or 20C (H), and plants had either 0 (-) or
2 developing fruits (+). Means with the same letter were not significantly
different at P<0.05 (LSD test); n=8.


145


450
400
350
300
250
200
150
100
50
0


7000

6000

E5000

a 4000
N
3000
O
2000

1000

0

250


ou
-o
25 C

20 o

15 _
E
10 c

5

0

5



c
3 3



2 E
3
1 o
(U


300

c 250
E
S200
N
= 150
O
100

50

0


0.5

0.4 r
CU

0.3 0

0.2
E
3
0.1
0
0.0

2.5

o
2.0

Cu
1.5

1.0 -
E

0.5

0.0


200

150

100












140

3 120
E
- 100

(D 80

u 60
LL A n


- +- +
18-19 38-40
DAT DAT


HOvary FW
O Diameter


+ + + +
18-19 38-40 18-19 38-40
DAT DAT DAT DAT
Harvest time x fruiting treatment combination


SOvary wall thickness
o Ovary wall transverse area
a r


- + +
18-19 38-40
DAT DAT


10 '
E
8 E
8
6

4
2

0


Figure 5-17. Effect of harvest time and presence/absence of developing fruits on ovary
size and ovary wall thickness and area in 'Legionnaire' bell pepper flowers
harvested at anthesis. Flowers were harvested at 18-19 days after treatment
(DAT) or 38-40 DAT and plants had either 0 (-) or 2 developing fruits (+).
Means with the same letter are not significantly different at P<0.05 (LSD test);
n=8.


146












9000
8000
7000
6000
5000
4000
3000
2000
1000
0


500
450
^400
E350
S300
u250
' 200
150
100
50
0

300

250

S200
E
S150
a)
N
. 100

50

0


250

200

150

100


+ +
18-19 38-40
DAT DAT
Harvest time x fruiting treatment


-+ +
18-19 38-40
DAT DAT
combination


+
18-19
DAT


"o
20
co

15 0
"-

10
E

5
0


- +
38-40
DAT


Figure 5-18. Effect of harvest time and presence/absence of developing fruits on ovary
wall cell size and cell number in 'Legionnaire' bell pepper flowers harvested at
anthesis. A) mesocarp, B) giant cells, C) epidermal tissues, D) endocarp.
Flowers were harvested at 18-19 days after treatment (DAT) or 38-40 DAT
and plants had either 0 (-) or 2 developing fruits (+).Means with the same
letter are not significantly different at P<0.05 (t-test).


147


*Cell size


- 0.5

- 0.4
ro
- 0.3 o
.-

- 0.2
E
S0.1 c
(D
0
0.0

2.5
"o
2.0

1.5 o
,,
1.0 .n
E

0
0.5
0.0-


b b


it


45

4

3

==
2E


0


-+
18-19
DAT


-+
38-40
DAT









CHAPTER 6
SUMMARY

Sweet peppers are an important vegetable crop whose fruits are mostly consumed

as fresh produce all year. Cultivated species of peppers were domesticated in tropical

and temperate areas, and are sensitive to low temperatures (Harlan, 1971; Eshbaugh,

1993). Pepper plants grown under cool night temperatures (<15C) develop deformed

flowers, including swollen flowers and ovaries (i.e., flowers and ovaries that are larger

than the ones developed under optimum temperature), resulting in a high percentage of

malformed fruits that are not marketable (Mercado et al., 1997c; Pressman et al.,

1998a; Pressman et al., 1998b; Aloni et al., 1999). Swollen ovaries also develop under

high source:sink ratios in pepper (Aloni et al., 1999). Under both conditions, i.e. low

night temperatures and high source:sink ratios, swollen flowers are reported to have

increased reducing sugars and starch concentration compared to normal (not swollen)

flowers (Aloni et al., 1999). Although several studies have been done, the relation

between assimilate availability and ovary swelling in pepper remains unclear.

Furthermore, the anatomical basis for ovary swelling and deformation under these

conditions is unknown. In this study, the central hypothesis was that both low night

temperature and high source:sink ratio increase ovary size and therefore ovary swelling

via effects on assimilate availability/accumulation and/or cell size/number.

The series of experiments were carried out under growth chamber conditions by

controlling night temperature at either 120C (low night temperature, LNT) or 200C (high

night temperature, HNT). The experiments also implied continuous leaf pruning to keep

plants at two main axis and continuous flower removal. In the first three experiments

(i.e., Experiments 1 and 2 in Chapter 3, and Experiment 1 in Chapter 4) plants did not


148









bear any developing fruits before and during the duration of the experiment. In two

experiments (Experiment 1 and 2 [same as Experiment 2 in Chapter 4] in Chapter 5), 2

fruits were allowed to set and ~6-10 days after fruit setting (petal fall), fruits were

removed in one treatment, while fruits on the other treatment were allowed to develop to

maturity.

The discussion of the results was addressed from a source-sink point of view. We

are aware that plant growth regulators might be masking or enhancing some responses,

since the experiments implied continuous leaf and flower pruning, and in some cases,

presence or absence of fruits. However, since we did not have any data on hormones,

such effects were considered as an integral component of the treatment effect.

It has been previously suggested that other factors such as hormones may also

influence the development of swollen ovaries (Polowick and Sawhney, 1985; Aloni et

al., 1995; Pressman et al., 1998b). one hypothesis is that low night temperature inhibits

the basal auxin transport from the flower bud to other parts of the plant, and then the

increased auxin concentration in the flower favors ovary growth (Aloni et al., 1995;

Pressman et al., 1998b). Presence/absence of fruits may also alter the hormonal

balance in the plant. It has been suggested that growth inhibition of a second flower,

and therefore a fruit, is controlled by both competition for limited assimilates (Ali and

Kelly, 1992; Marcelis and Baan-Hofman-Eijer, 1997) and by dominance due to the

production of plant growth regulators by the earlier developed fruit (Marcelis and Baan-

Hofman-Eijer, 1997).

Ultimately, from the results of this research, it is concluded that LNT induced ovary

swelling in different kinds of sweet pepper (cherry, elongated and blocky bell). Three to


149









four weeks of continuous low night temperature were required for maximum response,

suggesting that flowers must be exposed to LNT soon after flower initiation in order for

this response to occur. However, the minimum duration of exposure to LNT required to

induce this response is unknown, as the present research maintained LNT throughout

the course of flower development. Fruit removal (i.e. increasing the source:sink ratio)

also increased the incidence of swollen ovaries. Both LNT and fruit removal decreased

net CER without reducing plant growth, suggesting that excess availability of current

photosynthate may not be the mechanism that results in the increase in swollen ovaries

observed under both LNT and high source:sink conditions. HNT combined with high

source:sink ratio or LNT combined with low source:sink ratio can overcome the

detrimental effects of low night temperature or high source:sink ratio on ovary swelling

in pepper. Finally, although ovary swelling is induced by both LNT and by absence of

fruits, the mechanisms for the increase in ovary size, however, appear to be different.

LNT significantly increased pericarp thickness and transverse area, with only slight

increases in cell size and number. LNT also increased ovary wall reducing sugar and

starch concentration. Absence of fruits increased floral ovary size mainly through

increased cell size, with no effect on increasing ovary reducing sugar or starch

concentration.


150









APPENDIX
ADDITIONAL TABLES AND FIGURES

A) Tables

Table A-1. P-values for temperature (Temp), cultivar (CV), and days after treatment
(harvest) main effects and their interactions. Branch within each cultivar was
also considered. Number of days after anthesis (DAA) was used as covariate,
and the subject for repeated measures was branch within each plant.
(Chapter 3)
Source of DF Fresh Volume Diameter Length Ovary wall Length: FW: Vol.
variation weight (mm3) (mm) (mm) thickness diam. ratio
(mg) (mm) ratio
Temp 1 0.008 0.002 0.003 <0.001 0.004 0.59 <0.001
CV 5 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Temp x CV 5 0.03 0.03 0.03 0.007 0.19 <0.001 0.96
Harvest 4 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Temp x Harvest 4 <0.001 <0.001 <0.001 <0.001 0.009 0.29 0.84
CVx Harvest 20 <0.001 <0.001 <0.001 0.19 <0.001 0.01 0.006
Temp x CV x 20 0.003 0.004 0.07 0.008 0.43 0.05 0.49
Harvest
Branch(CV) 6 0.03 0.03 0.05 0.07 0.21 0.66 0.39
DAA 1 <0.001 <0.001 <0.001 <0.001 <0.001 0.17 <0.001
Branch(Plant) <0.001 <0.001 <0.001 0.004 0.02 0.02 0.88


Table A-2. P values for ovary, ovary wall (OW) and placenta fresh weight, ovary wall
fresh weight: ovary fresh weight ratio, ovary diameter and length, ovary
length/diameter ratio, and ovary wall thickness.z
FW (mg) OW FW: Diameter Length Length: OW
Source of Ovary OW Placenta ovary FW (mm) (mm) diameterthickness
variation ratio (mm)
Temperature 0.01 0.01 0.01 0.36 0.010 0.007 0.31 0.02
Cultivar (CV) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Temp*CV 0.001 0.01 <0.001 0.04 0.004 0.34 <0.001 0.29
Branch <0.001 <0.001 <0.001 0.03 <0.001 <0.001 0.17 0.005
Node <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.88 <0.001
Node*Branch 0.002 0.02 0.001 0.99 0.01 0.10 0.28 0.21
Temp*Node <0.001 <0.001 <0.001 0.92 <0.001 <0.001 0.62 <0.001
CV*Branch <0.001 <0.001 <0.001 0.32 <0.001 0.06 0.05 0.009
CV*Node <0.001 0.01 <0.001 <0.001 <0.001 0.007 0.01 0.37
CV*Node*Branch 0.03 0.01 0.05 0.07 0.07 0.11 0.37 0.41
Temp*CV*Branch 0.04 0.05 0.05 0.07 0.11 0.56 0.24 0.46
Temp*CV*Node <0.001 <0.001 0.003 0.04 <0.001 0.05 <0.001 0.28


z Factorial experiment with two night temperatures
harvested at anthesis from nodes 2 to 7.


and four cultivars. Flowers were


151










Table A-3. Number of days after beginning of temperature treatments at which flowers
from nodes 2 to 7 were harvested in four cultivars of sweet pepper. Means
S.E. (Chapter 3)
Night Node number
Cultivar temp (oC) 2 3 4 5 6 7
'Ariane' 12 4.50.5 11.50.8 20.71.5 28.61.0 35.10.9 40.40.8
20 4.90.5 11.30.8 18.41.6 25.91.4 31.91.1 36.71.6
'Banana 12 6.30.7 13.70.6 20.61.0 26.30.7 30.80.6 35.60.7
Supreme' 20 5.20.8 11.31.5 14.61.1 21.71.9 26.92.0 39.05.0
'Legionnaire' 12 8.50.8 16.90.9 24.61.1 31.61.1 36.60.8 42.40.7
20 5.40.3 12.70.9 25.82.0 31.21.1 37.71.0 41.41.4
'Red Cherry 12 1.50.2 7.40.6 14.31.1 19.91.1 27.01.1 32.11.0
Sweet' 20 2.20.3 6.10.5 11.11.0 15.70.7 22.70.9 27.60.7
ZNodes counted above the first branching node, i.e., flower from sympodial unit 1 was
node 1.

Table A-4. Effect of the interaction between days after treatment began (DAT) with night
temperature and cultivar on the intercellular CO2 (Ci, pmol mol- ) of sweet
pepper. Gas exchange was measured over a 45-day period after the
beginning of night temperature treatments. (Chapter 4)


Temperaturey
DAT 120C 200C P values LE(
0 296 278 0.05 2!
3 274 255 0.02 21
6 206 185 0.02 2(
9 217 230 0.12 2:
12 238 242 0.67 2
15 249 249 0.99 2
19 263 280 0.05 2
21 208 265 <0.001 2:
24 222 247 0.004 2:
27 233 248 0.09 2:
30 262 266 0.71 21
33 222 222 0.99 2:
36 208 217 0.31 2(
45 229 220 0.28 2:
ZGas exchange was measured over a 45-day period after the
temperature treatments.
YData were averaged across cultivars. n = 18.
xData were averaged across temperature treatments. n = 18.


Cultivarx
G RSC P values
91 283 0.35
67 262 0.50
05 186 0.03
22 225 0.71
45 236 0.28
44 254 0.24
77 267 0.23
38 235 0.74
38 231 0.45
39 242 0.67
64 264 0.94
20 224 0.64
06 219 0.12
21 227 0.45
beginning of night


152









Table A-5. P-values for ovary fresh weight, diameter and length, ovary length/diameter
ratio, and ovary wall thickness for a 2 factor experiment (2 night temperature
regimes, 2 levels of fruits) repeated over time (11 harvest intervals of 4 days
each). (Chapter 5)
Effect Fresh weight Diameter Length
Temperature (Temp) <0.001 <0.001 <0.001
Fruits <0.001 <0.001 <0.001
Temp*Fruits 0.02 0.08 0.08
Harvest interval (HI) <0.001 <0.001 <0.001
Temp*HI 0.04 0.36 0.76
Fruits*HI <0.001 <0.001 <0.001
Temp*Fruits*HI 0.57 0.16 0.20
Branch <0.001 <0.001 <0.001
DAA <0.001 <0.001 <0.001
z Pooled data from two experiments. Experiments were conducted with a factorial
design (two night temperatures and presence/absence of developing fruits). Flowers
were harvested at anthesis for 42 days in Experiment 1 and 38 days in experiment 2,
and grouped in 11 harvest intervals of 4 days.


153









Table A-6. P values for ovary characteristics, ovary wall thickness and ovary wall
transverse area, and thickness, area, cell number and cell size for mesocarp,
giant cells, epidermal tissues and endocarp for a 3 factor experiment (2 night
temperature regimes, 2 levels of fruits and 2 harvest times) (Chapter 5)
Sources of variation z
Tissues and parametersT F Tx F HT HT x F HT x THT xTx F Block


Ovary


Ovary wall


Mesocarp




Giant cells




Epidermal
tissues



Endocarp


FW
Diameter
Length

Thickness
Total area

Thickness
Total area
Cell number
Cell size

Thickness
Total area
Cell number
Cell size

Thickness
Total area
Cell number
Cell size

Thickness
Total area
Cell number
Cell size


0.001
0.01
0.05


0.001
0.001
0.001


0.001
0.02
0.001


0.02
0.58
0.01


0.09
0.05
0.05


0.83
0.63
0.90


0.73
0.43
0.80


0.05 0.001 0.14 0.01 0.02 0.06 0.74
0.01 0.001 0.03 0.03 0.01 0.08 0.77


0.04
0.01
0.28
0.23

0.35
0.14
0.79
0.64

0.28
0.04
0.61
0.05

0.29
0.06
0.12
0.60


0.01
0.001
0.11
0.01

0.01
0.001
0.55
0.01

0.04
0.001
0.09
0.001

0.14
0.01
0.03
0.25


0.04
0.01
0.14
0.65

0.55
0.98
0.03
0.07

0.83
0.40
0.81
0.26

0.20
0.06
0.09
0.89


0.05
0.11
0.24
0.01

0.01
0.01
0.29
0.01

0.23
0.37
0.04
0.01

0.31
0.64
0.92
0.28


0.04
0.02
0.90
0.04

0.06
0.02
0.83
0.18

0.23
0.09
0.69
0.02

0.53
0.33
0.39
1.00


0.01
0.03
0.13
0.63

0.95
0.98
0.26
0.97

0.73
0.91
0.28
0.29

0.74
0.95
0.41
0.15


z T, temperature; F, non-fruiting/fruiting treatment; HT, harvest time.


0.40
0.48
0.32
0.43

0.62
0.62
0.75
0.23

0.41
0.38
0.83
0.68

0.26
0.20
0.89
0.12


0.58
0.23
0.93

0.60
0.56

0.62
0.65
0.84
0.74

0.56
0.38
0.70
0.60

0.93
0.73
0.96
0.53

0.97
0.67
0.34
0.77


154










Ovary wall Placenta
Parameter Temp Fruits T x F Temp Fruits Tx F
Dry weight 0.06 0.001 0.16 0.06 0.001 0.16
Concentration
Glucose 0.01 0.95 0.09 0.73 0.57 0.24
Fructose 0.02 0.39 0.22 0.19 0.77 0.30
Sucrose 0.04 0.86 0.84 0.19 0.04 0.12
Starch 0.17 0.33 0.69 0.29 0.29 0.65
Reducing sugars 0.01 0.64 0.11 0.36 0.89 0.24
Red. Sugars + starch 0.03 0.50 0.76 0.25 0.55 0.72
Content
Glucose 0.03 0.06 0.10 0.11 0.00 0.05
Fructose 0.001 0.01 0.09 0.02 0.00 0.02
Sucrose 0.30 0.04 0.57 0.66 0.03 0.85
Starch 0.02 0.40 0.69 0.05 0.04 0.38
Reducing sugars 0.002 0.03 0.09 0.05 0.00 0.03
Red. Sugars + starch 0.005 0.13 0.31 0.03 0.01 0.09
ZFlowers were harvested 24 to 29 days after treatments started.


155


Table A-7. P-values for carbohydrate concentration and


content.z (Chapter 5)









B) Figures

100
90 Harvest
80 interval
>70 I
c 60
50
240 --I
LU-
30 IV
20 V
10
0
1 2 3 4 5 6 7 8 9 10 11 12
Relative node
Figure A-1. Distribution of harvested nodes per harvest interval. Nodes were counted
from the first harvested node on every stem. n = 160, 207, 211, 233, 273 for
harvest intervals I, II, III, IV, and V, respectively. (Chapter 3)


156










7.5


** **
7.0 -20 *** -

O *** *** ......
E 6.0 -. -



5.5

0.01
5.5
B

5.0 *** ** *
E *** **
4.5 **


4.0


0 .0 ..



Days after treatment

Figure A-2 Effect of night temperature and days after treatment on ovary diameter (A)
and length (B) of flowers at anthesis in sweet pepper 'Legionnaire'. Day
temperature was set to 22C. Pooled data of two experiments. Flowers were
continuously harvested during 42 days in Experiment 1, and 38 days in
Experiment 2. Means were averaged for each combination across
presence/absence of fruits treatments; n = 26 to 32 for 1-4 and 41-44 DAT,
and 35 to 86 for the remaining intervals. *, ** ***: Significant differences
between temperature regimes within each harvest interval at P<0.05, 0.01
and 0.001 respectively. (Chapter 5).


157











- -12C, non fruiting plants
- -12C, fruiting plants
--*--20C, non fruiting plants
-0-20C, fruiting plants ..- -
qT~-


- -..- =
-.- -


200

180

-160
E
z140

.-120

S100

2 80
LU
0o
8.5
8.0

E 7.5
D 7.0
E 6.5

n 6.0
5.5
0.0


5.5


-


N? W t


N


Days after treatment
Days after treatment


Figure A-3. Effect of night temperature, presence/absence of fruits and days after
treatment on ovary fresh weight (A) diameter (B) and length (C) of flowers at
anthesis in sweet pepper 'Legionnaire'. Day temperature was set to 220C.
Pooled data of two experiments. Flowers were continuously harvested during
42 days in Experiment 1, and 38 days in Experiment 2. Means were averaged
for each combination across presence/absence of fruits treatments. n = 5
tol5 for intervals 2 and 11, and from 14 to 48 for the remaining intervals. *
**, ***: Significant differences between temperature regimes within each
harvest interval at Ps0.05, 0.01 and 0.001 respectively. (Chapter 5)


158


B








..--0
,+ --
,(


E 5.0
E
(.-

)4.5
-J

4.0


0.0


C\ ~Jt~Z


,~C -~C ~









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of the pedicel. Journal of Experimental Botany, 56: 2713-2719.

Zhang SP, Scheller HV. 2004. Photoinhibition of photosystem I at chilling temperature
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BIOGRAPHICAL SKETCH

Nicacio Cruz-Huerta was born in Mexico. He is married and has a daughter. Being

raised in a family of farmers, his interest in plants started in his childhood. His formal

training on agriculture began in the middle and high school. He received his BS degree

in plant sciences from the Universidad Aut6noma Chapingo (Chapingo Autonomous

University), and his M Sc degree in plant physiology from the Colegio de Postgraduados

(Postgraduate College in Agricultural Sciences). He worked in plant physiology, with

special focus on gas exchange, source sink relationships, plant growth analysis, and

plant growth modeling. He was also the teaching assistant during several years in the

graduate course "Crop Physiology". Later, he obtained a scholarship from the Mexican

National Council for Science and Technology to start his PhD program in the

Horticultural Sciences Department at the University of Florida. His research focused on

studying how low night temperatures and source and sink ratios affect flower

development in bell pepper. After graduation, he plans to continue working on

physiology, production techniques, and modeling of horticultural crops.


176





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1 TEMPERATURE EFFECTS ON OVARY SWELLING IN SWEET PEPPER: PHYSIOLOGY AND ANATOMY By NICACIO CRUZ HUERTA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Nicacio Cruz Huerta

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3 To my wife Miriam and my daughter Diana, with love

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4 ACKNOWLEDGMENTS Many people and institutions have been involved and made possible my studies. I am expressing here my gratitude to them. My gratitude and appreciation go to my advisor Dr. Rebecca L. Darnell, for her continuous counseling, guidance, encouragement, help, and patience throughout my entire graduate program, and for the financi al support W ithout it, it would have been impossible to reach the final goal. I also thank Drs. Jeff Williamson, Karen Koch, Ken Boote and Don Huber, for their assistance in this project. What I have learned from each of them will make me a better profess or and researcher. I thank Steven Hiss for all the good advice and help during the experimental and laboratory phases. I thank Valerie Jones, for her help in the carbohydrate analysis. I thank Elio Jovicich, Paul Miller, and Mike McCaffery for all the good advice on how to deal with growth chambers and pepper pests and diseases under Florida conditions, and lab work. I also thank Drs. Tim Martin, Joseph Vu, Gloria Moore, Paul Lyrene, Charles Guy, Byung Ho Kang, and Mr. Dale, and Mrs. Kelley for letting me use their laboratories and equipment during my research and/or helping with their expertise. I extend my gratitude to all Horticultural Sciences Department staff, students and f aculty that in one way or another enriched my work and life during my doctoral program. I thank the institutions that financially supported me during my studies: the Mexican Council for Science and Technology (CONACYT), Colegio de Postgraduados, Horticultural Sciences Department of the University of Florida, and the FloridaMexico Ins titute.

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5 I am grateful to my parents, Arturo and Reina, my brothers and sisters and to the Cruz Diaz and Carrillo Moreno families for giving me the encouragement and their continuous support. I specially thank Paola, Oscar, Sonya, Jack, Patty Omar, Mireya Julio, Nancy Jose, Rebecca, Tim, Desire, Nadia, and their families for their friendship, help, and support when my family needed it the most. I thank Mexicans in Gainesville and all our new friends in Gainesville. Finally, my deepest gratitude goes to M iriam and Diana for making the hardest moments easier, and for being with me in this journey.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 2 LITERATURE REVIEW .......................................................................................... 18 General Description of the Plant ............................................................................. 18 Fruit Anatomy/Morphology ...................................................................................... 19 Fruit Growth and Development ............................................................................... 20 Flower Development ........................................................................................ 21 Flower Abscission and Fruit Set ....................................................................... 21 Fruit Growth ...................................................................................................... 23 Carbon Economy in the Fruit .................................................................................. 28 Respiratory Losses ........................................................................................... 28 Carbon Gain ..................................................................................................... 29 Fruit photosynthesis ................................................................................... 29 Imported carbon ......................................................................................... 31 Abnormal Flower and Fruit Development ................................................................ 39 3 LOW NIGHT TEMPERATURE INCREASES OVARY SIZE IN SWEET PEPPER .. 45 Introduction ............................................................................................................. 45 Material and Methods ............................................................................................. 48 Plant Material and Growing Conditions ............................................................ 48 Experiment 1 .................................................................................................... 50 Experiment 2 .................................................................................................... 51 Statistical Analysis ............................................................................................ 52 Results .................................................................................................................... 53 Experiment 1 .................................................................................................... 53 Experiment 2 .................................................................................................... 56 Discussion .............................................................................................................. 58 Conclusions ............................................................................................................ 64

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7 4 LOW NIGHT TEMPERATURE AFFECTS CARBON EXCHANGE RATES OF SWEET PEPPER LEAVES ..................................................................................... 76 Introduction ............................................................................................................. 76 Material and Methods ............................................................................................. 80 Plant Material and Growing Conditions ............................................................ 80 Experiment 1 .................................................................................................... 81 Experiment 2 .................................................................................................... 82 Data Analysis ................................................................................................... 83 Results .................................................................................................................... 83 Experiment 1 .................................................................................................... 83 Experiment 2 .................................................................................................... 86 Discussion .............................................................................................................. 88 Conclusions ............................................................................................................ 91 5 BELL PEPPER OVARY SWELLING DUE TO LOW NIGHT TEMPERATURE AND SOURCE SINK RATIO: OVARY WALL ANATOMY AND CARBOHYDRATES .............................................................................................. 105 Introduction ........................................................................................................... 105 Materials and Methods .......................................................................................... 107 Plant Material ................................................................................................. 107 Experimental Des cription ................................................................................ 108 Anatomy ......................................................................................................... 109 Carbohydrates ................................................................................................ 111 Data Analysis ................................................................................................. 113 Results .................................................................................................................. 114 Ovary Size ...................................................................................................... 114 Anatomy ......................................................................................................... 116 Carbohydrates ................................................................................................ 119 Discussion ............................................................................................................ 120 Conclusions .......................................................................................................... 127 6 SUMMARY ........................................................................................................... 148 APPENDIX ADDITIONAL TABLES AND FIGURES ................................................................ 151 REFERENCES ............................................................................................................ 159 BIOGRAPHICAL SKETCH .......................................................................................... 176

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8 LIST OF TABLES Table page 3 1 Relationship between days after treatment and harvest interval defined for statistical analysis in two groups of sweet pepper cultivars. ............................... 65 3 2 Number of flowers harvested at anthesis and number of nodes bearing flowers in six cultivars of sweet pepper grown at 22/20C or 22/12C day/night temperatures. ...................................................................................... 65 3 3 Main e ffect of night temperature during the preanthesis stage on ovary characteristics of sweet pepper flowers measured at anthesis. .......................... 66 3 4 Main effect of cultivar on characteristics of ovaries at flower anthesis in sweet pepper. ............................................................................................................... 66 3 5 Main effect of harvest interval on ovary characteristics of six cultivars of sweet pepper. ..................................................................................................... 67 3 6 Interaction of night temperature and cultivar on ovary characteris tics of sweet pepper flowers harvested at anthesis during the duration of the experiment or only during the last two harvest intervals. ........................................................... 68 3 7 Coefficients of conversion to estimate ovary volume at anthesis stage as a function of its fresh weight in six cultivars of sweet pepper grown under two night temperatures. ............................................................................................. 68 3 8 Main effect of night temperature during the preanthesis stage on ovary characteristics of sweet pepper flowers at anthesis. ........................................... 69 3 9 Main effect of cultivar on ovary characteristics of sweet pepper flowers at anthesis. ............................................................................................................. 69 3 10 Main effect of node on ovary characteristics of sweet pepper flowers at anthesis. ............................................................................................................. 70 3 11 Interaction of night temperature and cultivar on ovary characteristics of sweet pepper flowers harvested at anthesis. ................................................................ 70 3 12 P values of t test comparison between night temperatures of 12 and 20C within each cultivar and node for ovary fresh weight (FW), diameter (Diam.), and length. .......................................................................................................... 71 4 1 Effects of night temperature and cultivar on fresh weight of flower and flower parts, ovary diameter and ovary length in sweet pepper. ................................... 93

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9 4 2 P values for effects of night temperature (Temp), cultivar (CV), and days after treatment (DAT) and their interactions on leaf gas exchange parameters. ........................................................................................................ 93 4 3 Main effects of night temperature and cultivar on mean carbon exchange rate, stomatal conductance and intercellular CO2 in sweet pepper leaves measured six hours after lights were on. ............................................................ 94 4 4 Main effects of night temperature and cultivar on daily net photosynthesis, daily night respiration, and calculated daily total CO2 gain in sweet pepper leaves. ................................................................................................................ 94 4 5 Night temperature and cultivar effects on vegetative growth of sweet pepper at the end of the 45day experiment period. ....................................................... 95 4 6 Main effects of night temperature and presence/absence of fruits on fresh weight of the flower, flower parts, ovary diameter, and ovary length in Legionnaire bell pepper. ................................................................................... 95 4 7 P values for effects of night temperature (Temp), presence/absence of fruits (Fruits), and days after treatment (DAT) and their interactions on leaf gas exchange parameters in Legionnaire bell pepper. ............................................ 96 4 8 Main effects of night temperature and presence/absence of fruits on carbon exchange rate, stomatal conductance and intercellular CO2 in Legionnaire bell pepper leaves. ............................................................................................. 96 4 9 Night temperature and presence/absence of fruit effects on growth of Legionnaire bell pepper at the end of the 45 day experiment period. ............... 97 5 1 Dehydration and resin embedding steps for Legionnaire bell pepper ovaries previously fixed in FAA containing 50% ethanol. .............................................. 128 5 2 Main effects of night temperature and presence/absence of fruits on the fresh weight, diamet er, and length of the ovary in Legionnaire bell pepper flowers harvested at anthesis ....................................................................................... 128 5 3 Main effect of days after treatment on the fresh weight, diameter, and length of the ovary in Legionnaire bell pepper flowers harvested at anthesis. ........... 129 5 4 Main eff ects of night temperature and presence/absence of fruits on soluble sugar and starch concentration in ovaries (ovary wall and placenta) of Legionnaire bell pepper flowers harvested at anthesisz. ................................. 129 5 5 Main effects of night temperature and presence/absence of fruits on soluble sugar and starch content in ovaries (ovary wall and placenta) of Legionnaire bell pepper flowers harvest ed at anthesis. ....................................................... 130

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10 LIST OF FIGURES Figure page 2 1 Schematic drawing of pepper branching and flowering sequence. ..................... 43 2 2 Schematic diagram of a pepper plant. ................................................................ 44 2 3 Pepper fruit and its parts. ................................................................................... 44 3 1 Schematic drawing of pepper branching and flowering sequence. ..................... 72 3 2 Schematic drawing of a sweet pepper plant pruned to two main axes with lateral branches. ................................................................................................. 73 3 3 Ovary fresh weight of sweet pepper flowers at anthesis developed under 22/12C or 22/20C day/night temperatures for discrete harvest intervals. ........ 74 3 4 Ovary fresh weight of sweet pepper flowers harvested at anthesis from the second to the seventh nodes above the first flowering node in plants grown at day/night temperatures of 22/12C or 22/20C.. ............................................. 75 4 1 Effect of days after treatment began on net carbon exchange rate and stomatal conductance, and intercellular CO2 of sweet pepper leaves. .............. 98 4 2 Effect of night temperature and cultivar on net carbon exchange rate (CER) of sweet pepper leaves over time. ...................................................................... 99 4 3 Effects of night temperature and cultivar on stomatal conductance (gs) of sweet pepper leaves over time.. ....................................................................... 100 4 4 Effect of night temperature, cultivar, and days after treatments began on leaf respiration in sweet pepper plants during the day and night ........................... 101 4 5 Effect of days after treatment began on net carbon exchange rate, stomatal conductance, and intercellular CO2 of Legionnaire bell pepper leaves. ......... 102 4 6 Effect of night temperature and days after treatment began on net carbon exchange rate, stomatal conductance, and intercellular CO2 of Legionnaire bell pepper leaves. ........................................................................................... 103 4 7 Effect of presence or absence of devel oping fruits and days after treatment began on net carbon exchange rate and stomatal conductance of Legionnaire bell pepper leaves. ...................................................................... 104 5 1 Schematic representation of harvesting times for anatomical analysis in relation to fruit growth in the fruiting plants. ...................................................... 131

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11 5 2 Transverse cross section of the ovary wall of ovaries at flower anthesis in Legionnaire bell pepper. ................................................................................. 131 5 3 Relationship between the measured and the calculated area of a transverse section in the ovary of Legionnaire bell pepper. .............................................. 132 5 4 Interaction of night temperature and presence/absence of fruits on ovary characteristics of flowers harvested at anthesis in Legionnaire bell pepper. ... 133 5 5 Effect of night temperature and days after treatment on ovary fresh weight of flowers at anthesis in Legionnaire bell pepper. ............................................... 134 5 6 Effect of presence/absence of fruits and days after treatment on ovary size of flowers harvested at anthesis in Legionnaire bell pepper. .............................. 135 5 7 Effect of night temperature on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers at anthesis. ............................................... 136 5 8 Effect of night temperature on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. .................................. 137 5 9 Median cross section of ovaries in Legionnaire bell pepper flowers developed under two night temperatures. ........................................................ 138 5 10 Effect of presence or absence of fruits on ovary size and ovary wall thickness and area in Legionnaire bell pepper flower s harvested at anthesis. ............... 139 5 11 Effect of the presence or absence of developing fruits on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. .... 140 5 12 Median cross section of ovaries in Legionnaire bell pepper flowers developed in fruiting and nonfruiting plants. .................................................... 141 5 13 Effect of harvest time on the ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. .................................. 142 5 14 Effect of harvest time on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. ....................................................... 143 5 15 Interaction of night temperature and presence/absence o f fruits on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. ...................................................................................... 144 5 16 Interaction of night temperature and presence/absence of fruits on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. ........................................................................................................... 145

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12 5 17 Effect of harvest time and presence/absence of developing fruits on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. ...................................................................................... 146 5 18 Effect of harvest time and presence/absence of developing fruits on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. ....................................................................................................... 147

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TEMPERATURE EFFECTS ON OVARY SWELLING IN SWEET PEPPER: PHYSIOLOGY AND ANATOMY By Nicacio Cruz Huerta August 2010 Chair: Rebecca L. Darnell Cochair: Jeffrey G. Williamson Major: Horticultural Science In bell peppers ( Capsicum annuum L.), low night temperature and high source:sink ratios during the preanthesis stage cause swollen ovaries, i.e ., abnormally enlarged ovaries, resulting in low quality fruits. The p hysiological reasons for this ovary response are still unclear, but excess carbohydrate accumulation in ovaries has been implicated. In this research, it was found that low night temperature (LNT, 12C) induced ovary swelling in three types of sweet pepper s (cherry, elongated, and blocky bell), wit h the greatest response occurring in bell peppers. T hree to four weeks of continuous LNT were required for maximum response, which coincides with the timing required for flower bud initiation in pepper. This suggests that flowers must be exposed to LNT soon after initiation in order for this response to occur. A second set of experiments showed that both LNT and fruit removal ( i.e. increasing the source:sink ratio) increased the incidence of swollen ovaries in sweet pepper. F ruiting plants under LNT and nonfruiting plants under optimum night temperature (20C) produced an intermediatesized ovary suggesting that night temperature of 20C combined with high source:sink ratio or night temperature of 12C combined with low source:sink ratio can partially over come the

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14 detrimental effects of low night temperature or high source:sink ratio on ovary swelling in pepper. Both LNT and fruit removal decreased net carbon exchange rate without affecting total plant dry weight. This suggests that excess availability of c urrent photosynthate (via maintaining similar carbon exchange rates and reducing plant growth) is not the mechanism that results in the increase in swollen ovaries observed under both LNT and high source:sink conditions. Ovary carbohydrate analysis and anatomical analysis revealed marked differences between swollen ovaries produced under LNT vs swollen ovaries produced under high source:sink ratios. Low night temperature increased floral ovary reducing s ugar and starch concentration, while high source:sink ratios had no effect on ovary carbohydrate concentrations. Ovaries developed under LNT had thicker ovary walls and greater transverse area, with only slight increases in cell size and number. In contrast, ovaries developed under high source:sink ratios inc reased floral ovary size mainly through increased cell size. Finally, both LNT and high source:sink ratio increased ovary size through mechanisms that appear to be different.

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15 CHAPTER 1 1. INTRODUCTION Pepper ( Capsicum annuum L.) is an important cultivated species worldwide. World production is led by China, Mexico, Turkey, Indonesia, Spain and the U.S. The U.S. ranks sixth and produces 3.5% of the worlds sweet and pungent (chile) peppers (FAO, 2007) In 2007, harvested area in the U.S. was 21,974 ha for bell pepper and 9,996 ha for chile pepper, accounting for about 7.5% of the total harvested area of vegetable crops (USDA Economic Research Service, 2008) After California, Florida is the second largest pepper producing state (USDA Economic Research Ser vice, 2008) In 2007, harvested area in Florida was 7,365 ha of bell peppers (USDA National Agricultural Statistics Services, 2008) and ~1200 ha of hot peppers (Li et al. 2006) Peppers also have a cultural importance in countries like Mexico because o f their variety of uses. In addition to the traditional use of peppers in the cuisine of several cultures, peppers are also important as a source of natural pigments (paprika pepper), capsaicin (hot peppers, used in pharmaceutical industries and food preparation) (Surh and Lee, 1995; Duarte et al. 2004) antioxidants (colored bell peppers ) (Ishikawa, 2003; Mateos et al. 2003; Sun et al. 2007) and as ornamental plants (Stummel and Bosland, 2006) Bosland (1999) described peppers importance based on three aspects: historic, general use (fresh vegetable, condiment, ornamental), and medicinal use. Pepper fruit development depends on proper flower development, successful pollination, fertilization and fruit set, suitable fruit growth, and ripening in pigmented varieties. Therefore, favorable conditions for the proper growth and development of the sexual organs are a requirement to ensure high fruit set, yield and quality (Testa et al. 2002) Adverse environmental c onditions, such as extremes in temperature, light,

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16 and/or water availability negatively affect early flower development, fruit set and fruit growth. These effects can include altered pollen and/or ovary development and flower bud and flower abortion (Rylsk i and Spigelman, 1982; Polowick and Sawhney, 1985; Rylski, 1985; Aloni et al. 1991a; Mercado et al. 1997b; Mercado et al. 1997c; Aloni et al. 1999) Fruit size and uniform shape are two important characteristics that determine fruit quality. These char acteristics are determined by a number of factors, including assimilate accumulation in the developing flower/fruit and flower/fruit cell number and cell size. In turn, assimilate accumulation and cell number/size are determined by several factors, includi ng temperature and source:sink ratios. Previous research has shown that pepper grown under cool night temperatures develop a high percentage of malformed fruits that are not marketable. Low temperatures induce flower deformation, including swollen flowers and ovaries ( i.e. flowers and ovaries that are larger than the ones developed under optimum temperature), resulting in fruit malformation (Mercado et al. 1997c; Aloni et al. 1999) Swollen ovaries also develop under high source:sink ratios in pepper (Al oni et al. 1999) In both cases ( i.e. cool night temperatures and high source:sink ratios), swollen flower buds are reported to have increased reducing sugars and starch concentration compared to normal ( i.e. not swollen) flower buds (Aloni et al. 1999) Although several studies have been done, the relation between assimilate availability and ovary swelling in pepper remains unclear. Furthermore, the anatomical basis for ovary swelling and deformation under these conditions is unknown.

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17 The central hypothesis of this study is that both low night temperature and high source:sink ratio increase ovary size and therefore ovary swelling via effects on assimilate availability/accumulation and/or cell size/number. The objectives of this research were to determine the effects of night temperatures and source:sink ratios on: 1) ovary size in sweet pepper cultivars that differ in fruit size and shape; 2) net carbon exchange rates, growth, and ovary swelling in two cultivars of pepper; 3) ovary soluble sugar and starch concentration in bell pepper ovaries; and 4) cell number and cell size of bell pepper floral ovaries at anthesis.

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18 CHAPTER 2 2. LITERATURE REVIEW General Description of the Plant Pepper is the common name for several species from the genus Capsicum There are five botanical species that are commercially cultivated. Four of these species ( C. frutescens, C. chinense, C. baccatum and C. pubescens ) play a role in local economies but are not widely cultivated. C. annuum, which includes the long fruited bell var ieties, is the most widely cultivated around the world, both in temperate and tropical areas (Bosland, 1996; Wien, 1997) Bell pepper plants are herbaceous and either annual or perennial in their native habitat. Their shoot architecture is determined by their sympodial growth habit (Elitzur et al. 2009) Plant height varies from 50 to 90 cm under field conditions, but under greenhouse conditions, plant height may vary from 0.7 to 2.5 m. (Vilmorin Diaz, 1977; Nuez Vials et al. 1996) .The main stem bears 8 to 15 leaves forming a helix around the stem, with a 2/5 phyllotaxy, and terminates in a flower (Child, 1979) The two or three nodes below the terminal flower in the main stem bear sympodial shoots (Elitzur et al. 2009) or first lateral branches (Child, 1979) A sympodial shoot is composed of a leaf and the internode connected to the previous node. The sympodial shoot terminates in a flower, and contains a bud at the leaf axil that develops two opposite leaves. The two opposite leaves and the terminal flower from the preceding shoot compose a sympodial unit ( Figure 2 1 A). As the two leaves expand, the corresponding stem internodes elongate and carry up the leaves, which are finally located above the preceding flower ( Figure 2 1 B). Again, shoots end in a terminal flower, new leaves develop from the leaf axil, and internodes elongate ( Figure 2 2 A). After the first branching point, every new

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19 leaf axil below the floral terminus of a sympodial shoot develops two opposite new leaves. The cycle of development repeats itself and could, in theory, continue for an indefinite period of time. (Elitzur et al. 2009) .However, differential growth between the two leaves developing from the same axilar bud may result in asymetric sympodial development, i.e. one sympodial shoot has a smaller leaf and a shorter internode compared to the other shoot ( Figure 2 2 B). The suppression of one sympodial shoot that leads to the suppression of subsequent sympodial shoots may occur at any time. Such suppression is especially common in sympodia 3 and greater, and results in a great v ariability of final pepper plant architecture (Steer and Pearson, 1976; Child, 1979; Elitzur et al. 2009) Because of the flowering pattern, leaf and flower number ratio is close to one (Heuvelink and Marcelis, 1996) Fruit Anatomy/Morphology The pepper f ruit is a berry composed of three or four carpels (Steer and Pearson, 1976) The main parts are the peduncle, calyx, pericarp, placenta, and seeds ( Figure 2 3 ). The peduncle is comprised of the epidermis (rectangular square or oblong cells), hypodermis (one to three layers of cells that are partially lignified), cortex (parenchyma cells with irregular form), vascular tissue and pith (in large fruits). The peduncle has internal and external phloem tissue, and in the outer side of the external phloem tissue, phloem fibers occur singly, in pairs, or in small groups separated by parenchyma tissue. There is a ring of xylem tissue, broken by radial xylem rays (Parry, 1969; Rylski, 1986) The calyx is the connection between the peduncle and the pericarp and placenta itself (Rylski, 1986) The pericarp represents more than 80% of the fruits fresh weight and is the edible part. The pericarp is comprised of the epidermis, the hypodermis, the

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20 mesocarp (with chloroplasts), the inner mesocarp which includes fibrovascular bundles (xylem and phloem tissues), and the endocarp. Between the mesocarp and endocarp there are cells called giant cells, responsible for the blisters seen on the inner surface (Parry, 1969; Rylski, 1986) Pericarp thickness appears to be related to the number of cell layers in the ovary wall (Munting, 1974; Ali and Kelly, 1992) Fruit Growth and Development Fruit production is the final phase of a continuous physiological process that begins with successful pollination and fertilization of flowers and ends with fruit maturation and ripening. Fertilization is usually required for successful fruit set and development, otherwise the flower senesces and no fruit is formed (Gillaspy et al. 1993; Testa et al. 2002) In some cases, however, parthenocarpic fruits may develop due to genetic or environmental factors (e.g. extreme temperatures, low relative humidity) or by application of plant growth regulators to the flower (Fos and Nuez, 1996; Fos et al. 2000; Heuveli nk and Korner, 2001; Sato et al. 2001; Gorguet et al. 2005) In tomato, an increased level of auxins and gibberellins in the ovary can substitute for pollination and trigger fruit development, therefore those hormones are considered as the key elements i n parthenocarpic fruit development (Gorguet et al. 2005) Fruit shape, fruit size, pericarp thickness, and carpel number are important characteristics used to differentiate pepper types and varieties. In sweet pepper, fruit shape, fruit size and uniformity of carpels are the main determinants of fruit quality (Al oni et al. 1999) Fruit development comprises several stages, including flower and early ovary development, fruit abscission/fruit set, and fruit growth.

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21 Flower Development Flower initiation in bell pepper starts when the main stem reaches ~the sixth leaf (Choi and Gerber, 1992) At day/night temperatures of 28/15C, it takes approximately 30 days from the sixth leaf to anthesis of the first flower (Cruz Huerta, 2001) There is no specific information about the duration of cell division and cell enlargemen t in sweet pepper ovary. The ovary in a bell pepper flower comprises the ovary wall placental tissue, and the ovules, which will develop into seeds after fertilization. Based on morphological observations, Munting (1974) suggested that most cell division in the ovary takes places before anthesis, similar to some other crops. In cucumber ( Cucumis sativus L), for example, approximately 70% of cell division takes place before anthesis (Marcelis and BaanHofman Eijer, 1993) and in tomato ( Lycopersicon esculent um Mill.) fruit, cell division ends about two weeks after anthesis (Mapelli et al. 1978) Flower Abscission and Fruit Set Flower bud and flower abscission is a serious problem in sweet pepper, especially in the largefruited varieties. Consequences of flower abscission vary from delayed fruit production to low fruit yield depending on when the stress occurs (Wien, 1997) Factors that trigger flower abscission are high temperature (Bakker, 1989b; Turner and Wien, 1994a) low light intensity (Turner and Wien, 1994b, a) water stress (Wien, 1997) low relative humidity (Bakker, 1989a) developing fruits on the plant (Marcelis and BaanHofman Eijer, 1997; Marcelis et al. 2004) and biotic factors like pests and diseases (Johnstone et al. 2005) Of these facto rs, the most common cause of flower bud and flower abscission appears to be high temperature (> 21C during the night or > 32C during the day) (Rylski and Spigelman, 1982; Aloni et al. 1991b) as occurs in tomato (Sato et al.

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22 2001; Gorguet et al. 2005) In general, optimum temperature for fruit set in sweet pepper ranges from 22 to 26 C during the day and from 15 to 18 C during the night (Rylski and Spigelman, 1982; Bakker, 1989b; Bhatt and SrinivasaRao, 1993b; Turner and Wien, 1994a) Combinations o f high temperature with other stress factors increase abscission. For instance, high air temperature combined with low soil moisture (Cochran, 1936 cited by Wien, 1997) or low light intensity (Turner and Wien, 1994b) increased flower bud and flower absciss ion in pepper plants compared to high temperatures alone. Low night temperatures, on the other hand, cause low pollen fertility and swollen ovaries resulting in production of small, deformed and/or parthenocarpic fruits (Rylski and Spigelman, 1982; Polowic k and Sawhney, 1985; Pressman et al. 1998a; Aloni et al. 1999) Detailed description of the effects of low night temperature on flower and fruit development is given in the section Abnorm al Flower and Fruit Development on page 39 Factors related to flower bud and flower abortion in pepper can be g rouped into two main categories. The first group comprises those factors, such as extreme temperatures (Pressman et al. 1998a; Aloni et al. 2001) and low relative humidity (Bakker, 1989a) that impair pollination. Pollination and resultant seed number ar e important for fruit set. Well pollinated fruits grown under optimal conditions have between 150300 seeds (Aloni et al. 1999) although Marcelis and BaanHofman Eijer (1997) found that 50100 seeds/fruit was sufficient for maximal fruit set. Furthermore, they found that fruit set decreased as seed number fell below 50 seed per fruit. The second group comprises those factors that reduce carbohydrate availability. High temperature (Turner and Wien, 1994a) water stress (Aloni et al. 1991a) low light

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23 inte nsity (Turner and Wien, 1994b; Aloni et al. 1996) some pests or diseases (Johnstone et al. 2005) and developing fruits on the plant (Marcelis and BaanHofman Eijer, 1997; Marcelis et al. 2004) may reduce photosynthesis and/or sugar availability in pep per plant. Pepper cultivars that exhibit high flower abscission generally have a lower capacity to accumulate sugars and starch in their flowers compared to cultivars that exhibit low abscission (Aloni et al. 1996) Marcelis et al. (2004) reported that when source strength was decreased in pepper by shading, high plant density, or leaf pruning, the rate of flower bud abortion increased linearly. Similar results have been reported in other species. For example, i n maize ( Zea mays L), water deficits inhibit photosynthesis, and the decrease in photosynthate flux to the developing organs appears to trigger abortion (Boyer and Westgate, 2004) Under optimal growing conditions, the petals and ovary of pepper contain the highest amount of starch and sucrose per organ compared with the rest of flower structures, and the highest absolute and relative activity of sucrose synthase (Aloni et al. 1996) Although a continuous flux of sucrose into the developing flower is required for successful fruit set (Aloni et al. 1997) conditions that result in excess sugar accumulation in the flower bud may cause swollen ovaries (see Abnorm al Flower and Fruit Development on page 41) that develop into abnormal fruits. Fruit Growth The fruit growth period in sweet pepper depends on the cultivar and the temperature. Fruit reached the mature stage approximately 65 days after anthesis (DAA) in Mazurka (Marcelis and BaanHofman Eijer, 1995b) and Ariane (Cruz Huerta, 2001) but this period was 75 days in Domino (Tadesse et al. 2002) and more than 1 00 days in California (Pretel et al. 1995) Temperature also affects the fruit growth

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24 period. The growth period of pepper fruit Delphin decreased as daily mean temperatures increased, regardless of the day/night temperature amplitude (Bakker, 1989b) In tomato, Walker and Ho (1977) found that carbon import was inhibited by fruit cooling (5 C) and enhanced by fruit warming (35 C) compared with controls (25 C). Both fresh and dry matter accumulation in pepper follow a single sigmoid curve, but the pat tern differs among cultivars (Munting, 1974; Nielsen et al. 1991; Marcelis and BaanHofman Eijer, 1995b) In Mazurka (Marcelis and BaanHofman Eijer, 1995b) fruit fresh weight reached its maximum value between 40 and 45 DAA (stage of marketable green); but the fruit continued gaining dry weight until 60 65 DAA (stage of marketable red); Domino (Tadesse et al. 2002) required 56 days and 77 days, while California (Pretel et al. 1995) required 80 and 100 days to reach its maximum fresh and dry weight, respectively. The absolute growth rate in Mazurka fruit followed a bell shaped curve (Marcelis and BaanHofman Eijer, 1995b) In this cultivar, the maximum rate of fresh (>6 g d1) and dry (>0.4 g d1) weight gain occurred about the fourth week after anthesis. However, in Domino there was no period of peak growth because fruit fresh weight and volume were predominantly linear until 8 weeks after anthesis (Tadesse et al. 2002) Hall (1 977) reported the maximum rate of dry weight gain (0.2 g d1) occurred approximately 40 DAA, while the maximum relative growth rate (RGR, g DW g1 d1) occurred during the first days after anthesis. Based on the data reported by Marcelis and BaanHofman Ei jer (1995b) the maximum RGR in pepper fruit takes place in the first three days after anthesis, and Nielsen et al. (1991) found the maximum RGR during the first 11 days after anthesis.

PAGE 25

25 Growth preand post anthesis determines fruit shape and size in pepper. Fruit shape is defined primarily in the preanthesis stages of flower development, as a result of genetic and environmental control (Munting, 1974; Perin et al. 2002; Weiss et al. 2005) In bell pepper, as well as spherical and ovoid pepper cultivars, fruit shape is defined before pollination occurs, by the pattern and rate of cell division, most of which occurs before bloom (Munting, 1974; Aloni et al. 1999; Meijer and Murray, 2001) However, in long fruited pepper genotypes, fruit shape is defined by both cell division (pre and postanthesis) and cell elongation (postanthesis) (Munting, 1974) The shape of the ovary at anthesis may not be related to the final fruit shape (Munting, 1974; Wien, 1997) One parameter to measure the shape is the length:diam eter ratio (LDR). In bell peppers, the LDR in the ovary is about 0.9 and it increases to about 1.1 at fruit maturity. In long fruited genotypes, however, the ratio may change from about 1.0 to 2.2 or even as high as about 9 in mature fruits (Munting, 1974; Wien, 1997) During the first two weeks after anthesis, relatively higher growth rates were observed in the base of the ovary of long and ovoid pepper fruits compared to the growth rates in the tip. This basal growth was attributed to cell division and el ongation (Munting, 1974) Ultimately, final fruit shape and regularity is the result of processes that take place preanthesis (locule number) and postanthesis (uniformity of the locules in each fruit) (Rylski, 1986; Ali and Kelly, 1993; Wien, 1997; Aloni e t al. 1999; Perin et al. 2002) As with shape, final fruit size and weight (fresh and dry) are determined mainly by cell number at anthesis and cell elongation during anthesis and postanthesis (Munting, 1974; Coombe, 1976; Rylski, 1986; Ali and Kelly, 1993; Bertin et al. 2002) Smaller

PAGE 26

26 contributions to weight are due to cell division after anthesis and increases in cell solute concentration (Coombe, 1976) Fruit cell number and cell size are influenced by several factors, including water supply (Ho et al. 1987) and assimilate supply (Bertin et al. 2002) Cell expansion and therefore cell size is influenced by water relations (Ho et al. 1987) Mild water deficit (Ho et al. 1987) or high vapor pressure deficit (vpd) conditions (Leonardi et al. 2000) both of which decrease water import during cell elongation, may reduce final fruit size and fresh weight Sugar import; however, may not be affected, resulting in smaller fruit with increased soluble solids. Assimilate supply also influences fruit size by affecting source sink relations. Flowers and the small fruits just after anthesis represent a small sink (Archbold et al. 1982; Aloni et al. 1991b) In young pepper plants bearing one small fruit, one flower, and one flower bud, for example, the flower bud imports up to 3.6 mg of sucrose per day, which represents 3.2% of the exported sucrose, while the flower imports 3.7% and the fruit 45.9% of those carbohydrates. However, in fruitless plants of pepper, about 18.3% of the exported sucrose is allocated t o flowers and flower buds (Aloni et al. 1991b) Developing fruits however, rapidly become an important sink. In sweet pepper, when fruit growth rate is close to its maximum, fruit may represent up to 90% of the dry matter accumulation in the plant for a short period of time ( 80% for a period of 20 days (Hall, 1977) Over the entire development period, fruit represents between 30 and 65% of the dry matter accumulation (Nielsen and Veierskov, 1988; Bhatt and SrinivasaRao, 1993b; Cruz Huerta et al. 2005) Similar values have

PAGE 27

27 been found for tomato (Ho, 1984) Photosynthate accumulation in the fruit depends on sink strength rather than on the capacity of the peduncle to transport assimilates (Zhang et al. 2005) Small tomato fruits ( 20% of their maximum volume) imported carbon at an absolute rate nearly twice that of the larger fruits (8090% of its maximum volume) even though the peduncles phloem area in the smaller fruits was also smaller (Walker and Ho, 1977) Walker and Ho (1977) concluded that in tomato fruit, sink strength depends more on sink activity than on sink size, especially when the fruit is small. Presence of fruits also modifies dry matter distribution in the plant. Rapidly growing fruits decrease growth rate of all other organs (Hall, 1977) Ali and Kelly (1992) found that defruiting the first and second nodes in pepper plants increased the number of cell tiers in the ovary wall the final fruit weight, and pericarp thickness of the fruit in the third node, but had no effect on the fruit length or diameter. However, when the sink competition was alleviated in the flower bud stage, fruit length, diameter, weight and pericarp thickness were increased. Assuming the final cell size was similar in the fruits of all treatmen ts, these findings suggest that decreased competition during the entire period of ovary development increased both periclinal and anticlinal cell divisions in the ovary wall ; however, decreased competition in the later stages of growth increased only antic linal cell division. It remains unknown whether in pepper, cell number is more important than cell size in defining fruit size under carbohydrate limiting and nonlimiting conditions. Position of the fruit on the plant also influences final fruit size. The first fruits, which are borne in the lower part of the plant, are usually larger, and fruit size decreases as they are formed in the upper levels (Khah and Passam, 1992; Cruz Huerta, 2001)

PAGE 28

28 However, this influence seems to be related to fruit cell number (Bertin et al. 2002) Within a tomato truss, proximal fruits are bigger and already contain more cells at anthesis than distal ones (Bangerth and Ho, 1984) Bertin et al. (2002) found that the tomato fruit cell number was similar in fruits within the same truss under low sink competition conditions. However, when competition increased, cell number declined more in the distal than in proximal fruits. Even though the pepper fruit i s the main sink in the plant, its capacity to metabolize assimilates, and therefore to grow, seems to be affected by its previous assimilate supply (Koning and Marcelis, 1998) When sourcelimited fruits were changed to nonlimiting source conditions, it t ook 2 to 3 weeks before fruit growth rate was as fast as that from fruits growing continuously under nonlimiting conditions. Carbon Economy in the Fruit The assimilate supply to fruit is determined primarily by carbohydrate allocation, as carbon is one of the most important components of the fruits dry matter. Fruit carbon accumulation is the balance between carbon loss (via fruit respiration) and carbon gain (via fruit photosynthesis and imported carbon). Respiratory Losses Respiration rates per unit wei ght are higher in young fruits of sweet pepper compared with older fruits (Pretel et al. 1995; Tadesse et al. 2002) especially during the first three weeks of growth. This is similar to other fruits such as tomato (Xu et al. 1997) cucumber (Marcelis a nd Baan Hofman Eijer, 1995c) and blueberry (Birkhold et al. 1992) In some fruits, there may be a second peak in respiration when maximum fresh weight or maturation approaches. For example, in Domino bell pepper (Tadesse et al. 2002) CO2 production increased when fruit reached the maximum fresh weight;

PAGE 29

29 however, in California bell pepper (Pretel et al. 1995) CO2 liberation remained low until fruit was red. As an indirect way to measure respiration, Villavicencio et al. (2001) measured CO2 concentrati on inside the Camelot pepper fruit and found that the concentration increased from 33 to 66 mg L1 from the mature green stage to the red stage, and then decreased. Respiration losses in the fruits may represent an important proportion of the total carbon requirement. For instance, in cucumber, respiration losses accounted for 13 to 15% of the total cumulative carbon requirement, irrespective of temperature and number of fruits competing for assimilates (Marcelis and BaanHofman Eijer, 1995c) and in toma to, such losses are 25% (Tanaka et al. 1974a) In blueberry, respiration losses were 37% of the total carbon requirement (Birkhold et al. 1992) There is no specific information for pepper, but the respiratory costs might be lower than they are in tomato and cucumber because of carbon fixation in pepper fruit (Steer and Pearson, 1976) and the possibility of CO2 refixation (Bower et al. 2000) Carbon G ain Fruit photosynthesis Fruit carbon fixation or refixation has been reported for several species, particularly in the early stages of fruit development. In general, carbon fixation in fruit is 10 to 100 times lower than that of the respective leaves (Blanke and Lenz, 1989; Bower et al. 2000) and comprises only a small percentage of the total carbon requirement for fruit development. Steer and Pearson (1976) calculated that pepper fruits were able to fix up to 13% of the required carbon. Tomato fruit photosynthesis contributions ranged from 10 to 15% (Tanaka et al. 1974a; Hetherington et al. 1998) in cucumber the range was from 1 to 5% (Marcelis and BaanHofman Eijer, 1995a) and in blueberry about

PAGE 30

30 15% (Birkhold et al. 1992) Blueberry fruit photosynthesis supplied 50% of the carbon required during the first 10 days after flowering and 85% during the 5 days after petal fall (Birkhold et al. 1992) Some small fruits even exhibit net CO2 fixation under light conditions. Xu et al. (1997) found that small tomato fruits were net photosynthesizers under photosynthetic photon flux (PPF) greater than 300 mol m2 s1. Birkhold et al. (1992) found that blueberry fruits exhibited net photosynthesis from petal fall through fruit color break. In Cinnamomum camphora, CO2 refixation accounted for 22.9% of the carbon balance of the fruit (Ogawa and Takano, 1997) In addition, results of Smillie et al. (1999) suggested that the photosynthetic activity found in the calyx, green shoulder, pericarp and locular parenchyma of tomato fruits plays a s ignificant role in providing carbon assimilates to the fruit. Fruit photosynthesis differs from leaf photosynthesis in several aspects. Pepper fruit enzymatic activity of ribulose bisphosphate carboxylase oxygenase (Rubisco) on a fresh weight basis is only one fifteenth of the activity found in leaves (Steer and Pearson, 1976) Chloroplasts in the outer fruit layers are typically sun chloroplasts but evolve to shade chloroplasts in the inner tissues as light penetration decreases (Blanke and Lenz, 1989) Stomata in the fruit if present, are 10 to 100 times fewer in number than in the respective leaves (Blanke and Lenz, 1989) In pepper fruits no stomata were found (Blanke and Holthe, 1997) Blanke and Lenz (1989) and Blanke (1998) proposed that fruit photosynthesis is mainly intended to refix CO2 from mitochondrial respiration of predominantly imported carbon. Gas exchange through the fruit epidermis in fruits like apple and pepper is low, which favors higher CO2 concentrations in the center of the fruit, since seeds are an

PAGE 31

31 important source of respired CO2 (Blanke and Lenz, 1989; Blanke and Holthe, 1997; Villavicencio et al. 2001) Part of this CO2 may be refixed by phosphoenolpyruvate carboxylase (PEPC) and possibly phosphoenol pyruvate carboxykinase (PEPCk) by carboxylation and accumulation of malic acid in the vacuole (Blanke and Lenz, 1989) ; however, no diurnal variation in the pH has been measured. Pepper fruits remain green during most of the growing period. In addition, the large cavity and relatively thin pericarp may favor a higher light intensity throughout the tissue, increasing photosynthetic activity and possibly the capacity to refix more carbon than other fruits. Imported carbon Carbon imported by the fruit comes from either current photosynthesis or previously stored carbohydrates. There is no specific research in pepper addressing translocation of previously stored carbon to the fruit. In nonfruiting plants of pepper, the stem becomes the main sink, but there is no evidence that it stores soluble sugars or starch that are later used by the fruit (Hall and Milthorpe, 1978) In tomato, the stem is also a major sink for assimilates and it functions as a storage organ. However, there is little evidence to suggest tha t these assimilates are used in fruit production, except when the plant is in the final stages of its growth cycle, or if premature leaf loss occurs (Hocking and Steer, 1994) By far, the most important source of imported carbon to developing fruit comes f rom current photosynthesis. Although leaves supply the majority of photosynthate for fruit development (Steer and Pearson, 1976) green stems and petioles may also fix CO2. Xu et al. (Xu et al. 1997) found that net photosynthesis in petioles of young toma to leaves (5th and 10th node from the apex) was positive when PPF was higher than 100 mol m2 s1, but in petioles from older leaves (15 and 18th nodes) net CO2

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32 exchange was close to 0 even at higher PPF. In contrast, there was no net photosynthesis in the stem, but the higher the irradiance, the lower the rates of net CO2 efflux regardless of stem age, indicating some carbon fixation was occurring. Leaf and plant photosynthesis are affected by a variety of factors including leaf area, leaf age, presence of fruits, light intensity, and temperature. a) Leaf area. Pepper plants can compensate photosynthetically for certain amounts of defoliation. Defoliation of up to 50% in young pepper plants increased net photosynthesis in the remaining leaves such that f inal fruit fresh and dry matter were not affected (Bhatt and SrinivasaRao, 1993a) In tomato, partial leaf removal increased net assimilation rate of the remaining leaves, so fruit growth was not affected (Tanaka and Fujita, 1974) b) Leaf age. Photosynthetic rates vary with leaf age. Very young leaves have a negative carbon exchange rate and may represent the major sink for photosynthates manufactured within them (Tanaka et al. 1974b) In pepper California Wonder, maximum photosynthetic rate occurs when leaf area is ~ 10 cm2 (Steer, 1971) the time at which stomatal density is highest (Steer, 1972) The photosynthetic rate decreases slightly until the leaf reaches its maximum size, and then remains steady for a period of time (Steer, 1971) Hall (1977) reported that the photosynthetic rate of pepper leaves that expand during or immediately after anthesis in fruiting plants remains steady throughout fruit growth. Older leaves generally exhibit lower photosynthetic rates compared to younger leaves (Steer a nd Pearson, 1976) In tomato, Bolanos and Hsiao (1991) reported that maximum leaf photosynthesis was reached near the time of full expansion and declined steadily thereafter. Also in tomato, Xu et al. (1997) found that

PAGE 33

33 net photosynthesis of the 10th, 15th and 18th leaves from the apex were only 50, 21 and 7% of that of the 5th leaf, and suggested that leaves below the 18th node can be eliminated to improve air circulation and reduce humidity within the crop without affecting CO2 fixation. Photosaturation may also differ with leaf age, where old leaves photosaturate at lower light intensities. In addition, the surplus of photosynthates from young, upper leaves may depress the photosynthetic activity of old leaves (Tanaka et al. 1974a) c) Developing fruits Developing fruits may affect plant photosynthesis by decreasing leaf area and/or increasing leaf photosynthetic rates. Compared with nonfruiting plants, plants with developing fruits often have reduced total leaf area (due to fewer and smaller leaves) and increased life span of functional leaves (Hall, 1977; Klring et al. 1996) Decreases in leaf area of fruiting pepper plants compared to nonfruiting plants were significant as soon as 2 weeks after anthesis (Hall, 1977; Bhatt and Srinivasa Rao, 1989) By 80 DAA (2 weeks after maturation of first fruit), functional leaf area in fruiting plants was about 50% that of nonfruiting plants. However, in nonfruiting plants, leaf senescence and abscission rates were sevenfold or more greater than in fruiting plants (Hall, 1977) This phenomenon is not seen in all pepper cultivars. Bhatt and SrinivasaRao (1997) reported no differences in total leaf area in bell pepper with different fruit loads. Developing fruits may also affect photosynthesis by increasing l eaf CO2 assimilation rates or by causing a partial recovery of the leaf photosynthesis rate in older leaves (Hall and Brady, 1977) Leaf CO2 assimilation rates may increase up to 65% in fruiting plants compared to nonfruiting plants (Hall and Brady, 1977; Nilwik,

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34 1980; Bhatt and SrinivasaRao, 1989) or in the same plants before and after fruit set and development (Cruz Huerta et al. 2005) In addition, removing pepper fruits during rapid growth reduced the rate of CO2 assimila tion by up to 30% in the leaves close to the fruits, mainly due to changes in both the leaf and the intracellular resistance (Hall and Milthorpe, 1978) However, removal of the first two flowers or fruits in pepper plants had no consistent effect on the photosynthetic rates of fully expanded and exposed leaves (Bhatt and SrinivasaRao, 1997) Bertin et al. (2001) reported that decreasing sink competition during tomato fruit cell elongation decreased fruit dry matter content, suggesting that reduced sink strength inhibited leaf photosy nthetic rates. d) Light Light is one of the most important factors in plant productivity (Papadopoulos and Pararajasingham, 1997) It is generally accepted that most C3 species photosaturate between 500 and 1000 PPF (full sun light intensity is ~2000 PPF) (Taiz and Zeiger, 2006) ; however, there is great variation, even within species. For example, Xu et al. (1997) found that young tomato leaves reached their maximum photosynthetic rate between 750 and 800 mol m2 s1 while older leaves saturated at about 300 mol m2 s1. In pepper, E Jaimez et al. (2005) reported that light saturation in bell pepper grown in a greenhouse occurred ~ 1500 mol m2 s1 PPF. Decreasing light intensity from 1800 to 1000 mol m2 s1 PPF decreased photosynthesis ~20% on sunny days; however photosynthesis decreased ~40% on cloudy days due to higher stomatal resistance (E Jaimez et al. 2005) Photoperiod also affects plant productivity. Some Capsicum annuum genotypes have the ability t o acclimate to supplementary light, even to continuous light, by increasing chlorophyll content (Murage and Masuda, 1997) carboxylation capacity,

PAGE 35

35 electron transport capacity, and the light saturation point (Dorais et al. 1995) Demers et al. (1998) repor ted that continuous light increased plant growth (fresh and dry basis) and yield for the first five weeks in pepper compared to a 14 h photoperiod; however, after five weeks, growth under continuous light decreased,. The authors suggested that the negative effects of continuous light on plant growth were due to a limitation in photosynthate export, as evidenced by high leaf starch content and leaf deformation. However, Dorais et al. (1996) reported that after 15 weeks of growing bell pepper under extended photoperiod (18 h) or continuous lighting, fruit yield increased compared with plants grown under 8 or 12 h photoperiods, indicating increased photosynthate allocation to fruits. e) Temperature. Temperatures above or below the optimal range for photosynthesis reduce net photosynthesis and alter biomass partitioning, favoring vegetative over reproductive growth (Ahmed et al. 1993; Wang et al. 1997; Diczbalis and Menzel, 1998) Temperature effects on photosynthesis depend on several factors, including the plant organ exposed (root vs shoot), the time of the day (day vs night), and the duration of the event. Some species tolerate root temperatures up to 35C without decreasing photosynthetic rates (Du and Tachibana, 1994; Nada et al. 2003) ; however, root te mperatures above 38C generally result in severe decreases in photosynthesis (Du and Tachibana, 1994; Diczbalis and Menzel, 1998; Nada et al. 2003) The reduction in photosynthesis is initially caused by stomatal closure triggered by water stress (He et a l. 2001) or abscisic acid (ABA) accumulation (Nada et al. 2003) Other physiological disorders, including impaired activation of Rubisco and decreased chlorophyll content,

PAGE 36

36 also play an important role under prolonged high root temperatures (Du and Tachibana, 1994; Nada et al. 2003) Variable root temperatures (from 23C to 40C) decreased photosynthesis in lettuce (He et al. 2001) and dry matter accumulation in pepper (Dodd et al. 2000) However, Gosselin and Trudel (1986) reported that maximum dry weight in pepper was found at root temperatures of 24C, while maximum photosynthesis occurred at root temperatures of 36C. In tropical rambutan ( Nephelium lappaceum ), maximum photosynthetic rates occurred at 28C root temperature, and photosynthesis decreased significantly at lower (15C) or higher (38C) root temperatures (Diczbalis and Menzel, 1998) Excessively high shoot temperatures decrease photosynthesis and increase respiration, reducing carbohydrate availability (Taiz and Zeiger, 2006) Decreases in photosynthetic rates in C3 plants are often attributed to stomatal closure (Taiz and Zeiger, 2006) followed by the inactivation of Rubisco and/or denaturation of Rubisco activase, which facilitates the carbamylation and maintenance of Rubisco acti v ity (Hendrickson et al. 2008, and references there in) Low temperatures also reduce shoot photosynthesis. This effect is more serious in chilling sensitive species, such as cucumber, tomato, and sweet pepper (Li et al. 2003b) Studies in cucumber, sweet pepper (Li et al. 2003b) and Arabidopsis (Zhang and Scheller, 2004) showed that low temperature (4C) combined with low light intensity (100150 PPF) damaged PSI without damaging PSII. However, cold temperatures and high irradiances damaged PSII (Liu et al. 2001) Pepper plants that were coldhardened

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37 (5 d at 14C and 250 PPF) were more tolerant to chilling stress, but tolerance was only expressed by a faster recovery (Liu et al. 2001) Night shoot temperature also affects photosynthesis. Night temperatures between 15 and 25 C decrease photosynthesis in grapevine due to a delay in stomatal opening the next morning, although stomatal conductance recovers as temperatures increase during the day (Hendrickson et al. 2004a; Hendrickson et al. 2004b) Low night temperatures, especially below 10C may also decrease photosynthesis by decreasing ribulose1,5 bisphosphate (RuBP) regeneration and Pi availability for recycling (Hendrickson et al. 2004a; Hendrickson et al. 2004b) reducing activity of PSII (Sun dar and Reddy, 2000; Bertamini et al. 2005) and decreasing leaf chlorophyll content (Sundar and Reddy, 2000; Bertamini et al. 2005) Reduction in Rubisco amount and activity, as well as other photosynthetic/carbohydrate metabolizing enzymes, such as fructose bisphosphatase (FBPase), and sucrose phosphate synthase, may also occur in response to low night temperature (Sundar and Reddy, 2000; Ramalho et al. 2003; Hendrickson et al. 2004a; Hendrickson et al. 2004b; Bertamini et al. 2005) However, spe cies/cultivars tolerant to low night temperatures increase the activity of Rubisco and other photosynthetic enzymes, including phosphoenolpyruvate carboxylase, NADP malate dehydrogenase, NADP malic enzyme, and stromal FBPase (Du et al. 1999) In several t ropical crops, including sugar cane, bean, soybean, coffee, maize, and peanut (Wolfe, 1991; Ying et al. 2000, and references there in; Ramalho et al. 2003; Wang et al. 2004) one cold night may decrease photosynthetic rates 5 to 80%. Sweet pepper seems to be very sensitive to low night temperatures. Bhatt and SrinivasaRao (1993b) reported that decreasing night temperatures from 22 to 17C decreased net

PAGE 38

38 CO2 assimilation rates up to 30%. During maize grainfilling, one night of exposure to 4C decreased t he carbon exchange rate 14 to 30%, depending on the genotype (Ying et al. 2000) Greater reductions were found when maize plants were exposed to two or three cold nights at 4C. Maintaining plants in the dark at 15 to 18C for an hour before light exposur e reduced the negative effect of cold night temperature on maize photosynthesis (Ying et al. 2002) The reduction in maize leaf photosynthesis after cold exposure was linearly related to the incident PPF level the next day, but fluorescence and CO2 exchange readings suggest that the reduction in photosynthesis is not associated with photoinhibition (Ying et al. 2002) Similarly, Hendrickson et al (2004a; 2004b) found that chilling induced reduction in grapevine photosynthesis was not due to photoinhibiti on. Initial inhibition appeared to be related to stomatal closure, followed by a biochemical restriction due to limitation of RuBP regeneration and sucrose and starch synthesis limitation (endproduct limitation) (Hendrickson et al. 2004a; 2004b) Some sp ecies have the ability to acclimate to low temperatures. In such cases, exposure to low temperatures for long period (weeks) markedly increased the maximum activities of Rubisco, stromal and cytosolic fructose1,6 bisphosphatase, and sucrosephosphate synt hase, and increased RuBP regeneration and carboxylation in leaves of several species, leading to significant increases in whole plant photosynthetic capacity (Holaday et al. 1992; Hurry et al. 1994; Du et al. 1999; Yamori et al. 2005) Although net pho tosynthesis in Parthenium argentatum initially decreased when night temperature was decreased from 20C to 15C, photosynthetic rates began to recover after 30 days of low night temperatures, reaching values equal to or higher than initial values (Sundar and Reddy, 2000) In cotton, Singh et al (2005) reported that after 30

PAGE 39

39 days of growing plants under decreasing night temperatures from 16 to 11C (at day temperatures of 28C), CO2 assimilation, stomatal conductance, carboxylation capacity, electron transport capacity, and triosephosphate utilization capacity were similar between leaves developed under low night temperature and leaves developed on control plants (28/24C day/night) Mercado et al. (1997a) reported that sweet pepper plants grown under low night temperatures (14 C) showed improved chilling resistance upon exposure to night temperatures of 6 C compared with plants grown under higher night temperatures (20 C) Abnorm al Flower and Fruit Development In pepper, as in most horticultural crops, yield of high quality fruit is as important as total yield. Fruit weight, size, shape, firmness, color, and disease/pest incidence are used to evaluate pepper fruit quality (Aloni e t al. 1999; Navarro et al. 2002; Kissinger et al. 2005; Lim et al. 2007) Both final fruit size and shape in pepper are directly affected by early flower and fruit development (Ali and Kelly, 1992; Aloni et al. 1999) Early development, in turn, is very sensitive to several factors, including ambient temperature and carbohydrate availability in the plant. High temperatures induce flower abortion and impair pollen fertility in several crops, including tomato (Bertin, 1995; Sato et al. 2001; Sato et al. 2002) pepper (Polowick and Sawhney, 1985; Turner and Wien, 1994a; Aloni et al. 2001) and Brassica napus (Young et al. 2004) In tomato and pepper, high temperature stress also reduces pollen grain number and germination (Aloni et al. 2001; Sato et a l. 2004; Sato and Peet, 2005) In pepper, additional morphological changes are associated with high temperatures. Day/night temperatures of 28/23C decreased ovary diameter and increased style length compared to a day/night temperatures of 23/18C (Polowi ck and

PAGE 40

40 Sawhney, 1985) High temperature after anthesis also reduces fruit and seed growth and seed quality in chili pepper (Pagamas and Nawata, 2008) Low temperatures also induce abnormal flower and fruit development in bell pepper. In winter, cooler temperatures and shorter day lengths increase the percentage of small, flattene d parthenocarpic fruits. Pollen fertility is the main reason for this condition, but female organs (Polowick and Sawhney, 1985; Pressman et al. 1998a; Shaked et al. 2004) and other parts of the flower (Aloni et al. 1999) are also affected. Night temperatures of 15C or less (day temperature of 20 to 25C) increase ovary diameter in pepper without increasing locule number, creating swollen ovaries and malformed fruit (Polowick and Sawhney, 1985; Rylski, 1985; Bakker, 1989b; Aloni et al. 1999; Shaked et al. 2004) Low night temperatures also produce abnormalities in the petal and stamen, and reduce style length. The longer the duration of low night temperatures, the higher the percentage of swollen flowers and ovaries (Polowick and Sawhney, 1985; Aloni et al. 199 9) Aloni et al. (1999) found that 15 days at night temperatures of 12 or 18C resulted in 21 and 7% swollen ovaries, respectively By 49 days, these percentages increased to 78 and 14%, respectively. Cell volume in swollen ovaries is greater than that in normal ovaries, but cell number remains the same (Pressman et al. 1998b) suggesting that swollen ovaries develop after cell division ends. Although low night air temperatures result in swollen pepper ovaries and malformed fruit, low night root temperatures do not appear to have the same effect. Pepper plants grown at night air temperatures of 12C produced low fertility pollen and small fruits at root night temperatures of either 12 or 20C (Mercado et al. 1997c; Aloni

PAGE 41

41 et al. 1999) These findings sugge st that flower deformation and ovary swelling are favored by low air temperature during the night, regardless of root temperature. Very high day temperature may reduce negative effects that low night temperatures have on final fruit shape and size in pepper. Pressman et al. (2006) found that increasing the day temperature to ~ 35C compensated for some of the deleterious effects of low night temperature on vegetative and reproductive development, particularly on stamen and pollen development. However, no in formation on the effects of these high day temperatures on ovary development was reported. Excess carbohydrate availability during early flowering/fruiting also results in abnormal flower and fruit development in pepper. Increased source/sink ratio by fruit removal increases the proportion of swollen ovaries ( 7 mm diameter) compared with fruiting plants (Aloni et al. 1999) Fresh weight of swollen flowers that developed in defruited plants was three to four times that of the flowers on fruiting plants (Aloni et al. 1999) In addition, the percentage of swollen flowers was inversely related to the number of growing fruits on the plant, and directly proportional to the concentration (fresh weigh t basis) of reducing sugars and starch in the flowers devel oped in those plants (Aloni et al. 1999) Flowers that develop on defruited plants accumulated four fold more 14CO2 (600 vs 150 g d1) and fresh weight (180 vs 60 mg) than flowers that develop on fruiting plants (Aloni et al. 1991b; Aloni et al. 1999) These data suggest that excess assimilates are transported to flower buds on defruited plants, resulting in ovary swelling and deformation (Aloni et al. 1999) In summary, flower and ovary swelling in pepper is favored by exposing the shoot to low night temperature or by removing actively growing fruits. However, the

PAGE 42

42 physiological changes in the plant and flower buds that induce such swelling and deformation are still not clear

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43 Figure 2 1 Schematic drawing of pepper branching and flowering sequence. A) Meristematic stage. B) Well developed branch. A sympodial shoot bears a leaf (L1, blue), and terminates in a flower (F2). From the leaf axil, two leaves (L2) develop. The flower and the two leaves developed from the same sympodial shoot compose a sympodial unit (F2 and L2, green). L2 leaves are pushed up by internode elongation, so that the leaves appear above the preceding flower. The shoot terminates in a flower, which is part of the subsequent sympodial unit. From each of the L2 axils, two new leaves (L3, red) develop. Each color represents sympodial units of a different order (Drawing A based on micrograph from Elitzur et al. 2009) L2 F2 L2L3 L3 L1 L2 F2 L2 L3 L3 L1 A B

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44 Figure 2 2 Schematic diagram of a pepper plant. A) Pepper plant has a main stem with cotyledonary (C) and true leaves (L, yellow). The main stem ends in a terminal flower (FTF, first terminal flower, brown circle). The terminal flower and the first two leaves above it (brown) originate from the main stem and form the main stem sympodial unit. Each new sympodial unit is also composed of two leaves and a flower (circle), forming sympodial unit 1 (SU), 2 (SU2), and so forth. B) Asymmetric growth of the sympodial components results in different plant architecture. Figure 2 3 Pepper fruit and its parts ( Mod i fied from Rylski, 1986) C C FTF SU2 SU1 L L FTF SU1 SU2 B SU2 A SU1 Peduncle Placenta Calyx Seeds Pericarp

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45 CHAPTER 3 3. LOW NIGHT TEMPERATURE INCREASES OVARY SIZE IN SWEET PEPPER Introduction Pepper ( Capsicum annuum L) is an important vegetable c rop that belongs to the Solanaceae family and whose edible part is botanically a fruit Historically, peppers have been grown in the tropics as well as in temperate climates (Eshbaugh, 1993) Hot peppers are consumed fresh, dried or processed, mostly as spice, or as a source of food colorants or capsaicin. Sweet peppers, a genetic recessive nonpungent form, are an important green vegetable crop worldwide, especially in temperate regions (Esh baugh, 1993; Bosland, 1996) There is large variability in sweet pepper fruit size and shape, ranging from cherry (spherical, ~2.5 to 3.5 cm in diameter), to elongated (banana or cubanelle), to bell (blocky) peppers, which are the most important from an ec onomic standpoint (Bosland, 1996; Bosland, 1999) In the U.S., per capita consumption of bell peppers increased from 1 kg in 1970 to ~3 kg in the late 1990s and has remained steady during the last 10 years (USDA Economic Research Service, 2008) Pepper plants develop a main stem that bears leaves on the first 8 to 15 nodes and terminates in a flower. The two or three nodes preceding the terminal flower bear sympodial shoots (first lateral branches) (Child, 1979; Elitzur et al. 2009) Each sympodial shoot has a leaf, and terminates in a flower. Two new leaves emerge from the leaf axil below the floral terminus, and are pushed above the terminal flower by elongation of the new internode ( Figure 3 1 ). From every sympodial shoot, a new sympodial unit is formed, consisting of the two sympodial shoots, the flower in the vertex of the shoots ( i.e. the terminal flower from the preceding shoot), and the single

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46 leaf at the di stal end of each sympodial shoot ( i.e. two leaves due to the presence of two sympodial shots ). This cycle of development repeats itself and could, in theory, continue for an indefinite period of time (Elitzur et al. 2009) Thus, flowering and fruiting in pepper is continuous as long as plants are growing. Fruit shape, size and regularity are important quality components in sweet peppers. Final fruit shape is the result of genetic and environmental factors (Perin et al. 2002; Weiss et al. 2005) and it is defined in the early stages of fruit development (Munting, 1974) In bell, spherical, and ovoid fruited pepper varieties, shape definition takes place before pollination occurs (Munting, 1974; Aloni et al. 1999; Meijer and Murray, 2001) However, in l ong fruited varieties, fruit shape is defined during both the preanthesis and postanthesis stages (Munting, 1974) I n all genotypes, pollination and fertilization are key factor s in attaining the ideal size, shape and uniformity (Mercado et al. 1997c) Under optimal growth conditions, bell pepper fruit requires 150300 seeds to develop the uniform blocky shape (Aloni et al. 1999) After fertilization and fruit set in bell pepper, changes in assimilate supply can change the growth rate and therefore the size, but do not affect fruit shape (Aloni et al. 1999) Sweet peppers, as with other fresh fruits and vegetables, are a commodity in demand all year. In order to fill that market need, sweet pepper production during the winter has been developed in areas with warm or mild winters or even in cold winters under greenhouse conditions. However, w hen grown in cold winters under greenhouse conditions, bell peppers can develop up to 60% of flattened and deformed fruits, and thus unmarketable (Rylski, 1973; Al i and Kelly, 1993; Aloni et al. 1999)

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47 Several reports have previously addressed the effect of low night temperature (LNT) on flower and fruit development and malformation in sweet pepper. However, they have mainly focused on pollen quality (Mercado et al 1997b; Pressman et al. 1998a; Shaked et al. 2004) pollination (Mercado et al. 1997c; Pressman et al. 1998a) number of seeds (Rylski, 1973; Kato, 1989) and fruit quality (Rylski, 1973; Kato, 1989; Pressman et al. 2006) Only a few studies have focused on flower and ovary malformation and swelling, and these studies have been limited to effects in bell pepper (Pressman et al. 1998b; Aloni et al. 1999) Flower deformations due to LNT include abnormal and curled petals that do not fully expand, shorter stamens with low pollen content and low germinability, and larger ovaries with shorter styles compared with flowers developed under intermediate or high temperatures (Polowick and Sawhney, 1985; Rylski, 1985; Mercado et al. 1997b; Pressman et al. 19 98a; Aloni et al. 1999; Shaked et al. 2004) The ovary enlargement observed under cool temperatures is referred to as ovary swelling (Pressman et al. 1998b; Aloni et al. 1999) Ovaries developed under LNT are impaired in fruit development, even when pollinated with viable pollen, and result in smaller, irregularly shaped fruit compared with fruit developed under higher night temperatures (Pressman et al. 1998a) In addition to LNT, high source:sink ratio can also result in swollen ovaries in bell pepper (Aloni et al. 1999) High source:sink ratio can be caused by harvesting all the fruits or by a massive flower drop. When bell pepper plants bearing several fruits in different stages were artificially defruited, 90% of the flowers that reached anthesis 15 days after defruiti ng were swollen, compared to 20% in the nondefruited control. Although partial defoliation of completely defruited plants did not reduce ovary swelling,

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48 some reduction in ovary swelling was observed in subsequently formed flowers when two developing fruit s were allowed to remain on the plant (Aloni et al. 1999) Although the effects of LNT and high source:sink ratios on ovary swelling in bell peppers are documented, LNT and high source:sink ratio effects in other types of sweet peppers are unknown. In add ition, in pepper cultivars with blocky or spherical fruit, ovary shape and cell number are determined in the preanthesis stages; however, in long fruited cultivars, cell number and ovary shape are modified in the postanthesis stages (Munting, 1974) Thus, LNT and/or source sink ratio might affect ovary shape differently depending on the final fruit shape. Therefore, t he hypothesis tested in this work is that low night temperature increases ovary size and causes ovary swelling in a range of pepper cultivars differing in final fruit shape and size, but low source:sink ratio can counteract the effect of LNT on ovary swelling. The objectives were to determine the effects of LNT and two source sink ratios on ovary size in different sweet pepper cultivars and to determine the parts of the ovary that are most affected by LNT. Material and Methods Plant Material and Growing Conditions Seedlings of six (Experiment 1) or four (Experiment 2) sweet pepper varieties were germinated using a mix of commercial potting media (SunGro Metro Mix Ag Lite Mix1 1 Composition: Canadian Sphagnum peat moss (5565%), horticultural grade vermiculite, dolomite limestone, starter nutrient charge, and wetting agents () and perlite (4:1 v:v) in 40cell flats (60 cm3 each cell) in 1.4 m2 growth chambers (E15 Conviron, Winnipeg, Canada). Chamber temperature was set to 22/20C (day/night) and PPF was maintained between 450500 mol m2 s1 abo ve the canopy with a 14-www.sungro.com/products.php, 12/17/2008).

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49 hour photoperiod. Light was provided by fluorescent and incandescent lights. There was no control of CO2 concentration or relative humidity. Plants were watered with tap water until the beginning of expansion of the first true leaf (4 weeks after seeding), when fertigation began. The fertigation protocol differed with the experiment and is described below. Pests were controlled by using a combination of biological control (wasps for aphids) and chemical control. Insecticidal soap (Sa fer Brand Insect Killing Soap, Safer Brand, www.saferbrand.com 12/17/2008) dicofol (Dicofol 4E, Dicofol 42%, Makhteshim Agan of North America, NY, USA), or abamectin (Agri Mek 0.15EC, abamectin 2%, Syngenta Crop Protection, Greensboro, NC, USA) were sprayed as needed at onehalf the lowest recommended dosage until seedlings were transplanted to containers. Seedlings at about the sixthleaf stage (54 days after sowing) were transplanted into 1.5 L containers using the same media mix. Plants were continuously pruned once branching began. At the first branching point, two shoots were allowed to grow, forming the main stem sympodial unit. For the subsequent sympodial units in Experiment 1, the strongest shoot was allowed to grow (contributing to the main axis of the plant) and the other shoot was allowed to develop one sympodial unit (forming a lateral branch) before pruning ( Figure 3 2 A). In Experiment 2, onehalf of the plants were pruned as in Figure 3 2 A and the other half were pruned so that the less strong shoot was allowed to develop only the flower of the next sympodial unit before pruning ( Figure 3 2 B). Sympodial units formed on the main stem sympodial shoots were numbered SU1 (sympodial unit 1), which in turn bore SU2. Subsequent sympodial units were numbered 3, 4, n (see Elitzur et al. 2009) For the purpose of this study, nodes above the first

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50 branching point were also numbered according to their sympodial unit in order to describe each flowers position, i.e. node 1 (corresponding to SU1), node 2, node 3, etc. Flowers from the main stem and node 1 ( i.e. SU1) were removed at the flower bud stage. Expe riment 1 This experiment was designed to determine the response of six cultivars of sweet pepper to low night temperature. Four blocky bell type pepper cultivars (Ariane, Aristot le with X3R, Brigadier, Legionnaire), one elongatedfruit cultivar (Banana Supreme, length:diameter ratio ~4), and one cherry type cultivar (Red Cherry Sweet, rounded, 2.53.7 cm diameter ) were exposed to day temperatures of 22C and one of two night temperatures: 20 (high night temperature, HNT) or 12C (low night temperature, LNT). Several cultivars of bell pepper cultivars allowed to test differences among bell pepper cultivars, since previous work on ovary swelling as a response to night tem perature had been done mostly in Mazurka cultivar (Pressman et al. 1998a; Pressman et al. 1998b; Aloni et al. 1999) Plants were fertigated starting at about 6 weeks after seeding with 20N:6.7P:16.7K water soluble fertilizer (Scotts Sierra Horticultur al Products Company, Marysville, OH, www.scottsprohort.com ). Application rates were ~1 mg N per plant per day before transplanting (54 days after sowing) and from 15 to 50 mg N per plant per day after transplanting, depending on plant age. Starting at 7 weeks after transplanting, plants were fertigated as needed with the following nutrient solution (mM): 3.4 Ca(NO3)2, 1.8 KNO3, 1.6 KH2PO4, 0.3 KCl, 2.7 MgSO4, and (M): 50.2 FeEDTA, 3.1 CuSO4, 14.6 MnSO4, 64.8 H3BO3, 4.6 ZnSO4, 0.6 Na2MoO4 (modified from Cruz Huerta et al. 2005; and Jovicich, 2007) Plants were watered in the morning to increase root zone temperature in the LNT treatment (Ying et

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51 al. 2002) so that day temperature was similar in the root and shoot in both LNT and HNT Three plants per cultivar were randomly distributed in each growth chamber. Night temperature treatments began just before anthesis of flowers in node 3 (Ariane and Aristotle), 4 (Brigadier, Legionnaire and Banana Supreme) or 5 (Red Cherry Sweet). Once treatments started, all the flowers at anthesis were continuously harvested during a 39day period in Brigadier, Legionnaire, Banana Supreme and Red Cherry Sweet and a 64day period in Ariane and Aristotle. The flower harvest period was prolonged in these latter two cultivars due to a delay in their growth and development after transplanting. Plants did not bear any fruit before and during the experiment. Flower harvests occurred every 23 days during the first 50 d ays and every day during the last 13 days. A minimum of 66 flowers per night temperature and cultivar combination were harvested. The flower position in the plant ( i.e., node number above first terminal flower and whether it was in the main axis or in a lateral branch) was recorded. Ovary fresh weight, volume (measured by water displacement), diameter and length were recorded. Ovaries were cut in half and ovary wall thickness was also measured. Experiment 2 Based on the results from the first experiment, four cultivars, Ariane, Legionnaire, Banana Supreme, and Red Cherry Sweet, were selected for Experiment 2. T he criteria used to select the cultivars were 1) contrasting responses to LNT in bell pepper s (Ariane and Legionnaire), and 2) contrasting fruit shape and size (bell, elongated and cherry fruited varieties). Two night temperatures regimes, high (HNT, 20C) and low (LNT, 12C), and two levels of source supply (high and low) were studied. Sourc e supply was modified either by leaving the complete sympodial unit on

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52 every lateral shoot ( Figure 3 2 A), or removing the leaves and leaving only the flower ( Figure 3 2 B). Therefore, starting in sympodial unit 2, high source supply treatment had twice as many leaves as the low source supply treatment did., i.e. 8 vs. 4 leaves per sympodial unit, as shown in Figure 3 2 Each growth chamber was divided into 3 blocks (based on variation in light intensity within the growth chamber observed in Experiment 1) and within each block 8 plants were randomly placed (4 cultivars and 2 levels of source supply), with one plant per treatment using a completely randomized block design. Treatments began just before anthesis of flowers on the second sympodial unit (node 2) The first terminal flower and flowers from node 1 were removed. Flowers were harvested every day at anthesis for up to 45 days, from the second to the seventh nodes above the first terminal flower. As in Experiment 1, plants did not bear any fruit. The position of every harvested flower in the plant was recorded. Ovary fresh weight (FW), volume, diameter, and length were recorded. Ovary wall thickness and FW were also measured, as was placenta FW. Statistical Analysis Data from Experiment 1 were grouped i nto five harvest intervals ( Table 3 1 ) For Brigadier, Legionnaire, Banana Supreme, and Red Cherry Sweet, harvest intervals were 8 days, and for Ariane and Aristotle, harvest intervals were 13 days, since development was delayed in these two cultivars. Data were analyzed as a mixed model with repeated measures using cultivar, night temperature and harvest period as main factors, axis ( i.e. main axis or later al branch) nested within each plant as the repeated measures subject, and days after anthesis as the covariate.

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53 Experiment 2 originally included three main factors (4 cultivars, 2 temperature regimes, 2 levels of source supply). Additionally, node number ( number of nodes counted above the first flowering node) and the axis within the plant were included in the model during the analysis. After the initial statistical analysis, it was found that the source supply did not significantly affect the parameters ev aluated, either as main or interactive effects. Thus, this factor was not considered in the final analysis. Data from experiment 2 were also analyzed as a mixed model with repeated measures, using temperature and cultivar as the factors of the main model, and the node number and axis (main axis, lateral branch) of the harvested flower as components of the sub model. The subject for the repeated measures was an individual plant. Data were analyzed using SAS (SAS Institute Inc., 2008) and mean separation was done using Tukey Kramer (multiple LSmeans) or t test (two means). Multiple LSMeans letter grouping was done using a SAS macro developed by Saxton (1998) Results Experiment 1 Approximately 30 f lowers per plant reached anthesis and were harvested during the experiment, with no significant difference in the number of flowers among cultivars ( Table 3 2 ). Low night temperature (LNT) decreased the number of flowers compared with high night temperature ( HNT ) (27 vs 33 flowers respectively). Within each cultivar, temperature reduced the number of flowers only in Ariane and Legionnaire ( P The decrease in flowers harvested at LNT compared wit h HNT was accompanied by a concomitant decrease in number of nodes under LNT ( Table 3 2 ).

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54 All ovary parameters measured were affected by temperature, cultivar, and har vest interval as main effects. Interaction of temperature with cultivar and harvest interval was significant in most cases, except for ovary wall thickness and fresh weight:volume ratio ( Table A 1 Appendix ). For fresh weight and volume, all interactions were significant, whereas for the rest of the variables, one or more interactions were nonsignificant. The covariate days after anthesis (DAA) and the repeating subject component (branch within each plant) were also significant for most of the cases. Ovary FW, volume, diameter, length, and ovary wall thickness were greater in flowers developed under LNT compared with flowers developed under HNT ( Table 3 3 ). However, shape was not affected, as indicated by the length:diameter ratio. There were significant differences among cultivars in ovary size ( Table 3 4 ). Blocky varieties (bell peppers Ariane, Aristotle, Brigadier, and Legionnaire) developed larger ovaries than the cherry (Red Sweet Cherry) or elongated (Banana Supreme) cultivars Within the bell pepper types, Ariane and Aristotle developed smaller ovaries than Legionnaire, possibly due to the delayed growth. Differences in ovary volume and diameter were similar to those in ovary FW; however, length in some blocky cultivars (Aristotle and Brigadier) was less than in the nonblocky cultivars (Banana Supreme and Red Cherry Sweet), resulting in significant differences in the length:diameter ratio (0.70 vs 1.1, respectively). Ovary wall thickness was greater in cherry and elongatedfruited cultivars compared with bell fruited cultivars. Ovary fresh weight and volume of flowers harvested at anthesis remained constant during the first two harvest intervals ( i.e. 16 DAT Brigadier, Legionnaire, Banana Supreme and Red C herry Sweet and 26 days in Ariane and Aristotle see

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55 Table 3 1 for further details) ( Table 3 5 ), which generally corresponded to the first and second nodes harvested ( Figure A 1 Appendix ). Subsequently, ovary FW increased significantly at a rate of ~6 mg per harvest interval, and ovary volume increased at ~7 mm3 per interval. Similar patterns were observed with diameter and length. Ovary wall thickness, however, decreased initially, then increased, but did not reach the initial values. Ovary shape at anthesis (as indicated by the length:diameter ratio) remained constant during the first four harvest intervals, but was more elongated in the final interval. There was a significant interaction between temperature and cultivar on ovary fresh weight, volume, diameter, length and length:diameter ratio ( Table 3 6 ). When comparing the average effect of the night temperature treatment on ovary characteristics, significant differences were detected only in Aristotle and Legionnaire. However, when only the flowers harvested during the last two harvest intervals of the experiment (harvest intervals IV and V ) are considered, significant differences were found in Aristotle, Legionnaire, Brigadier, and Banana Supreme ( Table 3 6 ). The relationship between ovary fresh weight and volume was linear (volume = b ranged from 1.04 to 1.09 in bell peppers, slightly less than 1.00 in Red Cherry Sweet, with intermediate values for Banana Supreme ( Table 3 7 ). Thus, Red Sweet Cherry ovaries were denser than ovaries of bell pepper cultivars. There was a significant threeway interaction (temperature x cultivar x harvest interval) on ovary characteristics ( Table A 1 ). There was no effect of night temperature during the first thr ee weeks of treatment ( i.e. harvest intervals I and II for Aristotle and

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56 Ariane and I to III for Brigadier, Legionnaire, Banana Supreme, and Red Cherry Sweet) in any cultivar ( Figure 3 3 ). Subsequently, however, fresh weight of ovar ies from flowers at anthesis in three out of four bell pepper varieties (Aristotle, Brigadier, and Legionnaire), and in the long fruited var iety (Banana Supreme) increased under LNT compared with HNT. However, in this experiment, ovary FW of Ariane (bell), and Red Cherry Sweet (cherry), did not significantly increase under LNT compared with HNT. In general, three to four weeks of LNT wer e required to significantly increase ovary fresh weight compared to HNT treated plants. In four out of six cultivars, ovary FW did not change under HNT throughout the harvest intervals. Experiment 2 Ovary characteristics of Ariane, Banana Supreme, Leg ionnaire, and Red Cherry Sweet were dependent on night temperature, cultivar, and their interaction ( Table A 2 in Appendix ), but were not dependent on number of l eaves at the node ( i.e. potential source supply differences), as indicated earlier. Fresh weight of ovary, placenta and ovary wall were significantly greater in flowers developed under LNT compared with those developed under HNT ( Table 3 8 ). Ovary diameter and length were 18% and 15% greater, respectively, under LNT compared with HNT, while ovary wall thickness was 10% greater in ovaries developed under LNT. Length:diam eter and ovary wall fresh weight:total fresh weight ratios were similar in both LNT and HNT. The ovaries at anthesis were larger in the bell pepper types (Ariane and Legionnaire) than in the banana or cherry types ( Table 3 9 ). In addition, there were differences in the percent of the total ovary FW that was contributed by the ovary wall In Ariane and Legionnaire, the ovary wall FW represented ~ 50% of the total FW,

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57 while in Banana Supreme and Red Cherry Sweet, the ovary wall represented ~65% of the total FW. There was also a significant correlation between the ovary fresh weight and ovary wall thickness for each genotype (r2 0.63 for all four genotypes; P In general, flowers from the lowest level nodes had the smallest ovaries, with ovary FW increasing steadily from 35 mg in node 2 to 56 mg in node 7 ( Table 3 10), representing ~ a 60% increase. The ovary wall and placenta also increased by ~60%, while the diameter and length increased by 18% and 15%, respectively, as node level increased. Interactions between night temperature and cultivar were significant for ovary, ovary wall and placenta FW, and for ovary diameter, but not for ovary length ( Table A 2 in Appendix ). Over the course of the experiment, plants grown under LNT produced flowers with greater ovary and ovary wall FW compared to plants grown under HNT. The increase in ovary FW under LNT ranged from 50% in Banana Supreme to 76% in Legionnaire ( Table 3 11). Similar results were obtained in ovary wall FW, where increases ranged from 47% (Red Cherry Sweet) to 85% (Legionnaire). However, LNT increased placenta FW only for bell peppers (Ariane and Legionnaire), not for the banana or cherry cultivars. The ovaries from plants grown under LNT increas ed in both diameter and length, except for the long fruited cultivar, which increased only in length. When data from nodes 5 to 7 only are considered, the increase in ovary, ovary wall and placenta FW under LNT is even more dramatic, resulting in greater ovary swelling under LNT compared with HNT. Although ovary size increased as node number increased ( Table 3 10), there were differences among cultivars and temperatur e treatments in this response ( Figur e 3 4 ).

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58 Ovary size increased with node number in all cultivars under LNT, while ovary size increased with node number under HNT onl y in Legionnaire. Ariane and Legionnaire (bell type) responded faster to night temperature treatments than Banana Supreme and Red Cherry Sweet. In Legionnaire, for example, LNT significantly increased the ovary size compared with HNT starting at node 2, which was harvested 8 days after the beginning of treatment (DAT) ( Table A 3 Appendix ). In contrast, in Red Cherry Sweet, significant differences between temperature treatments were not found until node 5 (20 DAT). In Ariane, differences in ovary FW due to night temperature treatments began by node 4 (21 DAT, Table A 3 Appendix ). However, maximum ovary swelling was reached after node 5 for all cultivars (20 DAT in Red Cherry Sweet to 31 DAT in Legionnaire). Although ovary FW increased more than diameter and length at LNT compared with HNT within each cultivar ( Table 3 11), both diameter and length responded as quickly as FW to night temperature treatments ( Table 3 12 ). In fact, in Banana Supreme and Red Cherry Sweet, significant differences in length were found earlier ( i.e. node 4) than were differences in fresh weight and diameter ( i.e. node 5). Dis cussion Most of the studies involving LNT on ovary and fruit development have been on large fruited pepper cultivars, including bell type (Rylski, 1973; Polowick and Sawhney, 1985; Bhatt and SrinivasaRao, 1993b; Mercado et al. 1997b; Mercado et al. 1997c; Pressman et al. 1998a; Pressman et al. 1998b; Aloni et al. 1999; Shaked et al. 2004) Limited work has been done on small fruited sweet pepper cultivars (Kato, 1989) other types of peppers such as cayenne and jalapeno (Shaked et al. 2004) or other species of Capsicum (Mercado et al. 1997c) and these studies focused on pollen development and fertility, but not on the ovary development

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59 In the present work, low night temperature increased ovary diameter and fresh weight in bell pepper flowers, confirming previous results (Polowick and Sawhney, 1985; Pressman et al. 1998b; Aloni et al. 1999) Final ovary diameter of the bell pepper culti vars used in the present experiments ranged from 5.7 to 6.8 mm under LNT, and from 4.3 to 6.4 mm under HNT, and these values are within the ranges reported previously (Pressman et al. 1998b; Aloni et al. 1999; Shaked et al. 2004) Ovary swelling occurred in all bell, long fruited and cherry pepper types under LNT. A previous study (Shaked et al. 2004) reported that night temperatures of 10C increased ovary diameter of sweet bell and hot long fruited cayenne pepper flowers at anthesis compared with ovar ies developed at night temperatures of 20C; however, there was no such effect of LNT on ovary diameter of jalapeno peppers. Our results indicate that under LNT conditions, the increase in ovary fresh weight is proportionately greater than the increase in ovary diameter or length. For example, in Experiment 2, there was a 61% increase in ovary FW and only a 15 to 18% increase in ovary diameter and length under LNT compared with HNT. Most previous studies have measured night temperature effects on ovary diam eter, not fresh weight or length (Polowick and Sawhney, 1985; Aloni et al. 1999) Our results suggest that LNT effects on ovary swelling in peppers, and subsequent effects on fruit malformation, may be better predicted by looking at effects on ovary FW in bell peppers. However, in long fruited cultivars such as Banana Supreme, ovary length may also be a good indicator of ovary swelling, as length increased more rapidly than did FW or volume in response to LNT compared with HNT.

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60 Analysis of continuous har vests over time as a main effect, i.e. harvest intervals in Experiment 1 and nodes in Experiment 2, revealed that flowers harvested at the beginning of the experiments had smaller ovaries than the flowers harvested at the end of the experiments ( Table 3 5 Table 3 10 ). However, when data were analyzed by cultivar and temperature treatment, there were no significant differences in ovary size among harvest intervals ( Figure 3 3 ) or nodes ( Figur e 3 4 ) in ovaries developed under HNT, except for Ariane and Aristotle in Experiment 1 and Legionnaire in Experiment 2. Thus, the increase in ovary size over time (harvest intervals or nodes), as observed in the present work, is primarily due to the effect of LNT. In Experiment 1, Ariane and Aristotle were the two cultivars that suffered delayed growth when transplanted. It is possible that the stress of transplanting reduced the carbohydrate availability in both cultivars for a period of time after transplant, resulting in smaller flowers. Carvalho and Heuvelink (2001) reported that increasing assimilate availability increased flower size in chrysanthemum. In pepper, plants bearing growing fruits developed ov aries with thinner ovary wall compared to the ovaries from plants without fruits, and the authors suggested that assimilate availability limited ovary wall thickness in fruiting plants (Ali and Kelly, 1992) Although ovary size was not recorded by Ali and Kelly (1992) the high correlation between ovary wall thickness and ovary fresh weight in our work (r2 = 0.63 to 0.70, P <0.001; data not shown) suggests a similar limitation in assimilate availability may have occurred for Ariane and Aristotle in Exper iment 1. In general, sweet pepper cultivars developing under LNT required from three to four weeks to develop swollen ovaries. In the main stem of bell pepper, floral initiation occurs between the sixth and the seventh leaf and is independent of temperature (Choi

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61 and Gerber, 1992) At day/night temperatures of 28/15C, it takes ~ 4 weeks from the sixth leaf to anthesis of the first flower (Cruz Huerta, 2001) Thi s suggests that flower buds must be exposed to LNT within the first week after flower bud initiation in order to develop a large percentage of swollen ovaries. The exception to this was observed in Legionnaire bell pepper in Experiment 2, in which signif icant increases in ovary size occurred after ~ one week of LNT compared with HNT. Even so, ~30 days of LNT were required for maximum increases in ovary size to be manifested in Legionnaire. Although a critical period for LNT exposure was revealed in our work ( i.e. within the first week after flower bud initiation), the duration of LNT exposure required for development of swollen ovaries was not examined, as plants were exposed to low or high night temperatures continuously throughout the experiments. It i s clear; however, that the percentage of flowers that exhibit ovary swelling increases as the duration of the LNT exposure increases. This has been demonstrated previously in bell pepper (Aloni et al. 1999) and reflects the continuous floral initiation nature of pepper. Previous work on ovary swelling in pepper focused only on the whole ovary. Our data show that the ovary wall FW: total ovary FW ratio varied among types of cultivars, averaging ~65% for the nonbell pepper cultivars (Banana Supreme and R ed Cherry Sweet) and ~50% for the bell pepper cultivars (Ariane and Legionnaire). However, LNT did not change the ratios, indicating that the increase in ovary FW is proportionally similar for ovary wall The placenta FW, however, was affected by night temperature in bell and cherry type peppers, but not in long fruited. Therefore, increased ovary FW in bell and cherry type peppers caused by LNT was due to increased ovary wall and placenta FW, and in Banana Supreme, was mainly due to ovary wall incre ase.

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62 Although LNT treatments increased ovary size in both experiments, there were differences between experiments, as indicated above. Ovaries in Experiment 1 were larger than in Experiment 2, regardless of cultivar or temperature treatments. Differences i n ovary size between experiments may in part be explained by harvest frequency (every 3 days in Experiment 1 vs every day in Experiment 2), and in part by plant density (13 and 17 plants/m2, for Experiments 1 and 2 respectively). Previous studies have reported that higher plant densities produce smaller fruits (Cebula, 1995; Cruz Huerta et al. 2009) and that smaller fruits are correlated with smaller ovaries (Ali and Kelly, 1992) .Therefore it is likely that flowers developed at higher plant densities had s maller ovaries. Relative response to LNT was also different between Experiments 1 and 2. In Experiment 1, ovary FW and volume were ~10 to 12% greater when developed under LNT compared with those developed under HNT ( Table 3 3 ) and ~20% greater when only the last two harvest intervals were considered ( Table 3 6 ). In Experiment 2, FW of ovaries developed under LNT were ~60% greater than that of ovaries developed under HNT ( Table 3 8 ) or greater when data from nodes 5 to 7 within each cultivar were considered ( Table 3 11 ). The smaller difference in ovary FW between LNT and HNT in Experiment 1 may have been due to longer exposure of ovaries to HNT before harvest, resulting in faster growth, as has been reported by Rylski (1972) As indicated, ovaries in Experiment 1 were harvested every three days, while those in Experiment 2 wer e harvested every day. Thus, ovaries in the HNT treatment in Experiment 1 were exposed to a longer duration of high temperatures, and likely grew faster than did ovaries under LNT, reducing the differences in ovary FW between treatments. There was also a m arkedly slower response to LNT with respect to

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63 increases in ovary FW with time in Experiment 1 compared with Experiment 2, particularly in Legionnaire, Red Cherry Sweet, and Ariane. This may again reflect the larger ovary size and increased time betw een flower harvests in Experiment 1 compared with Experiment 2. Although Aloni et al (1999) reported that a high source:sink ratio also favors ovary swelling in bell peppers, we did not find that in the present work. The difference in leaf number we maint ained (by pruning) may not have been great enough to result in a significant difference in carbohydrate supply to ovaries. Alternatively, the high plant density in Experiment 2 (17 plants/m2) may have resulted in sufficient leaf shading such that increasing the number of leaves in a sympodial unit from four to eight did not increase whole plant photosynthesis and therefore carbohydrate supply. It is unknown, however, if under lower plant densities leaves developed under low night temperature conditions accl imate and reach similar photosynthetic rates as leaves developed under high night temperatures, as has been found in other species (Singh et al. 2005) The most visible response of bell pepper to low night temperatures during the preanthesis stages is def ormed fruits caused by few or no seeds (Rylski, 1986; Pressman et al. 1998a) and the formation of swollen ovaries (Rylski, 1972; Polowick and Sawhney, 1985; Pressman et al. 1998b; Aloni et al. 1999; Shaked et a l. 2004) The absence of seed or reduction in seed number is caused primarily by lack of successful pollination/fertilization (Kato, 1989; Mercado et al. 1997b; Mercado et al. 1997c; Shaked et al. 2004) Shaked et al. (2004) and Pressman et al. (2006) reported that LNT during the 4 days prior to anthesis in bell peppers reduced pollen carbohydrate concentrations, thereby decreasing pollen viability and germinability. This may explain

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64 why in commercial operations even 2 or 3 nights of low temperature causes defor mation of the fruits developed from flowers that were exposed to LNT prior to anthesis (E. Jovicich, personal communication). Conclusions Low night temperature (12C) induced ovary swelling in three types of sweet pepper (cherry, elongated, and blocky bell ), with the greatest response occurring in bell peppers. Three to four weeks of continuous low night temperature were required for maximum response. This timing coincides with the time required for flower bud initiation in pepper and suggests that flowers must be exposed to LNT soon after initiation in order for this response to occur. However, the minimum duration of exposure to LNT required to elicit this response is unknown, as the present research maintained LNT throughout the course of flower development. Decreasing the source supply by leaf removal did not influence ovary size, regardless of the temperature regime. However, plant density may have resulted in plant to plant shading such that differences in whole plant photosynthesis (and therefore sourc e:sink ratios) did not occur. Additional work on night temperature x source:sink ratio is necessary to further test the hypothesis that both factors interact to affect ovary swelling in sweet pepper.

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65 Table 3 1 Relationship between days after treatment and harvest interval defined for statistical analysis in two groups of sweet pepper cultivars. Days after treatment z Harvest interval Group 1 Group 2 I 1 8 1 13 II 9 16 14 26 III 17 24 27 39 IV 25 32 40 52 V 33 40 53 65 z Group 1: Brigadier, Legionnaire, Banana Supreme, and Red Cherry Sweet; Group 2: Ariane and Aristotle. Table 3 2 Number of flowers harvested at anthesis and number of nodes bearing flowers in six cultivars of sweet pepper grown at 22/20C or 22/12C day/night temperatures. Number of flowers/plant z Number of nodes Night temperature (C) Night temperature (C) Cultivar 12 20 P values y Mean x 12 20 P values Mean Ariane 25.0 33.3 0.04 29.2 a w 6.3 8.7 0.01 7.5 ab Aristotle 31.3 36.0 0.29 33.7 a 8.5 9.7 0.16 9.1 a Brigadier 26.7 28.0 0.82 27.3 a 6.8 7.2 0.69 7.0 b Legionnaire 22.0 30.3 0.04 26.2 a 5.5 8.0 <0.01 6.8 b Banana Supreme 30.0 37.3 0.12 33.7 a 7.3 8.8 0.08 8.1 ab Red Cherry Sweet 27.3 34.0 0.13 30.7 a 6.2 8.3 0.01 7.3 b Mean v 27.1 33.2 <0.01 30.1 6.8 8.4 <0.01 7.6 z All f lowers were continuously harvested at anthesis during a 39day period for Brigadier, Legionnaire, Banana Supreme and Red Cherry Sweet and a 64day period for Ariane and Aristotle. y P values were generated from the temperature treatment data within each cultivar; n=3 for flower number per plant and 6 for node number. x Means were averaged for each cultivar across both temperature treatments; n=6 for flower number per plant and 12 for node number. w Means with the same letter in the same column are not significantly different (Tukey Kramer, P v Means averaged across all cultivars; n=18 for flower number per plant and 36 for node number.

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66 Table 3 3 Main effect of night temperature during the preanthesis stage on ovary characteristics of sweet pepper flowers measured at anthesis Temperature treatments y Parameter z 22/12C 22/20C P value Fresh weight (mg) 84 x 76 0.008 Volume (mm 3 ) 88 79 0.002 Diameter (mm) 5.5 5.3 0.003 Length (mm) 4.3 4.2 <0.001 Length:diameter ratio 0.82 0.82 0.59 Ovary wall thickness (mm) 0.59 0.57 0.004 z Flowers were continuously harvested at anthesis during a 39day period for Brigadier, Legionnaire, Banana Supreme and Red Cherry Sweet and a 64day period for Ariane and Aristotle. y Day/night temperatures. x Means were averaged for each temperature treatment across all cultivars and harves t intervals; n= 597 and 487 for 22/20C and 22/12C, respectively. Table 3 4 Main effect of cultivar on characteristics of ovaries at flower anthesis in sweet pepper. Cultivar z Ovary FW (mg) Volume (mm 3 ) Diameter (mm) Length (mm) Length: Diameter ratio Ovary wall thickness (mm) Ariane 80 c y,x 86 c 5.6 c 4.1 c 0.74 b 0.56 b Aristotle 91 bc 96 bc 5.9 bc 3.9 d 0.66 c 0.55 b Brigadier 101 ab 107 ab 6.1 ab 4.2 bc 0.68 c 0.56 b Legionnaire 112 a 117 a 6.4 a 4.4 a 0.69 c 0.56 b Banana Supreme 48 d 49 d 4.1 d 4.5 a 1.08 a 0.61 a Red Cherry Sweet 48 d 46 d 4.1 d 4.4 ab 1.07 a 0.62 a z Flowers were continuously harvested at anthesis during a 39day period for Brigadier, Legionnaire, Banana Supreme and Red Cherry Sweet and a 64day period for Ariane and Aristotle. y Means were averaged for each cultivar across both temperatur e treatments and all harvest intervals. For fresh weight, n = 173, 200, 158, 152, 192, and 171 for Ariane, Aristotle, Brigadier, Legionnaire, Banana Supreme, and Red Cherry Sweet, respectively. For the remaining variables, n was equal to or greater than it was for FW. x Means with the same letter in the same column are not significantly different (Tukey Kramer, P

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67 Table 3 5 Main effect of harvest interval on ovary characteristics of six cultivars of sweet pepper. Harvest interval z y Fresh weight (mg) Volume (mm3) Diameter (mm) Length (mm) Length:diameter ratio Ovary wall thickness (mm) I 70 d x, w 74 d 5.2 c 4.1 b 0.84 a 0.60 a II 73 d 76 d 5.3 c 4.2 b 0.82 a 0.58 b III 79 c 82 c 5.3 bc 4.3 ab 0.84 a 0.56 c IV 86 b 89 b 5.4 b 4.3 a 0.82 a 0.56 c V 92 a 97 a 5.7 a 4.3 a 0.79 b 0.58 b z Flowers were continuously harvested at anthesis during a 39day period for Brigadier, Legionnaire, Banana Supreme and Red Cherry Sweet and a 64 day period for Ariane and Aristotle. y Harvest intervals were either 8 (39 days total) or 13 (64 days total) days, depending on cultivar (see details on Table 3 1 ) x Means were averaged for each harvest interval across cultivar and temperature treatment; n = 160, 207, 211, 233, and 273 for intervals 1 to 5, respectively. For fresh weight, volume, FW:volume ratio, and ovary wall thickness in interval 1, n = 122. w Means with the same letter in the same column are not significantly different (Tukey Kramer, P

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68 Table 3 6 Interaction of night temperature and cultivar on ovary characteristics of sweet pepper flower s harvested at anthesis during the duration of the experiment or only during the last two harvest intervals. Fresh weight (mg) Volume (mm 3 ) Diameter (mm) Length (mm) Night temperature (C) Cultivar z y 12 20 P value x 12 20 P value 12 20 P value 12 20 P value All harvest intervals w ARN 75 86 0.10 81 91 0.17 5.4 5.7 0.10 4.1 4.1 0.74 ATS 101 80 0.003 108 84 0.002 6.2 5.6 0.002 4.0 3.8 0.11 BRI 1 04 97 0.30 112 101 0.14 6.3 6.0 0.14 4.2 4.1 0.48 LEG 119 105 0.05 126 109 0.03 6.6 6.2 0.07 4.5 4.3 0.09 BAS 5 4 42 0.10 55 42 0.10 4.3 4.0 0.18 4.7 4.2 <0.001 RCS 5 0 46 0.57 48 44 0.57 4.2 4.0 0.24 4.4 4.4 0.72 H arvest intervals IV and V v ARN 87 97 0.22 94 103 0.34 5.7 6.0 0.15 4.2 4.2 1.00 ATS 116 79 <0.001 125 83 <0.001 6.5 5.5 <0.001 4.0 3.8 0.18 BRI 1 22 97 0.01 132 102 0.004 6.6 6.0 0.01 4.4 4.1 0.03 LEG 1 33 112 0.02 141 117 0.02 6.8 6.4 0.12 4.6 4.3 0.01 BAS 6 5 46 0.03 68 46 0.03 4.5 4.1 0.07 5.1 4.2 <0.001 RCS 6 1 52 0.34 57 50 0.46 4.5 4.2 0.17 4.6 4.4 0.10 z Flowers were continuously harvested at anthesis during a 39day period for BRI, LEG, BAS and RCS and a 64day period for ARN and ATS. y ARN=Ariane; ATS = Aristotle; BRI =Brigadier; LEG = Legionnaire; BAS = Banana Supreme; RCS = Red Cherry Sw eet. x P values were generated from the temperature treatment data within each cultivar. w Means were averaged for each cultivar across all harvest intervals; n = 75 to 107, depending on temperature treatment and cultivar. v Last two harvest intervals corresponded to 25 to 39 days after treatments began for BRI, LEG, BAS and RCS or 40 to 64 days after treatments began for ARN and ARS; n = 30 to 64, depending on temperature treatment and cultivar. Table 3 7 Coefficients of conversion to estimate ovary volume at anthesis stage as a function of its fresh weight in six cultivars of sweet pepper grown under two night temperatures Volume (mm3) = b 2>0.99) Temperature tr eatment (C) Cultivar 22/12 22/20 Average Ariane 1.09 1.07 1.08 Aristotle 1.07 1.05 1.06 Brigadier 1.08 1.05 1.07 Legionnaire 1.06 1.04 1.05 Red Cherry Sweet 0.97 0.96 0.96 Banana Supreme 1.05 1.00 1.03 Average 1.07 1.04 1.06

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69 Table 3 8 Main effect of night temperature during the preanthesis stage on ovary characteristics of sweet pepper flowers at anthesis Night temperature z Ovary FW (mg) Ovary wall FW (mg) Placenta FW (mg) Ovary wal l FW:total FW Diameter (mm) Length (mm) Length: diameter ratio Ovary wall thickness (mm) 12C 58** y,x 30** 27* 0.56* 4.5* 3.8* 0.91 0.51 20C 36 19 17 0.55 3.8 3.3 0.90 0.46 z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7. y *, ** Means are significantly different (t test) at P P x Means were averaged across four cultivars and six nodes; n = 514 for 12C and 401 for 20C. Table 3 9 Main effect of cultivar on ovary characteristics of sweet pepper flowers at anthesis Cultivar z y Ovary FW (mg) Ovary wall FW (mg) Placenta FW (mg) Ovary wall FW:total FW Diameter (mm) Length (mm) Length: diameter ratio Ovary wall thickness (mm) ARN 61 a x, w 28 a 32 a 0.47 c 4.9 a 3.5 b 0.70 c 0.47 b LEG 62 a 29 a 33 a 0.46 c 4.9 a 3.4 b 0.69 c 0.46 b BAS 33 b 22 b 11 b 0.65 a 3.4 b 3.9 a 1.15 a 0.50 a RCS 32 b 20 b 12 b 0.63 b 3.3 b 3.5 b 1.06 b 0.52 a z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7. y ARN =Ariane; LEG = Legionnaire; BAS = Banana Supreme; RCS = Red Cherry Sweet x Means were averaged for each cultivar across both temperature treatments and nodes. w Means with the same letter in the same column are not significantly different (Tukey Kramer, P Supr eme, and Red Cherry Sweet, respectively.

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70 Table 3 10. Main effect of node on ovary characteristics of sweet pepper flowers at anthesis Node z y Ovary FW (mg) Ovary wall FW (mg) Placenta FW (mg) Ovary wall FW:total FW Diameter (mm) Length (mm) Length: diameter ratio Ovary wall thickness (mm) 2 35 e x ,w 18 e 16 d 0.55 bc 3.8 e 3.3 c 0.91 a 0.46 c 3 39 d 22 d 17 d 0.58 a 3.9 d 3.3 c 0.90 a 0.48 bc 4 46 c 24 c 21 c 0.56 ab 4.1 c 3.5 b 0.91 a 0.48 ab 5 51 b 26 b 25 b 0.54 c 4.2 b 3.7 a 0.91 a 0.50 a 6 54 ab 28 ab 26 ab 0.54 c 4.4 ab 3.7 a 0.90 a 0.50 a 7 56 a 29 a 27 a 0.54 bc 4.5 a 3.8 a 0.90 a 0.51 a z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7. y Days after treatment at which nodes were harvested: 2: 29; 3: 617; 4: 1126; 5: 16 31; 6: 2238; 7: 2842, depending on the cultivar. x Means were averaged across all four cultivars and two temperature treatments; n = 179, 154, 154, 161, 148, 119 for nodes 2, 3, 4, 5, 6, and 7, respectively. w Means with the same letter in the same column are not significantly different (Tukey Kramer, P Table 3 11. Interaction of night temperature and cultivar on ovary character istics of sweet pepper flowers harvested at anthesis. Ovary FW (mg) Ovary wall FW (mg) Placenta FW (mg) Diameter (mm) Length (mm) Night temperatures (C) CV z y 12 20 P value 12 20 P value 12 20 P value 12 20 P value 12 20 P value Nodes 2 7 ARN 74 x 47 <0.001 w 35 22 <0.001 39 26 <0.001 5.4 4.5 <0.001 3.7 3.2 <0.001 LEG 79 45 <0.001 37 20 <0.001 42 25 <0.001 5.4 4.3 <0.001 3.6 3.1 <0.001 BAS 39 26 0.04 26 17 0.01 13 9 0.21 3.5 3.2 0.13 4.2 3.5 <0.001 RCS 38 25 0.04 23 16 0.03 15 9 0.06 3.6 3.1 0.007 3.8 3.3 <0.001 Nodes 5 7 ARN 88 v 52 <0.001 40 22 <0.001 48 29 <0.001 5.8 4.6 <0.001 4.0 3.3 <0.001 LEG 93 49 <0.001 43 21 <0.001 50 28 <0.001 5.8 4.5 <0.001 3.8 3.1 <0.001 BAS 45 27 0.01 30 18 0.01 15 9 0.09 3.7 3.2 0.04 4.5 3.6 <0.001 RCS 48 25 <0.001 29 16 <0.001 19 9 0.01 4.0 3.1 <0.001 4.1 3.3 <0.001 z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7. yCV=cultivars. ARN: Ariane, LEG: Legionnaire, BAS: Banana Supreme, RCS: Red Cherry Sweet. x Means were averaged for each cultivar across all nodes harvested; n = 67 to 134, depending on temperature treatment and cultivar. w P values were generated from the temperature treatment data within each cultivar. v Means were averaged for each cultivar across nodes 5, 6 and 7; n = 23 to 68, depending on temperature treatment and cultivar.

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71 Table 3 12. P values of t test comparison between night temperatures of 12 and 20C within each cultivar and node for ovary fresh weight (FW), diameter (Diam.), and length. Cultivar z, y ARN LEG BAS RCS Node FW Diam. Length FW Diam. Length FW Diam. Length FW Diam. Length 2 0.86 x 0.97 0.42 0.04 0.02 0.04 0.49 0.58 0.04 0.76 0.61 0.98 3 0.15 0.04 0.22 <0.001 <0.001 0.03 0.06 0.15 <0.001 0.76 0.97 0.23 4 <0.001 <0.001 <0.001 <0.001 <0.001 0.004 0.10 0.10 0.002 0.23 0.13 0.003 5 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.08 0.08 <0.001 0.004 <0.001 <0.001 6 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.01 0.14 <0.001 <0.001 <0.001 <0.001 7 <0.001 <0.001 0.002 <0.001 <0.001 0.007 0.08 0.51 <0.001 <0.001 <0.001 <0.001 z Flowers were continuously harvested at anthesis for up to 45 days from nodes 2 to 7. y ARN =Ariane; LEG = Legionnaire; BAS = Banana Supreme; RCS = Red Cherry Sweet x In all cases, P values 0.05 mean that ovaries developed at 12C had significantly greater fresh weight, diameter or length than the ovaries developed at 20C.

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72 Figure 3 1 Schematic drawing of pepper branching and flowering sequence. A) Meristematic stage. B) Well developed branch. The sympodial shoot bears a leaf (L1, blue), and terminates in a flower (F2). From the leaf axil, two leaves (L2) develop. The flower and the t wo leaves developed from the same sympodial shoot compose a sympodial unit (F2 and L2, green). L2 leaves are pushed up by internode elongation, so that the leaves appear above the preceding flower. The shoot terminates in a flower, which is part of the subsequent sympodial unit. From each of the leaf 2 axils, two new leaves (L3, red) develop. Each color represents sympodial units of a different order (Drawing A based on micrograph from Elitzur et al. 2009) L2 F2 L2L3 L3 L1 L2 F2 L2 L3 L3 L1 A B

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73 Figure 3 2 Schematic drawing of a sweet pepper plant pruned to two main axes with lateral branches. Two shoots were allowed to grow at the first branching to form the mainstem sympodial unit (SU, brown). A) Starting in SU1 (blue), the strongest shoot was allowed to grow and the second shoot was limited to development of one sympodial unit (one flower and two leaves) by pruning. This pruning system was used in all the plants in Experiment 1 and half of the plants in Experiment 2. B) The strongest shoot was allowed to grow and the second shoot was pruned so that only the flower of the next sympodial unit remained. This system was used in half of the plants in Experiment 2. In both A and B, flowers from the main stem and first SU were rem oved before treatment started. SU1= blue; SU2 = green; SU3= red, SU4= white. A B

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74 Days after treatment Figure 3 3 Ovary fresh weight of sweet pepper flowers at anthesis developed under 22/12C ( ----) or 22/20C ( intervals. Plants did not bear any fruit before and during the experiment Means with the same letter in the same temperature treatment and cultivar are not significantly different (Tukey Kramer, P between temperature treatments within a harvest interval for each cultivar are significantly different at P P P n values for harvest intervals: 1st : 7 20; 2nd : 11 25; 3rd : 1 2 23; 4th : 12 29; 5th : 14 35. bc c bc,** b,*** a,*** ab ab ab b a 1 13 14 26 27 39 40 52 53 64 'Aristotle b b b a,* a,*** a a a a a 30 50 70 90 110 130 Fresh weight (mg) 'Brigadier' b b ab ab,* a,*** a a a a a 'Legionnaire' b b ab ab,* a,** a a a a a 30 50 70 90 110 130 1 8 9 16 17 24 25 32 33 39 Fresh weight (mg) 'Banana Supreme' b ab ab ab a a a a a a 1 8 9 16 17 24 25 32 33 39 'Red Cherry Sweet' b b ab a a b b ab ab, a 30 50 70 90 110 130 1 13 14 26 27 39 40 52 53 64 Fresh weight (mg) 'Ariane'

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75 Node above first branching Figur e 3 4 Ovary fresh weight of sweet pepper flowers harvested at anthesis from the second to the seventh nodes above the first flowering node in plants grown at day/night temperatures of 22/12C ( ----) or 22/20C ( bear any fruit. Means with the same letter in the same temperature treatment and cultivar are not significantly different (Tukey Kramer, P Means b etween temperature treatments within a cultivar and node are significantly different at P P P n values for the temperature treatments: 22/12C: 1523; 22/20C: 8 24, excluding Banana Supreme node 2, where n=2. Days after treatment at which nodes were harvested: 2: 29; 3: 6 17; 4: 1126; 5: 1631; 6: 22 38; 7: 2842, depending on the cultivar. c c b,*** ab,*** a, *** ab, *** a a a a a a 0 10 20 30 40 50 60 70 80 90 100 Fresh weight (mg) 'Ariane' b ab ab a a,** a a a a a a a 'Banana Supreme' d,* c,*** b,*** ab,*** a,*** a,*** b ab ab ab ab a 0 10 20 30 40 50 60 70 80 90 100 2 3 4 5 6 7 Fresh weight (mg) 'Legionnaire' c c bc ab,** a,*** a,*** a a a a a a 2 3 4 5 6 7 'Red Cherry Sweet'

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76 CHAPTER 4 4. LOW NIGHT TEMPERATURE AFFECTS CARBON EXCHANGE RATES OF SWEET PEPPER LEAVES Introduction Pepper ( Capsicu m annuum L) is an herbaceous species that belongs to the family Solanaceae (Eshbaugh, 1993) and its fruit is one of the most consumed vegetables worldwide (Mateos et al. 2003) Pepper fruits, especially sweet peppers, are an excellent source of essential nutrients, especially vitamin C, carotene, calcium and antioxidant compounds (LopezHernandez et al. 1996; Mateos et al. 2003; Sun et al. 2007; Mateos et al. 2009) Cultivated species of peppers were domesticated in tropical and temperate areas in Mexico, Central America and South America (Bolivia), and are sensitive to low temperatures (Harlan, 1971; Eshbaugh, 1993) Low temperature is one of the major factors limiting the productivity and geographical distribution of many plant species (Li et al. 2 003a) Low temperature effects on physiology and growth have been studied in several agronomic and horticultural crops (Koscielniak, 1993; Gibson and Mullen, 1996; Boese et al. 1997; Wang et al. 1997; Roussopoulos et al. 1998; Sukhvibal et al. 1999; Venema et al. 1999a; Adams et al. 2001; Hendrickson et al. 2004a; Hendrickson et al. 2004b; Singh et al. 2005; Limin and Fowler, 2006) including pepper (Mercado et al. 1997b; Pressman et al. 1998b; Aloni et al. 1999; Li et al. 2004; Pressman et a l. 2006) One of the more striking effects of low temperature, particularly low night temperature, on pepper growth is an increase in swollen floral ovaries and subsequent deformed fruit (Polowick and Sawhney, 1985; Rylski, 1985; Mercado et al. 1997b; Pr essman et al. 1998a; Aloni et al. 1999; Shaked et al. 2004)

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77 Among other physiological effects, low temperatures reduce photosynthetic rates. Day temperatures of 67C may cause rapid stomatal closure in a variety of plant species (Boese et al. 1997; W ilkinson et al. 2001) and subsequent photoinhibition (Liu et al. 2001; Li et al. 2003a; Li et al. 2004; Zhang and Scheller, 2004) Photosynthetic enzyme activity may also be reduced under low temperatures (KingstonSmith et al. 1997) On the other hand, low night temperatures ( i.e. below 1015C) delay the start and slow the rate of stomatal opening the following morning, as temperatures increase (Hendrickson et al. 2004a; Hendrickson et al. 2004b) Night temperatures below 15C may also decrease photosynthesis by decreasing ribulose1,5 bisphosphate regeneration and Pi availability for recycling (Hendrickson et al. 2004a; Hendrickson et al. 2004b) reducing activity of PSII (Sundar and Reddy, 2000; Bertamini et al. 2005) and decreasing leaf chlorophyll content (Sundar and Reddy, 2000; Bertamini et al. 2005) Reduction in Rubisco amount and activity, as well as other photosynthetic/carbohydrate metabolizing enzymes, such as fructose bisphosphatase and sucrose phosphate synthas e, may also occur in response to low night temperature (Sundar and Reddy, 2000; Ramalho et al. 2003; Hendrickson et al. 2004a; Hendrickson et al. 2004b; Bertamini et al. 2005) Low night temperature (5C) followed by high irradiance (1900 PPF) also dec reases photosynthesis by increasing photoinhibition (Bertamini et al. 2006) Tropical crops, including pepper, are especially sensitive to low night temperature, and one cold night of 4 to 12C may decrease photosynthetic rates 5 to 80% (Van deDijk, 1985 ; Van deDijk and Maris, 1985; Wolfe, 1991; Janssen et al. 1992; Boese et

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78 al. 1997; Ying et al. 2000, and references therein; Li et al. 2003a; Ramalho et al. 2003; Wang et al. 2004) Bhatt and SrinivasaRao (1993b) reported that net CO2 exchange rate s in sweet pepper were 6 to 30% higher at night temperatures of 22C compared with 17C. Some species have the capacity to acclimate to low temperatures. In such cases, exposure to low temperatures for several days may markedly increase the maximum activit ies of Rubisco, stromal and cytosolic fructose1,6 bisphosphatase, and sucrosephosphate synthase in leaves, RuBP regeneration and carboxylation, and lead to significant increases in whole plant photosynthetic capacity (Holaday et al. 1992; Hurry et al. 1994; Du et al. 1999; Yamori et al. 2005) In pepper, plants that were cold acclimated (5 days at 14C and 250 PPF) exhibited greater photosynthetic rates compared to nonacclimated plants upon subsequent exposure to low temperatures (4C, 1200 PPF) (Liu et al. 2001) Respiration rates are also affected by low temperature, with rates in both the light and the dark increasing as temperature increases up to about 40C (Li n and Markhart, 1990; Taiz and Zeiger, 2006) In the short term, respiration is highly responsive to ambient temperatures; however, in the long term, temperature acclimation can occur. Tjoelker et al. (1999) reported that respiration in five species of high temperature acclimated plants (30/24C day/night) was lower than that of low temperature acclimated plants (18/12C day/night) when measured at temperatures between 12 and 30C. Net carbon exchange rates (CER) and ovary sw elling in pepper are affected not only by temperature, but also by source:sink ratios. Net CER increased in newly

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79 developed leaves of fruiting compared with nonfruiting pepper (Cruz Huerta et al. 2005) In bell pepper, fruiting decreased vegetative growt h rates compared to nonfruiting plants (Hall and Brady, 1977; Hall and Milthorpe, 1978; Bhatt and SrinivasaRao, 1989) since growing fruits represent up to 90% of sink demand (Hall, 1977) Defruiting, which rapidly increases the source:sink ratio, increased the incidence of flower deformation and swollen ovaries in pepper (Aloni et al. 1999) The incidence of swollen ovaries in bell pepper due to low night temperature or high source:sink ratio has been correlated with increased ovary carbohydrate concentration in the flower bud (Aloni et al. 1999) Under low night temperature, pepper plants exhibit slower growth rates, resulting in decreased shoot dry weight compared with plants grown under higher night temperatures (Mercado et al. 1997a) In som e species, leaves developed under low night temperature may acclimate to such conditions, resulting in carbon exchange rates as high as in plants growing under warmer temperatures (Singh et al. 2005) Therefore, similar photosynthetic rates and slower gro wth rates under low night temperatures may cause excess carbohydrate accumulation in floral ovaries, resulting in swelling and deformed fruits. Similarly, developing fruits in bell pepper are a strong sink, and defruiting pepper plants increases starch con centration in stems (Hall and Milthorpe, 1978) which may also result in an excess carbohydrate supply to floral ovaries. The hypothesis tested in the present experiments is that sweet pepper leaves developed under low night temperatures acclimate to such conditions and have similar carbon exchange rates as leaves developed under high night temperatures. This, combined with reduced growth under low night temperatures, increases the incidence of swollen or deformed ovaries in

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80 pepper flowers. A second hypothesis tested is that fruiting decreases the incidence of swollen ovaries, regardless of night temperature. The objectives were to 1) measure net CER, growth, and ovary swelling in two cultivars of pepper grown under low and high night temperatures and 2) det ermine fruiting effects on net CER, growth, and ovary swelling in plants grown under two night temperatures. Material and Methods Plant Material and Growing Conditions The experiments were carried out using two types of sweet pepper with contrasting fruit size and fruit shape Legionnaire bell pepper (Experiments 1, 2, and 3) and Red Cherry Sweet cherry pepper (Experiment 1). Seeds were germinated in a mix of peat moss, vermiculite, and dolomite limestone (SunGro MetroMix Ag Lite Mix2Plants were fertigated as needed with the following nutrient solution (mM): 3.4 Ca(NO3)2, 1.8 KNO3, 1.6 KH2PO4, 0.3 KCl, 2.7 MgSO4, and (M): 50.2 FeEDTA, 3.1 CuSO4, 14.6 MnSO4, 64.8 H3BO3, 4.6 ZnSO4, 0.6 Na2MoO4 (modified from Cruz Huerta et al. 2005; and Jovicich, 2007) Pests were controlled as described in Chapter 3. ) and grown in 1. 4 m2 growth chambers (Mod E15, Conviron, Winnipeg, Canada) Chamber temperature was set to 22/20C (day/night), and PPF was maintained between 450500 mol m2 s1 above the canopy with a 14hour photoperiod, as described in Chapter 3. Seedlings at about t he sixth leaf stage (~ 50 days after sowing) were transplanted to 1.5 L containers, using the same growing media for germination, and maintained in the growth chambers throughout the experiments. 2 Composition: Canadian Sphagnum peat moss (5565%), horticultural grade vermiculite, dolomite limestone, starter nutrient charge, and wetting agents ( www.sungro.com/products.php, 12/17/2008).

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81 Plants were pruned to two main axes, with one lat eral sympodial unit (one flower and two leaves) on every node in the main axis, as described in Chapter 3 ( Figure 3 2 A). Two experiments were conducted to measure net CER and biomass accumulation under low (12C) vs high (20C) night temperatures (Experiments 1 and 2) and different fruit loads (Experiment 2). Experiment 1 Eighteen seedlings each of Legionnaire and Red Cherry Sweet were selected when flowers in nodes 3 or 4 were at anthesis (~20 days after transpl anting). Growth chamber temperatures were set to 22C day, with night temperatures of either 20 (high night temperature, HNT) or 12C (low night temperature, LNT). Each growth chamber was divided into three blocks to reduce PPF variation and three plants of each cultivar were randomly placed within each block. All the flowers were removed; therefore plants did not bear any fruit. Gas exchange measurements were performed using a portable photosynthesis system (LI 6400, Licor, Lincoln, NE, USA) on the most r ecent mature leaf, which was the fourth or fifth from the apex. Measurements included net CER, stomatal conductance (gs), intercellular CO2 concentration (Ci), and leaf dark respiration (RD, day and night). Net CER measurements were performed about six hours after lights were on and were done every three days during a 36day period after treatments started, with a final measurement at 45 days. This period of time ensured that several new leaves in each axis completely developed under the temperature treatme nts. Based on data reported by Choi and Gerber (1992) and Cruz Huerta (2001) sweet pepper leaves take four to five weeks from the beginning of formation until full expansion. Respiration measurements during the day were performed 60 to 90 seconds after net CER

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82 measurements were completed, by covering the top of the assimilation chamber with an opaque card, and during the night before the dark period ended. Respiration measurements during the day were performed only twice at the beginning of the experiment (1 and 3 days after treatment, DAT) since no difference among treatments was found. Night respiration rates were measured at 13, 27 and 47 DAT. Fresh weight of flowers and flower parts (ovary, calyx, and petals) at anthesis and ovary diameter and length were recorded on flowers harvested from 30 to 37 DAT. At the end o f the experiment, leaf area, dry weight per organ, plant height, and number of internodes were also recorded. Experiment 2 Twenty four Legionnaire seedlings were selected ~30 days after transplanting and flower buds from the mainstem sympodial unit (first bifurcation) and sympodial unit 1 (node 1) were removed. Flowers on nodes 2 and 3 were handpollinated, allowing one fruit per axis (2 fruits per plant) to develop beyond petal fall, the indicator of fruit set. About ten days after petal fall, when the fruits were rapidly growing (Marcelis and BaanHofman Eijer, 1995b) night temperature (HNT vs LNT) and source:sink ratio treatments began. Fruits were either allowed to grow or were removed i n order to vary the source:sink ratio, resulting in a 2 x 2 factorial (two night temperatures and two source:sink ratios) with six replications. As in Experiment 1, net CER measurements were performed using a portable LI 6400 photosynthesis system. Measurements were done every three to four days during a 41day period on one to three of the most recently matured leaves per plant. Photosynthesis was measured about six hours after lights were on. Measurement on day 19 was not considered in the final analysis since plants were under water stress

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83 when measured. Fresh weight of flowers and flower parts at anthesis and ovary diameter and length were recorded on flowers harvested from 30 to 36 DAT. Organ dry weight, plant leaf area, and plant height were recorded at end of the experiment. Data Analysis Data from all experiments were analyzed as a completely randomized block design, with repeated measures, except for the dry weight in Experiments 1 and 2. The subject for repeated measures was the individual plant. Co mparisons performed were night temperatures, days after beginning of treatment (Experiments 1 and 2), cultivars (Experiment 1), and source:sink ratio (Experiment 2). Data from the two measurement dates for day respiration were pooled and analyzed as a 2x2 factorial (night temperature x cultivars). Night respiration data were analyzed as a factorial (night temperature x cultivars x measurement dates) with repeated measures over time. Results Experiment 1 Low night temperature increased flower, flower parts, and ovary fresh weight and ovary size, compared to HNT ( Table 4 1 ). Flower fresh weight in the LNT was 32% greater than in the HNT. As expected, bell pepper had larger flowers and ovaries than cherry type pepper. The interaction between cultivar and night temperature was significant for total flower fresh weight, calyx fresh weight, and ovary diameter and length. After four weeks of night temperature treatments, ovaries of Red Cherry Sweet exhibited a proportionately greater increase in size under LNT compared with HNT than did ovaries of Legionnaire. However, we selected Legionnaire for the subsequent experiments due to the more rapid response of this cult ivar to LNT compared to the slower initial response in Red Cherry Sweet (Chapter 3).

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84 Carbon exchange rate was significantly influenced by the cultivar, DAT, and interactions between DAT x cultivar and DAT x temperature ( Table 4 2 ). Stomatal conductance was influenced by DAT and interactions where DAT was a factor, while Ci was influenced only by DAT and the interaction of DAT x cultivar. Net CER was significantly higher in Red Cherry Sweet than Legionnaire over the course of the experiment ( Table 4 3 ); however, night temperature had no effect on net CER, stomatal conductance, or intercellular CO2. Regardless of cultivar, CER decreased over time ( Figure 4 1 A). There were two periods where CER decreased rapidly; the first occurring 6 to 9 DAT and the second occurring 24 to 27 DAT. Stomatal conductance followed a similar pattern over time ( i.e. DAT) as net CER, although with more pronounced decreases ( Figure 4 1 B). The intercellular CO2 concentration (Ci) also decreased with time. Ci decreased rapidly from 287 to 196 mol mol1 during the first 6 DAT. After the initial decrease, Ci increased to reach a peak at 19 DAT, followed by a second peak at 30 DAT, before finally decreasing to ~220 mol mol1 after 33 DAT ( Figure 4 1 C). Although the main effect of night temperature on net CER was not significant ( Table 4 3 ); there was a significant interaction of DA T x temperature on net CER ( Figure 4 2 A). Three days after the beginning of the experiment, plants under LNT had significantly greater CER than those under HNT. After that, however, plants under HNT had either similar or significantly greater CER than those under LNT. Differences in net CER between cultivars were found between 15 and 21 DAT and at the end of the experiment (between 36 and 45 DAT), with Red Cherry Sweet exhibiting greater CER than Legionnaire ( Figure 4 2 B).

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85 The interaction between DAT x night temperature on stomatal conductance ( Figure 4 3 A) was similar to that observed for net CER. Stomatal conductance was higher under LNT compared with HNT the first three days after treatments began, but were significantly lower between 21 and 33 DAT. Significant differences in stomatal conductance between cultivars were found only at 15 and 19 DAT ( Figure 4 3 B). The initial decrease in gs, which was similar in both cultivars, was followed by a transient increase, with a greater increase observed in Legionnaire compared with Red Cherry Sweet ( Figure 4 3 B). The increase in gs in Red Cherry Sweet correlated with the increase observed in CER ( Figure 4 2 B); however, no such correlation was observed in Legionnaire. Internal CO2 concentrations were significantly higher under LNT compared with HNT during the first 6 days of treatment and significantly lower from day 19 to day 24 ( Table A 4 Appendix ), which corresponded with patterns observed in both CER ( Figure 4 2 A) and gs ( Figure 4 3 A). Although the interaction between DAT and cultivar was significant for Ci, differences between cultivars were observed only at 6 DAT, where Ci in Legionnaire was significantly greater than in Red Cherry Sweet ( Table A 4 Appendix ). During the day, leaf respiration was not affected by night temperature (P=0.71), but was affected by cultivar (P 0. 04, Figure 4 4 A, B). No interaction between night temperature and cultivar was found (P=0.14). During the night, leaf respiration was significantly reduced by LNT co mpared to HNT ( P 0.001). Night leaf respiration was lower in Legionnaire bell pepper compared to Red Cherry Sweet. Night respiration was affected by DAT, increasing from 0.67 mol m2 s1 at 13 DAT to 0.85 mol m2 s1 at

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86 47 DAT ( P 0.008). There were no significant interactions among night temperature, cultivar and DAT on night respiration rate (data not shown). Assuming constant CER during the day and uniform respiration rates during the night, carbon balance for leaves was calculated ( Table 4 4 ). At the end of the experiment, sweet pepper plants under LNT gained less carbon during the day, and although they respired less during the night, LNT plants gained less carbon than did HNT plants during a 24h period. There were no significant effects of cultivar or the interaction of night temperature x cultivar. LNT reduced leaf number and specific leaf area compared to HNT, but there was no significant difference in total leaf area ( Table 4 5 ). No significant temperature effects were found in plant or organ dry weight. Plants growing under LNT, however, were shorter due to shorter internodes and had smaller main stem diameter compared to plants growing under HNT. There were significant differences in leaf number, leaf area, dry weight, and plant height between cultivars, but there were no significant interactions between night temperature and cultivar. Experiment 2 Both LNT and reduced sink demand (i.e, nonfruiting plants) increased fresh weight of the flower, flower parts, and the ovary diameter compared to HNT ( Table 4 6 ). Ovary length was affected only by night temperature. Both LNT and the absence of fruit significantly decreased CER and gs in Legionnaire bell pepper (Tables 4 7 and 4 8 ). Intercellular CO2 concentration was decreased under LNT, but was not affect ed by the presence or absence of fruit.

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87 In general, CER, gs, and Ci decreased over time ( i.e. DAT). A dramatic decrease in CER (26%), gs (58%), and Ci ( 23% ) was observed within 6 days after the beginning of night temperature treatments ( Figure 4 5 A, B, and C). After this initial decrease, there was little statistical difference in CER, gs, or Ci with time, although there was some variability. Initia l values of CER at 3 DAT in Experiment 2 were similar to the initial rates in Experiment 1 (1112 mol m2 s1), and to the rates previously reported by Cruz Huerta (2005) at fruit set of the first fruit (12 14 mol m2 s1) Gas exchange parameters were s ignificantly influenced by the interaction between night temperature and days after treatment ( Figure 4 6 A, B, and C). However, CER was significantly lower under LNT compared with HNT, except for two measurements dates (22 and 24 DAT) ( Figure 4 6 A). Stomatal conductance and intercellular CO2 concentration ( Figure 4 6 B) responses were very similar to that of CER. There were no significant interactions between night temperature x fruiting or DAT x fruiting on gas exchange parameters. Initially net CER was similar between fruiting treatments and decreased with time until ~6 DAT. By 15 DAT, CER in nonfruiting plants decreased markedly, resulting in significantly lower CER compared with fruiting plants throughout the rest of the experiment ( Figure 4 7 ). LNT reduced the specific leaf area, but not leaf number or total leaf area ( Table 4 9 ). No significant temperature effects were found on plant or organ dry weight. Plants growing under LNT were shorter due to fewer nodes compared to plants growing under HNT. Fruiting plants had decreased leaf number per plant and greater specific leaf area compared to the nonfruiting plants, but leaf area was not affected ( Table 4 9 ) Non -

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88 fruiting plants had increased leaf, stem and root DW compared t o fruiting plants. However, due to the obvious differences in fruit DW, total plant DW was not affected by the presence/absence of fruits. No interactions between night temperature and fruiting treatment were found for any of the parameters recorded. Discu ssion Low night temperatures ( i.e. less than 15C) and/or low sink demand ( i.e. presence of developing fruits) induced larger flowers and swollen ovaries in our study, as has been previously reported (Polowick and Sawhney, 1985; Aloni et al. 1999; Shaked et al. 2004) Previous results (Aloni et al. 1999) showed that ovary swelling in bell pepper was correlated with increased concentration of reducing sugars and starch in the flower bud. Since carbohydrate concentration and content ultimately depends on plant photosynthesis, we quantified leaf carbon exchange rates, dry weight accumulation, and incidence of ovary swelling in pepper plants grown under low vs high night temperature and different source:sink ratios. In the present work, sweet pepper leaves d eveloped under LNT exhibited lower net CER compared with leaves developed under HNT. These results do not support our hypothesis that leaves fully developed under LNT conditions acclimate and maintain photosynthetic rates as high as those from leaves that developed under HNT. Our results are in contrast to previous reports on tomato (Van deDijk and Maris, 1985) cotton (Koniger and Winter, 1993; Singh et al. ) guayule ( Parthenium argentatum Gray) (Sundar and Reddy, 2000) and common bean ( Phaseolus vulgari s L) (Wolfe, 1991; Wolfe and Kelly, 1992) In these species, leaves that developed under low night temperatures exhibited CER similar to or greater than CER of leaves developed under warm night conditions. However, our results are in agreement with work in other tropical

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89 and subtropical plants, where low night temperature (6 to 10C below the control) decreased CER between 20 and 50% (Wolfe, 1991; Bruggemann et al. 1992; Diczbalis and Menzel, 1998; Venema et al. 1999b) In our experiments, CER and gs decr eased over time, independently from night temperature, cultivar, or presence/absence of developing fruits. In general, CER decreased significantly in the first 69 days after treatments began. The decrease in CER over time was accompanied by decreases in b oth gs and Ci concentrations, suggesting stomatal limitation to CER. This is in agreement with work by Hall and Milthorpe (1978) who reported that decreased leaf CER in bell pepper was correlated with decreased leaf and intracellular conductance. Both net CER and night respiration were reduced by LNT compared to HNT. The lower respiration during the dark period partially compensated for the carbon balance during a 24h period. However, considering only the leaf tissue, carbon gain during a 24h period was higher under HNT compared to LNT. Whole plant respiration was not calculated since only leaf respiration data were available. However, on a whole plant basis, it is likely that differences in net carbon gain between LNT and HNT were negligible since no dif ferences in DW between LNT and HNT were found. Frantz et al. (2004) reported that when varying night temperature from 17 to 32C in rapidly growing lettuce, tomato and soybean, respiration rates during the night increased 2 to 4% per Celsius degree. Howeve r, photosynthetic rates remained steady or decreased slightly as night temperature increased and dry mass accumulation was not affected. Although LNT decreased photosynthetic rates w ithout decreasing total plant or organ dry weight, plant height, stem diam eter and specific leaf area were reduced

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90 compared with plants grown under HNT. Similarly, Mercado (1997a) reported that cool day/night temperatures (25/14C) significantly decreased stem height and specific leaf area of bell pepper compared with plants grown under warm day/night temperatures (29/20C). Fruit removal (Expt. 2) decreased net CER compared to no fruit removal, supporting previous work in pepper (Hall and Milthorpe, 1978) and tomato (Hucklesby and Blanke, 1992) and other species (Setter et al. 1980; Fujii and Kennedy, 1985; Schaffer et al. 1987; Gucci et al. 1991; Gucci et al. 1995) indicating that increasing the source:sink ratio decreased leaf net CER This decrease was likely due to nonstomatal rather than stomatal limitations, as fruit r emoval decreased CER without affecting Ci. The most likely nonstomatal limitation is end product inhibition (Layne and Flore, 1995; Pieters et al. 2001; Sawada et al. 2001) Plants with low sink strength exhibit decreased sucrose synthesis due to low demand from the rest of the plant (Pieters et al. 2001) Low sucrose synthesis reduces the recycling of Pi to the chloroplast, ATP synthesis and RUBP regeneration, and therefore, photosynthesis (Pieters et al. 2001) Under sink limited conditions, leaf sta rch con centration increases and specific leaf area decreases (Nederhoff et al. 1992) as was found in our work. In addition, plant manipulations that cause sugars to accumulate can decrease the expression of photosynthetic genes and upregulate genes for i mproved C metabolism, increasing, among others, sucrose metabolism and starch accumulation (Koch, 1996) which also explain the decrease in CER due to fruit removal. Although fruit removal decreased net CER overall, initially there was no effect of the presence or absence of fruit on CER, as CER in both treatments decreased during

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91 the first 6 DAT and significant differences between fruiting treatments were not manifested until after ~15 DAT. The initial decrease in CER observed in fruiting plants in our stu dy is in c ontrast to work by Cruz Huerta et al. (2005) who reported that CER in pepper increased 40 to 50% between fruit set and 14 days after fruit set, and by VandeDijk and Maris (1985) who reported that net CER increased over time in fruiting tomat o plants. In our experiment, two developing fruits may not have resulted in sufficient sink demand initially to cause the increase in CER observed in previous work, where fruit load was greater (Bhatt and SrinivasaRao, 1997; Cruz Huerta et al. 2005) How ever, once fruit development progressed, differences in CER between fruiting and nonfruiting plants became more apparent. Additionally, the increase in root, stem, and leaf DW observed in nonfruiting compared with fruiting plants may have occurred quickl y in response to the defruiting, thus establishing new sinks in the nonfruiting treatment and partially masking early effects of defruiting on net CER. Conclusions We hypothesized that LNT and/or high source:sink ratio would increase the incidence of swol len ovaries in pepper either through a combination of maintaining net CER under conditions of reduced growth rates (LNT), or by reducing collective fruit demand for assimilates (high source:sink ratio). Both of these conditions would theoretically result i n excess assimilate availability and increased incidence of swollen ovaries. We found that sweet pepper leaves developed under LNT exhibited lower CER than leaves developed under HNT, and thus were unable to acclimate to night temperature conditions. Addit ionally, plant dry weight accumulation was not affected by night temperature treatments. Thus, although LNT did increase the incidence of swollen ovaries, the mechanism was not through maintenance of net CER in combination with

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92 reduced growth rate. On the other hand, fruit removal ( i.e. increasing the source:sink ratio) did decrease net CER and increase the incidence of swollen ovaries. This suggests that excess availability of current photosynthate may not be the mechanism that results in the increase in s wollen ovaries observed under both LNT and high source:sink conditions.

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93 Table 4 1 Effects of night temperature and cultivar on fresh weight of flower and flower parts, ovary diameter and ovary length in sweet pepper Fresh weight (mg) Ovary Ovary Flower Ovary Calyx Petals diameter (mm) length (mm) Night temperature z y 12C 287*** 84** 61** 134** 5.3** 4.4*** 20C 217 57 46 107 4.6 3.7 Cultivar x Legionnaire 295*** 87*** 60*** 140*** 5.6*** 3.9*** Red Cherry Sweet 209 55 48 101 4.3 4.2 Night temp x cultivar 12C Legionnaire 323 a w 98 a 65 a 152a 5.8 a 4.1b 12C Red Cherry Sweet 251 b 70 b 58 b 117b 4.7c 4.6a 20C Legionnaire 267 b 76 b 55 b 129b 5.4 b 3.7c 20C Red Cherry Sweet 167 c 39 c 38 c 85c 3.8 d 3.7c P values ns *** ns *** zFlowers at anthesis were harvested from 3037 days after treatments began. yMeans were averaged across cultivars, n = 94 for 12C and 103 for 20C. xMeans were averaged across temperature, n = 101 for Legionnaire and 96 for Red Cherry Sweet. wMeans with the same letters were not significantly different (Tukey Kramer, P 0.05), n=48, 46, 53, and 50 for 12C Legionnaire, 12C Red Cherry Sweet, 20C Legionnaire, and 20C Red Cherry Sweet, respectively. ns= non significant. *, **, *** Asterisks indicate means are significantly different (t test) at P 0.05, P 0.01, P 0.001, respectively. Table 4 2 P values for effects of night temperature (Temp), cultivar (CV), and days after treatment (DAT) and their interactions on leaf gas exchange parameters. Source of variation z Carbon exchange rate (mol m 2 s 1 ) Stomatal conductance (mol m 2 s 1 ) Intercellular CO 2 (L L 1 ) Temp 0.11 0.06 0.25 CV 0.01 0.99 0.61 Temp x CV 0.64 0.07 0.21 DAT <0.001 <0.001 <0.001 DAT*CV <0.001 <0.001 <0.001 DAT*Temp 0.01 0.05 0.31 DAT*Temp*CV 0.47 0.05 0.44 Block(Temp) y 0.13 0.39 0.13 Plant x 0.03 0.08 0.03 zDegrees of freedom: 1 for Temp, CV, and Temp x CV; 13 for DAT, DAT*CV, DAT*Temp, and DAT*Temp*CV. yRandom effect in the model. xThe individual plant was the subject for the repeated measures component.

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94 Table 4 3 Main effects of night temperature and cultivar on mean carbon exchange rate, stomatal conductance and intercellular CO2 in sweet pepper leaves measured six hours after lights were on. Carbon exchange rate (mol m 2 s 1 ) Stomatal conductance (mol m 2 s 1 ) Intercellular CO 2 (mol mol 1 ) Night temperature z 12C 8.3 0.14 238 20C 9.2 0.16 243 Cultivar y Legionnaire 8.5* 0.15 240 Red Cherry Sweet 9.1 0.15 241 zMeans were averaged across cultivars and days after treatment, n = 252. yMeans were averaged across temperature and days after treatment, n = 252. *Asterisks indicate means are significantly different (Tukey Kramer, P 0.05). Table 4 4 Main effects of night temperature and cultivar on daily net photosynthesis, daily night respiration, and calculated daily total CO2 gain in sweet pepper leaves. Carbon budget (mmol CO 2 d 1 per plant) z Daily net photosynthesis Daily night respiration Daily net CO 2 gain Night temperature y 12C 59* 5** 54* 20C 90 10 80 Cultivar x Legionnaire 70 8 62 Red Cherry Sweet 79 7 72 zData were calculated for leaf tissue only, using plant total leaf area, net CER during the day, and night respiration rates at the end of the experiment (4547 DAT). yMeans were averaged across cultivars, n = 18. xMeans were averaged across temperature, n = 18. *, **Asterisks indicate means are significantly different (ttest) at P 0.05) or P 0.01, respectively.

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95 Table 4 5 Night temperature and cultivar effects on vegetative growth of sweet pepper at the end of the 45day experiment period. 1. Leaf Leaf SLA z DW (g) Plant Stem Node Internode no. area (cm 2 ) Leaf Stem Root Total height (cm) diam. (mm) number length (cm) Night temp y 12C 68 2273 149 15.2 20.0 10.8 49.3 69 12.1 10.7 4.4 20C 74 2612 184 14.2 24.0 10.7 52.5 83 12.9 11.2 5.4 P values 0.03 0.06 0.006 0.13 0.13 0.72 0.31 0.006 0.05 0.16 0.005 Cultivar x LEG 60 2741 170 16.2 22.4 11.3 52.6 80 13.6 9.1 4.8 RCS 82 2144 163 13.2 21.6 10.2 49.1 72 11.5 12.8 5.0 P values 0.01 0.001 0.13 0.003 0.28 0.001 0.07 0.003 <0.001 <0.001 0.12 zSLA: specific leaf area (cm2 g1). yMeans were averaged across cultivars, n = 18. xLEG: Legionnaire, RCS: Red Cherry Sweet. Means were averaged across temperatures, n = 18. Table 4 6 Main effects of night temperature and presence/absence of fruits on fresh weight of the flower, flower parts, ovary diameter, and ovary length in Legionnaire bell pepper Fresh weight (mg) Ovary Ovary Flower Ovary Petals Calyx Stamen Ovary wall diameter (mm) length (mm) Night temperature z, y 12C 451** 171** 148* 95* 26** 73** 7.5** 5.2** 20C 352 121 128 71 21 51 6.5 4.6 Fruits x Non fruiting plants 431** 156** 146* 94** 25** 66* 7.2** 4.9 Fruiting plants 372 136 130 72 22 58 6.8 4.9 zFlowers at anthesis were harvested from 3036 days after treatments began. yMeans were averaged across fruiting treatment, n = 53 and 52 for 12C and 20C, respectively. xMeans were averaged across temperature, n = 60 and 45 for nonfruiting and fruiting plants, respectively. *, **Asterisks indicate means are significantly different (t test) at P 0.01 or P 0.001, respectively.

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96 Table 4 7 P values for effects of night temperature (Temp), presence/absence of fruits (Fruits), and days after treatment (DAT) and their interactions on leaf gas exchange parameters in Legionnaire bell pepper. Source of variation z Carbon exchange rate ( mol m 2 s 1 ) Stomatal conductance (mol m 2 s 1 ) Intercellular CO 2 ( mol mol 1 ) Temp 0.006 <0.001 <0.001 Fruits 0.003 0.02 0.17 Temp*Fruits 0.33 0.26 0.92 DAT <0.001 <0.001 <0.001 DAT*Temp <0.001 <0.001 < 0 001 DAT*Fruits 0.12 0.30 0.88 DAT*Temp*Fruits 0.70 0.87 0.63 Block(Temp) 0.20 0.45 0.47 Plant 0.09 0.21 0.40 zDegrees of freedom: 1 for Temp, Fruits, and Temp*Fruits, 11 for DAT, DAT*Temp, DAT*Fruits, DAT*Temp*Fruits; not available for Block(Temp) (random factor) or Plant (subject for the repeated measures). Table 4 8 Main effects of night temperature and presence/absence of fruits on carbon exchange rate, stomatal conductance and intercellular CO2 in Legionnaire bell pepper leaves Carbon exchange rate ( mol m 2 s 1 ) Stomatal conductance (mol m 2 s 1 ) Intercellular CO 2 ( mol mol 1 ) Night temperature z, y 12C 8.4* 0.10*** 212*** 20C 10.2 0.15 238 Fruits x Non fruiting plants 8.7** 0.11* 222 Fruiting plants 9.9 0.13 227 zMeasurements were done six hours after lights were on and every three to four days during a 41day period on one to three of the most recently matured leaves per plant. yMeans were averaged across fruiting treatment and days after treatment, n = 144. xZero or two developing fruits per plant. Means were averaged across temperature and days after treatments n = 144. *, **, ***Means are significantly different (t test) at P 0.05, P 0.01, and P 0.001, respectiv ely.

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97 Table 4 9 Night temperature and presence/absence of fruit effects on growth of Legionnaire bell pepper at the end of the 45day experiment period. Leaf Leaf SLA z DW (g) Plant Node Internode no. area (cm 2 ) Leaf Stem Root Fruits y Total height (cm) no. length (cm) Night temp x 12C 79 3187 106 30.0 35.3 17.4 18.9 108 75 10.5 4.8 20C 83 3549 135 26.6 38.6 17.3 19.1 107 84 12.1 5.0 P values 0.60 0.31 0.001 0.21 0.29 0.95 0.95 0.93 0.01 0.03 0.30 Fruits w Non fruiting y 86 3500 114 30.7 41.5 18.6 6.4 105 82.0 11.8 4.9 Fruiting 76 3236 126 25.9 32.4 16.0 31.6 110 76.9 10.8 4.8 P values 0.03 0.12 0.05 0.003 0.001 0.002 0.001 0.12 0.01 0.01 0.76 zSLA: specific leaf area (cm2 g1). yIn nonfruiting plants, the fruits were harvested at the beginning of the experiment. xData were averaged across fruiting treatments, n = 12. wData were averaged across temperatures, n = 12.

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98 Days after treatment Figure 4 1 Effect of days after treatment began on (A) net carbon exchange rate (CER) and (B) stomatal conductance (gs), and (C) intercellular CO2 (Ci) of sweet pepper leaves Gas exchange was measured six hours after lights were on and over a 45day period after the beginning of night temperature treatments in two cultivars. Data were averaged across night temperatures and cultivars. Means with the same letters were not significantly dif ferent (Tukey Kramer, P ab a a c c bc bc c c d d d d d 4 5 6 7 8 9 10 11 12 13 14 0 10 20 30 40 50 CER ( mol m 2 s 1 ) a ab cde def cd c b c cde def de ef f f 0.0 0.1 0.2 0.3 0 10 20 30 40 50 gs (mol m 2 s 1 ) a bc g ef de cd ab de de de bc ef fg ef 150 170 190 210 230 250 270 290 0 10 20 30 40 50 Ci (mol mol 1 ) A C B

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99 Days after treatment Figure 4 2 Effect of (A) night temperature and (B) cultivar on net carbon exchange rate (CER) of sweet pepper leaves over time. Gas exchange was measured six hours into the light period and over a 45day period after the beginning of night temperature treatments. *, **, ***Means between temperature treatments (A) or cultivars (B) within each measurement time were significantly di fferent at P P P ** *** * ** 4 5 6 7 8 9 10 11 12 13 14 0 10 20 30 40 50 CER (mol m 2 s 1 ) 12 C 20 C ** *** 0 10 20 30 40 50 'Legionnaire' 'Red Cherry Sweet' A B

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100 Days after treatment Figure 4 3 Effects of (A) night temperature and (B) cultivar on stomatal conductance (gs) of sweet pepper leaves over time. Measurements were done six hours after lights were on and over a 45day period after the beginning of night temperature treatments. *, **, ***Means between temperature treatments (A) or cultivars (B) within each measurem ent time were significantly different at P P P *** *** *** *** 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0 10 20 30 40 50 gs (mol m 2 s 1 ) 12 C 20 C 0 10 20 30 40 50 'Legionnaire' 'Red Cherry Sweet' B A

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101 Night temperature ( C) Cultivar DAT Figure 4 4 Effect of (A) night temperature, (B) cultivar, and (C) days after treatments began (DAT) on leaf respiration in sweet pepper plants during the day ( night ( RCS: Red Cherry Sweet, LEG: Legionnaire. For day respir ation (A and B), n = 30; for night respiration (A and B), n=54; and for DAT (C), n = 36. *, **Means are significantly different at P P In (C), means with the same letters were not significantly different (Tukey Kramer, P ** 0 1 2 12 20 12 20 Leaf respiration (mol m 2 s 1 ) LEG RCS LEG RCS b b a 13 27 47 A B C

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102 Days after treatment Figure 4 5 Effect of days after treatment began on (A) net carbon exchange rate (CER), (B) stomatal conductance (gs), and (C) intercellular CO2 (Ci) of Legionnaire bell pepper leaves. Measurements were done six hours after lights were on, and over a 41day period after the beginning of night temperature and fruiting treatments. Data were averaged across night temperatures and fruiting treatment s. Means with the same letters were not significantly different (Tukey Kramer, P a bc b b bc bc d bc cd d bc bc 4 5 6 7 8 9 10 11 12 13 14 0 10 20 30 40 CER ( mol m 2 s 1 ) a bcde bc b bcd cde e bcd bcde de b b 0 0.1 0.2 0.3 0 10 20 30 40 g s (mol m 2 s 1 ) a cd bc de bcd ef f bcd bcd bcd b bcd 150 175 200 225 250 275 300 0 10 20 30 40 Ci ( mol mol 1 ) A B C

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103 Days after treatment Figure 4 6 Effect of night temperature and days after treatment began on (A) net carbon exchange rate (CER), (B) stomatal conductance (gs), and (C) intercellular CO2 (Ci) of Legionnaire bell pepper leaves. Measurements were done six hours after lights were on, and over a 41day period after the beginning of the treatments. Data were averaged across fruiting treatments. *, **, *** Means between the two night temperature treatments within each measuring time were significantly different at P P P respectively, n=12. ** ** ** *** *** ** *** 4 5 6 7 8 9 10 11 12 13 14 0 10 20 30 40 CER ( mol m 2 s 1 ) 12 C 20 C ** *** *** ** *** ** ** *** 0.0 0.1 0.2 0.3 0 10 20 30 40 g s (mol m 2 s 1 ) *** *** *** ** ** *** 150 175 200 225 250 275 300 0 10 20 30 40 Ci ( mol mol 1 ) A B C

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104 Days after treatment Figure 4 7 Effect of presence or absence of developing fruits and days after treatment began on (A) net carbon exchange rate (CER) and (B) stomatal conductance (gs) of Legionnaire bell pepper leaves. Measurements were done six hours after lights were on, and over a 41day period after the beginning of the treatments. Data were averaged across night temperature treatments. *, **, *** Means between the two night temperature treatment s within each measuring time were significantly different at P respectively. For each mean, n = 12. * ** ** 4 5 6 7 8 9 10 11 12 13 14 0 10 20 30 40 50 CER ( mol m 2 s 1 ) Fruits + Fruits 0.0 0.1 0.2 0.3 0 10 20 30 40 50 gs (mol m 2 s 1 ) A B

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105 CHAPTER 5 5. BELL PEPPER OVARY SW ELLING DUE TO LOW NI GHT TEMPERATURE AND SOURCE SINK RATIO: OVARY WALL ANATOMY AND CARBOHYDRATES Introduction In pepper ( Capsicum annuum L.), as in all horticultural crops, high quality yield is as important as total yield. Fruit weight, size, shape, pericarp thickness, and carpel number and regularity are used to evaluate pepper fruit quality paramet ers and to differentiate pepper types and varieties (Aloni et al. 1999; Navarro et al. 2002; Kissinger et al. 2005; Lim et al. 2007) In bell and spherical peppers, both shape and size are primarily determined at early preanthesis stage, while in elong ated fruits, shape is determined in both pre and post anthesis (Munting, 1974) Early flower development, in turn, is very sensitive to several factors, including low ambient temperature and carbohydrate availability (Ali and Kelly, 1992; Tomer et al. 199 8; Aloni et al. 1999) Low night temperatures affect bell pepper production. In winter, lower temperatures and shorter day lengths increase the percentage of small, flattened and parthenocarpic fruits. Poor pollination is the main reason for such problems but malformation of female organs (Polowick and Sawhney, 1985; Pressman et al. 1998a; Shaked et al. 2004) and other parts of the flower (Aloni et al. 1999) also play a role. In tomato, low night temperature decreases ovule number in one or more locules in some cultivars, causing malformed fruits (Tomer et al. 1998) Night temperatures of 15C or less increase ovary diameter in pepper without increasing locule number, creating swollen ovaries and malformed fruit (Polowick and Sawhney, 1985; Rylski, 1985; Bakker, 1989b; Aloni et al. 1999; Shaked et al. 2004) The longer the duration of low night temperatures, the higher the percentage of swollen flowers and ovaries (Polowick and Sawhney, 1985; Aloni et al. 1999) or the greater the

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106 swelling (Chapter 3). For instance, under night temperature of 12C, the percentage of swollen flowers, and therefore ovaries, increased from 21% at 15 days after treatment to 78% at 49 days after treatment, compared with an increase in swollen flowers from 7 to 14% in plants growing at night temperatures of 18C (Aloni et al. 1999) Evidence indicates that flower deformation, including ovary swelling, is favored by low shoot temperature during the night, regardless of root temperature (Polowick and Sawhney, 1985; Mercado e t al. 1997c; Aloni et al. 1999) Increased source/sink ratio also increases the proportion of swollen ovaries. Aloni et al. (1999) compared fruiting with defruited pepper plants and found that defruiting increased fresh weight of flowers three to four ti mes that of the flowers on fruiting plants, resulting in swollen ovaries and malformed fruits. In addition, the percentage of swollen flowers was inversely related to the number of growing fruits on the plant, and directly proportional to the concentration of reducing sugars and starch in the flowers developed in those plants (Aloni et al. 1999) Flowers that developed on defruited plants accumulated more 14CO2 (600 vs 150 g d1) and fresh weight (180 vs 60 mg) than flowers that developed on fruiting plants (Aloni et al. 1991b; Aloni et al. 1999) These data suggest that excess assimilates are transported to flower buds on defruited plants, resulting in ovary swelling and deformation (Aloni et al. 1999) Limited work has been done on the anatomical aspects of swollen ovaries in pepper. Pressman et al. (1998b) reported that swollen ovaries of bell pepper that developed in response to cool night temperatures had larger cells than normal ovaries, but cell number was similar, suggesting that swollen ovaries develop after cell division ends. However, our findings (Chapter 3) suggest that in order to reach the maximum

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107 swelling effect, the cool night temperature stimulus needs to start during the first week af ter flower initiation, a stage in which active cell division is taking place (Munting, 1974) Flower and ovary swelling is favored by exposing the shoot to low night temperature or by increasing the source:sink ratio. However, the interaction between night temperature and sourcesink modification on ovary swelling is unknown. For these reasons, the hypothesis tested in this study was that high night temperature (HNT) combined with high source:sink ratio or low night temperature (LNT) combined with low sourc e:sink ratio can overcome the detrimental effects of LNT or high source: sink ratio on ovary swelling. The specific objectives of this study were to 1) investigate whether ovary size is increased by high source:sink ratio under warm night temperature, and reduced by low source:sink ratio under low night temperature, 2) investigate the combined effect of night temperature and source:sink ratio on cell number and cell size of ovary wall at flower anthesis, and 3) determine the ovary wall and placenta content and concentration of soluble sugars and starch in bell pepper ovaries developed under two night temperature regimes and two source:sink ratios. Materials and Methods Plant Material Legionnaire bell pepper seedlings were grown in a growth chamber (E 15 Conviron, Winnipeg, Canada) set to 22C/20C (day/night temperature), 400500 mol m2 s1, and a14h photoperiod. At the sixthleaf stage, seedlings were transferred to 1.5 L containers using commercial growing media. Plants were trimmed to two axes, as described in Chapter 3 (Figure 32A). Starting in sympodial unit 1, the strongest shoot was allowed to grow and the second shoot was limited to development of one

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108 sympodial unit (one flower and two leaves) by pruning. Flower buds from the main stem (firs t terminal flower) and the first node in the two axes ( i.e. sympodial unit 1) were removed. Experimental Description A 2 x 2 factorial experiment (two night temperatures regimes and two source:sink ratios) was carried out twice. Night temperatures were 20C (HNT) and 12C (LNT) with the day temperature set to 22C. To establish two different source:sink ratios, one fruit was allowed to set in the second or third node of each of the two axes in all plants. Five to 12 days after petal fall (depending on the experiment), fruits on the plants in one treatment were removed, while fruits on the other treatment were allowed to develop to maturity. All subsequent flowers and small fruits were removed to avoid further fruit set. These treatments resulted in low sour ce:sink (fruiting plants, +F) or high source:sink (nonfruiting plants, F) ratios. This resulted in four treatment combinations (HNT + F, HNT F, LNT + F, LNT F). The night temperature and source:sink ratio treatments started five to twelve days after petal fall, when fruits on fruiting plants exhibited maximum relative growth rate, according to Marcelis et al. (1995b) Each treatment combination was replicated six times in a completely randomize block design. All open flowers were removed when the treatments started. Subsequently, all flowers at anthesis were harvested for sampling, starting the day after treatments began. Two experiments were performed. Experiment 1 was designed to determine the contribution of cell number and cell size to ovary size i n the four treatment combinations. Temperature treatments began 5 to 8 days after petal fall, when one half of the plants were defruited (high source:sink ratio) Flowers from nodes 4 or 5 and the subsequent nodes were harvested daily for 42 days after treatments started (DAT).

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109 Diameter, length and fresh weight of the ovary were recorded and ovaries were fixed in a formalinacetic acid ethanol (FAA) mixture for anatomical studies (see below). Experiment 2 was designed to determine carbohydrate concentration and content in the ovary wall and placenta in the four treatment combinations. Developing fruits were between 8 and 12 days after petal fall when the defruiting was performed and the n ight temperature treatments started. Flowers from Experiment 2 were harvested at anthesis, starting in node 5 and 6, and flowers were harvested daily for 38 days. Fresh weight, diameter, and length of the ovary were recorded. Anatomy Ovaries were individually fixed in FAA. Based on the ovary size data (see below), an anatomical analysis was performed three times : at 1 2 DAT, at 18 19 DAT, and at 3840 DAT (see Figure 5 1 ) A common sampling was used at the beginning of the experiment, since it was the initial control with no treatment effect. For every treatment combination and harvest time the mean fresh weight of all flowers harvested was calculated. Then, four flowers with fresh weights closest to the mean FW for each treatment and harvest time were selected for anatomical analysis. Samples were dehydrated and resin embedded. First, samples were transferred from the FAA to 50% ethanol to rinse the formalin and acetic acid from the FAA. In order to facilitate tissue infiltration, the bottom and top sections of the ovary were cut, and only the middle section was processed. Samples were then dehydrated by gradually increasing ethanol from 50% to 100%, before placing in 1 00% acetone (see Table 5 1 ). Resin embedding steps were done under partial vacuum. Dehydration and resin embedding were done using a microprocessor controlled microwave oven equipped with a ColdSpot temperature control system (PELCO BioWave Laboratory

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110 Tissue Processing System, Ted Pella, Inc. Redding, CA, USA), to speed the process. Microwave power output was set to 180 W, and ColdSpot water bed temperature was 24C. Spurr resin3Following resin embedding, samples were cut in four pieces, and 700 nm slices were cut from one piece using a cryoultramicrotome (Leica Ultracut UCT, (cycloaliphatic epoxide resin [ERL 4221, 4.10g], diglycidyl ether of polypropylene glycol [DER 736, 1.43g], nonenyl succinic anhydride [NSA, 5.90g] [ Ted Pella, Inc. and PELCO International, Redding, C A] and dimethylaminoethanol [DMAE, 0.1g] [Electron Microscopy Sciences, Hartfield, PA]) was used. After resin embedding, the samples were put in molds. Molds were kept overnight under vacuum to remove air bubbles. Samples were then polymerized in an oven at 60C for 24 hours. www.leica.com ). Slices were stained with an aqueous solution of 1% toluidine Blue O and 1% sodium borate, and mounted with immersion oil. Slides were observed in an Olympus BH2 RFCA fluorescence microscope (Olympus, Melville, NY). Images were taken with a digital camer a Q Imaging Retiga 2000R and Q Capture Pro Ver 5.1.1.14 for Win (Q Imaging, Surrey, BC, Canada), and analyzed using ImageJ software (Rasband, 2010) after calibration. Ovary wall thickness, thickness of each ovary wall tissue layer (epidermis, hypodermis, m esocarp, and endocarp), number of cell layers in the ovary wall and cell size of the epidermis, hypodermis, mesocarp, giant, and endocarp cells were measured ( Figure 5 2 ). Average cell size in each tissue was measured by drawing around a set of cells, measuring the area, and determining cell number with the Cell Counter plug in (Kurt De Vos. University of Sheffield, Academic Neurology, England). There were no differen ces between hypodermis and epidermis 3 http://www.tedpella.com/technote_html/ 18300 4221%20TN.pdf ; 3/2/2010.

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111 cell size, therefore for the final analysis, both tissues were analyzed as epidermal tissues. No information was recorded for the vascular bundles. Transverse section area of the ovary wall and ovary wall tissue layers was calculated with the circle and the ring area formula (ring area = (r1 r2)2, where r1 equals diameter divided by 2, and r2 equals r1 minus tissue thickness). The section area was then corrected with a constant for shape irregularity. To find the constant, 23 ovaries ranging from 23 to 249 mg FW (3.5 9.1 mm in diameter and 2.75.3 mm in length) were photographed using a binocular microscope. Total area was then measured from monochromatic images using ImageJ software (Rasband, 2010) and a linear regression (y = b x, y = measured area, b = slope, and x = calculated area) between measured and calculated area was generated to determine the slope ( Figure 5 3 ). Carbohydrates Carbohydrates were analyzed in flowers harvested in Experiment 2 during the fourth week after the beginning of treatment (261 DAT, nodes 8 to 10 above first terminal flower), when the maximum difference in ovary size was found. Glucose, fruc tose, sucrose and starch were analyzed in the ovary wall and placenta of the ovary. Harvested flowers were maintained on ice while ovary fresh weight, length and diameter were recorded. The ovary was then separated into ovary wall and placenta and stored at 80C (Harris. Kendro Laboratory Products, Asheville, NC, USA). Samples were lyophilized (Virtis Model 10MR TR, The Virtis Co., Gardiner, NY, USA) for 72 hours. Samples were subsequently stored at 20C in sealed containers until analysis For soluble sugar and starch analysis, samples were weighed and ground in liquid nitrogen. Samples (1025 mg) were dissolved in 1 mL 80% ethanol, shaken for 20 min at 200 rpm in an orbital shaker (Fisher Scientific Model 361; U.S.A.), and centrifuged at

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112 1380 x g for 10 minutes. The supernatant was removed, and the pellet was reextracted with ethanol. The supernatants of the two extractions were combined and total volume measured. The supernatants were cleared with activated charcoal to remove pigments and centrifuged at high speed (13,250x g, 4 min). Soluble sugar recovery from samples was above 95%, as determined by a 14C external standard. Soluble sugars were analyzed with an enzymatic method (modified from Outlaw Jr. and Tarczynski, 1984) Three enzyme systems (ES) were used: 1) ESA hexokinase (E.C. 2.7.1.1, 0.0565 units/ L) and glucose6 phosphate dehydrogenase (E.C. 1.1.1.49, 0.0808 units/ L) to measure glucose; 2) ESB ESA and phosphoglucoisomerase (EC 5.3.1.9, 0.0741 units/ L) to measure glucose and fructose; and 3) ESC ESB and invertase (EC 3.2.1.26, 0.7712 units/ L) to measure glucose, fructose and sucrose. The enzymes were diluted in 1% w/v bovine serum albumin (BSA, Sigma A6003), 1 M HCl, and 40 mM tris(hydroxymethyl) aminomethane (Fisher Scientif ic BP152 500, pH: 1011.5). For each ES, an aliquot of the cleared sample (30 L), the assay cocktail (1 mL), and the enzyme system (10 L) were mixed and incubated at room temperature for 60 to 90 min, before reading absorbance at 339 nm in a spectrophotom eter (UV 1800, Shimadzu Corp., Kyoto, Japan). The assay cocktail contained the following components: 170 mM N,N Bis(2 hydroxyethyl) 2 aminoethanesulfonic acid (BES, Sigma B4554), 65 mM KOH; 15 mM MgCl2, 1.5 mM EDTA, 2 mM NADP, 5 mM ATP, and 0.015% w/v BSA. Sample pellets were analyzed for starch using amyloglucosidase (E.C. 3.2.1.3, 50 units/sample) and measuring resultant glucose equivalents. Pellets were boiled in 2 ml 0.2N KOH for 30 minutes, then acidified with 1 ml of 1M acetic acid. After cooling, 1 m L

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113 of amyloglucosidase solution (50 units/mL in 0.2M calcium acetate buffer, pH 4.5) was added and samples were incubated for 18 hours at 37C. Volume was recorded and the digest was centrifuged for 10 min at 1975 x g. An aliquot of the supernatant was remo ved and glucose was quantified. Percent recovery, as estimated using a 14C external standard, was above 95%. Data Analysis Ovary size data from the two experiments were pooled and analyzed together using mixed models and the SAS program, (SAS Institute Inc., 2008) Flower harvests were grouped into 4day intervals, resulting in 11 groups for Experiment 1 and 10 groups for Experiment 2. D ata from 5 8 DAT was only from Experiment 2 and data from 41 44 DAT was only from Experiment 1. Night temperature regime, source:sink ratio (presence/absence of fruits), days after harvest (grouped in 4day intervals) and branch (flowers from the main axis or lateral branches) were considered fixed effects. Blocks within each experiment and the experiments (1 and 2) were c onsidered as random effects, and every plant within each experiment was the repeated measures unit. The anatomy data were analyzed in two phases. A preliminary analysis of variance performed as a 2 x 2 x 2 factorial (2 night temperatures, 2 source:sink rat ios, 2 harvest times [ i.e. without considering the control]) indicated that the temperature x harvest time and the temperature x source:sink ratio x harvest time were not significant ( Table A 6 Appendix ). The final analysis was done as a randomized block design, where all combinations were handled as treatments ( i.e. 9 treatments: 23 factorial + control). Main effects and specific comparisons were performed based on the preliminary data and by using contrasts. No comparisons were performed for the non significant

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114 interactions. Carbohydrate data were analyzed as a 2 x 2 factorial using a completely randomized block design with 6 replications. Results Ovary Size Low night temperature significantly increased ovary fresh weight, diameter, and length ( Table 5 2 ; Table A 5 Appendix ). Fresh weight increased about 25%, while diameter and length increased 9 and 6%, respectively. The presence or absence of fruits also affected ovary size parameters. In nonfruiting plants, ovary fresh weight of flow ers at anthesis was 35% greater than those from fruiting plants. Both ovary diameter and length also increased in nonfruiting compared with fruiting plants. Since flowers at anthesis were harvested over 3842 days after temperature treatments started, ovaries were grouped into 10 (Exp. 2) or 11 (Exp. 1) 4day harvest intervals. Harvest timing significantly influenced ovary size parameters. In general, ovary FW increased as harvesting intervals proceeded ( Table 5 3 ). Significant increases in ovary FW were observed beginning at 1316 DAT and maximum size was reached at 2528 DAT. Based on ovary FW, ovaries could be classified into three groups: 1 to 12 DAT 13 to 24 DAT, and 25 to 42 DAT. Ovary diameter increased in a similar pattern as fresh weight, with significant increases observed beginning at 912 DAT. Ovary length was less responsive; ovaries harvested from 1 through 20 DAT were similar in length and shorter than ov aries harvested from 25 through 44 DAT There was a significant interaction between night temperature and presence/absence of fruits on ovary fresh weight ( P 0.02), but not on diameter or length ( P 0.08). Ovary fresh weights in nonfruiting plants under LN T (L ) were the largest,

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115 averaging 149 mg; while fresh weights were smallest in the HNT with fruits (H+), averaging 90 mg ( Figure 5 4 ). A similar pattern was observed in ovary diameter and length, although the interaction was not significant. The interaction between night temperature and harvest interval was significant for ovary FW ( Figure 5 5 ), but not for diameter ( P 0.36) or length ( P 0.76) ( Table A 5 and Fig ure A 2 Appendix ). Ovary fresh weight increased ~42 mg (42%) between harvest intervals 14 DAT and 3336 DAT in plants developed under LNT, while fresh weight increased ~25 mg (28%) in ovaries developed under HNT during the same intervals. Ovary si ze was also affected by the interaction between days after treatment and fruiting treatments, which was highly significant for the three parameters ( Figure 5 6 ). Starting at 5 8 DAT the ovaries developed on nonfruiting plants had greater FW, diameter and length than the ovaries developed on fruiting plants. On the nonfruiting plants, ovary FW, diameter, and length increased steadily from 14 DAT through 2528 DAT, then decreased between 33 36 and 4144 DAT (t test, P <0.04 for 33 36 DAT vs 3740 DAT and P <0.003 for 3336 DAT vs 41 44 DAT ). In fact, pair wise comparisons revealed that ovary FW and diameter were significantly greater as early as 5 8 DAT compared to 1 4 DAT (t test, P 0.01). In fruiting plants; however, there was a significant reduction in ovary FW and length during the first 5 to 6 harvest intervals compared to the first harvest interval (t test, P 0.001), while ovary diameter remained constant. Subsequently, ovary FW, length, and diameter increased, and by 2528 DAT (FW and length) or 2932 DAT (diameter), were significantly larger than observed at harvest intervals 1720 or 2124 DAT (t test, P 0.001).

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116 Anatomy Ovaries used for the anatomical study were a subsample of the total number harvested, as described above. Ovaries developed under LNT were significantly larger compared with ovaries developed under HNT ( Figure 5 7 A), in accordance with data presented in Table 5 2 LNT also increased the ovary wall thickness and total transverse area in the ovary midsection ( Figure 5 7 B). Based on total transverse area of the ovary wall mesocarp tissue accounted for 72% of the area, followed by giant cells (16%), epidermal tissue (8%) and endocarp (4%). Although there were differences between LNT and HNT in ovary FW, diameter, and ovary wall transverse area ( Figure 5 7 ), there were no differences in cell size or cell number in mesocarp or giant cells between the two night temperature treatments (Figures 5 8 and 5 9 ). There was also no difference in the number of cell layers i n the mesocarp (22.0 1.1 vs 20.3 0.7 for LNT and HNT, respectively, data not shown). Epidermal cells were larger under LNT compared to HNT ( P 0.04; 214 vs 190 m2), but there were no differences in cell number (Figures 5 8 and 5 9 ). There was also no significant effect of night temperature on endocarp cell size or cell number. O vary FW and diameter in nonfruiting plants were significantly greater compared with ovaries developed on fruiting plants ( Figure 5 10), as previously shown in Table 5 2 Ovary wall thickness was also greater in ovaries of nonfruiting compared with ovaries of fruiting plants (564 vs 444 m), which in combination with the greater diameter, resulted in increased total transverse area (12.11 vs 7.73 mm2 for nonfruiting and fruiting, respectively). Increases in ovary size in nonfruiting compared with fruiting plants were due to increased cell size in mesocarp, giant cell, and epidermal tissues, which comprised 96%

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117 of the ovary wall ( Figure 5 11 A, B, C; Figure 5 12). Cell number was significantly greater in ovary endocarp tissue of nonfruiting compared with fruiting plants. There was also a small, but nonsignificant, increase in cell number in mesocarp (P 0.09) and epidermal tissues (P 0.07) of ovaries in nonfruiting plants. Nonfruiting plants also had a greater number of cell layers in the mesocarp compared to the fruiting plants (23.01. 1 vs 19.40.6 for F and +F plants, respectively). Ovary FW increased between 1819 DAT and 3840 DAT, while ovary diameter did not increase ( Figure 5 13). Ovary wall thickness at 18 19 DAT (550 m) was greater than at 3840 DAT (458 m), resulting in a significant increase in ovary wall area at 1819 DAT compared with the other two harvest times Greater ovary fresh weight at 3840 DAT may be due to greater ovary leng th in that time (4.3 mm) compared to 1 2 DAT (3.9 mm) and 1819 DAT (4.1 mm) (P 0.004, data not shown) Mesocarp and giant cell size was greater in 18 19 DAT than in 3840 DAT ( Figure 5 14A, B). However, mesocarp cell number increased from 17,100 to 22,000 per cross section area in the ovary wall between 1 2 DAT and 38 40 DAT ( P 0.06). Regression analysis indicated a significant increase in giant cell number (cell numb er = 314.30 + 2.63 x DAT; r2 = 0.99; P 0.01) with days after treatment A similar trend was observed in mesocarp cell number (cell number = 17165 + 129.4 DAT; r 2 = 0.95, P 0.10), and the increase in cell number may account for the increased ovary FW in 3840 DAT compared with 1819 DAT There was a significant interaction between night temperature and presence/absence of fruit on ovary growth and anatomy (Figures 5 15 and 5 16, Table A 5 Appendix ). Flowers developed under LNT in nonfruiting p lants developed the

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118 largest ovaries ( Figure 5 15), while fruiting plants under both night temperatures developed the smallest ovaries. This differs slightly from the average ovary FW determined using the entire dataset ( Figure 5 4 ), where ovary FW in fruiting plants under HNT were significantly smaller than ovaries developed under all other conditions. Although the trend observed in Figure 5 15 is similar to that observed in Figure 5 4 the difference is likely due to the smaller sample size used in the anatomical studies, as well as the slight difference i n ovary harvesting times between the anatomical study (Experiment 1) and the pooled data (Experiments 1 and 2) that was used for ovary growth measurements. In the mesocarp, cell size was largest in nonfruiting plants under LNT and smallest in fruiting pla nts under HNT ( Figure 5 16A). There were no differences due to temperature, but only due to presence or absence of developing fruits. Mesocarp cell number, however, w as greater in nonfruiting plants under LNT than in the rest of the combinations ( P 0.06). The number of mesocarp cell layers followed the same trend, with greater number of layers in the nonfruiting plants under LNT (25.0 1.6) compared with the rest of the treatment combinations (19.1 0.6, 20.7 0.7 and 19.8 1.2 for L+, F and F+, respectively). Size of giant cells was affected only under HNT, where nonfruiting plants had larger giant cells than did fruiting plants ( Figure 5 16B). Such differences in cell size were compensated for in cell number, so that the total area of giant cells was similar in the ovaries of both fruiting and nonfruiting plants under HNT (data not shown). There were also significant interactions between harvest time and presence/absence of fruit. Ovary FW and diameter increased at both 1819 DAT and

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119 3 8 40 DAT in nonfruiting plants, compared to fruiting plants ( Figure 5 17A). The smallest ovaries were harvested from fruiting plants at 1819 DAT in accordance with what was previously described in Figure 5 6 Although there were no differences in ovary FW or diameter between nonfruiting plants at 18 19 DAT and 3 8 40 DAT ovary wall thickness was greatest in nonfruiting plants at 1819 DAT compared with the other treatments, resulting in greater ovary wall transverse area ( Figure 5 17B). Since there were no differences in ovary length between 1819 DAT and 3 8 40 DAT (4.5 and 4.6 mm, respectively, P 0.50) in nonfruiting plants, the similar ovary FW and diameter between these two harvest times suggests that the placenta in 3840 DAT ovaries was larger than those of 18 19DAT ovaries. In fruiting plants, there were no differences in diam eter, but ovary FW was greater at 3840 DAT due to a greater length at 38 40 DAT (4.1 mm) compared to the diameter at 1819DAT (3.6 mm, P ). Differences in ovary wall transverse area are likely due to the increased cell size of the mesocarp, giant cells and epidermal tissues in nonfruiting plants at 18 20 DAT compared to the rest of the treatment combinations ( Figure 5 18 ). There were no differences between harvest times within fruiting and nonfruiting plants for mesocarp cell number. There was, however, a trend towards increased cell number in mesocarp, epidermal tissue and endoc arp of nonfruiting compared with fruiting plants. Carbohydrates Low night temperature (LNT) increased glucose, fructose and starch concentration and decreased sucrose concentration in the ovary wall compared to HNT ( Table 5 4 ). In contrast, soluble sugar and starch concentrations in the placenta were not affected by night temperature. In general, there were no differences in soluble sugar or starch concentrations in t he ovary wall or in placenta between fruiting and nonfruiting plants,

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120 with the exception of sucrose in the placenta. There were no significant interactions between night temperature and presence/absence of fruits on carbohydrate concentrations ( Table A 7 Appendix ). Total content of reducing sugars and starch was also greater in the ovary wall of ovaries developed under LNT compared to ovaries in the HNT ( Table 5 5 ). Sucrose content; however, was similar, since the increased DW under LNT was compensated by the decreased sucrose concentration. In the placenta, fructose and starch content was greater und er LNT compared with HNT; however, sucrose and glucose contents were not affected. Overall, total content of soluble sugars and starch was 74% greater under LNT than under HNT. Soluble sugar content was higher in ovary wall and placenta of nonfruiting com pared with fruiting plants ( Table 5 5 ). Starch content was also higher, although the increased starch in ovary wall of nonfruiting compared with fruiting plants was not significant. Overall, carbohydrate content was 31% greater in nonfruiting plants compared with fruiting plants. No significant interaction was found between night temperature and presence/absence of fruits on carbohydrate content in the ovary wall or p lacenta. Discussion Ovary size (and therefore the incidence of swollen ovaries) of Legionnaire bell pepper increased under LNT compared with HNT, and in nonfruiting (high source:sink ratio), compared to fruiting (low source:sink ratio) plants, confirmi ng previous reports for bell and other sweet pepper cultivars (Chapter 3; Pressman et al. 1998b; Aloni et al. 1999; Shaked et al. 2004) In the present study, ovary FW (which is comprised of ovary wall FW and FW of the inner placental tissues located in the cavity) increased 25% to

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121 35% under LNT compared to HNT and nonfruiting compared with fruiting treatments, respectively. Such increases are comparatively smaller than the values reported by Aloni et al. (1998b; 1999) who reported increases of 100 to 300% in ovary FW of bell peppers under LNT or nonfruiting conditions. The reasons for the quantitative differences in effects of night temperature or source:sink ratio on ovary size of bell pepper between the two studies may be due to differences in culti var, plant age, or environmental conditions used. Ovary size under LNT increased due to a significant increase in ovary wall thickness and increased transverse area, without significant increases in cell size or cell number. However, there was a trend towards larger cells in all ovary wall tissues of floral ovaries developed under LNT compared with HNT, and a trend towards increased cell number in the mesocarp and endocarp. Taken together, these increases resulted in significant increases in ovary FW, diame ter, length, and ovary wall thickness and transverse area in floral ovaries developed under LNT compared with HNT. The trend in increased cell size observed in the present study is supported by results from a previous study where LNT increased cell size i n mesocarp and giant cells in bell pepper compared with HNT (Pressman et al. 1998b) In contrast to the effect of night temperature on ovary swelling, the increase in floral ovary FW in nonfruiting compared with fruiting plants observed in the present st udy was clearly due to significant increases in cell size of the mesocarp, giant cells and epidermal cells. Actively growing fruits are a much stronger sink for assimilates compared to flowers (Ali and Kelly, 1992; Marcelis and BaanHofman Eijer, 1997) an d the presence of developing fruits likely reduced the source supply available for floral

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122 development, resulting in decreased cell size and ovary FW, and therefore the incidence of swollen ovaries compared with nonfruiting plants. Similarly, the observed decrease in the number of cell layers in floral ovary mesocarp in fruiting compared with nonfruiting plants is also a response to increased sink demand of fruiting plants, as observed by Ali and Kelly (1992) Differences in ovary FW, diameter, and length due to night temperature were found within the first four days after treatment. Responsiveness of Legionnaire bell pepper in the two experiments reported here was similar to the previous response (Chapter 3), where significant differences due to night t emperature were found in the first week. In that experiment, we found that most sweet pepper cultivars required three to four weeks of LNT for induction of maximum ovary swelling and concluded that flowers must be exposed to LNT soon after floral initiatio n begins. Legionnaire bell pepper appears more sensitive than other sweet pepper types to LNT induced ovary swelling and does not appear to require exposure during the first week after flower bud initiation. Further, although Munting (1974) reported that cell division is the main process occurring during floral development in pepper prior to anthesis, our results indicate that cell size is most affected in Legionnaire preanthesis in response to LNT or source:sink modification. Thus, there may be genoty pic differences among pepper cultivars in the intervals of cell division and cell enlargement during flower development due to the different rates of cell division between genotypes, specially elongated vs blocky or spherical (Munting, 1974) The combined effects of low night temperature and source:sink ratio on ovary swelling in sweet pepper has not been studied previously. In the present research, floral

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123 ovary FW was greatest in nonfruiting plants under LNT and smallest in fruiting plants under HNT, sugg esting that night temperature and source:sink effects on ovary FW are cumulative. As hypothesized, fruiting plants under LNT and nonfruiting plants under LNT had intermediate ovary size. The increased ovary FW in nonfruiting plants under LNT was due prim arily to both increased mesocarp cell number and size, with additional influences from increased epidermal cell size and increased endocarp cell number. On the other hand, the decreased ovary FW exhibited by fruiting plants under HNT compared with other tr eatments was due to decreased size of mesocarp and giant cells. Although ovary FW and diameter were greater in nonfruiting compared with fruiting plants throughout the duration of the experiment the re sponse over time ( i.e. over DAT) was clearly different between fruiting and non fruiting plants In fruiting plants, floral ovary FW decreased during the first 24 days after treatments started, after which, ovary FW increased. The decrease was likely due to the high sink demand of the growing collective fru its. During the first third to the first half of the fruit growth period, bell pepper fruit reaches the maximum absolute growth rate, gaining over 80% of its final fresh weight and volume, and over 90% of its final diameter and length (Hall, 1977; Nielsen et al. 1991; Marcelis and BaanHofman Eijer, 1995b) Flower buds and flowers, on the other hand, represent the weakest sink in bell pepper plant (Turner and Wien, 1994a) The sink demand of growing bell pepper fruits decreases ~ 35 days after anthesis (Ni elsen et al. 1991; Marcelis and BaanHofman Eijer, 1995b) which coincides with the increase in floral ovary FW observed in the present study. In nonfruiting plants, on the other hand, floral ovaries increased in size during the first seven harvest inter vals ( i.e. 28 days after treatments started) then remained stable,

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124 before decreasing during the last two harvest periods. Initial increase in ovary size is likely due to the lack of fruiting and therefore increased source supply to flowers. As discussed i n Chapter 3 and based on previous reports (Choi and Gerber, 1992; Cruz Huerta, 2001) the time from flower initiation to anthesis is ~ 4 weeks in bell pepper. Thus, maximum size of floral ovaries in nonfruiting plants was reached ~28 days after treatment s began, since these were flowers that developed fully under the nonfruiting conditions. The decrease in floral ovary size in nonfruiting plants at the end of the experiment may be related to a decreased photosynthetic rate in the nonfruiting treatment as previously reported in pepper (Chapter 4; Hall and Milthorpe, 1978) and tomato (Hucklesby and Blanke, 1992) Anatomical analysis of a subsample of flowers harvested at discrete intervals ( i.e. 1 2 DAT, 18 19 DAT and 38 40 DAT) revealed that ovary wal l transverse area and cell size of mesocarp, giant cells, and epidermis were significantly greater in ovaries harvested at 1819 DAT in nonfruiting plants compared with 3840 DAT in nonfruiting or either harvest time in fruiting plants. This correlates well with the larger ovary FW at 1819 DAT non fruiting compared with either harvest time in fruiting plants. This does not explain, however, why there was no difference in ovary FW in flowers harvested at 3840 DAT vs 18 19 DAT in nonfruiting plants. Since there were no differences in ovary length between 18 19 and 3840 DAT in nonfruiting plants, the similar ovary FW and diameter between the harvest times suggests that the placenta at 38 40 DAT ovaries was larger than those of 18 19 DAT ovaries. This is supported by previous data showing that placenta FW increases and the ratio of ovary wall FW:total ovary FW decreases with harvest interval (Chapter 3).

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125 The increase in the ovary wall transverse area in ovaries harvest ed at 3840 DAT of non fruiting plants compared with ovary wall transverse area of fruiting plants at both harvest times may be due to the sum of the non significant increases in cell numbers of the mesocarp, epidermis, and endocarp. Changes in source:sink ratios may affect fruit size by regulating cell division as early as petal and sepal differentiation, and before carpel initiation (Brukhin et al. 2003; Baldet et al. 2006) Our results suggest that reducing fruit load initially increased ovary wall cel l size, but later, as the fruits became weaker sinks (Marcelis and BaanHofman Eijer, 1995b) the effect on cell size of initiating flowers was negligible. The increase in ovary FW in harvest at 3840 DAT compared with harvest at 1819 DAT in fruiting plan ts was not reflected in significant increases in cell size in ovary wall tissues. However, there was a trend towards increasing cell number in all ovary wall tissues in fruiting plants at 3840 DAT compared with 1819 DAT This, combined with the greater ovary length, likely resulted in greater ovary FW at 38 40 DAT compared with harvested flowers at 18 19 DAT on fruiting plants. Carbohydrate accumulation was also affected by both night temperature and source:sink ratio. As with effects on ovary cell size and number; however, the effects of night temperature and source:sink ratio on carbohydrate accumulation as related to ovary size differed. Low night temperature and high source:sink ratio both increased floral ovary size, but the increase in ovary size was associated with increased ovary carbohydrate concentration only in the low night temperature, not the high source:sink, treatment. The increased reducing sugar and starch concentration in floral ovaries

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126 developed under LNT compared with HNT may be due to decreased night respiration at the lower temperature, as observed earlier in this work (see Chapter 4). Non fruiting conditions, even though they also caused ovary swelling, did not increase ovary sugar or starch concentrations. This is in contrast to work by Aloni et al. (1991b; 1999) who found increased carbohydrate concentrations in flower buds of nonfruiting compared with fruiting bell pepper. In their work, Aloni et al. (1999) used plants bearing five fruits that resulted in floral ovary weights that were onethird to onehalf the weight of ovaries on nonfruiting plants. In our work, the plants had only two developing fruits, resulting in the development of floral ovaries with an average 25% decrease in FW compared with floral ovaries on nonfruiting plants. It may be that under the conditions of our experiment, the increased source:sink ratio on the nonfruiting plants was sufficient to cause increase ovary size and therefore carbohydrate content, but not sufficient to increase storage of carbohydrate and therefore increase concentration. Our results are in accordance with previous reports that both LNT and high source:sink ratio increase ovary size, and therefore, ovary swelling. Our hypothesis that LNT combined with low source:sink ratio or HNT combi ned with high source:sink ratio can overcome the detrimental effects of LNT or high source:sink ratio on ovary swelling is also supported by our results. However, it appears that the mechanisms by which floral ovary size increases, resulting in increases i n swollen ovaries, differs depending on whether the swollen ovaries occur in response to LNT or in response to excess source supply. Ovary swelling caused by LNT is associated with an increased concentration of reducing sugars and starch in the ovary wall but not in the placental tissues, as well as with slight increases in both cell size and cell number in the ovary

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127 wall In contrast, ovary swelling caused by high source:sink ratio seems to be most clearly associated with increased cell size in the mesocarp, giant cells and epidermal cells, with negligible effects on carbohydrate concentration. Conclusions Both fruiting plants under LNT and nonfruiting plants under HNT produced an intermediatesized ovary, which supports our hypothesis that HNT combined with high source:sink ratio or LNT combined with low source:sink ratio can overcome the detrimental effec ts of low night temperature or high source:sink ratio on ovary swelling in pepper. Ovary fresh weight, diameter and length of flowers at anthesis were increased by both low night temperature (12C) as well as by absence of fruits. The mechanisms for the in crease in ovary size, however, appear to be different. LNT significantly increased ovary wall thickness and transverse area, with only slight increases in cell size and number. Low night temperature also increased floral ovary reducing s ugar and starch concentration. Absence of fruits increased floral ovary size mainly through increased cell size, with no effect on increasing ovary reducing sugar or starch concentration.

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128 Table 5 1 Dehydration and resin embedding steps for Legionnaire bell pepper ovaries previously fixed in FAA containing 50% ethanol. Step Product Processing time z Infiltration time y Dehydration Ethanol 50% 45 seconds 1 min. 75% 45 seconds 1 min. 95% 45 seconds 1 min. 100% 45 seconds 1 min. Acetone 100% 45 seconds Acetone 100% Overnight Spurr resin embedding Resin: Acetone 30:70 3 min. 5 min. 50:50 3 min. 5 min. 70:30 3 min. 5 min. 100:0 3 min. 5 min. 100:0 Overnight zTime that the samples were processed in a PELCO BioWave Laboratory Tissue Processing System with ColdSpot ( Ted Pella, Inc. Redding, CA, USA) at 180 W and waterbed at 24C. yAfter every step, samples were left on the bench top for various times to improve infiltration. Table 5 2 Main effects of night temperature and presence/absence of fruits on the fresh weight, diameter, and length of the ovary in Legionnaire bell pepper flowers harvested at anthesis Fresh weight (mg) Diamet er (mm) Length (mm) Night temperature z y 12C 128* 6.6* 4.5* 20C 102 6.0 4.3 Fruits x Non fruiting plants 132* 6.7* 4.6* Fruiting plants 98 5.9 4.2 zPooled data of two experiments. Flowers were continuously harvested for 42 days in Experiment 1 and 38 days in Experiment 2. yMeans were averaged across fruiting treatments and harvest intervals; n= 597 and 492 for 20C and 12C, respectively. xMeans were averaged across temperature treatments and harvest intervals; n= 595 and 494 for nonfruiting and fruiting treatments, respectively. Asteri sks indicate means are significantly different at P 0.001.

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129 Table 5 3 Main effect of days after treatment on the fresh weight, diameter, and length of the ovary in Legionnaire bell pepper flowers harvested at anthesis Days after treatment z y Fresh weight (mg) Diameter (mm) Length (mm) 1 4 97 e x, w 5.8 g 4.3 c 5 8 89 e 5.8 fg 3.9 d 9 12 101 de 6.0 ef 4.3 c 13 16 108 cd 6.2 de 4.3 bc 17 20 104 de 6.2 cde 4.3 bc 21 24 118 bc 6.4 bcd 4.5 ab 25 28 128 ab 6.6 ab 4.6 a 29 32 132 a 6.7 a 4.6 a 33 36 130 a 6.6 ab 4.6 a 37 40 129 ab 6.6 ab 4.6 a 41 44 128 ab 6.6 abc 4.6 a zPooled data of two experiments. Flowers were continuously harvested during 42 days in Experiment 1 and 38 days in Experiment 2. yOvaries were grouped at 4 day interval s. Data from 5 8 DAT were from Experiment 2 only; data from 4144 DAT were from Experiment 1 only. xMeans were averaged for each 4 day interval across night temperature and fruiting treatments; n = 136, 42, 155, 106, 93, 87, 96,121,127, 93, and 33 for intervals 14 DAT to 41 44 DAT respectively. wMeans with the same letter in the same column are not significantly different (Tukey Kramer, P Table 5 4 Main effects of night temperature and presence/absence of fruits on soluble sugar and starch concentration in ovaries ( ovary wall and placenta) of Legionnaire bell pepper flowers harvested at anthesisz. Ovary wall (g/g DW) Placenta (g/g DW) Glu y Fru Suc Starch Glu Fru Suc Starch Temperature x 12C 31.9** 31.9* 27.9* 56.9* 62.8 72.9 45.7 99.6 20C 26.2 26.3 35.6 44.8 61.1 65.8 50.5 87.4 Fruits w Non fruiting 29.0 30.1 31.4 46.6 63.4 68.6 44.2* 87.4 Fruiting 29.1 28.2 32.0 55.1 60.5 70.1 52.1 99.6 zFlowers were harvested 24 to 29 days after treatments started. yGlu: glucose, Fru: fructose, Suc: sucrose. *, **: Significant differences at P 0.05 and 0.01 respectively. xMeans were averaged across fruiting treatments, n =12. wMeans were averaged across temperature treatments, n = 12.

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130 Table 5 5 Main effects of night temperature and presence/absence of fruits on soluble sugar and starch content in ovaries ( ovary wall and placenta) of Legionnaire bell pepper flowers harvested at anthesis Ovar y wall content (g/flower) Placenta content (g/flower) Glu y Fru Suc Starch Glu Fru Suc Starch Temperature z x 12C 540** 533*** 466 986* 976 1098* 658 1489* 20C 314 319 411 552 771 813 626 1071 Fruits w Non fruiting 492 501* 498* 844 1095** 1158** 729* 1494* Fruiting 362 350 380 694 653 753 554 1066 zFlowers were harvested 24 to 29 days after treatments started. yGlu: glucose, Fru: fructose, Suc: sucrose. *, **: Significant differences at P 0.05 and 0.01, respectively. xMeans were averaged across fruiting treatments, n =12. wMeans were averaged across temperature treatments, n = 12.

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131 Figure 5 1 Schematic representation of harvesting times for anatomical analysis in relation to fruit growth in the fruiting plants. Dry weight; Fresh weight. Day 0 represent anthesis. Flower initiation occurs four weeks before anthesis, and petal fall occ urs ~3 4 days after anthesis. Treatments started ~6 days after petal fall. Flowers used in the anatomical analysis reached their anthesis at 12, 1819 or 3840 days after treatment (DAT). Figure 5 2 Transverse cross section of the ovary wall of ovaries at flower anthesis in Legionnaire bell pepper. Tissues: A) epidermis and hypodermis, B) mesocarp, C) giant cells, D) endocarp, E) ovule, F) placenta tissue, G) vascular bundle. 0 20 40 60 80 100 30 20 10 0 10 20 30 40 50 60 Relative weight (%) Flower initiation Time (days) Petal fall 1 2 DAT 1819 DAT 3840 DAT 100 m A B C D E F G Anthesis

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132 Figure 5 3 Relationship between the measured and the calculated area of a transverse section in the ovary of Legionnaire bell pepper. A) Cross section area was calculated with the average diameter (arrow), then the area was measured from monochromatic images with imageJ (Rasband, 2010) B) Linear regression between calcul ated and measured area. 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Measured area (mm 2 ) Calculated area (mm 2 ) y = 1.0952 x r2=0.995 P value < 0.001 n=23 1 mm A c = r 2 B A

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133 Temperature x fruiting treatment combination Figure 5 4 Interaction of night temperature and presence/absence of fruits on ovary characteristics of flowers harvested at anthesis in Legionnaire bell pepper. Night temperature was either 12 (L) or 20C (H), and plants had either 0 ( ) or two developing fruits (+). Data for the two experiments were pooled for analysis. Flowers were continuously harvested for 42 days in Experiment 1 and 38 days in Experiment 2. Means were averaged for each combination across harvest intervals; n = 265, 227, 330 and 267 for L, L+, H and H+ treatments, respectively. Means with the same letter in the same parameter are not significantly different (Tukey Kramer, P a b b c a b b c a bc b c 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 140 160 L L+ H H+ L L+ H H+ L L+ H H+ Diameter or length (mm) Fresh weight (mg) Ovary FW Diameter Length

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134 Days after treatment Figure 5 5 Effect of night temperature and days after treatment on ovary fresh weight of flowers at anthesis in Legionnaire bell pepper. Flowers were harvested dail y for 42 days in Experiment 1 and for 38 days in Experiment 2 ; data were grouped every 4 days. Data for the two experiments were pooled for analysis. Data from 5 8 DAT was from Exp. 2 only; data from 41 44 DAT was from Exp. 1 only. Means were averaged for each combination across fruiting treatments; n = 26 to 32 for 1 4 and 4144 DAT and 35 to 8 6 for the remaining intervals. *, **, ***: Significant differences between temperature regimes within each harvest interval at P 0.05, 0.01 and 0.001, respectively. *** *** *** *** *** *** *** *** ** 60 70 80 90 100 110 120 130 140 150 160 Fresh weight (mg) 12 C 20 C 0

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135 Days after treatment Figure 5 6 Effect of presence/absence of fruits and days after treatment on ovary size of flowers harvested at anthesis in Legionnaire bell pepper. A) Ovary fresh weight; B) diameter; C) length. Flowers were harvested daily for 42 days in Experiment 1 and 38 days in Experiment 2; data were grouped every 4 days. Data for the two experiments were pooled for analysis. Data from 5 8 DAT was from Exp. 2 only; data from 41 44 DAT was from Exp. 1 only. Means were averaged for each combination across night temperature treatments; n = 22 to 3 1 for intervals 5 8 DAT and 4144 DAT and 39 to 90 for the remaining intervals. *, **, ***: Significant differences within each harvest interval at P 0.05, 0.01 and 0.001, respectively. *** *** *** *** *** *** *** *** 70 80 90 100 110 120 130 140 150 160 Fresh weight (mg) Non fruiting plants Fruiting plants 0 ** *** *** *** *** *** *** *** *** 5.0 5.5 6.0 6.5 7.0 7.5 Diameter (mm) 0.0 ** *** *** *** *** *** *** *** *** 3.5 4.0 4.5 5.0 Length (mm) 0.0 B A C

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136 Night temperature ( C) Figure 5 7 Effect of night temperature on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers at anthesis. A) ovary size; B) ovary wall thickness and ovary wall transverse area. *, **, ***: Means between temperature treatments are significantly different at P .05, P P *** *** 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 140 12 20 12 20 Diameter (mm) Fresh weight (mg) Ovary FW Diameter A ** 0 2 4 6 8 10 12 0.0 0.1 0.2 0.3 0.4 0.5 0.6 12 20 12 20 Area (mm 2 ) Thickness (mm) Ovary wall thickness Ovary wall transverse area B

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137 Night temperature Figure 5 8 Effect of night temperature on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. A) mesocarp; B) giant cells; C) epidermal tissues; D) endocarp. Means between temperature treatments are significantly different at P 0.05; n = 16. 0 5 10 15 20 25 0 50 100 150 200 250 300 350 400 Cell number (thousands) Cell size (m 2 ) Cell size Cell number A 0.0 0.1 0.2 0.3 0.4 0.5 0 1000 2000 3000 4000 5000 6000 Cell number (thousands) Cell size (m 2 ) B 0 1 2 3 4 5 0 50 100 150 200 250 12 20 12 20 Cell number (thousands) Cell size (m 2 ) C 0.0 0.5 1.0 1.5 2.0 0 50 100 150 200 250 12 20 12 20 Cell number (thousands) Cell size (m 2 ) D

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138 Figure 5 9 Median cross section of ovaries in Legionnaire bell pepper flowers developed under two night temperatures. A, C, E: low night temperature, B, D, F: high night temperature; A, B: total ovary wall ; C, D: outer ovary wall ; E, F: inner ovary wall ; ep, epidermis; hp, hypodermis; me, mesocarp; vb, vascular bundle; gc, giant cell; en, endocarp; ov, ovule. Note the thicker ovary wall under LNT compared to HNT but no difference in cell size. A B ep me hp gc gc vb me me me ep hp D C E F gc gc ov ov 100 m 100 m 25 m 25 m 25 m 25 m en en ov

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139 Fruiting treatment Figure 5 10. Effect of presence (+F) or absence ( F) of fruits on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. A) ovary size, B) ovary wall thickness and ovary wal l transverse area. +F: 2 developing fruits per plant. *, **, ***: Means between fruiting treatments are significantly different at P P P respectively; n = 16. *** *** 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 140 F +F F +F Diameter (mm) Fresh weight (mg) Ovary FW Diameter A ** 0 2 4 6 8 10 12 0.0 0.1 0.2 0.3 0.4 0.5 0.6 F +F F +F Area (mm 2 ) Thickness (mm) Ovary wall thickness Ovary wall transverse area B

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140 Fruiting treatment Figure 5 11. Effect of the presence (+F) or absence ( F) of developing fruits on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. A) mesocarp; B) giant cells; C) epidermal tissues; D) endocarp. +F: 2 developing fruits per plant. *, **, ***: Means between fruiting treatments are significantly different at P P P *** 0 5 10 15 20 25 0 50 100 150 200 250 300 350 400 450 Cell number (thousands) Cell size (m 2 ) Cell size Cell number A ** 0.0 0.1 0.2 0.3 0.4 0.5 0 1000 2000 3000 4000 5000 6000 7000 Cell number (thousands) Cell size (m 2 ) B *** 0 1 2 3 4 5 0 50 100 150 200 250 F +F F +F Cell number (thousands) Cell size (m 2 ) C 0.0 0.5 1.0 1.5 2.0 2.5 0 50 100 150 200 250 F +F F +F Cell number (thousands) Cell size (m 2 ) D

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141 Figure 5 12. Median cross section of ovaries in Legionnaire bell pepper flowers developed in fruiting and nonfruiting plants. A, C, E: non fruiting plants; B, D, F: fruiting plants (2 developing fruits per plant) ; A, B: total ovary wall ; C, D: outer ovary wall ; E, F: inner ovary wall ; ep, epidermis; hp, hypodermis; me, mesocarp; vb, vascular bundle; gc, giant cell; en, endocarp; ov, ovule. Note the thicker ovary wall and larger cells in the mesocarp and giant cells in the nonfruiting compared to the fruiting plants. A B ep me hp gc gc vb me me me ep hp D C E F gc gc ov ov 100 m 100 m 25 m 25 m 25 m 25 m en en vb

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142 Days after treatment Figure 5 13. Effect of harvest time on the ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. A) Ovary size, B) Ovary wall thickness and ovary wall transverse area. Means with the sam e letter are not significantly different at P 0.05 (LSD test); n = 4 for 12 DAT n = 16 for 1819 and 38 40 DAT b b a a a a 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 Diameter (mm) Fresh weight (mg) Ovary FW Diameter A ab a b b a b 0 2 4 6 8 10 12 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Area (mm 2 ) Thickness (mm) Ovary wall thickness Ovary wall transverse area B

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143 Days after treatment Figure 5 14. Effect of harvest time on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. A) mesocarp; B) giant cells; C) epidermal tissues; D) endocarp. Means with the same letter are not significantly different at P 0.05 (LSD test); n = 4 for 12 DAT n = 16 for 1819 and 3840 DAT ab a b a a a 0 5 10 15 20 25 0 50 100 150 200 250 300 350 400 450 Cell number (thousands) Cell size (m 2 ) Cell size Cell number A ab a b a a a 0.0 0.1 0.2 0.3 0.4 0.5 0 1000 2000 3000 4000 5000 6000 7000 Cell number (thousands) Cell size (m 2 ) B a a a a a a 0 1 2 3 4 5 0 50 100 150 200 250 Cell number (thousands) Cell size (m 2 ) C a a a a a a 0.0 0.5 1.0 1.5 2.0 0 50 100 150 200 250 Cell number (thousands) Cell size (m 2 ) D

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144 Temperature x fruiting treatment combination Figure 5 15. Interaction of night temperature and presence/absence of fruits on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. Night temperature was either 12 (L) or 20C (H), and plants had either 0 ( ) or 2 developing fruits (+). Means with the same letter are not significantly different at P 0.05 (LSD test); n=8. a c b c a c b c 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 140 160 L L+ H H+ L L+ H H+ Diameter (mm) Fresh weight (mg) Ovary FW Diameter A a b b b a c b c 0 2 4 6 8 10 12 14 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 L L+ H H+ L L+ H H+ Area (mm 2 ) Thickness (mm) Ovary wall thickness Ovary wall transverse area B

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145 Temperature x fruiting treatment combination Figure 5 16. Interaction of night temperature and presence/absence of fruits on ovary wall cell size and cell number in Legionnaire bell pepper flowers harvested at anthesis. A) mesocarp, B) giant cells, C) epidermal tissues, D) endocarp. Night temperature was either 12 (L) or 20C (H), and plants had either 0 ( ) or 2 developing fruits (+). Means with the same letter were not significantly dif ferent at P 0.05 (LSD test); n=8. a bc ab c a b b b 0 5 10 15 20 25 30 0 50 100 150 200 250 300 350 400 450 Cell number (thousands) Cell size (m 2 ) Cell size Cell number A ab abc a c ab ab b a 0.0 0.1 0.2 0.3 0.4 0.5 0 1000 2000 3000 4000 5000 6000 7000 Cell number (thousands) Cell size (m 2 ) B a b b b a a a a 0 1 2 3 4 5 0 50 100 150 200 250 300 L L+ H H+ L L+ H H+ Cell number (thousands) Cell size (m 2 ) C a a a a a b b b 0.0 0.5 1.0 1.5 2.0 2.5 0 50 100 150 200 250 L L+ H H+ L L+ H H+ Cell number (thousands) Cell size (m 2 ) D

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146 Harvest time x f ruiting treatment combination Figure 5 17. Effect of harvest time and presence/absence of developing fruits on ovary size and ovary wall thickness and area in Legionnaire bell pepper flowers harvested at anthesis. Flowers were harvested at 1819 days after treatment (DAT) or 38 40 DAT and plants had either 0 ( ) or 2 developing fruits (+). Means with the same letter are not significantly different at P 0.05 (LSD test); n=8. a c a b a b a b 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 140 Diameter (mm) Fresh weight (mg) Ovary FW Diameter A a b b b a c b c 0 2 4 6 8 10 12 14 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Area (mm 2 ) Thickness (mm) Ovary wall thickness Ovary wall transverse area B + + + + 1819 3840 1819 38 40 DAT DAT DAT DAT + + + + 1819 3840 18 19 3840 DAT DAT DAT DAT

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147 Harvest time x fruiting treatment combination Figure 5 18. Effect of harvest time and presence/absence of developing fruits on ovary wall cell size and cell number in Legi onnaire bell pepper flowers harvested at anthesis. A) mesocarp, B) giant cells, C) epidermal tissues, D) endocarp. Flowers were harvested at 1819 days after treatment (DAT) or 38 40 DAT and plants had either 0 ( ) or 2 developing fruits (+).Means with the same letter are not significantly different at P 0.05 (t test). a b b b ab b a ab 0 5 10 15 20 25 0 50 100 150 200 250 300 350 400 450 500 Cell number (thousands) Cell size (m 2 ) Cell size Cell number A a b b b a a a a 0.0 0.1 0.2 0.3 0.4 0.5 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Cell number (thousands) Cell size (m 2 ) B a b b b ab b a ab 0 1 2 3 4 5 6 0 50 100 150 200 250 300 Cell number (thousands) Cell size (m 2 ) C a a a a a b ab ab 0.0 0.5 1.0 1.5 2.0 2.5 0 50 100 150 200 250 Cell number (thousands) Cell size (m 2 ) D + + + + 1819 3840 1819 38 40 DAT DAT DAT DAT + + + + 1819 3840 1819 38 40 DAT DAT DAT DAT

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148 CHAPTER 6 1. SUMMARY Sweet peppers are an important vegetable crop whose fruits are mostly consumed as fresh produce all year. Cultivated species of peppers were domesticated in tropical and temperate areas and are sensitive to low temperatures (Harlan, 1971; Eshbaugh, 1993) Pepper plants grown under cool night temperatures (<15C) develop deformed flower s, including swollen flowers and ovaries ( i.e. flowers and ovaries that are larger than the ones developed under optimum temperature), resulting in a high percentage of malformed fruits that are not marketable (Mercado et al. 1997c; Pressman et al. 1998a; Pressman et al. 1998b; Aloni et al. 1999) Swollen ovaries also develop under high source:sink ratios in pepper (Aloni et al. 1999) Under both conditions, i.e. low night temperatures and high source:sink ratios, swollen flowers are reported to have increased reducing sugars and starch concentration compared to normal ( not swollen) flowers (Aloni et al. 1999) Although several studies have been done, the relation between assimilate availability and ovary swelling in pepper remains unclear. Furthermore, the anatomical basis for ovary swelling and deformation under these conditions is unknown. In this study the central hypothesis was that both low night temperature and high source:sink ratio increase ovary size and therefore ovary swelling via effects on assimilate availability/accumulation and/or cell size/number. The series of experiments were carried out under growth chamber conditions by controlling night temperature at either 12C (low night temperature, LNT) or 20C (high night temperature, HNT). The experiments also implied continuous leaf pruning to keep plants at two main axis and continuous flower removal. In the first three experiments ( i.e., Experiments 1 and 2 in Chapter 3, and Experiment 1 in Chapter 4) plants did not

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149 bear any developing fruits before and during the duration of the experiment. In two experiments (Experiment 1 and 2 [same as Experiment 2 in Chapter 4] in Chapter 5), 2 fruits were allowed to set and ~610 days after fruit setting (petal fall), fruits were removed in one treatment, while fruits on the other treatment were allowed to develop to maturity The discussion of the results was addressed from a sourcesink point of view. We are aware that plant growth regulators might be masking or enhancing some responses, since the experiments implied continuous leaf and flower pruning, and in some cases, presence or absence of fruits. However, since we did not have any data on hormones, such effects were considered as an integral component of the treatment effect. It has been previously suggested that other factors such as hormones may also influence the devel opment of swollen ovaries (Polowick and Sawhney, 1985; Aloni et al. 1995; Pressman et al. 1998b) one hypothesis is that low night temperature inhibit s the basal auxin transport from the flower bud to other parts of the plant, and then the increased auxi n concentration in the flower favor s ovary growth (Aloni et al. 1995; Pressman et al. 1998b) Presence/absence of fruits may also alter the hormonal balance in the plant. I t has been suggested that growth inhibition of a second flower and therefore a fr uit is controlled by both competition for limited assimilates (Ali and Kelly, 1992; Marcelis and BaanHofman Eijer, 1997) and by dominance due to the production of plant growth regulators by the earlier developed fruit (Marcelis and BaanHofman Eijer, 19 97) Ultimately, from the results of this research, it is concluded that LNT induced ovary swelling in different kinds of sweet pepper (cherry, elongated and blocky bell). Three to

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150 four weeks of continuous low night temperature were required for maximum r esponse, suggesting that flowers must be exposed to LNT soon after flower initiation in order for this response to occur However, the minimum duration of exposure to LNT required to induce this response is unknown, as the present research maintained LNT t hroughout the course of flower development. Fruit removal ( i.e. increasing the source:sink ratio) also increased the incidence of swollen ovaries. Both LNT and fruit removal decreased net CER without reducing plant growth, suggesting that excess availabili ty of current photosynthate may not be the mechanism that results in the increase in swollen ovaries observed under both LNT and high source:sink conditions. HNT combined with high source:sink ratio or LNT combined with low source:sink ratio can overcome t he detrimental effects of low night temperature or high source:sink ratio on ovary swelling in pepper. Finally, although ovary swelling is induced by both LNT and by absence of fruits t he mechanisms for the increase in ovary size, however, appear to be different. LNT significantly increased pericarp thickness and transverse area, with only slight increases in cell size and number. LNT also increased ovary wall reducing sugar and starch concentration. Absence of fruits increased floral ovary size mainly through increased cell size, with no effect on increasing ovary reducing sugar or starch concentration.

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151 APPENDIX ADDITIONAL TABLES AND FIGURES A) Tables Table A 1 P values for temperature (Temp), cultivar (CV), and days after treatment (harvest) main effects and their interactions. Branch within each cultivar was also considered. Number of days after anthesis (DAA) was used as covariate, and the subject for repeated measures was branch within each plant. (Chapter 3) Source of variation DF Fresh weight (mg) Volume (mm3) Diameter (mm) Length (mm) Ovary wall thickness (mm) Length: diam. ratio FW : Vol. ratio Temp 1 0.008 0.002 0.003 <0.001 0.004 0.59 <0.001 CV 5 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Temp x CV 5 0.03 0.03 0.03 0.007 0.19 <0.001 0.96 Harvest 4 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Temp x Harvest 4 <0.001 <0.001 <0.001 <0.001 0.009 0.29 0.84 CV x Harvest 20 <0.001 <0.001 <0.001 0.19 <0.001 0.01 0.006 Temp x CV x Harvest 20 0.003 0.004 0.07 0.008 0.43 0.05 0.49 Branch(CV) 6 0.03 0.03 0.05 0.07 0.21 0.66 0.39 DAA 1 <0.001 <0.001 <0.001 <0.001 <0.001 0.17 <0.001 Branch(Plant) <0.001 <0.001 <0.001 0.004 0.02 0.02 0.88 Table A 2 P values for ovary, ovary wall (OW) and placenta fresh weight, ovary wall fresh weight: ovary fresh weight ratio, ovary diameter and length, ovary length/diameter ratio, and ovary wall thickness.z FW (mg) OW FW: Diameter Length Length: OW Source of variation Ovary OW Placenta ovary FW (mm) (mm) diameter ratio thickness (mm) Temperature 0.01 0.01 0.01 0.36 0.010 0.007 0.31 0.02 Cultivar (CV) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 < 0.001 Temp*CV 0.001 0.01 <0.001 0.04 0.004 0.34 <0.001 0.29 Branch <0.001 <0.001 <0.001 0.03 <0.001 <0.001 0.17 0.005 Node <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.88 <0.001 Node*Branch 0.002 0.02 0.001 0.99 0.01 0.10 0.28 0.21 Temp*Node < 0.001 <0.001 <0.001 0.92 <0.001 <0.001 0.62 <0.001 CV*Branch <0.001 <0.001 <0.001 0.32 <0.001 0.06 0.05 0.009 CV*Node <0.001 0.01 <0.001 <0.001 <0.001 0.007 0.01 0.37 CV*Node*Branch 0.03 0.01 0.05 0.07 0.07 0.11 0.37 0.41 Temp*CV*Branch 0.04 0.05 0.05 0.07 0.11 0.56 0.24 0.46 Temp*CV*Node <0.001 <0.001 0.003 0.04 <0.001 0.05 <0.001 0.28 z Factorial experiment with two night temperatures and four cultivars. Flowers were harvested at anthesis from nodes 2 to 7.

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152 Table A 3 Number of days after beginning of temperature treatments at which flowers from nodes 2 to 7 were harvested in four cultivars of sweet pepper. Means S.E. (Chapter 3) Night Node number z Cultivar temp (C) 2 3 4 5 6 7 Ariane 12 4.50.5 11.50.8 20.71.5 28.61.0 35.10.9 40.40.8 20 4.90.5 11.30.8 18.41.6 25.91.4 31.91.1 36.71.6 Banana 12 6.30.7 13.70.6 20.61.0 26.30.7 30.80.6 35.60.7 Supreme 20 5.20.8 11.31.5 14.61.1 21.71.9 26.92.0 39.05.0 Legionnaire 12 8.50.8 16.90.9 24.61.1 31.61.1 36.60.8 42.40.7 20 5.40.3 12.70.9 25.82.0 31.21.1 37.71.0 41.41.4 Red Cherry 12 1.50.2 7.40.6 14.31.1 19.91.1 27.01.1 32.11.0 Sweet 20 2.20.3 6.10.5 11.11.0 15.70.7 22.70.9 27.60.7 zNodes counted above the first branching node, i.e. flower from sympodial unit 1 was node 1. Table A 4 Effect of the interaction between days after treatment began (DAT) with night temperature and cultivar on the intercellular CO2 (Ci, mol mol1) of sweet pepper. Gas exchange was measured over a 45day period after the beginning of night temperature treatmentsz. (Chapter 4) Temperature y Cultivar x DAT 12C 20C P values LEG RSC P values 0 296 278 0.05 291 283 0.35 3 274 255 0.02 267 262 0.50 6 206 185 0.02 205 186 0.03 9 217 230 0.12 222 225 0.71 12 238 242 0.67 245 236 0.28 15 249 249 0.99 244 254 0.24 19 263 280 0.05 277 267 0.23 21 208 265 <0.001 238 235 0.74 24 222 247 0.004 238 231 0.45 27 233 248 0.09 239 242 0.67 30 262 266 0.71 264 264 0.94 33 222 222 0.99 220 224 0.64 36 208 217 0.31 206 219 0.12 45 229 220 0.28 221 227 0.45 zGas exchange was measured over a 45day period after the beginning of night temperature treatments. yData were averaged across cultivars. n = 18. xData were averaged across temperature treatments. n = 18.

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153 Table A 5 P values for ovary fresh weight, diameter and length, ovary length/diameter ratio, and ov ary wall thickness for a 2 factor experiment (2 night temperature regimes, 2 levels of fruits) repeated over time (11 harvest intervals of 4 days each). (Chapter 5) Effect Fresh weight Diameter Length Temperature (Temp) <0.001 <0.001 <0.001 Fruits < 0.001 <0.001 <0.001 Temp*Fruits 0.02 0.08 0.08 Harvest interval (HI) <0.001 <0.001 <0.001 Temp*HI 0.04 0.36 0.76 Fruits*HI <0.001 <0.001 <0.001 Temp*Fruits*HI 0.57 0.16 0.20 Branch <0.001 <0.001 <0.001 DAA <0.001 <0.001 <0.001 z Pooled data from two experiments. Experiments were conducted with a factorial design (two night temperatures and presence/absence of developing fruits). Flowers were harvested at anthesis for 42 days in Experiment 1 and 38 days in experiment 2, and grouped in 11 harvest intervals of 4 days.

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154 Table A 6 P values for ovary characteristics, ovary wall thickness and ovary wall transverse area, and thickness, area, cell number and cell size for mesocarp, giant cells, epidermal tissues and endocarp for a 3 factor experiment (2 night temperature regimes, 2 levels of fruits and 2 harvest times ) (Chapter 5) Sources of variation z Tissues and parameters T F T x F HT HT x F HT x T HT x T x F Block Ovary FW 0.001 0.001 0.001 0.02 0.09 0.83 0.73 0.58 Diameter 0.01 0.001 0.02 0.58 0.05 0.63 0.43 0.23 Length 0.05 0.001 0.001 0.01 0.05 0.90 0.80 0.93 Ovary wall Thickness 0.05 0.001 0.14 0.01 0.02 0.06 0.74 0.60 Total area 0.01 0.001 0.03 0.03 0.01 0.08 0.77 0.56 Mesocarp Thickness 0.04 0.01 0.04 0.05 0.04 0.01 0.40 0.62 Total area 0.01 0.001 0.01 0.11 0.02 0.03 0.48 0.65 Cell number 0.28 0.11 0.14 0.24 0.90 0.13 0.32 0.84 Cell size 0.23 0.01 0.65 0.01 0.04 0.63 0.43 0.74 Giant cells Thickness 0.35 0.01 0.55 0.01 0.06 0.95 0.62 0.56 Total area 0.14 0.001 0.98 0.01 0.02 0.98 0.62 0.38 Cell number 0.79 0.55 0.03 0.29 0.83 0.26 0.75 0.70 Cell size 0.64 0.01 0.07 0.01 0.18 0.97 0.23 0.60 Epidermal Thickness 0.28 0.04 0.83 0.23 0.23 0.73 0.41 0.93 tissues Total area 0.04 0.001 0.40 0.37 0.09 0.91 0.38 0.73 Cell number 0.61 0.09 0.81 0.04 0.69 0.28 0.83 0.96 Cell size 0.05 0.001 0.26 0.01 0.02 0.29 0.68 0.53 Endocarp Thickness 0.29 0.14 0.20 0.31 0.53 0.74 0.26 0.97 Total area 0.06 0.01 0.06 0.64 0.33 0.95 0.20 0.67 Cell number 0.12 0.03 0.09 0.92 0.39 0.41 0.89 0.34 Cell size 0.60 0.25 0.89 0.28 1.00 0.15 0.12 0.77 z T, temperature; F, non fruiting/fruiting treatment; HT harvest time

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155 Table A 7 P values for carbohydrate concentration and content.z (Chapter 5) Ovary wall Placenta Parameter Temp Fruits T x F Temp Fruits T x F Dry weight 0.06 0.001 0.16 0.06 0.001 0.16 Concentration Glucose 0.01 0.95 0.09 0.73 0.57 0.24 Fructose 0.02 0.39 0.22 0.19 0.77 0.30 Sucrose 0.04 0.86 0.84 0.19 0.04 0.12 Starch 0.17 0.33 0.69 0.29 0.29 0.65 Reducing sugars 0.01 0.64 0.11 0.36 0.89 0.24 Red. Sugars + starch 0.03 0.50 0.76 0.25 0.55 0.72 Content Glucose 0.03 0.06 0.10 0.11 0.00 0.05 Fructose 0.001 0.01 0.09 0.02 0.00 0.02 Sucrose 0.30 0.04 0.57 0.66 0.03 0.85 Starch 0.02 0.40 0.69 0.05 0.04 0.38 Reducing sugars 0.002 0.03 0.09 0.05 0.00 0.03 Red. Sugars + starch 0.005 0.13 0.31 0.03 0.01 0.09 zFlowers were harvested 24 to 29 days after treatments started.

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156 B) Figures Figure A 1 Distribution of harvested nodes per harvest interval. Nodes were counted from the first harvested node on every stem. n = 160, 207, 211, 233, 273 for harvest intervals I II, III IV and V respectively. (Chapter 3) 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 9 10 11 12 Frequency Relative node I II III IV V Harvest interval

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157 Days after treatment Fig ure A 2 Effect of night temperature and days after treatment on ovary diameter (A) and length (B) of flowers at anthesis in sweet pepper Legionnaire. Day temperature was set to 22C. Pooled data of two experiments. Flowers were continuously harvested during 42 days in Experiment 1, and 38 days in Experiment 2. Means were averaged for each combination across presence/absence of fruits treatments ; n = 26 to 32 for 1 4 and 4144 DAT and 35 to 8 6 for the remaining intervals *, **, ***: Significant differences between temperature regimes within each harvest interval at P 0.05, 0.01 and 0.001 respectively. (Chapter 5). *** *** *** *** *** *** *** *** ** 5.0 5.5 6.0 6.5 7.0 7.5 1 4 5 8 9 12 13 16 17 20 21 24 25 28 29 32 33 36 37 40 41 44 Diameter (mm) 12 20 0.0 ** *** ** ** *** *** *** ** ** 3.5 4.0 4.5 5.0 5.5 Length (mm) 0.0 A B

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158 Days after treatment Figure A 3 Effect of night temperature, presence/a bsence of fruits and days after treatment on ovary fresh weight (A) diameter (B) and length (C) of flowers at anthesis in sweet pepper Legionnaire. Day temperature was set to 22C. Pooled data of two experiments. Flowers were continuously harvested during 42 days in Experiment 1, and 38 days in Experiment 2. Means were averaged for each combination across presence/absence of fruits treatments. n = 5 to15 for intervals 2 and 11, and from 14 to 48 for the remaining intervals. *, **, ***: Significant differ ences between temperature regimes within each harvest interval at P 0.05, 0.01 and 0.001 respectively. (Chapter 5) 60 80 100 120 140 160 180 200 Fresh weight (mg) 12 C, non fruiting plants 12 C, fruiting plants 20 C, non fruiting plants 20 C, fruiting plants 0 A 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Diameter (mm) 0.0 B 3.5 4.0 4.5 5.0 5.5 Length (mm) 0.0 C

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176 BIOGRAPHICAL SKETCH Nicacio Cruz Huerta was born in Mexico. He is married and has a daughter. Being raised in a family of farmers, his interest in plants started in his childhood. His formal training on agriculture began in the middle and high school. He received his BS degree in plant sciences from the Universidad Autnoma Chapingo (Chapingo Autonomous University), and his M Sc degree i n plant physiology from the Colegio de Postgraduados ( Post graduate College in Agricultural Sciences). He worked in plant physiology, with special focus on gas exchange, source sink relationships, plant growth analysis, and plant growth modeling. He was al so the teaching assistant during several years in the graduate course Crop Physiology Later, he obtained a scholarship from the Mexican National Council for Science and Technology to start his PhD program in the Horticultural Sciences Department at the University of Florida. His research focused on studying how low night temperatures and source and sink ratios affect flower development in bell pepper. After graduation, he plans to continue working on physiology, production techniques, and modeling of hor ticultural crops.