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Influence of Water Stress, Nonstructural Carbohydrates and Free Amino Acids on Control of Root and Shoot Growth of Ligus...

Permanent Link: http://ufdc.ufl.edu/UFE0041868/00001

Material Information

Title: Influence of Water Stress, Nonstructural Carbohydrates and Free Amino Acids on Control of Root and Shoot Growth of Ligustrum Japonicum Thunb.
Physical Description: 1 online resource (161 p.)
Language: english
Creator: Silva, Dilma
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: acids, amino, architecture, carbohydrates, control, free, growth, japonicum, ligustrum, nonstructural, ratio, rhizotron, root, shoot, stress, water
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: 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 INFLUENCE OF WATER STRESS, NONSTRUCTURAL CARBOHYDRATES AND FREE AMINO ACIDS ON CONTROL OF ROOT AND SHOOT GROWTH OF Ligustrum japonicum Thunb. By Dilma Daniela Silva August 2010 Chair: Richard C. Beeson, Jr. Major: Horticultural Science Environmental Horticulture A more profound knowledge of the factors affecting root and shoot post-transplant growth would enable more conscientious decisions on plant management and on practices to be adopted. The experimental system required to impose precise drought treatments, and to make possible observation of natural growth of Ligustrum japonicum Thunb. under favorable conditions was developed. Influence of different intensities and duration of water stress on plant growth was tested. Plant architecture was described for undisturbed conditions with and without moderate water stress. Interactions between labile forms of carbon and nitrogen within different meristem tissues, and control of growth initiation or cessation was studied in a whole plant approach. The final rhizotron designed allowed exceptional root observation, provided a near-uniform profile of soil moisture, and was easily manageable for precise long-term data acquisition. This rhizotron had eight independent viewing/sampling windows and held 0.16 m3 of soil. An electric powered root separator was developed that sped sample preparation for root dry mass determination with a capacity of 40 L of container substrate or 32 kg of sandy soil. No water was required and a four-fold reduction in total processing time was achieved with a > 98% root mass recovery. Excessive water availability resulted in marked reductions of carbon allocation towards roots, consequently luxury shoot growth was observed. Constant or intermittent moderate water stress resulted in total biomass reductions of approximately 20%, which disproportionally reduced shoot mass, particularly diminishing leaf number and size. Prolonged, severe water stress resulted in a 40% reduction of total biomass. This reduction was also disproportionally greater for shoot biomass production, with smaller reductions in root biomass production. Plant responses to water stress differed with time of exposure and degree of stress. Low correlation of root-to-shoot ratio and irrigation frequency suggested that root-to-shoot ratio may not be the best indicator of water status during the plant growing period. Following transplant into rhizotrons, root growth began before shoot growth and the first flush of shoot growth was mostly basipetal. Water stress altered shoot architecture by enhancing apical dominance. As plants adapted to the stress imposed, indeterminate growth was triggered more often in meristematic regions of terminal buds. At the second flush, which occurred later in the stress treatment, old buds expanded more frequently than the newly formed apex lateral bud. Temporal variations of moisture caused by wetting and drying cycles resulted in continuous growth for portions of the root system, with quiescent periods observed for shoot growth. Conversely, continuous high moisture levels resulted in roots exhibiting quiescent periods in some plants. Patterns of shoot and root growth varied considerably between these clonal plants, which may be an important consideration for analyses of populations of woody plants. Free amino acid levels at the shoot tip were more decisive for initiation of meristem growth or quiescence than the quantity of total nonstructural carbohydrates or nonstructural carbohydrate-to-free amino acid ratio. In roots, this ratio was a good predictor of root growth. Valine, leucine, tyrosine, cysteine, metionine, and arginine increased significantly with bud set, compared with growing shoot tips. Root tips contained abundant fructose, stachyose, and myoinositol. Mannitol was the major transport sugar and glutamine, valine and histidine were the main free amino acids transported in xylem fluid. Water stress resulted in increases in the concentration of some amino acids in growing shoo tips, such as arginine, valine, and histidine, and especially valine in developing buds at the beginning of root flush. The results observed from these experiments propose important considerations for woody plant management. Factors such as pruning, frequency of irrigation, and timing of fertilization and transplant can affect decisions for optimal plant growth. Some examples are the fact that water stress can increase apical dominance; thus, increasing the necessity for pruning, if the intent is a plant to be used as a bush rather than as a small tree. Well-irrigated plants have basipetal branching in the beginning of the growing season, which can also affect pruning decisions. Wetting and drying cycles result in continuous growth of portions of the root system; thus, establishment can be beneficiated by temporal variations of soil moisture. Fertilization during the growing period previous to transplant into the landscape is an important factor for buildup of internal reserves to support initial root growth and bud burst in the beginning of the growing season. These observations suggest that woody plant management can benefit from appropriate decision making of pruning, frequency of irrigation, and timing of fertilization and transplant.
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 Dilma Silva.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Beeson, Richard C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041868:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041868/00001

Material Information

Title: Influence of Water Stress, Nonstructural Carbohydrates and Free Amino Acids on Control of Root and Shoot Growth of Ligustrum Japonicum Thunb.
Physical Description: 1 online resource (161 p.)
Language: english
Creator: Silva, Dilma
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: acids, amino, architecture, carbohydrates, control, free, growth, japonicum, ligustrum, nonstructural, ratio, rhizotron, root, shoot, stress, water
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: 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 INFLUENCE OF WATER STRESS, NONSTRUCTURAL CARBOHYDRATES AND FREE AMINO ACIDS ON CONTROL OF ROOT AND SHOOT GROWTH OF Ligustrum japonicum Thunb. By Dilma Daniela Silva August 2010 Chair: Richard C. Beeson, Jr. Major: Horticultural Science Environmental Horticulture A more profound knowledge of the factors affecting root and shoot post-transplant growth would enable more conscientious decisions on plant management and on practices to be adopted. The experimental system required to impose precise drought treatments, and to make possible observation of natural growth of Ligustrum japonicum Thunb. under favorable conditions was developed. Influence of different intensities and duration of water stress on plant growth was tested. Plant architecture was described for undisturbed conditions with and without moderate water stress. Interactions between labile forms of carbon and nitrogen within different meristem tissues, and control of growth initiation or cessation was studied in a whole plant approach. The final rhizotron designed allowed exceptional root observation, provided a near-uniform profile of soil moisture, and was easily manageable for precise long-term data acquisition. This rhizotron had eight independent viewing/sampling windows and held 0.16 m3 of soil. An electric powered root separator was developed that sped sample preparation for root dry mass determination with a capacity of 40 L of container substrate or 32 kg of sandy soil. No water was required and a four-fold reduction in total processing time was achieved with a > 98% root mass recovery. Excessive water availability resulted in marked reductions of carbon allocation towards roots, consequently luxury shoot growth was observed. Constant or intermittent moderate water stress resulted in total biomass reductions of approximately 20%, which disproportionally reduced shoot mass, particularly diminishing leaf number and size. Prolonged, severe water stress resulted in a 40% reduction of total biomass. This reduction was also disproportionally greater for shoot biomass production, with smaller reductions in root biomass production. Plant responses to water stress differed with time of exposure and degree of stress. Low correlation of root-to-shoot ratio and irrigation frequency suggested that root-to-shoot ratio may not be the best indicator of water status during the plant growing period. Following transplant into rhizotrons, root growth began before shoot growth and the first flush of shoot growth was mostly basipetal. Water stress altered shoot architecture by enhancing apical dominance. As plants adapted to the stress imposed, indeterminate growth was triggered more often in meristematic regions of terminal buds. At the second flush, which occurred later in the stress treatment, old buds expanded more frequently than the newly formed apex lateral bud. Temporal variations of moisture caused by wetting and drying cycles resulted in continuous growth for portions of the root system, with quiescent periods observed for shoot growth. Conversely, continuous high moisture levels resulted in roots exhibiting quiescent periods in some plants. Patterns of shoot and root growth varied considerably between these clonal plants, which may be an important consideration for analyses of populations of woody plants. Free amino acid levels at the shoot tip were more decisive for initiation of meristem growth or quiescence than the quantity of total nonstructural carbohydrates or nonstructural carbohydrate-to-free amino acid ratio. In roots, this ratio was a good predictor of root growth. Valine, leucine, tyrosine, cysteine, metionine, and arginine increased significantly with bud set, compared with growing shoot tips. Root tips contained abundant fructose, stachyose, and myoinositol. Mannitol was the major transport sugar and glutamine, valine and histidine were the main free amino acids transported in xylem fluid. Water stress resulted in increases in the concentration of some amino acids in growing shoo tips, such as arginine, valine, and histidine, and especially valine in developing buds at the beginning of root flush. The results observed from these experiments propose important considerations for woody plant management. Factors such as pruning, frequency of irrigation, and timing of fertilization and transplant can affect decisions for optimal plant growth. Some examples are the fact that water stress can increase apical dominance; thus, increasing the necessity for pruning, if the intent is a plant to be used as a bush rather than as a small tree. Well-irrigated plants have basipetal branching in the beginning of the growing season, which can also affect pruning decisions. Wetting and drying cycles result in continuous growth of portions of the root system; thus, establishment can be beneficiated by temporal variations of soil moisture. Fertilization during the growing period previous to transplant into the landscape is an important factor for buildup of internal reserves to support initial root growth and bud burst in the beginning of the growing season. These observations suggest that woody plant management can benefit from appropriate decision making of pruning, frequency of irrigation, and timing of fertilization and transplant.
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 Dilma Silva.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Beeson, Richard C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041868:00001


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INFLUENCE OF WATER STRESS, NONSTRUCTURAL CARBOHYDRATES AND
FREE AMINO ACIDS ON CONTROL OF ROOT AND SHOOT GROWTH OF Ligustrum
japonicum Thunb.


















By

DILMA DANIELA SILVA


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 Dilma Daniela Silva

































To my mom, Guiomar, and to my son, Nicolas









ACKNOWLEDGMENTS

Successful completion of this work would not have been possible without the

support, encouragement and assistance of many people.

First and foremost, I would like to take the opportunity to convey my sincere

appreciation to Dr. Richard C Beeson Jr, my advisor and committee chair, for believing

on my potential and allowing me the opportunity to pursue a dream. His many hours of

guidance are appreciated, especially for the many hours Dr Beeson endured reading

and editing this dissertation.

Second, I would like to thank my committee members Drs. Karen E Koch, Michael

E Kane, Peter C Andersen, and Timothy A Martin for their guidance, expertise,

constructive suggestions, and review of the manuscripts. All are greatly appreciated.

Each member's contributions were unique, teaching me the importance of utilizing

diverse perspectives, all of which substantially improved the finished result.

I am grateful to Christopher Fooshee and Heidi Savage for their thoughts,

contributions and reviews to this dissertation. Special thanks also goes to those who

shared some of the many hours I spent building rhizotrons, planting, collecting data,

washing roots or HPLC-fighting: Brian Pearson, Donald Cox, Edward Tillman, Fabieli

Lanes, and Julie Brooks.

I will always have tender memories of my time at MREC. I would also like to thank

Ana Willianms, Angelica Cretu, Eliandro Costa, Gregory Alexander, Mary Risner, and

Patricia Ramos for their friendship and encouragement.









TABLE OF CONTENTS

page

A C K N O W LE D G M E N T S ......... ......................... ......... ......................................... 4

LIST O F TA BLES ............... ........ ................................................... ...... ........ 7

L IS T O F F IG U R E S .......................................................................................................... 9

ABSTRACT ........... ...... ..... .................................... 12

CHAPTERS

1 INTRODUCTION ...................................................................... ......... .......... ......... 16

Partitioning of Photosynthates ........................................................................... 16
Episodic G row th ................................. .. ..... .... ....................... 18
Induction and Control of Shoot and Root Growth .................... .................. 20
Root-to-Shoot Ratio .... .................................................................................. 21
Signaling System s in Plants..................... ....................... .................... 24

2 AN ELEVATED LARGE-VOLUME RHIZOTRON FOR EVALUATING ROOT
GROWTH UNDER NATURAL SOIL MOISTURE CONDITIONS ............................ 28

In tro d u c tio n .................................................................................................... 2 8
M material and M ethods ...................... ........ ........................ .. 29
Stage 1 Designs for containment and observation ports .............. ............... 29
Stage 2 Designs for drainage systems allowing natural-soil profiles ............. 31
Results and Discussion.......................................... ............... 33

3 DEVELOPMENT AND EVALUATION OF A ROTARY ROOT SEPARATOR......... 46

In tro d u c tio n .................................................................................................... 4 6
M material and M methods ................................... ............. ..... ............... 47
Results and Discussion.......................................... ............... 49

4 EFFECTS OF WATER STRESS ON PLANT GROWTH OF Ligustrum
japonicum Thunb. .................................................. 53

In tro d u c tio n ................................................... ....................................... 5 3
M material and M methods ...................................... ............. ..... ............... 55
E x pe rim e nt 1 ............... ................... ...................................................... .. 5 5
E x pe rim e nt 2 ............... ................... ...................................................... 5 7
E x p e rim e nt 3 .................................................. 5 8
Results and Discussion.......................................... ............... 60
Soil moisture management............................ ......... 60
E x pe rim e nt 1 ............... ................... ...................................................... .. 6 2









Experiment 2 .................. ......... .......... ......... 63
Experiment 3 ................ ......... .......... ......... 66

5 DEVELOPMENT OF SHOOT ARCHITECTURE OF Ligustrumjaponicum
Thunb. IN RESPONSE TO SOIL MOISTURE. ................................................ 82

Introduction .................................................................................................. ........ 82
Material and Methods ........................................ ......... ..... .. ........... 84
Growth conditions and experimental design............................ .... ........... 84
G row th m easurem ents ................................... ...................... .... ........... 85
Results and Discussion................. .... .. ..... ....................... 86
Growth under well-irrigated conditions (2 day wetting and drying cycles) ........ 86
Effect of intermittent water stress on growth ............... ................................... 90
Root and shoot growth patterns ................................................. ............... 93
C conclusions ................................................................ .. ..... ......... 97

6 PATTERNS OF FREE AMINO ACIDS AND NON-STRUCTURAL
CARBOHYDRATES ASSOCIATED WITH EPISODICAL GROWTH OF
Ligustrum japonicum Thunb .............................................................. ............... 112

Introduction ................................................................ ... .... ... ..... 112
M materials and M ethods...................................................................... 115
Growth conditions and experimental design............................................... 115
G row th m easurem ents ............................................................ ...... ....... 116
B iochem ical analyses ...................... ....... ......... .. ............................ 116
R e su lts a nd D iscussio n ................................ ... ... .................................. 12 1
Patterns of faa and tnc under well-irrigated conditions.............. ........... 121
Influence of water stress in patterns of faa and tnc ............... ................. 125
C o n c lus io n s ............. ......... .. .. ......... .. .. ......... ................................ 12 7

7 SUMMARY AND CONCLUSIONS................ ............................ 137

APPENDIX DIFFERENCES BETWEEN METABOLITES ANALYZED IN
C H A P T E R 6 ................................................................................................ 1 4 1

LIST OF REFERENCES .. ................................. ........................................... 146

BIOGRAPHICAL SKETCH .................................................. 161









LIST OF TABLES


Table page

3-1 Root ball volume and substrate/soil of root samples. .................................. 51

3-2 Dry mass of root samples and yield achieved using the rotary root separator. .. 51

4-1 Mean percent volumetric water content (%VWC) achieved in Experiments 1,
2 and 3 determined after recalculation using equations based on prolonged
dual measurement of VWC using Digital TDT and ECH20 probes..................... 73

4-2 Experiment 1. Dry mass (g) of components of L. japonicum grown at different
constant volumetric water contents in rhizotrons. Moisture probes remained
near original roots ball for the duration of the experiment. Masses between
treatments were pooled within harvest. ........... .................... .......... ........ 73

4-3 Experiment 1. Leaf area, allometric relationships and percentage of
allocation in L. japonicum grown in rhizotrons at constant volumetric water
contents. Results between treatments were pooled within harvest. ................ 74

4-4 Experiment 2. Dry mass (g) of components of L. japonicum grown in
rhizotrons at constant volumetric water contents .................... ....... ............ 74

4-5 Experiment 2. Leaf area, allometric relationships and percentage of
allocation in L. japonicum grown in rhizotrons at constant volumetric water
contents ............. .......... ....................................................... 75

4-6 Experiment 3. Dry mass (g) of components of L. japonicum grown in
rhizotrons at variable volumetric water content......................... ........ ........... 76

4-7 Experiment 3. Leaf area, allometric relationships and percentage of
allocation in L. japonicum grown in rhizotrons at variable volumetric water
content ............. .. ..... .................... ......... ........... ............77

5-1 Percentages of bud outgrowth and dormancy of neoformed buds on
branches expanded during 100 days of undisturbed growth by L. japonicum
grown in large rhizotrons in 2009............................ ................. .. ............... 99

5-2 Number of growing points recorded during 100 days of undisturbed growth of
L. japonicum grown in large rhizotrons in 2009. .............................. 100

5-3 New branch distribution of L. japonicum grown in large rhizotrons in 2009,
during 100 days of undisturbed growth......... ...... ....... .... ............... 101

5-4 Single stem and internode length (cm) of new branches produced during 100
days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009. 102









5-5 Total length (cm) of new branches produced during 100 days of undisturbed
growth of L. japonicum grown in large rhizotrons in 2009.............................. 102

5-6 Number of stems and leaves per stem per plant produced during 100 days of
undisturbed growth of L. japonicum grown in large rhizotrons in 2009............. 103

A-1 Differences between metabolites analyzed as 2 x 2 x 5 factorial, with
irrigation frequency, harvest and tissue as treatments described in Chapter 6. 141









LIST OF FIGURES


Figure page

2-1 Prototype rhizotron with Ligustrum japonicum 2 months after transplanting
into the rhizotron............. .... ....... .. ......... ............. ........ ....... 40

2-2 Drainage systems tested on rhizotrons, ......... .... ........... ....... ...... ... 41

2-3 Schematic diagram of a rhizotron.............. ........... ................ .. ............... 42

2-4 One-year-old Ligustrumjaponicum transplanted in a rhizotron, window open
just for dem onstration. ............ ......... ....... ..................................... 43

2-5 Temperature of air and substrate immediately next to single PVC door
(substrate: average of four thermocouples, and air: average of two
therm couples) ................................. ................................ .............. 43

2-6 Camera positioning frame used to take pictures mounted on rhizotron.............. 44

2-7 Percent volumetric water content (% VWC) observed during drainage trial
(average of actual values for 3 repetitions)......... ........... ......... ....... .. 45

3-1 A) Root separator. B) Detail showing internal basket. C) Detail showing
reducing transm mission and m otor ............... .................................................. 52

4-1 Percent contribution of each plant component to the total dry mass of
L. japonicum grown under different substrate moisture levels............................ 78

4-2 Daily stomatal conductance, Dgs (A) and shoot water potential, /T (B) of
L. japonicum grown under irrigation varying between saturation and 19.8 and
22.3% VWC for moderartely-stressed and well-irrigated plants respectively
on the day of minimum and maximum water stress in Exp. 3 .......................... 79

4-3 Correlation among root and shoot dry mass ................ ............. ............... 80

4-4 Correlation for Exp. 2 among A%VWC (differential between saturation and
the triggering volumetric water content) and (A) root dry mass, (B) shoot dry
mass, (C) leaf dry mass, and (D) leaf area. ......... .. ....................... .............. 81

5-1 Organs of L. japonicum at different growth stages. ....................................... 104

5-2 Diagram showing branch orders and bud positions of L. japonicum............... 105

5-3 Different organs of L. japonicum ................................................................... 106









5-4 Number of new leaves and leaf area of L. japonicum at variable volumetric
water content (2 day irrigation cycle, well-irrigated plants and 7 day irrigation
cycles, moderately-stressed plants) for 2009 plants................ ............. ... 107

5-5 Growth patterns of L. japonicum grown in rhizotrons at constant high
substrate volumetric water content (well-irrigated plants) in 2008 .................. 108

5-6 Growth patterns of L. japonicum grown in rhizotrons at variable volumetric
water content (2 day irrigation cycle, WP) for 2009. .................................. 109

5-7 Growth patterns of L. japonicum grown in rhizotrons at severe stress, left
column, and moderate stress, right column (severely-stressed and stressed
plants, respectively) in 2008. ... ................ ................. ............... 110

5-8 Growth patterns of L. japonicum grown in rhizotrons at variable volumetric
water content (7 day irrigation cycle, stressed plants) for 2009..................... 111

6-1 Total nonstructural carbohydrates, free amino acids, and total nonstructural
carbohydrate-to-free amino acid ratio of L. japonicum shoot tissues of plants
grown in rhizotrons at variable volumetric water content (2 or 7 day irrigation
cycles, well-irrigated and moderately-stressed plants, respectively). ............. 129

6-2 Free amino acids and nonstructural carbohydrates of L. japonicum shoot
tissues of plants grown in rhizotrons at variable volumetric water content (2
day irrigation cycles, well-irrigated plants). ...... ............... ......................... 130

6-3 Total nonstructural carbohydrates, free amino acids, and total nonstructural
carbohydrate-to-free amino acid ratio of L. japonicum root tissues of plants
grown in rhizotrons at variable volumetric water content (2 or 7 day irrigation
cycles, well-irrigated and moderately-stressed plants, respectively). ............. 131

6-4 Free amino acids and nonstructural carbohydrates of L. japonicum root
tissues of plants grown in rhizotrons at variable volumetric water content (2
day irrigation cycles, well-irrigated plants). ...... ............... ......................... 132

6-5 Free amino acids and nonstructural carbohydrates of L. japonicum xylem
fluid extracted from plants grown in rhizotrons at variable volumetric water
content (2 or 7 day irrigation cycles, well-irrigated plants or moderately-
stressed plants, respectively). ............... .......... ................................... 133

6-6 Total nonstructural carbohydrates, free amino acids, and total nonstructural
carbohydrate-to-free amino acid ratio of L. japonicum xylem fluid extracted
from plants grown in rhizotrons at variable volumetric water content (2 or 7
day irrigation cycles, well-irrigated plants and moderately-stressed plants,
respectively). ............. ... ............................. ... .................. 134









6-7 Free amino acids and nonstructural carbohydrates of L. japonicum shoot
tissues grown in rhizotrons at variable volumetric water content (7 day
irrigation cycles, moderately-stressed plants)......................... ........ ........... 135

6-8 Free amino acids and nonstructural carbohydrates of L. japonicum root
tissues grown in rhizotrons at variable volumetric water content (7 day
irrigation cycles, moderately-stressed plants)......................... ........ ........... 136









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

INFLUENCE OF WATER STRESS, NONSTRUCTURAL CARBOHYDRATES AND
FREE AMINO ACIDS ON CONTROL OF ROOT AND SHOOT GROWTH OF Ligustrum
japonicum Thunb.

By

Dilma Daniela Silva

August 2010

Chair: Richard C. Beeson, Jr.
Major: Horticultural Science Environmental Horticulture

A more profound knowledge of the factors affecting root and shoot post-transplant

growth would enable more conscientious decisions on plant management and on

practices to be adopted. The experimental system required to impose precise drought

treatments, and to make possible observation of natural growth of Ligustrumjaponicum

Thunb. under favorable conditions was developed. Influence of different intensities and

duration of water stress on plant growth was tested. Plant architecture was described

for undisturbed conditions with and without moderate water stress. Interactions between

labile forms of carbon and nitrogen within different meristem tissues, and control of

growth initiation or cessation was studied in a whole plant approach.

The final rhizotron designed allowed exceptional root observation, provided a

near-uniform profile of soil moisture, and was easily manageable for precise long-term

data acquisition. This rhizotron had eight independent viewing/sampling windows and

held 0.16 m3 of soil. An electric powered root separator was developed that sped

sample preparation for root dry mass determination with a capacity of 40 L of container









substrate or 32 kg of sandy soil. No water was required and a four-fold reduction in total

processing time was achieved with a >98% root mass recovery.

Excessive water availability resulted in marked reductions of carbon allocation

towards roots, consequently luxury shoot growth was observed. Constant or intermittent

moderate water stress resulted in total biomass reductions of approximately 20%, which

disproportionally reduced shoot mass, particularly diminishing leaf number and size.

Prolonged, severe water stress resulted in a 40% reduction of total biomass. This

reduction was also disproportionally greater for shoot biomass production, with smaller

reductions in root biomass production. Plant responses to water stress differed with time

of exposure and degree of stress. Low correlation of root-to-shoot ratio and irrigation

frequency suggested that root-to-shoot ratio may not be the best indicator of water

status during the plant growing period.

Following transplant into rhizotrons, root growth began before shoot growth and

the first flush of shoot growth was mostly basipetal. Water stress altered shoot

architecture by enhancing apical dominance. As plants adapted to the stress imposed,

indeterminate growth was triggered more often in meristematic regions of terminal buds.

At the second flush, which occurred later in the stress treatment, old buds expanded

more frequently than the newly formed apex lateral bud. Temporal variations of

moisture caused by wetting and drying cycles resulted in continuous growth for portions

of the root system, with quiescent periods observed for shoot growth. Conversely,

continuous high moisture levels resulted in roots exhibiting quiescent periods in some

plants. Patterns of shoot and root growth varied considerably between these clonal









plants, which may be an important consideration for analyses of populations of woody

plants.

Free amino acid levels at the shoot tip were more decisive for initiation of

meristem growth or quiescence than the quantity of total nonstructural carbohydrates or

nonstructural carbohydrate-to-free amino acid ratio. In roots, this ratio was a good

predictor of root growth. Valine, leucine, tyrosine, cysteine, metionine, and arginine

increased significantly with bud set, compared with growing shoot tips. Root tips

contained abundant fructose, stachyose, and myoinositol. Mannitol was the major

transport sugar and glutamine, valine and histidine were the main free amino acids

transported in xylem fluid. Water stress resulted in increases in the concentration of

some amino acids in growing shoo tips, such as arginine, valine, and histidine, and

especially valine in developing buds at the beginning of root flush.

The results observed from these experiments suggest important considerations for

woody plant management. Since it appears that water stress can increase apical

dominance; the necessity for pruning may increase if the plant is to be used as a shrub

rather than as a small tree. Conversely, well-irrigated plants show basipetal branching in

the beginning of the growing season, which can also affect pruning decisions. Wetting

and drying cycles result in continuous growth of portions of the root system; thus,

establishment can benefit by temporal variations of soil moisture. Fertilization during the

growing period prior to transplant into a landscape is an important factor for building up

internal nutrient reserves to support initial root growth and bud expansion at the

beginning of the growing season. These observations suggest that woody plant









management can benefit from appropriate decision making of pruning, frequency of

irrigation, and timing of fertilization and transplant.









CHAPTER 1
INTRODUCTION

Transplanted in-ground trees undergo a severe physiological shock because their

capacity for water absorption is greatly decreased. This is the result of injury to roots,

loss of small absorbing roots, and disruption of the previously established contact of the

root system with a large volume of soil (Kozlowski and Pallardy 2002). Furthermore,

textural differences between the substrate used for container grown plants and

surrounding soil after transplant diminish water availability for root absorption. Shrubs

and trees usually do not have a root growth rate high enough to rapidly replace lost or

damaged roots, or to generate new root growth immediately after transplanting into new

soil. As a result, plants normally experience water stress and establishment is delayed

or not achieved in extreme cases. This requires additional water, fertilizer and extended

care. During the establishment period, roots are expanding out into the landscape soil,

and shoots and trunk grow more slowly than before transplanting (Gilman 2002) or

discontinue growth until the root system is established and capable of supplying

sufficient amounts of water to above-ground organs (Ongaro and Leyser 2008).

Partitioning of Photosynthates

Partitioning of photosynthates can be influenced by environmental stimuli (Engels

1994; Rogers et al. 1996). Earlier views, as described by Thornley (1972), considered

growth and partitioning to depend on two plant processes, transport and utilization.

However, more recent models have added substrate supply (Campagna and Margolis

1989); substrate sources (Thornley 1998); water stress (Chaves and Oliveira 2004) and

nutrient availability (Coleman et al. 2004). Campagna and Margolis (1989) stated that

allocation is likely to result from combined influences of substrate supply, transport and









utilization. As a plant grows, equilibrium is maintained between root and shoot growth

(Campagna and Margolis 1989). Bloom et al. (1985) describe this mechanism well,

stating that when a particular below-ground resource, such as nitrogen, limits growth

more than other resources such as light, plants that optimize growth would allocate

more carbon to the roots to absorb the limiting resource. Woody plants that grow on

poor sites generally allocate a greater proportion of photosynthates to root production

than do those that grow on good sites (Kozlowski and Pallardy 2002). The resultant

changes have obvious impacts on water-absorbing capacity in comparison with

transpiration by above-ground structures (Kozlowski and Pallardy 2002). Decreasing

availability of water, nitrogen, or both, reduces the leaf/root carbon partitioning ratio

(Running and Gower 1991). Factors affecting photosynthesis can also strongly affect

water and nutrient acquisition (Eissenstat and Van-Rees 1994).

Nitrogen plays a central role in plant metabolism, as a constituent of the

chlorophyll molecule and as a key component of enzymes used in photosynthesis,

respiration and growth process (Margolis and Waring 1986). The balance between

nitrogen and carbon availability is important in determining the relative allocation of dry

matter production between roots and shoots. Ericsson (1995) concluded that the

internal balance between labile (easily converted) nitrogen and carbon in roots and in

the shoots determines how dry matter is partitioned in a plant. Ericsson's work showed

that an increased allocation of dry matter to below-ground parts was associated with a

greater amount of starch in the tissues. Depletion of stored carbohydrates occurred

under all conditions in which root development was inhibited. Three different dry matter

allocation patterns were observed depending on which specific mineral nutrients were









withheld in each instance. Reduction in atmospheric carbon dioxide concentration led to

a strong decrease in root growth. However C02 enrichment had no effect on dry matter

partitioning.

Campagna and Margolis (1989) used manipulation of liquid fertilizer and CO2

enrichment of the air during greenhouse production of black spruce seedlings. They

found a strong correlation (r2 > 0.90) between tnc:faa and carbon allocation to shoots

and roots. When the relative balance between biochemically-available forms of carbon

was high in relation to the biochemically-available form of nitrogen, rates of protein

synthesis and other metabolic processes were affected favoring root growth. When

tnc:faa was low, shoot growth was favored. The authors also stated that while the ratio

of carbon to nitrogen has been known to affect root-to-shoot ratio over relatively long

periods of time, the carbon-to-nitrogen ratio may also influence short-term episodic

growth patterns. Furthermore, it was suggested that the carbon (C) to nitrogen (N)

balance offers a partial explanation of why root growth predominates in the later part of

growing season. When shoot growth either slows down or stops, the tnc:faa increases

because the carbohydrate sink in leaves declines, corresponding to a predominance of

root growth.

Episodic Growth

Many woody species display an episodic growth habit by fluctuating between

periods of rapid shoot growth and slow root growth versus periods with the inverse

pattern. Relatively little is known about the changing carbon allocation patterns in

species with episodic growth cycles (Dickson et al. 2000a). Kuehny, et al. (1997)

observed alternate flushes of root and shoot growth of approximately 20 days in

Ligustrum japonicum.









The control of vegetative growth involves close interdependence between roots

and shoots (Kozlowski and Pallardy 1997). It has been hypothesized that episodic shoot

and root growth is controlled by hereditary factors (Morrow 1950), and is influenced by

several non-hereditary factors including the carbon-to-nitrogen ratio within a plant

(Campagna and Margolis 1989); competition for carbohydrates among the various

tissues (Eissenstat and Van-Rees 1994; Kozlowski and Pallardy 1997; Kuehny et al.

1997); N fertilization (Huche-Thelier et al. 2006; Kuehny et al. 1997); changes in soil

water content (Kozlowski and Pallardy 2002) and temperature fluctuations (Lyr and

Garbe 1995; Reich et al. 1980).

Plant growth can be characterized by cell proliferation, organ elongation and mass

accumulation. Meristematic regions can be active, but not exhibit visible growth for a

period of time. Growth measurement method may influence perception of organ growth.

For instance, length measurement may not be followed by proportional dry mass

increase, which occurs with maturation. Thus differences in measurement method can

also be a factor in observation of episodic growth and cause disparities between

experimental results.

In northern red oak (Quercus rubra L.), an episodic species, upper leaves of a

branch allocated most 14C-photosynthate upward during leaf and shoot growth, while

lower leaves supplied more 14C to lower stem and roots (Dickson et al. 2000a). Starch

and sugar storage in leaves, stems, and roots during lag and bud growth stages

indicate a feedback response to decreasing sink strength and temporary storage of both

starch and sugar in these plant tissues (Dickson et al. 2000b). Kuehny and Halbrooks

(1993) determined episodic growth of Ligustrumjaponicum plants by measuring total









shoot and root fresh weight nondestructively. Kuehny et al. (1997) concluded that

changes in endogenous N concentrations and allocation patterns in L. japonicum were

linked to the control of episodic shoot and root growth.


Induction and Control of Shoot and Root Growth

Mechanisms by which plants modulate their growth rate, cell division, and sink

strength in response to environmental and developmental conditions are unknown, but

are presumed to involve meristems (Kuehny et al. 1997). A meristem is a group of

undifferentiated, actively dividing plant cells. They include formation of roots and shoots.

Individual meristematic cells are dividing, expanding and differentiating. These highly

dynamic cells are somehow coordinated in root or shoot meristems (Grandjean et al.

2004). The result can affect developmental patterning (Clark 2001) or developmental

programs (Lough and Lucas 2006) in plants. Cockcroft, et al. (2000) concluded that cell

division is the main determinant of meristem activity and overall growth rate. They

proposed that control of plant growth rate is achieved through regulation of the cell

phase G1. Tax and Durbak (2006) described the structure of the shoot apical meristem

and summarized experimental and molecular data for signaling. Plant meristems can

also be influenced by cytokinins (Werner et al. 2001). Francis and Halford (2006)

reviewed the literature on nutrient sensing in plant meristems and gave special

emphasis to the components of sugar sensing/signaling pathways.

Woody plants growth and development are regulated by interactions of their

heredity and environment as they influence the availability of resources (carbohydrates,

hormones, water and mineral nutrients) at meristematic sites (Kozlowski and Pallardy

1997). Shoot growth largely is determined by genetic factors and environmental factors









such as light, water, temperature, mineral supply, composition of above and below-

ground atmospheres, physical and chemical soil properties, insects, other plants, and

animals (Calfee 2003). Specific shoot activity depends upon environmental factors such

as photosynthetic photon flux and carbon dioxide concentration. Similarly, specific root

activity depends upon factors such as root temperature, concentration of nutrients in the

soil and water uptake (Engels 1994; Thornley 1972). Salaun, et al. (2005) observed that

N was translocated in the xylem fluid of Ligustrum ovalifolium L. predominantly in the

form of amino acids. Prior to bud break arginine and, later, glutamine accounted for the

principal components of nitrogen mobilization. However, of the 20 common amino acids

only five were analyzed in this study. Also working with Ligustrum ovalifolium, Salaun

and Charpentier (2001) found that in December, arginine followed an increasing

gradient from the roots to the trunk and decreased from the lower to the upper parts of

stems. The more fertilization the plants received, the higher the arginine content.

Storage content in trunks amounted to 45% of total plant arginine.

A high carbon-to-nitrogen ratio promotes root development, and, conversely, a low

carbon-to-nitrogen ratio promotes shoot development (Koch 1997). Previous research

by Beeson indicated that high N availability from biocompost incorporated into the soil at

transplanting reduced root growth into landscape soil, while stimulating shoot growth.

Conversely low N in landscape soils of unfertilized control plants stimulated root growth,

while severely restricting new shoot growth (Beeson, unpublished data).

Root-to-Shoot Ratio

The root-to-shoot ratio, defined as dry mass of roots divided by dry mass of

shoots, depends upon partitioning of photosynthates (Engels 1994; Rogers et al. 1996).









The proportion of above-ground and below-ground mass can be altered by responses of

genes that affect uptake, assimilation and storage (Koch 1997).

The balance between root and shoot growth is expressed by k, the variation of dry

mass of shoots divided by the variation of dry mass of roots over a given period of time.

In other words, k represents the relative partitioning of growth over a specific period of

time. In 1972, Thornley published a model which describes root-to-shoot ratio in

vegetative plants and how they respond to changes in root and shoot activity. In this

model a plant has only two components, roots and shoots, and growth is dependent

upon the supply of carbon (by shoots) and nitrogen (by roots). Later, Thornley (1998)

refined this model to accommodate influence of ontogenesis, scaling, within-plant

transport resistances, hormones and active transport.

Water deficits and mineral nutrient deficiencies promote greater relative allocation

of photosynthates to root growth. This ultimately results in plants that have higher root-

to-shoot ratio and greater capacity to absorb water and minerals relative to shoots that

must be supported (Kozlowski and Pallardy 2002). One of the adaptative mechanisms

found in plants to avoid water stress is to produce high root-to-shoot ratio, which permits

better exploitation of water reserves to replace transpirational losses.

Many plants respond to water stress with an increase in root-to-shoot ratio, usually

attributed to an decrease in shoot growth (Bachelard 1986; Sharp and Davies 1979;

Steinberg et al. 1990). However some authors attribute the change in root-to-shoot ratio

under water shortage mainly to an adaptative improvement (drought tolerance)

genetically inherited and argue that leaf growth slows down, causing a decrease in

foliage area and intercepted solar radiation (Farrell et al. 1996; Osorio et al. 1998).









Ultimately this relative decrease of foliage could represent a decrease in tnc:faa and

invert the relationship observed by Campagna and Margolis (1989) in well-irrigated

plants.

In response to drought, or limited soil moisture availability over extended periods

of time, plant water status was proposed to be more strongly influenced by changes in

root-to-shoot partitioning and root density rather than the interaction of soil moisture

content with stomatal conductance (Thornley 1996). Indeed, this has been verified

through many experiments in the past 15 years relating to physiological responses of

woody ornamentals transplanted into landscape situations (Beeson, pers. comm.).

Johnson et al. (1991) developed a water submodel that includes root-to-shoot message

control of stomatal conductance for incorporation into mechanistic plant growth models.

However, McMillin and Wagner (1995) observed that the influence of water stress in

root-to-shoot ratio are dependent on stage of development, thus age may influence

establishment as well.

Studies with C14 and C13 indicate a significant difference between the amounts of

C transported to different plant organs. Fifty percent of assimilates produced in young

cereal plants was rapidly transported below-ground, of which 50% was respired. Less

than 5% of the fixed carbon went to root exudates. When subjected to water stress, the

allocation of C13 to roots increased so that at flowering 38% of shoot C was below-

ground compared to 31% in well-irrigated plants. C02 enrichment increased the

proportion of root to total mass by 55%. While increasing air temperature by a mean of

3 C decreased the proportion of roots from 0.093 in the cool treatment to 0.055 in the

warm treatment (Engels 1994). Campagna and Margolis (1989) reported similar results









in black spruce seedlings. As tnc:faa increased, k decreased during seedling

development. Keyes and Grier (1981) reported that in low-productivity sites

Pseudotsuga menziesii trees partitioned 36% of total net primary production to fine

roots but only 8% when in high-productivity sites. Kuehny, et al. (1997) found that when

N is limiting whole-plant growth, more carbohydrates are translocated to below-ground,

thus facilitating root growth, and also stored in the shoots, where it would aid future

episodes of growth.

Cheng and Fuchigami (2002) working with apple trees observed that about 50% of

tree N content was remobilized to support new shoot and leaf growth. They concluded

that the amount of reserve N remobilized for new growth in spring was proportional to

tree N status, and was unaffected by current N supply in the soil.

Woody plants transplanted into landscapes normally extend substantial roots into

the soil before shoot growth returns to pre-transplant levels. This pre-dominance of root

growth overrides normal cycles of episodical shoot and root growth.

Signaling Systems in Plants

Higher plants are sessile organisms, thus the ability to adapt to their environment

is a key feature for survival. Environmental stimuli, such as light, water or nutrient

availability can be sensed in one organ of a plant and the information can be transmitted

to other organs or cells through the use of chemical signals that trigger plant responses.

The evolutionary development of a long-distance communication network in higher

plants reflects the need to communicate environmental inputs, sensed by mature

organs, to meristematic regions of the plant (Sack and Holbrook 2006). Signaling

pathways utilize a complex network of interactions to orchestrate biochemical and

physiological responses such as flowering, fruit ripening, germination, photosynthetic









regulation, and shoot or root development (Mulligan et al. 1997), and allows an optimal

response to environmental conditions (Genoud et al. 2001). Recent studies have

identified new molecular components required for proper shoot meristem activity, and

they have revealed that complex, intercellular communication pathways play important

roles in coordinating meristem function (Fletcher and Meyerowitz 2000; Haecker and

Laux 2001). Kepinski (2006) addressed the mechanistic links between hormone

signaling and developmental processes. Thum et al. (2003) reported interaction of

carbon methabolism with blue, red, and far-red-light signaling. Sugar and especially

amino acid signaling in plants is in the early stages of research, with the majority of it

being done with herbaceous rather than woody species.

Sugar-regulated expression provides a mechanism for control of resource

distribution among tissues and organs (Koch 1996). Hartig and Beck (2006) discussed

crosstalk between auxin, cytokinins, and sugars in the plant cell cycle and point out the

degree of tissue and cell specificity that exists for signal interactions, even in

physiologically similar processes like cell division. Gene responses to sugars in a sink

organ can depend on the pathway used for sucrose import into a given tissue (Koch

2004; Koch and Zeng 2002). In plants, different sugar signals are generated by

photosynthesis and carbon metabolism in source and sink tissues (Rolland et al. 2006).

Sucrose cleavage in cells can be catalyzed by two enzymes invertase (forming

fructose + glucose) or the reversible enzyme, sucrose synthase (forming fructose +

UDP-glucose). Thus, action of invertase generates twice as much substrate for hexose-

based sensing (Koch 2004). ABA accumulation during water stress may often function

to help maintain shoot as well as root growth, rather than to inhibit growth as is









commonly believed (Sharp and LeNoble 2002). The activity of acid vacuolar invertase

was highly correlated with xylem fluid ABA concentration (Trouverie et al. 2003) and

glucose directly affects ABA biosynthesis (Chaves and Oliveira 2004) by induction of

transcription of genes for this hormone biosynthesis (Cheng et al. 2002).

Intensity of remobilization and use of reserves in woody species may be different

than that observed in herbaceous plants. Furthermore, labile C and N pools in perennial

species are maintained not only by root absorption and shoot production, but by break

down of reserves as well. Changes in plant carbohydrate status can lead to a wide array

of responses at the whole-plant or gene-expression levels. Certain genes are

downregulated, and others upregulated by sugars affecting the balance between above

and below ground processes, including storage, utilization, photosynthesis,

remobilization, export, relative source/sink activity and growth (Koch 1996). Cell

differentiation and the cell cycle can also be strongly affected by sugar availability (Koch

1996). Nitric oxide acts as a signaling molecule, in particular by mediating the effects of

hormones and other primary signaling molecules in response to environmental stimuli

(Chaves and Oliveira 2004). Lough and Lucas (2006) discussed phloem-mediated

transport of macromolecules as components of an integrated long-distance signaling

network, with emphasis on proteins and RNA species.

'Crosstalk' between signals for availability of C and N was discussed by Koch

(1997) who concluded that interactive effects on gene expression are possible, although

the points of interface are unclear. The same author also stated that the form of both N

and C can also be important in the response of photosynthetic genes to C to N balance,









particularly since sugars and amino acids can have markedly different effects than

unassimilated C02, NO3, or even metabolically generated NH4 and NO3.

Amino acid signaling research in plants is only in its early stages. Glutamate

applied externally to root tips caused inhibition of growth (Sivaguru et al. 2003) and

inhibited lateral root formation and outgrowth (Walch-Liu and Forde 2007). Nitrate

counteracted Glu, thus stimulate root branching and main root growth. External nitrate

and Glu were proposed to interact to modulate root growth (Forde and Walch-Liu 2009).

However, internal Glu concentrations have not been cited to cause the same effects.

Excessive external concentrations of one amino acid in relation to others can inhibit

growth, with the only exception being Gin, which has the ability to counteract growth

inhibition caused by the excessive amino acid (Singh and Shaner 1995). An example is

Val-mediated inhibition to growth due to isoleucine (Iso) starvation (Bonner and Jensen

1997).









CHAPTER 2
AN ELEVATED LARGE-VOLUME RHIZOTRON FOR EVALUATING ROOT GROWTH
UNDER NATURAL SOIL MOISTURE CONDITIONS.

Introduction

Although root growth is central to overall plant performance, the study of natural

root development has remained a challenge due to the difficulty of observation.

Attempts to observe roots over time date back to at least the early 1900s (McDougall

1916). However, most methods used to study root development are extremely time

consuming and tedious (Calfee 2003).

A rhizotron is a device for observing plant roots non-destructively over time

(Garrigues et al. 2006). Root observation facilities described by Soileau et al. (1974),

Karnok (1982), and Taylor and Bohm (1976) consisted of underground laboratories

with transparent windows. Root observation windows installed in native soil (Gallandt et

al. 1990; McDougall 1916; Metcalfe et al. 2007) were less elaborate, but also below-soil

level. A different approach was the use of transparent tubes in the greenhouse by

Schoene and Yeager (2006; 2007), or more elaborated tubes developed by

Bland (1990). The majority of research on root development has relied on narrow

observation boxes usually made with plexiglass (Boukcim et al. 2001; Busch et al. 2006;

Devienne-Barret et al. 2006; Garrigues et al. 2006; James et al. 1985; Misra 1999;

Stepniewski et al. 1991; Ugoji and Laing 2008; Wiese et al. 2005). Although these

boxes and tubes enabled easier greenhouse study of root growth, the volume of soil

exploited by roots was severely reduced, narrowing applicability to seedlings or cuttings.

Moreover, normal root architecture was biased in these types of chambers because

roots were forced to grow in narrow spaces, forming an artificial root arrangement.

Wright and Wright (2004) developed a star-shaped rhizotron with eight glass panels,









suitable for greenhouse or field use that overcame many of the biases of the past.

However, it did not fully mimic in-ground conditions. A large rhizotron, designed around

concepts of enhanced root observation and tracking strategies would enable

researchers to study growth of whole mature root systems as they develop.

Understanding the effects of environmental factors and cultural practices on root growth

of mature plants would be of great benefit for applied and fundamental goals ranging

from ecology to agriculture, landscaping, and forestry.

The objective of this study was to design and test several alternative strategies for

above-ground rhizotrons that could enhance observation and recording of undisturbed,

natural root growth of woody plants. A primary goal was to mimic in-ground conditions,

including minimum physical restrictions and enhanced drainage (the latter being

especially valuable for testing effects of soil moisture deficits on root growth).

Material and Methods

Rhizotron designs tested here proceeded in sequence, each modified based on

results of the one immediately preceding it.

Stage 1 Designs for containment and observation ports

A prototype rhizotron was constructed to evaluate different materials and designs

for root observation and sampling. The shape was that of a star with four-arms radiating

from a central rectangular box (Figure 2-1). This star-shaped rhizotron enclosed 0.18 m3

of soil, measured 2.1 m across, and was 0.31 m deep at the tip of the arms. The central

box was 0.25 x 0.25 x 0.37 m tall. The bottom of each arm was sloped towards the

center box to facilitate drainage and to reduce perched water tables common in flat

bottom containers (Bilderback and Fonteno 1987; Spomer 1980). The bottom of the

center box consisted of a plastic mesh (12.5 mm square) over which woven ground









cover (Lumite Inc., Gainesville, GA) was placed to support the substrate. The frame of

this prototype rhizotron was welded from angle-iron strips (L-shaped cross-sections [18

x18 mm] of 3mm iron), with narrower width (12.5 x 12.5 mm) at the top of the box to

facilitate transplanting root balls (typically 11.4- L from standard nursery containers). To

facilitate observation and drainage, the rhizotron was supported by four legs (60 cm), at

the tip of each arm.

Each lateral (side of the arm of the rhizotron) was used to evaluate different

systems for non-disruptive visualization of roots while holding substrate in place. Four

different door designs were evaluated: 1) a single door the length of a lateral held by

two brass hinges (2-1A); 2) a single sliding panel the length of a lateral (Figure 2-1 B);

3) two independent doors each held by two brass hinges (Figure 2-1C); and 4) a side

panel with two smaller doors attached to panel (Figure 2-1D). For some configurations,

1.25 cm square wire mesh was placed in the lateral to hold substrate in place.

Two materials were tested for doors: 2.2 mm clear plexiglass and 8 mm Thinwall

polycarbonate (Lexan, General Eletric, Fairfield, CT). In addition, different methods to

seal doors were tested for avoidance of to avoid moisture loss and capacity to remain

secured. Rhizotron arms were covered with opaque woven ground cover (Lumite Inc.)

to maintain darkness of the root environment.

In May 2006, a Ligustrumjaponicum Thunb. from an 11.4-L container was

transplanted into the rhizotron and grown for five months. Irrigation was supplied daily

with three bubbler emitters per arm (Model Shrubbler 3600; Antelco, Longwood, FL).

Ground cover was removed from the arms only for root recording and monitoring. Roots

were traced using permanent markers on the plexiglass door models. For polycarbonate









doors, transparency sheets (20 x 25 cm) were placed on the side of a soil profile for

tracing roots.

Stage 2 Designs for drainage systems allowing natural-soil profiles

Two additional drainage systems were next compared to the original design for

their capacity to reduce or eliminate excess irrigation. Results from Stage 1

(Figure 2-2A) indicated that excess water percolates downward until an impervious

surface or perched water table is encountered (as typical in containers and in natural

soil profiles). In the elevated rhizotrons tested here, the 0.6-m air gap between the

plastic mesh and the ground below formed an impervious surface, resulting in perched

water tables within the rhizotrons. To simulate in-ground soil moisture characteristics,

these perched water tables had to be eliminated.

Two additional drainage systems were thus developed, with the goal of better

simulating in-ground moisture characteristics. The hanging water column system

(Figure 2-2B) was intended to remove excess water from the substrate to a set tension

level. For this, a 24-cm diameter polyethylene funnel was installed and sealed with

silicone caulk underneath the center box, and filled with medium texture sand of a finer

texture than the substrate above. A 60-cm length of Tygon R-3603 laboratory tubing

(6.3 mm internal diameter) was attached to the bottom of the funnel. The lower end was

elevated 7 cm to establish a hanging water column. The third was a wick system

(Figure 2-2C), which consisted of a superabsorbent felt, covered with perforated black

polyethylene landscape fabric (WeedBlock, Easy Gardner Products, Inc., Waco, TX)

installed under the substrate. The felt was the bottom layer of the capillary system

Aquamat (Soleno Textiles Inc., Laval, Quebec, Canada) and was hypothesized to wick

excess water from the substrate. Resulting fluid was envisioned to drip and evaporate









from the mat hanging below the rhizotron center. A WeedBlock layer was added to

prevent roots from growing into the felt below.

The three drainage systems were evaluated for 12 days (four irrigation cycles at

three day intervals), with three replications (each a separate rhizotron). Rhizotrons were

filled with a commercial substrate composed of Canadian sphagnum peat moss,

processed pine bark, perlite, vermiculite, starter nutrients, wetting agents, and dolomitic

limestone (Mix #4, Conrad Fafard Inc., Agawam, MA). Soil moisture sensors, EC-5

ECH20 probes (Decagon Devices Inc., Pullman, WA) were used to characterize soil

moisture profiles. Sensors were calibrated for the substrate prior to use. Calibration was

performed by placing sensors at a uniform depth in 0.03 m3 of substrate in a white vinyl

cylinder (50-cm diameter). A time domain transmissometry sensor (Digital TDT, model

ACC-SEN-TDT, Acclima, Inc., Meridian, ID) was included for measurement of

volumetric water content (VWC). The cylinder was filled with an excessive volume of

water, and then allowed to drain and dry naturally while positioned on top of native,

sandy soil. Millivolts were recorded for each senor concurrent with the volumetric water

content measured by the TDT every 15 minutes for 6 days using a datalogger and

multiplexer (CR1OX and a 16-32 multiplexier, respectively, Campbell Scientific, Inc.,

Logan, UT). Calibration equations were quadratic for each sensor when including

moisture ranges above field capacity (Microsoft Office 2007, Microsoft Corp. Redmond,

WA). Each rhizotron replication had three ECH20 probes installed to monitor substrate

moisture, at the center of one arm, center of the center box, and bottom of the center

box. Two thermocouples monitored air temperature. Four additional thermocouples

were installed to monitor substrate temperature 1 cm away from the door material.









Volumetric water content was analyzed as a 3 x 3 factorial, with drainage system and

probe position as treatments with three replications using SAS (version 9.1, SAS

Institute, Cary, NC).

Results and Discussion

After five months of growth in the prototype rhizotron, the L. japonicum plant had

expanded its roots only 75% of the length of a rhizotron's arm. It was concluded that the

arms could be shortened to reduce cost and overall floor space while still achieving

research goals. Final rhizotron's central box was 0.25 x 0.25 x 0.35 m tall, and laterals

were decreased from 0.93 m to 0.78 m in length. This reduced the overall width of a

rhizotron from 2.1 m to 1.76 m. The slope of the bottom of each arm increased slightly

(6.4% to 6.6%). Framework of the final rhizotron was constructed with galvanized sheet

metal (Figure 2-3 5 x 3.8 x 5 cm U-shape, 5 x 5 cm V-shape, and 5 x 2 cm L-shaped).

Only the top and bottom squares of the center box (25 x 25 cm) were made of 2-cm flat

iron strips (Figure 2-3). The pieces were welded and painted to avoid corrosion.

Expanded white PVC board (6 mm, Kommerling Inc., Huntsville, AL) was used in the

bottom of the arms, which were sealed on the corners with silicon chalk. Rhizotrons

were raised 0.6 m above the ground by wooden legs. The final rhizotron design

(Figure 2-4) enclosed 0.16 m3 of soil, measured 1.76 m across, 0.30 m deep in the tip of

the arms and 0.35 m deep in the center.

Of the door types evaluated, a single door (Figure 2-1A), attached with two hinges

proved most effective. It allowed easiest access for root observation, recording, and

measurement of roots. Once wet, the substrate was stable and remained in place when

the door was open. The wire mesh on the side of the arm was found to be unnecessary.

Although offering good stability, sliding panels (Figure 2-1 B) were not practical. When









slid open, friction crumbled the substrate into the tracks causing subsequent mechanical

problems. The center post used to help seal double doors (Figure 2-1C) obscured roots

and made recording root growth by digital images troublesome. Moreover, opening and

closing two doors for root observations was more time consuming. Likewise, side

panels with two doors (Figure 2-1 D) were more difficult to operate and close and offered

no advantages.

Investigation of materials for door construction indicated several advantages for

white 6-mm expanded PVC board (Kommerling Inc.). This was opaque, waterproof,

insulating, and sufficiently rigid to retain lateral shape of rhizotrons arms when filled with

the substrate. These were attached at their basal edge with two 5-cm brass hinges.

Doors were held in closed position by two 7-cm wooden buttons attached to the frame

with machine bolts. Both other materials evaluated in the prototype doors were rigid

enough for use, however, roots were observed with much more detail with open doors,

thus negating the need for transparency. Even though plexiglass has been the material

of choice for many researchers, moisture accumulation inside the panels can make

observation through them difficult. An additional problem associated with clear

plexiglass was glare, which makes digital photographs of the roots unfeasible. Thinwall

polycarbonate was light weight and readily accessible. However, it did not allow root

observation through doors, and permitted light penetration to roots unless the system

was covered. A better solution for the rhizotron doors was the expanded PVC board. It

was inexpensive, light weight, sturdy, easy to cut and manipulate, and had the added

advantage of being opaque. This eliminated the need for covers. The white color of the

board also reflected radiation, thus aiding heat management. The difference in









temperature between substrate immediately next to PVC door and air varied between

-4.50 C and 40 C during the course of the day. Substrate temperature varied less than

air temperature (Figure 2-5 cooler during the day and warmer during the night).

To limit evaporation from the substrate surface, a perforated black polyethylene

landscape fabric (WeedBlock) was cut to fit and placed on top of the substrate. Irrigation

was provided by placing a spray stake (model green 22500-001120, Netafim Irrigation,

Inc., Fresno, CA) at the tip of each arm pointing inward. The angle created by the

irrigation spray approximated the internal angle on the arms. The four sprays of water

slightly overlapped at the center, providing a uniform coverage.

Although a number of authors report the use of permanent markers for root tracing

(Boukcim et al. 2001; Metcalfe et al. 2007; Misra 1999; Schoene and Yeager 2006;

2007; Stepniewski et al. 1991), we found that for accurate recording of so many roots

they were impractical. Each rhizotron offered eight surfaces where roots could be

observed. Digital photography, followed by software analysis, provided a much more

precise account of root system development. Serial photography can also allow for

further analyses of morphological characteristics, various root orders, and root geometry

(Wiese et al. 2005). However, for accurate analysis, serial photographs must be taken

at exactly the same location and distance each time. Additionally, the camera that was

used for all digital photography (DSC-W170, Sony Corp. of America, Montvale, NJ)

would not cover the entire rhizotron surface at the widest lens setting. Moving the

camera further away was not an option due to close spacing between rhizotrons, and

loss of detail from increasing the lens-to-subject distance. To address this challenge,









two overlapping images were obtained for each panel using a device that would assure

consistent positioning.

To achieve this, a camera positioning frame (Figure 2-6) was developed to hold

the camera at a constant distance and location for time-series digital recording of root

growth. The positioning frame was constructed using a 2.8 x 2.8 x 61 cm magnetic bar

(Ningbo Ketian Magnet Co., Zhejiang Province, China) that could be centered on the

top edge of each lateral of an arm of the rhizotron. From the center of this magnetic bar,

an aluminum bar (2.5 x 0.5 cm) was bent into a C-shape with 900 turns at the distal

ends so that it braced the frame against the open PVC door. The distance of the arm to

the vertical leg of the "C" was 0.63 m. A 12-mm square aluminum bar 16 cm long was

attached perpendicularly to the vertical portion of the "C" using a single screw within a

10-cm section of 1.8-cm aluminum c-channel so that it would pivot from one side of the

vertical leg to the other within the c-channel. The vertical position of the pivot arm was

such that the optical center of the camera would point directly at the vertical face of the

rhizotron. The camera was connected to this perpendicular bar using one screw

enabling the camera to face the rhizotron from the left or right. Up to two pictures were

taken of each profile, with each accounting for a little more than half, and overlapping at

the center of the profile. This permitted digital fusion of the pictures. A mark was placed

on the rhizotron frame at the center of each of the four sides of the profile and a red

mark was placed exactly at the profile center to facilitate fusion of pictures when

necessary.

This flexibility of locations for the camera enabled a shorter distance from the soil

profile to the camera, and resulted in high resolutions and close spacing between









rhizotrons. Initially, only a single image was required to encompass the early root

growth. The second picture per profile (from the distal half of the profile) was necessary

only on later stages, after roots had grown past the halfway mark.

Rhizotron facilities usually devise methods for drainage (Huck and Taylor 1982;

Karnok and Kucharski 1982; Soileau et al. 1974), but persistence of perched water

tables has not been appraised or addressed in previous studies of root chamber models

(James et al. 1985; Ugoji and Laing 2008; Wright and Wright 2004). Nonetheless,

unimpeded drainage, especially removal of perched water tables, was a critical

objective for the rhizotrons described here. Soil moisture sensors were installed to

evaluate the effectiveness of the evaluated methods for removing perched water.

The simple mesh drainage system (Figure 2-2A) was unable to remove excess

water efficiently. In this system, the substrate at the bottom of the rhizotron was

consistently at a significantly higher moisture level than the other two locations,

indicating impeded drainage. For example, the bottom, arm and center positions

averaged 52.7, 39.4 and 27.9 %VWC, respectively, two hours before irrigation (Figure

2-7A). Moreover, substrate located in the arm remained at considerably higher moisture

levels than that in the center of the rhizotron. The hanging water column system (Figure

2-2B) worked relatively well at draining water (Figure 2-7B). This system however was

not able to remove as much water from arm and bottom positions as from substrate

located at the center (bottom, arm and center had an average of 37.2, 36.7 and 26.8

%VWC 2 hours before irrigation, respectively). Substrate moisture also appears to have

established equilibrium more quickly, and at somewhat lower VWC with the hanging









water column (approximately 12 hours after irrigation) compared to the mesh system at

the same locations (arm and center did not reach equilibrium).

Best results were obtained with the wick system (Figure 2-2C). This system came

into equilibrium more slowly than the hanging water column system (18 and 12 hours,

respectively), but maintained substrate moisture levels more uniformly between all

positions than did the other two systems (Figure 2-7C difference of %VWC between

bottom and center of 27.8, 9.9 and 4.7, for simple mesh, hanging water column and

wick systems respectively). Also, volumetric water content was significantly lower at

equilibrium and less variable (30-35 %VWC) than was that of the other two systems

(26-37 %VWC for hanging water column and 28-53 %VWC for simple mesh), thus was

considered more characteristic of landscape soils.

The majority of root observation devices reported in the literature allow limited

observation (one to two ports only), regardless of whether these rhizotrons force root

growth through narrow spaces or are installed in natural settings. The rhizotron

described here minimizes physical restriction to normal root growth, development and

distribution, and offers eight viewing profiles, greatly enhancing the study of the root

system as a whole. The structure is sturdy enough to support the large substrate

volume, and can be increased in size to accommodate larger root balls or for

experiments of longer duration. With the large rhizotron size, the root system developed

by L. japonicum after 5 months of growth showed no root restriction. Roots visible in the

window had good spatial separation, improving root analyses by digital photography or

visual observation. Root sampling for biochemical assay or root recording was fast,

efficient and minimally disturbing since each door could be opened independently









With removal of the perched water table, the system provides a good simulation of

in-ground soil environment. Easy adaptations to this model can enable it to be used in

split root trials or as a lysimeter. The rhizotron can be divided in the center box by the

same expanded PVC board used on the doors and provide up to four different

quadrants for split root studies. These rhizotrons can thus be used to investigate the

effect of diverse variables on root growth. Examples could include partial root drying,

soil texture (Bengough et al. 2006), fertilizers (Boukcim et al. 2006; Drew 1975) or

agricultural chemicals (Tsakaldimi and Ganatsas 2006).











alg;~~~~ L.~ls~r I


Figure 2-1. Prototype rhizotron with Ligustrum japonicum 2 months after transplanting
into the rhizotron. Doors of rhizotron open only for demonstration. A) Single
door. B) Sliding panel. C) Double doors. D) Side panel with two doors.
















Figure 2-2. Drainage systems tested on rhizotrons, i: top view and ii: bottom view.
A) Simple mesh. B) Hanging water column. C) Wick system.


r-;


Z1L~1 -










White Expanded PVC Board


Soil Profile Viewing Port
Soil Profile Viewing Port


u
10


E




I 76cm
*j----------


E
o


N


E
0
Co


I_ 25 cm


Figure 2-3. Schematic diagram of a rhizotron. A) Side view of open and closed doors
(left and right, respectively). B) Top view with details of materials used on
construction (left of rhizotron: galvanized sheet metal and bottom: flat iron).






































Figure 2-4. One-year-old Ligustrumjaponicum transplanted in a rhizotron, window open
just for demonstration. Windows enable root visualization and easy
sampling.



I I I I I



S i 29 I I



27 I I I I
2* I I Ii I I
I I t II ;




I III

17 tWII I


15 I

13 ,
121 122 123 124 125 126 127 128 129 130 131 132
Air -Substrate Day
Figure 2-5. Temperature of air and substrate immediately next to single PVC door
(substrate: average of four thermocouples, and air: average of two
thermocouples).





























Figure 2-6. Camera positioning frame used to take pictures mounted on rhizotron.











065 -



5>60 -
35 A


50

45

40

35

I I
30

60

55 -

50 -

45 -

460 -

35

30 T

60

55 -

50 -

45

40

35-

30 _-

25 .. .
40 42 44 46 48 51
Day
Arn -Bottom Center

Figure 2-7. Percent volumetric water content (% VWC) observed during drainage trial
(average of actual values for 3 repetitions). A) Simple mesh. B) Hanging
water column. C) Wick system.









CHAPTER 3
DEVELOPMENT AND EVALUATION OF A ROTARY ROOT SEPARATOR

Introduction

Quantitative studies of plant roots have remained a consistent challenge.

Extraction of roots from soil and debris of large samples for biomass quantification is

time consuming and tedious (Calfee 2003). This tends to limit research to small

experiments and small sample sizes, especially for woody plants with extensive root

systems. Analysis of entire root systems from the plants is particularly important,

because woody plants allocate a large portion of their resources to root development

(up to 69%,Vogt et al. 1995). Still, most knowledge of woody plants roots is based on

partial root extraction from soil cores, and subsequent estimation of the entire root

system. Roots from a single plant can be distributed differently in the surrounding soil,

growing more in wet pockets of soil or with higher levels of fertility (Michelakis et al.

1993; Stevens and Douglas 1994; Wilkin et al. 2006). Larger sample sizes or samples

of entire root systems would represent a more accurate account of a plant's root

system.

The most common method used for root isolation has been hand picking and

washing with jets of water over a sieve (Prathapar et al. 1989). Machines for isolating

roots with an elutriation system have also been reported (Benjamin and Nielsen 2004;

Brown and Thilenius 1976; Carlson and Donald 1986; Fribourg 1953; Smucker et al.

1982). Unfortunately, these approaches were designed for small sample sizes,

especially core soil samples. They also required large volumes of water. A drawback of

elutriation systems is that final separation of roots from debris is made more difficult by

adhesion of particles to themselves and roots. Additionally, changes in color make root









identification and separation more difficult during final hand separation. Benjamin and

Nielsen reported that up to 20 hours was necessary to clean roots from samples of 993

cm3 soil after machine washing.

The objective of the research reported here was to develop a fast, mechanical

system to separate roots from large sample volumes, with maximal root recovery,

minimal root damage, and little to no water use.

Material and Methods

A root separator was developed to speed root isolation for dry mass determination

(Figure 3-1). Mechanical separation is provided by a rotating cylinder that removes

small particles. The root separator consists of an external cylinder with four 38 x 20 cm

openings at the lower end. The cylinder was made of aluminum that was closed on one

end (79 cm tall and 46 cm in diameter) where it was attached to a right angle gear

reducer (1:20, model 13-175-20-R, Worldwide Electric, Rochester, NY). This was

belt-driven by a small electric motor (model SKH 47KR383 GS, 110 VAC, 0.75 hp, 1720

rpm, General Electric, Milwaukee, WI, Figure 3-C). The motor was shielded by a sheet

metal box (46 x 30 x 20 cm). A 1.5 x 1.5 cm wire mesh was placed on the outside of the

cylinder to serve as a secondary sieve. The upper end of the cylinder was supported by

two rubber tires (A tube type, 25.4 cm tall x 7.6 cm wide, World Caster & Equipment

Manufacturing, Inc, Lilburn, GA) and hub assemblies attached to the heavy metallic

base for stability. The cylinder was set at a 200 angle from the base. Samples, up to 40

L, were placed into a removable, internal basket (50 cm tall and 45 cm in diameter) of 6

x 6 mm wire mesh. Cylinder rotation speed was adjustable. After preliminary trials, the

rotational speed was established at 20 rpm to minimize root damage. During machine

use, particles smaller than wire mesh openings dropped through both the basket and









the openings at the bottom of the cylinder, then were channeled aside by a plexiglass

ramp (100 x 40 cm). After rotation, the basket was removed from the cylinder, and the

remaining material was transferred to a sieve (3 x 3 mm) where roots were

hand-separated from remaining large pieces of substrate and debris. Roots were rinsed

only after isolation, thus significantly reducing the use of water.

This root separator was used to isolate roots of individual Ligustrumjaponicum

Thum. grown in rhizotrons (Chapter 2) holding 0.16 m3 of substrate (Mix #4, Conrad

Fafard Inc., Agawam, MA). Twenty four plants were grown between May and October of

2007, and 24 more between March and June of 2008. Entire root systems were cleaned

utilizing the washing and hand-picking method in the 2007. In 2008, mechanical

isolation using the rotary separator preceded hand-picking. For the washing and hand-

picking method used in 2007, rhizotron contents after shoot removal were placed on top

of 3 x 3 mm sieves and roots were washed with 0.35 MPa water and hand-picked. For

mechanical separation, rhizotron contents were separated into four parts (ca. 40 L

each), and then rotated for 5 to 10 min each, depending on substrate moisture level

estimated visually (drier substrate needed less time). After rotation, roots were

removed and placed on the sieves used for hand-picking. They were rinsed with water,

and then oven dried at 650 C until constant dry mass was obtained.

To calculate the percentage of recovery (yield) from the root separator, root

samples from five woody plant species were isolated using the machine. Entire root

balls of container grown plants of Viburnum odoratissimum Ker Gawl., L. japonicum,

Illicium parviflorum Michx., and Magnolia grandiflora L. 'Little Gem' (Table 3-1) were

sampled for root isolation. Additionally, plants of Ilex cornuta Lindl. & Paxt. transplanted









into sandy soil, were also sampled. Of these plants, one fourth of the roots that

extended beyond the original root ball were used for root isolation. Rotation time was

varied based on root ball size and substrate moisture level. Rotation was stopped when

roots where visibly free of most small particles and remaining roots were cleaned as

described above for L. japonicum grown in rhizotrons. To determine total root mass,

substrate that fell from the cylinder was placed on the sieve and any additional root

pieces were collected by hand. Yield was calculated as percentage of root mass dry

recovered from the basket compared to the total dry root mass. Root yield from the five

plant species were compared using a completely randomized design, with 3

replications. Percent yield data were arcsin-square-root-transformed for data analysis.

Data analysis was accomplished using SAS (version 9.1, SAS Institute, Cary, NC).

Results and Discussion

Manual root washing of a 0.16 m3 sample required up to 16 man-hours (data not

shown). In contrast, mechanical isolation followed by hand-picking was completed in

approximately 4 man-hours. The time required for each sample to be processed with

the root separator was adjusted depending on substrate moisture content. Root

systems of L. japonicum from 24 rhizotrons were processed using mechanical isolation,

followed by hand-picking. This saved an estimated 288 man-hours (96 man-hours

versus 384 man-hours for hand washing in 2008 and 2007 experiments, respectively,

estimation based on 16 man-hours for hand picking and 4 man-hours for mechanical

isolation). The substrate medium selected for the rhizotron experiment was chosen to

minimize root cleaning time. However, tests of the container root balls included

substrates principally composed of composted pine bark (< 25 mm). Results from these

and the sandy soil suggested that total cleaning time can be diminished independently









of the root matrix composition. Samples processed for yield determination included

plants produced in substrates with different particle sizes and components. The

substrate for magnolia, anise, and viburnum root balls was composed mainly of large

pine bark pieces (up to 2.5 cm), which makes root separation much more difficult. The

sandy soil had a much finer texture than the rhizotron substrate. Plants selected had

different root morphology (fine versus coarse roots). Viburnum roots were coarse and

most resistant to breakage. L. japonicum roots were smaller in diameter than viburnum

roots, but larger than anise, magnolia or holly roots. Holly roots had the finest texture.

Roots of anise were the most tender of the species tested, and easily broken, yet yield

of anise roots was very high (Table 3-2). Yield of all species were high (Table 3-2), with

viburnum representing the lowest yield at 98.4% of total root dry mass.

The root isolation process worked best when the substrate or soil had low water

content. Time required for isolation depended on soil particle size, moisture level, root

density and morphology. To avoid root breakage, especially fine lateral roots, attention

was directed to minimize duration of rotation to no longer than necessary. Fine root

recovery was good for all species tested, and observationally better than root washing

by hand. High pressure water can break fine roots which are lost through the sieve.









Table 3-1. Root ball volume and substrate/soil of root samples.
Common Scientific Plant age Root ball
Substrate or soil type
name name (year) volume (L)
Viburnum Viburnum odoratissimum 2.0 11.4 commercial substrate z
Ligustrum Ligustrumjaponicum 3.0 11.4 commercial substrate Y
Holly Ilex cornuta 'Burfordii' 1.5 40.0 soil
Anise Illicium parviflorum 1.0 1.5 commercial substrate z

Magnolia gnola grandiflora 1.0 1.5 commercial substrate
'Little Gem'
z Pine bark fine, Florida sedge peat, sand (Florida Potting Soil, Inc., Apopka, FL).
Y Peat moss, pine bark, perlite, and vermiculite (Mix 4, Conrad Fafard Inc., Agawam, MA)
x Tavares-Millhopper fine sand

Table 3-2. Dry mass of root samples and yield achieved using the rotary root separator.
Common Root ball Rotation Time hand Dry mass Yield
name volume (L) time (min) Picking (min) roots (g) (%)

Viburnum 11.4 2 45 281.0 az 98.46 cz
Ligustrum 11.4 2 60 194.0 b 99.58 b
Holly 40 1 15 23.9 c 99.86 ab
Anise 1.5 0.25 5 5.8 c 99.85 ab
Magnolia 1.5 0.25 5 6.5 c 99.90 a
z Means of 3 repetitions. Means within columns not followed by the same letter are significant at
P<0.05 (Fisher's Least Significant Difference).































Figure 3-1. A) Root separator. B) Detail showing internal basket. C) Detail showing
reducing transmission and motor.









CHAPTER 4
EFFECTS OF WATER STRESS ON PLANT GROWTH OF Ligustrumjaponicum
THUNB.

Introduction

Plant responses to water shortage vary with severity as well as with duration of the

stress imposed (Farooq et al. 2009). The balance between dehydration and

photosynthetic activity is enabled by adaptation, acclimation and short-term

physiological regulation (Beikircher and Mayr 2009). Water stress can cause changes

from a subcellular to a whole plant level (Maseda and Fernandez 2006); however, the

major contributor to dehydration avoidance for a given species will vary depending on

the stress magnitude and duration. Plant adjustments to water availability may include

changes in aquaporin activity (Tyerman et al. 1999); abscisic acid (ABA) mediated

stomatal closure (Schraut et al. 2004); changes in morphological and anatomical

features such as change in leaf angle, deposition of cuticle, shedding of leaves, and

shifts in allocation of resources between roots and shoots (Abrams 1990; Kozlowski and

Pallardy 2002; Maseda and Fernandez 2006; Mishio 1992); deposition of dehydrins

(Pelah et al. 1997; Wisniewski et al. 2006) and osmotic adjustment (Kozlowski and

Pallardy 2002).

Constant or slowly imposed water stress can inhibit photosynthesis due to

stomatal closure (Saccardy et al. 1996), and reduced plant size (height, biomass, leaf

area, Maseda and Fernandez 2006). Water equilibration by whole organs can be on the

order of hours or even days. However, under conditions which result in very large and

rapid changes in extracellular water potential, such as moistening of soil following a

prolonged drought, aquaporins in root cells may play an important role in rapid water

absorption (Tyerman et al. 1999).









Cell enlargement is the cellular process most sensitive to drought stress. Plants

subjected to long-term water stress tend to slow shoot growth more than root growth.

Increased drought tolerance occurs with the onset of osmotic adjustments, following

wetting and drying cycles (Kozlowski and Pallardy 2002). Abiscisic acid is a prominent

contributor to maintenance of capacity for expansion of cells behind root apices under

severe water stress (Sharp et al. 2004). This enables root growth towards unexploited

soil. Over time, a larger root system can exploit a greater volume of soil, thus increasing

water absorption potential. Moreover, this switch in root-to-shoot ratio helps avoid water

stress because, proportionally, the volume of shoots that transpire will be smaller than

the volume of roots for water absorption.

McMillin and Wagner (1995) observed that the influence of water stress on root-to-

shoot ratio is dependent on stage of development. In addition, root-to-shoot ratio is

modified by natural growth patterns. Many woody species, such as L. japonicum, show

an episodic growth habit by fluctuating between periods of rapid shoot growth and slow

root growth, versus periods with the inverse pattern. Relatively little is known about the

changing carbon allocation patterns in species with episodic growth cycles (Dickson et

al. 2000a).

The objective of this research was to characterize and compare the effects of

water stress on dry matter partitioning and growth of L. japonicum exposed to: 1)

constant non-moisture-limiting conditions; 2) severe water stress; 3) constant low levels

of plant available water; 4) constant high levels of plant available water; 5) simulated

natural wetting and drying cycles of two days; and 6) simulated natural wetting and

drying cycles of seven days. Comparisons of plant growth among the experiments









described here provide an opportunity to view plant growth responses to water stress in

similar settings, therefore avoiding misleading comparisons between species,

developmental stages and growth conditions.

Material and Methods

Twenty-four star-shaped rhizotrons were constructed as described in Chapter 2

(Figure 2-3). Briefly, each rhizotron had four arms and held 0.16 m3 of substrate. They

were 1.76 m across, 0.30 m deep at the end of each arm and 0.35 m deep in the center.

Rhizotrons resided in an open-side greenhouse with a double polyethylene roof, under

natural light, located in Apopka, FL. Clonal Ligustrumjaponicum plants were selected

from a local nursery (Jon's Nursery, Eustis, FL) to ensure homogeneous size and

health. One plant was transplanted into each rhizotron using a commercial substrate

composed of Canadian sphagnum peat moss, processed pine bark, perlite, vermiculite,

starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc.,

Agawan, MA). Irrigation was supplied using a spray stake (model green 22500-001120,

Netafim Irrigation, Inc., Fresno, CA) at the tip of each arm pointing inward. Three

experiments, conducted consecutively over three years, examined shoot and root

growth under well-irrigated conditions and several different scenarios of water stress.

Experiment 1

Rhizotrons were used without the wick drainage system described in Chapter 2,

and thus had poor drainage. Plants from 11.4-L containers were transplanted into

rhizotrons in May 2007 and grown for 186 or 217 days. Substrate moisture was

managed based on measurements made with a soil moisture sensor (EC-5 ECH20

probe, Decagon Devices Inc., Pullman, WA). Five ECH20 probes were used to read a

series of 10 moisture levels determined gravimetrically based on a mass of substrate









oven dried at 700 C and a single calibration curve was developed based on average

readings (Nemali et al. 2007). One probe per rhizotron was installed at time of

transplant, adjacent to the original root system and half way down in the soil column,

and remained in place throughout the experiment. Irrigation was managed using a

datalogger (CR10X, Campbell Scientific, Inc., Logan, UT) connected to a AM16-32

multiplexer and two SDM-CD16AC remote relay controls (Campbell Scientific, Inc.). The

datalogger was programmed to query each probe every 60 minutes. If the volumetric

water content (VWC) at the probe was below the set point established for each

rhizotron, irrigation would be activated for 30 seconds. Times were established based

on trial and error to allow for substrate re-hydration around a probe. Plant available

water (PAW) was calculated for each individual rhizotron as the drained saturation

volume measured by the ECH20 probes minus available water of air dried substrate.

Drained saturation was determined by pouring 1 L of water around the newly inserted

probe, and then averaging VWC measured beginning from five until seven hours after

drenching. Treatments consisted of applying irrigation when PAW dropped below 30%

(moderately-stressed plants) and 70% (well-irrigated plants), with two harvest times as

the blocks, with 4 repetitions per block.

Stomatal conductance (gs) of three sun-exposed, fully expanded leaves from the

first growth flush of the current growing season of each plant was measured at 169 days

after transplant (DAT), between 1000 and 1400 hours with a steady-state porometer

(LI-1600; LI-COR, Lincoln, NE). Leaf area, dry mass of transplanted root ball, new roots,

leaves and stems were measured at final harvest. Entire root systems were cleaned









utilizing the washing and hand-picking method (Prathapar et al. 1989). Plant material

was oven dried at 650 C until constant dry mass was obtained.

Experiment 2

The wick drainage system was installed on rhizotrons as described in Chapter 2

prior to initiating this experiment, thus rhizotrons had good drainage. Plants from 11.4-L

containers were transplanted into rhizotrons in March 2008 and grown for 116 days.

After a 23-day acclimatization phase, plants were subjected to two treatments of

constant moisture levels (30% and 70 % PAW), with 12 replications each. Substrate

moisture level was managed based on substrate measurements as described for Exp.

1. Prior to initiation, sensors were re-calibrated against a time domain transmissometry

sensor (Digital TDT, model ACC-SEN-TDT, Acclima, Inc., Meridian, ID). The Digital

TDT was placed horizontally in the center of a 0.6-m polyvinyl ring made from landfill

liner material which was placed directly on porous ground cloth covering a native sand

soil base. The ring was 30-cm tall with the white side facing outward. Prior to placement

of the TDT, about 8 cm of the rhizotron substrate was placed in the bottom of the ring

and lightly packed. ECH20 probes were placed horizontally around the TDT, separated

from the TDT and other probes by at least 5 cm. Additional substrate was placed on top

of the sensors and lightly compacted to approximately 5-cm depth. Water from an onsite

well (electrical conductivity <0.2 ds/cm) was gently added to the substrate to a depth of

about 10 cm. Data was then collected from probes every 30 minutes for 13 days until

VWC had declined to approximately 20%. Data from each probe was analyzed by

regression (Microsoft Office Excel 2007, Microsoft Corp. Redmond, WA) against the

VWC recorded by the TDT to fit the best line (regression line for most probes was

quadratic).









Initially, ECH20 probes were placed close to the edge of a root ball and half way

down the soil column as in Exp. 1. Subsequently, probes were moved at 35-day

intervals to the edge of newly extended root tips along one arm of a rhizotron for all

rhizotrons. Plant available water was calculated as in Exp. 1 each time a probe was

relocated. Irrigation was managed as described for Exp. 1, but ran for 1.5 minutes each

event.

Leaf area and dry mass of roots, leaves and stems were measured at final

harvest. The mechanical root separator described in Chapter 3 was used to isolate

roots for the entire root ball. Plant material was oven dried at 650 C until constant dry

mass was obtained.

Experiment 3

Plants grown in 3.8-L containers were transplanted into each rhizotron in February

2009 and grown for 138 or 159 days. Entire rhizotrons were irrigated daily until

saturation for 41 days for plant acclimatization. Thereafter, irrigation was withheld for six

days before the start of irrigation treatments. Treatments consisted of two irrigation

frequencies, once a week for 10 minutes (12 L of water, moderately-stressed plants)

and every other day for 5 minutes (6 L of water, well-irrigated plants), with 12

replications each. Irrigation was initiated at 1930 hours each time. To characterize

variations of substrate moisture imposed by treatments, %VWC was measured every 5

min by an ECH20 probe and averages were recorded every 30 minutes. Probes were

re-calibrated against the Digital TDT before placement as described in Exp. 2. ECH20

probes were placed close to the root system and half way down in the soil column, 18

DAT, and then relocated at 41 and 84 DAT to the edge of newly-extended root tips. At

84 DAT, a second sensor, a Digital TDT, was installed in each rhizotron at the middle of









the arm, 20 cm from the ECH20 probe, to also monitor substrate moisture content.

ECH20 probe measurements from the entire experiment were corrected using

equations developed from regressions made of TDT versus ECH20 probe

measurements for the time both sensors were installed simultaneously. Saturation was

calculated as the average of measurements recorded from 5 to 7 hours after an

irrigation event (2 hours, 4 readings). The lowest achieved %VWC was the average of

the 2 hours before an irrigation event.

Stomatal conductance was measured with a steady-state porometer (LI-1600) at

99, 101, and 105 DAT. Measurements were taken between sunrise and sunset, in

intervals of 2.25 hours, on three sun-exposed, fully-expanded leaves from the first

growth flush of the current growing season chosen at random on each plant replication

(5 leaves per treatment). Stomatal conductance was measured for well-irrigated plants

at 1 and 2 days after irrigation (DAI), and for moderately-stressed plants at 1, 3 and 7

DAI. Daily stomatal conductance (Dgs) was developed as previously described for daily

accumulative water stress (Beeson 1992a). Daily stomatal conductance for each plant

was integrated each day by calculating the area under a gs curve of each repetition.

This parameter permitted simple comparison between the treatments, which

represented the potential quantity of water transpired during the day on a leaf area

basis.

Shoot water potentials were determined in six plants of each treatment 143 and

144 DAT at predawn and midday with a pressure chamber (Model 3000; Soil Moisture

Equipment Corp., Santa Barbara, CA) using compressed nitrogen. Measurement days

corresponded to one day before and one day after irrigation. Measurements were made









on individual twigs (ca. 10 cm long). Leaf area, number of leaves, and dry mass of

roots, leaves and stems of old and new growth were measured at final harvest. Plants

were harvested twice (138 and 159 DAT). Six plants from each treatment were

harvested at root flush (increasing root growing points, Chapter 6) and six at shoot flush

(increasing shoot growing points) at the termination of the experiment. The mechanical

root separator described in Chapter 3 was used to isolate roots for the entire root ball.

Plant material was oven dried at 650 C, until constant dry mass was obtained.

Results and Discussion

Soil moisture management

Over the course of the three experiments described here, ECH20 probe

measurements were found to vary up to four-fold among probes. For this reason, a

Digital TDT was used in conjunction with each of the ECH20 probe on Exp. 3 to develop

individual calibration equations for each ECH20 probe. TDT measurements of VWC are

similar to TDR (Blonquist et al. 2005; Burke et al. 2005; Harlow et al. 2003). TDR

sensors are considered the most accurate measurement of soil VWC, and the standard

by which other soil moisture sensors are evaluated (Blonquist et al. 2005). The

calibration equations represented a 30 day period comprising several irrigation cycles.

Calibration equations (r2 = 0.72 to 0.96) were used to re-calibrate ECH20 probe

measurements for all three experiments.

Each experiment was intended to provide a moisture deficient condition in one

treatment (30% PAW) and a well-irrigated condition in the other (70% PAW). The

irrigation control system used for Experiments 1 and 2 was developed to impose a set

VWC, and maintain substrate moisture levels close to the set-point throughout the

duration of the experiment. The system compensated for plant size and microclimate









effects on water content of the substrate. Nonetheless, water availability experienced by

the plants should be viewed in the light that probes used to control irrigation produced

highly variable values among rhizotrons. Thus, substrate moisture readings of each

experiment were reevaluated to provide more precise definition of the treatments for

comparisons and correct data interpretation.

Percent VWC in Experiments 1 and 2, and drained saturation and lowest achieved

%VWC in Exp. 3 were analyzed as a split-plot design (version 9.1, SAS Institute, Cary,

NC), with repeated measurements over time. Mean recalculated %VWC which occurred

during the three experiments were still different between treatments (Table 4-1).

Although measurements from Exp. 1 resulted in significant differences, 21.2 and

31.3 %VWC for moderately-stressed and well-irrigated plants respectively, poor

drainage from rhizotrons resulted in abundant water availability for plant growth for both

treatments. Insufficient removal of excess irrigation from the rhizotron resulted in the

occurrence of a perched water table at the bottom, which the ECH20 probe, installed at

one arm, was not able to detect (Chapter 2).

The ECH20 probe-measured saturation VWC was above the average and high in

some Exp. 2 rhizotrons. When this occurred in rhizotrons designated for 70% PAW, the

effect was negligible for most parameters measured; only leaf area was significantly

different between rhizotrons with dissimilar levels of saturation VWC. However, when

this occurred in rhizotrons designated for an irrigation trigger VWC of 30% PAW

(moderately-stressed plants), differentials between saturation (mean 46.1% VWC) and

the triggering VWC (mean 23.0% VWC) were 1.8 times higher for some

moderately-stressed plant replications than others. This resulted in irrigation occurring









only once or not at all in some moderately-stressed treatment rhizotrons after the initial

acclimation period. Additionally, each time a sensor was relocated; it was watered-in to

ensure close contact with the substrate to prevent erroneous measurements. This

created a localized increase in substrate moisture around the probe, thus further

delaying irrigation. The result of the differentiated irrigation was an unintended, but

unique subgroup of the moderately-stressed treatment which died near the end of the

experiment. These plants were designated as severely-stressed plants and analyzed as

an independent third treatment.

Experiment 1

Although averaged half-hour %VWC recorded by ECH20 probes during the

experiment was higher (P<0.05) for the well-irrigated plants than for

moderately-stressed plants (31.30 and 21.17%, respectively, Table 4-1), total plant dry

mass was not significantly different (P<0.05) between the two treatments at each

harvest. Also, as discussed above, all plants were exposed to excessive moisture due

to the perched water table discovered in rhizotrons (Chapter 2). Data of the two

treatments is presented polled (Table 4-2 and 3). All plants directed over 70% of mass

allocation to shoots (Figure 4-1, Table 4-3). Root growth was limited due to ample water

availability for root absorption even in the moderately-stressed treatment. Biomass

accumulation of all plant components was similar between treatments within each

harvest (data not shown) although %VWC was different at the sensor site (Table 4-1).

Allometric relationships were not affected in the treatment moderate-stress. Mean gs at

midday was similar (P>0.05) 138.5 and 132.8 mmol m-2 sec-1 for well-irrigated and

moderately-stressed treatments, respectively.









Stationary single capacitance probe measurements did not represent PAW

throughout the entire growing period in a large soil volume, especially for the low VWC

treatment. As roots elongated into unexplored substrate, they could exploit areas of high

VWC, since the entire substrate was moistened at each irrigation event. Furthermore,

water depletion in the soil region around the probe increased with time due to

accumulation of roots around it, which increased irrigation frequency (data not shown).

Additionally, later tests revealed the presence of a perched water table as discussed in

Chapter 2. These results suggested that irrigation control using single probes in large

soil volumes should be more accurate if the probe was relocated as new roots

expanded into unexplored substrate. Variation on water distribution within the soil

exploited by the root system should also be considered. Hedley and Yule (2009)

concluded that the accuracy of predicting VWC in a large native soil area was limited by

spatial variability of the soil moisture pattern within the area.

Experiment 2

Moderate constant stress (moderately-stressed plants) resulted in significantly

smaller biomass production in all plant components (Table 4-4). After 100 days under

severe stress conditions, all plants were dead. Dry mass of severely-stressed plants

was lower than for moderately-stressed and well-irrigated plants (Table 4-4). Allocation

to roots and root-to-shoot ratio (Table 4-5) was higher in severely-stressed plants than

in plants of the other two treatments. The high root-to-shoot ratio obtained in Exp. 2

noticeably contrasts with results from Exp. 1 (Figure 4-1). The average ratio was 0.36

between treatments and harvests of Exp. 1, where water availability was at excess

levels, versus 0.87, 0.75, and 0.72 for severely-stressed, moderately-stressed and

well-irrigated plants respectively in Exp. 2. Stabler and Martin (2006) studying









Caesalpiinia pulcherrima, found that frequent irrigation (every 2 days) decreased root-

to-shoot ratio by ca. 40% compared with moderate irrigation (every 5 days), and by

approximately 48% compared with infrequent irrigation (every 10 days). Here root-to-

shoot ratio was 21% less for well-irrigated plants compared to severely-stressed plants.

Root-to-shoot ratio appears to be very species- and perhaps situation-dependent.

Irrigation cycles of 2 days resulted in root-to-shoot ratio of 0.25 for Pittosporum tobira

and 0.49 for Viburnum odorissimum (Scheiber et al. 2007); 0.80 and 1.0 for Citrus

sinensis grown in sand and peat, respectively (Sanchez and Syvertsen 2009); 0.53 for

Caesalpinia pulcherrima, and 0.43 for Cercidium floridum (Stabler and Martin 2006).

Yeager et al. (1990) testing growth responses of L. japonicum to vesicular-arbuscular

mycorrhizae inoculation observed root-to-shoot ratio of 0.95 and 1.25 in container-

grown plants inoculated and noninoculated respectively.

Differences between the first two experiments are due to: 1) the success in

creating a more natural percolation of water through the entire substrate column; and

2) relocation of moisture probes to compensate for root elongation into very moist

substrate. Average %VWC was significantly different for all three treatments in Exp. 2

(Table 4-1). Dissimilarities between results observed in Experiments 1 and 2

demonstrate the preferential allocation to root growth by plants under chronic water

stress, and preferential allocation towards shoots under luxurious water conditions.

Plants in the severely-stressed treatment overcame the imbalance between

transpiration and water absorption through changes in allometric relationships (Maseda

and Fernandez 2006; Reddy et al. 1998). Specific leaf area was diminished by 31.5% in

severely-stressed plants compared with moderately-stressed plants (Table 4-5),









indicating these plants produced smaller and thicker leaves to reduce transpiration by

the canopy, as reported for other species (Abrams 1990; Fernandez et al. 2002;

Montague et al. 2000; Zwieniecki et al. 2002). The ratio of leaf area to root dry mass

(leaf area-to-root dry mass), a comparison between the transpiring surface and the

absorption mass, was also decreased by 45.8% compared with well-irrigated plants

(Table 4-5).

Water stress reduced whole plant biomass, but affected root growth to a lesser

extent than leaf growth (Table 4-4). Severe stress caused a reduction of 33.7% in root

biomass production compared to well-irrigated plants, while chronic water stress

provoked by moderately-stressed treatment resulted in a 26.4% reduction. In contrast

total leaf area was severely decreased by severe water stress, 64.2% and 26.4%

reductions for severely- and moderately-stressed plants (respectively) compared to

well-irrigated plants.

Death of severely-stressed plants was the result of insurmountable high water

stress. Plant water loss cannot exceed the maximum supply through roots indefinitely

(Jackson et al. 2000). Root growth occurred during the early period of the experiment

through the first relocation of the moisture probe (as evidenced by the count of root

growing points, Chapter 5). Root growth was limited after relocation of the probes. With

increased water stress, turgor pressures required for root growth apparently were

insufficient to expand root tips of these plants through dry substrate to reach into moist

substrate only centimeters away. High water stress was associated with slow root

growth in Quercus virginiana (Beeson 1992b). Moisture distribution in the substrate

profile was observed to be uneven at 100 DAT. Substrate located close to root systems









was visually dry, while substrate located towards the tips of each arm of a rhizotron

were wet. As observed in other species, plants that were able to explore further into the

substrate were able to survive, even under low VWC, such as observed in the

moderately-stressed treatment (Table 4-1). Volaire et al. (1998) concluded that an

important trait associated with survival of perennial grasses under prolonged drought

was the ability to develop deep root systems. Deep root systems enabled Quercus spp.

to maintain relatively high predawn water potentials during drought (Abrams 1990).

Hibiscus glaber avoided severe stress by developing a dense deep root system (Mishio

1992).

Experiment 3

When L. japonicum plants were subjected to wetting and drying cycles, the

moderately-stressed treatment predominately affected aboveground components of a

plant, particularly by diminished leaf growth. Compared to the well-irrigated treatment,

the moderately-stressed treatment resulted in reductions of 28.9% and 32.4% in dry

mass of new leaves harvested at root flush and at shoot flush respectively (Table 4-6);

30.7% in the number of new leaves when in shoot flush (Table 4-7); and 31.8% and

30.3% in leaf area of plants in root flush and shoot flush, respectively (Table 4-7).

Reduced leaf growth was the main contributor to reductions of 27.3% and 29.4% in the

dry mass of new shoots produced by plants on root and shoot flush, respectively (Table

4-6). Biomass reductions caused by water stress are well documented. For example,

grasses responded to drought with a 60% biomass reduction (Fernandez et al. 2002),

Caesalpinia pulcherrima responded with up to 36% shoot biomass reduction (Stabler

and Martin 2006), and Pittosporum tobira responded with up to 34% shoot biomass

reductions (Scheiber et al. 2007). In experiments described here, the biggest decrease









in shoot biomass was observed in severely-stressed plants of Exp. 2, which exposed

plants to low VWC for a prolonged period (40.4% compared with well-irrigated plants,

Table 4-4). Comparison of biomass shrinkage suffered by the moderately-stressed

treatment in Experiments 2 and 3 (constant exposure to 16.5% VWC versus intermittent

exposure to 19.8% VWC) exhibits similar levels (ca. 20% compared with respective

well-irrigated plants).

Partitioning of resources in the stressed and well-irrigated treatments were similar

(averaged allocation to shoots of 59.1% on root flush and 56.1% on shoot flush, Table

4-7), and occurred in similar levels of those observed in Exp. 2 (averaged 57.7%

allocation to shoots, Table 4-5), but shoot allocation was much lower than observed in

Exp. 1 (averaged 73.7%, Figure 4-1). In Exp. 1, dry mass distribution between above-

and below-ground portions of the plant is clearly toward promoting shoot growth, while

in Exp. 2 and 3 it is more balanced (Figure 4-1). Kozlowski and Pallardy (2002)

reviewed evidence that drought promotes relative allocation of photosynthates to root

growth. In our experiments, however, only severe stress resulted in preferred allocation

to roots.

Specific leaf area of plants of Exp. 3 was consistent between treatments (Table 4-

7), and was similar to the levels observed in moderately-stressed and well-irrigated

treatments of Exp. 2 (Table 4-5). Stress treatment not only diminished dry mass, but

also leaf size (Table 4-6). Old leaves of plants in this experiment had an average leaf

area of 8.53 cm2 (leaf area /number of leaves, Table 4-7). The average size for new

leaves at root flush was 11.73 cm2 for moderately-stressed plants and 13.34 cm2 for

well-irrigated plants; however, by the second harvest, new leaves of









moderately-stressed plants had increased to similar size of those growing in

well-irrigated plants (13.34 and 13.81 cm2 for moderately-stressed and well-irrigated

plants, respectively). Similarly, despite lower leaf area:root caused by moderate stress

treatment at the root flush harvest, by the second harvest this difference was no longer

significant. This comes as evidence that moderately-stressed plants were close to, or

had established a balance between transpiring area and absorbing mass, which if in a

landscape, the plants would have been considered established. Ilex cornuta, irrigated

every 7 days, took 24 weeks to reach similar levels of those observed in plants irrigated

every 2 days of cumulative water stress, thus being considered established (Scheiber et

al. 2007).

Daily stomatal conductance (Dgs) of moderately-stressed plants was lower than

well-irrigated plants at the day of maximum water stress during Exp. 3 (7 and 2 DAI for

moderately-stressed and well-irrigated plants, respectively, Figure 4-2A). However, Dgs

values were similar between irrigation treatments at 3 DAI for moderately-stressed

plants and 2 DAI for well-irrigated plants (5,983 and 6,344 mol m-2 day-', respectively).

Thus, there was sufficient water available near the end of the experiment to moderately-

stressed plants for normal transpiration for at least the first three days. This explains

some of the differences in dry mass. Within flushes, which were separated by 21 days,

there were no differences in root dry mass between treatments (Table 4-6). However,

there were differences in shoot dry mass, with more mass during shoot flush for the

well-irrigated plants than for moderately-stressed plants due to new shoot growth, with

new leaves contributing the most.









Predawn shoot water potentials were more negative for moderately-stressed than

well-irrigated plants, whether on the day before irrigation (maximum water stress) or on

the day after irrigation (minimum water stress, 10.5 hours after irrigation, Figure 4-2B).

Midday water potentials were similar between treatments each day, but were

significantly different between days. More negative water potentials of

moderately-stressed plants would result in smaller increases in shoot elongation

(Beeson 1992a) compared with root elongation, and could explain why there were few

differences in dry mass of plant components at root flush, yet significant differences in

new and total shoot growth, and new and total leaf mass at shoot flush (Table 4-6). In

Pinus pinaster, increased root growth was attributed to cell wall loosening at moderate

water stress (0.15 MPa), with inhibition of root growth attributed to severe water stress

(0.45 and 0.66 MPa) due to the inability of the plants to maintain turgor (Triboulot et al.

1995).

Exp. 3 subjected plants to wetting and drying cycles, which are more common in a

natural landscape setting. A plant under high water availability has the opportunity to

expand growing tissues, and implement strategies to tolerate drought that plants under

constant stress do not. Severely-stressed plants mainly directed resources towards root

growth, and had a severe impairment of shoot expansion, thus their photosynthate

production required to sustain root growth was in turn limited. Additionally, long-term

stress experienced by severely-stressed plants likely impaired net photosynthesis due

to stomata closure. Saccardy et al. (1996) concluded that the drop in photosynthesis

rate caused by water stress was mainly due to stomatal closure when a maize plant

dehydrates slowly, such as severely- and moderately-stressed plants of Exp. 2, while it









was mainly due to inhibition of non-stomatal processes when a plant is rapidly

dehydrated, such as in Exp. 3. Severely-stressed plants experienced slow dehydration,

with the initial high %VWC in these rhizotrons, 35 days were generally required to

decrease the %VWC to ca. 17%. Thus, inhibition of photosynthesis due to water stress

was likely constant in severely-stressed plants due to stomatal closure. Kanechi et al.

(1996) reported 65% of the limitation of photosynthesis in coffee after rapid dehydration

was due to non-stomatal processes (water potential declined from -1.0 to -4.0 over the

course of one week). The moderately-stressed plants in the Exp. 3 likely experienced

rather rapid dehydration since the %VWC declined to an average of 19.8% within a

week after irrigation. Recently it has been reported that transplanted woody species

undergo a severe water stress unless they receive irrigation every 2 days (Scheiber et

al. 2007).

Correlations between root dry mass and shoot dry mass were consistently high in

all three experiments (Figure 4-3). In Exp. 2, A%VWC (differential between saturation

and the triggering volumetric water content) was what ultimately dictated frequency of

irrigation, and in Exp. 3, it represented the frequency of irrigation. Although measured

biomass had strong correlations with A%VWC in Exp. 2 (Figure 4-4), root-to-shoot ratio

did not correlate well with A%VWC (r = 0.56, data not shown). Correlation of root-to-

shoot ratio and A%VWC in Exp. 3 was also only moderate (r = 0.6). Overall, lower

relationships observed for root-to-shoot ratio and A%VWC suggests that root-to-shoot

ratio may not be the best indicator of water status during the growing period of the plant.

Root-to-shoot ratios calculated from Exp. 2 and 3 were comparatively similar and higher

relative to those observed in Exp. 1. Actual percent contributions of roots to total









biomass for moderately-stressed and well-irrigated plants of Exp. 2 and 3 were

generally around 42%, and varied marginally between these two water moisture levels

(Figure 4-1), yet there were substantial differences in root and shoot dry mass quantities

between treatments in Exp. 2 (Figure 4-3 and 4-4, Table 4-4).

This contrasts with suggested relationships of root-to-shoot ratio in the literature.

Johnson et al. (1991) developed a water submodel that included root-to-shoot message

control of gs for incorporation into mechanistic plant growth models. Kozlowski and

Pallardy (2002) suggested that water deficits ultimately result in plants that have higher

root-to-shoot ratio and greater capacity to absorb water and minerals relative to shoots

that must be supported. Thornley (1996) proposed that in response to limited soil

moisture availability over extended periods of time, plant water status is more strongly

influenced by changes in root-to-shoot partitioning and root density than the interaction

of soil moisture content with gs.

The increase in root-to-shoot ratio is an adaptive response to water stress

(Chaves et al. 2003; Jackson et al. 2000), however, our results indicate that a severe

stress is needed to induce this response in L. japonicum plants. The balance between

root dry mass and shoot dry mass, namely root-to-shoot ratio, was influenced by water

availability in our results; however, it should not be viewed as a sole indicator of plant

water availability during the growth period. As previously observed for C4 species

(Sharp et al. 2004), root growth is less sensitive to water stress than shoot growth.

L. japonicum (C3) maintained root growth even with slow dehydration to ultimately fatal

levels, even though shoot growth was severely impaired. Constant, moderate levels of

water stress also reduced plant growth as whole; however, shoot growth, though









restricted, still occurred. This enabled these plants to survive since photosynthates

required for root growth could be supplied. Intermittent exposure to moderate levels of

water stress reduced plant biomass by similar amounts to that achieved by continuous

exposure. However, the wetting and drying cycles enabled plants under stress to

achieve greater carbon assimilation than plants under constant water stress, which led

to establishing a normal balance between absorbing and transpiring surfaces by the end

of the study.

Accurately imposing and measuring water stress in mature woody plants in a large

scale, while keeping other variables as constant as possible, is not an easy task. Soil

moisture measurements can be deceiving, thus a system has to be evaluated as a

whole for best performance. The final experimental system described here appears to

have achieved this goal. Future research delving into the study of water stress or other

growth variables of perennial plants may find this system valuable.









Table 4-1. Mean percent volumetric water content (%VWC) achieved in Experiments 1,
2 and 3 determined after recalculation using equations based on prolonged
dual measurement of VWC using Digital TDT and ECH20 probes.
Severely-stressed Moderately-stressed Well-irrigated
plants plants plants
Exp. 1 21.17 bz 31.30 a

Exp. 2 16.48 c 17.44 b 32.55 a

Exp. 3
Drained saturation 25.03 a 25.06 a
Low achieved %VWC 19.80 b 22.33 a
Differential 5.22 a 2.68 b
z Means within rows not followed by the same letter are significant at P<0.05 (Fisher's Least
Significant Difference).

Table 4-2. Experiment 1. Dry mass (g) of components of L. japonicum grown at different
constant volumetric water contents in rhizotrons. Moisture probes remained
near original roots ball for the duration of the experiment. Masses between
treatments were pooled within harvest.
Harvest 1 Harvest 2
186 DATz 217 DAT
Shoot 958.5 by 1461.1 a
Leaves 529.5 b 868.6 a
Stem 429.0 b 592.6 a

Total roots 331.5 b 539.4 a
Old roots 201.6 b 274.1 a
New roots 129.9 b 265.3 a

Plant 1290.0 b 2000.6 a
z Days after transplant.
Y Means representative of 4 replications. Means within rows not followed by the same letter are
significant at P<0.05 (Fisher's Least Significant Difference).
x Roots within the transplanted root ball.
w Roots grown during the duration of experiment.









Table 4-3. Experiment 1. Leaf area, allometric relationships and percentage of
allocation in L. japonicum grown in rhizotrons at constant volumetric water
contents. Results between treatments were pooled within harvest.
Harvest 1 Harvest 2
186 DATz 217 DAT
Leaf area (cm2) 43,587 ay 55,073 a


Specific leaf area (cm2 g-1)
Root-to-shoot ratio (g g-l)
Root dry mass-to-leaf dry mass ratio (g g-l)
Leaf area-to-root dry mass ratio (cm2 g-1)


82.92 a
0.34 a
0.57 a
132.03 a


62.22
0.37
0.63
101.25


% Allocation to shoots 74.44 a 73.05 a
% Allocation to roots 25.56 a 26.95 a
z Days after transplant.
Y Means representative of 4 replications. Means within rows not followed by the same letter are
significant at P<0.05 (Fisher's Least Significant Difference).

Table 4-4. Experiment 2. Dry mass (g) of components of L. japonicum grown in
rhizotrons at constant volumetric water contents.


Severely-stressed
plants
100 DATz
217.24 cY
116.88 c
100.36 c

187.40 b


Moderately-stressed
plants
116 DAT
308.52 b
165.44 b
143.08 b

233.72 b


Well-irrigated
plants
116 DAT
396.01 a
215.38 a
180.63 a

282.49 a


Plant 404.64 c 542.24 b 678.50 a
z Days after transplant.
Y Means of 5, 5, and 12 replications for severely-stressed, moderately-stressed, and
well-irrigated plants, respectively. Means within rows not followed by the same letter are
significant at P<0.05 (Fisher's Least Significant Difference).


Shoot
Leaves
Stem

Roots









Table 4-5. Experiment 2. Leaf area, allometric relationships and percentage of
allocation in L. japonicum grown in rhizotrons at constant volumetric water
contents.
Severely-stressed Moderately-stressed Well-irrigated
plants plants plants
100 DATz 116 DAT 116 DAT
Leaf area (cm2) 4,205 cy 8,649 b 11,750 a

Specific leaf area (cm2 g-) 35.86 b 52.37 a 54.44 a
Root-to-shoot ratio (g g-1) 0.87 a 0.75 b 0.72 b
Root dry mass-to-leaf dry
mass ratio (g g-1) 1.61 a 1.40 b 1.32 b
Leaf area-to-root dry mass
ratio (cm2 g-) 22.70 b 37.73 a 41.94 a

% Allocation to shoots 53.69 b 57.09 a 58.37 a
% Allocation to roots 46.31 a 42.91 b 41.63 b
z Days after transplant.
Y Means of 5, 5, and 12 replications for severely-stressed, moderately-stressed, and
well-irrigated plants, respectively. Means within rows not followed by the same letter are
significant at P<0.05 (Fisher's Least Significant Difference).









Table 4-6. Experiment 3. Dry mass (g) of components of L. japonicum grown in
rhizotrons at variable volumetric water content.


Moderately-stressed plants
138 DATz


Shoot flush


58.1
67.8
125.9


55.2
25.4
80.7


113.4
93.2
206.6


96.1
79.8
175.9


Well-irrigated plants
159 DAT


Root flush


57.5
75.4
132.8


48.5
25.6
74.1


106.0
101.0
207.0


84.3
47.4
131.7


Shoot flush


58.1
100.2
158.2


69.7
31.8
101.4


127.7
131.9
259.7


98.6
87.4
186.1


Plant 300.0 c
z Days after transplant.
Y Means of 6 replications.


382.5 ab 338.7 bc 445.7 a

Means within rows not followed by the same letter are significant at


P<0.05 (Fisher's Least Significant Difference).


Root flush


Leaves
Old
New
Total

Stem
Old
New
Total

Shoot
Old
New
Total

Root
Old
New
Total


53.5
53.6
107.0


46.3
19.8
66.1


99.7
73.4
173.1


82.8
44.1
126.9









Table 4-7. Experiment 3. Leaf area, allometric relationships and percentage of
allocation in L. japonicum grown in rhizotrons at variable volumetric water
content.


Moderately-stressed
plants
138 DATz


Root flush


Shoot flush


Well-irrigated
plants
159 DAT
Root flush Shoot flush


Old leaves
Number of leaves
Leaf area (cm2)
Specific leaf area (cm2 g-1)

New leaves
Number of leaves
Leaf area (cm2)
Specific leaf area (cm2 g-1)

Total leaves
Number of leaves
Leaf area (cm2)
Specific leaf area (cm2 g-1)

Allometric relationships
Root-to-shoot ratio (g g-1)
Root dry mass-to-leaf dry
mass ratio (g g-1)
Leaf area-to-root dry mass
ratio (cm2 g-1)


% Allocation
to shoots
to roots
z Days after transplant.


288.2
2,570
48.3


288.8
3,386
63.2


577.0
5,957
55.8


317.8
2,734
47.1


284.6
3,949
59.1


602.4
6,683
53.4


0.75 ab 0.85 a
1.21 ab 1.39 a


322.0
2,528
44.4


372.3
4,967
66.4


694.3
7,495
56.9


0.64 b 0.72 b
0.99 c 1.18 bc


47.30 b 38.76 b 57.77 a 46.10 b


57.43
42.57


Y Means of 6 replications. Means within rows
P<0.05 (Fisher's Least Significant Differenci


54.08
45.92


61.25
38.75


not followed by the same letter are significant at


323.2
2,824
48.5


410.7
5,669
56.7


733.8
8,493
53.6


58.28
41.72












100% 100% 100%

80% 80% 80% -

60% 60% 60%

40% 40% 40%

20% 20% -20%

0% 0% 0%
Harvest Harvest2 Severely- Moderately- Wall- Rootflush Shootflush Rootflush Shootflush
stressed stressed irrigated
Excessive moisture plants plants plants Moderately-stressed Well-irrigated
plants plants
90 New roots M Stem Roots M Stem M Leaves E New Root [] New Stem n New Leaves
Old roots M Leaves Old Root HOld Stem MOld Leaves

Figure 4-1. Percent contribution of each plant component to the total dry mass of
L. japonicum grown under different substrate moisture levels. Experiment 1:
moisture probes remained near original roots ball for the duration of the
experiment. Experiment 2: constant VWC due to consistent relocation of
moisture probes. Experiment 3: irrigation varied between saturation and 19.8
and 22.3% VWC for moderately-stressed and well-irrigated plants
respectively. Plants harvested during root and shoot flush.


Experiment 2


Experiment 3


Experiment 1










minimum water stress maximum water stress
7000 a A
a a a A
6000
b
S5000

S4000

E 3000

o 2000

1000

0
0 --

-o2 B

-OA a a
b b
-0.6

I-


a
-1.4 a


-1.8
6am 1pm 6am 1pm

minimum water stress maximum water stress

U Well-irrigated plants ] Moderately-stressed plants

Figure 4-2. Daily stomatal conductance, Dgs (A) and shoot water potential, YT (B) of
L. japonicum grown under irrigation varying between saturation and 19.8 and
22.3% VWC for moderartely-stressed and well-irrigated plants respectively on
the day of minimum and maximum water stress in Exp. 3. Each bar
represents the mean of fifteen daily curves of Dgs (5 plants x 3 leaves) and 6
replications of YT. Bars not followed by the same letter within each time are
significant at P0.05 (Fisher's Least Significant Difference). Bars followed by
within each treatment and time are significant at PO.05 (Fisher's Least
Significant Difference).










700
650
600
550
500
450
400
350
300
250
200
550


Shootdry mass (g)


A A



Al


r = 0.85


Shootdry mass (g)


250
230
210 -
190
170
150
lU-
130 -
110
90 -
70
50


0 A


r=0.79


X Severely-streseed
plants

0 Moderately-streseed
plants

A Well-irrigated
plants


100 150 200 250 300 350
Shootdry mass (g)
Figure 4-3. Correlation among root and shoot dry mass. (A) Experiment 1, (B)
Experiment 2 and (C) Experiment 3. The correlation coefficient (r) is included
for each relationship (all samples pooled).


r = 0.88

800 1050 1300 1550


350
330
310
290
270
250
230
210
190
170
150


A














A A


400 -
41-
B 350 -

S300 -
E

- 250 -
o
r 200

150


A%VWC


0 0


r= -0.88


510 -
450 -
400 -
35U -
YU10 -
250 -
200 -
150


A %VWC


r= -0.90


10 20
A%VWC


10 20
A %VWC


X Severely-stressed plants

0 Moderately-stressed plants

A Well-irrigated plants

Figure 4-4. Correlation for Exp. 2 among A%VWC (differential between saturation and
the triggering volumetric water content) and (A) root dry mass, (B) shoot dry
mass, (C) leaf dry mass, and (D) leaf area. The correlation coefficient (r) is
included for each relationship (all samples pooled).


S A r=-0.72
_


B









r=-0.87

30


1.8 -
16 -
1.4 -
1.2 -
1.0 -
0. -
0.6 -
0.4
02












CHAPTER 5
DEVELOPMENT OF SHOOT ARCHITECTURE OF Ligustrumjaponicum Thunb. IN
RESPONSE TO SOIL MOISTURE.

Introduction

The process of shoot branching is an important determinant of a plant's shape.

Shoot branching is the process by which axillary buds, located on the axil of a leaf,

develop and form new flowers or branches. Bud outgrowth is regulated by the

interaction of environmental and endogenous signals, such as plant growth regulators.

These interacting factors have a major effect on shoot system architecture (Ongaro and

Leyser 2008).

Depth of bud dormancy has been proposed to be related to abscisic acid (ABA)

levels (Tamura et al. 1993) and water status of the bud (Arora et al. 2003). Prolonged

exposure to drought can trigger ABA responses in shoot meristems, such as inhibition

of leaf production, growth and development (Kuang et al. 1990; Sauter et al. 2001).

Growth restrictions result in meristems losing relatively little water during water stress

(Arora et al. 2003). Frugis et al. (2001) demonstrated that overexpression of a gene in

lettuce associated with accumulation of specific types of cytokinins, caused alterations

to plant architecture, which changed from determinate to indeterminate leaf growth.

Overall plant form is achieved by regulation of initiation and outgrowth of axillary

meristems (Kerstetter and Hake 1997). The tendency of a dormant lateral bud to

outgrow is regulated by a network of variables (Waldie et al. 2010). Among the most

important variables are age of the bud relative to its initiation, and the zone of the stem

in which the bud develops (basal versus aerial branching, Napoli et al. 1999). Roles of

cytokinin as promoter and auxin as an inhibitor of lateral bud outgrowth have long been

known and studied in branches with decapitated terminal bud. However, it has been









suggested that these two plant growth regulators may not be the only factors in lateral

bud breakage of dormancy during undisturbed growth. A network of shoot and root

signals that regulates branching has been proposed (Thomas and Hay 2009). A

branching signal has been postulated to exist, which moves in the direction of root-to-

shoot (Beveridge 2000). The identity of this signal is still unknown, although a strong

candidate has recently emerged (Waldie et al. 2010).

Borchert (1975) observed variations of shoot growth patterns between young and

mature plants of Quercus palustris Muenchh. Patterns ranged from a series of flushes

characterized by determinate growth to longer flushes characterized by indeterminate

growth. Branch position or geometry alone was not sufficient to account for the various

growth patterns existing between species; other factors, such as timing of shoot growth,

also affected shoot architectural pattern (Napoli et al. 1999).

Napoli et al. (1999) noted that research on dormancy and shoot growth has mainly

focused on herbaceous plants; thus, all concepts are not necessarily applicable to

woody species. Developing a clearer understanding of bud dynamics and their type-

specific contributions under undisturbed conditions, is a necessary prerequisite for

predicting their responses under disturbed conditions (Zhang et al. 2009). Many

researchers have studied shoot architecture, but have given exclusive attention to shoot

growth by itself. Recently a root signal was proposed (Waldie et al. 2010). However, the

influence of relative growth of roots and shoots are still obscure. Relationships between

physiological processes and environmental conditions or plant development are often

confounded by variation on genetic, environmental or ontogenetic factors (Hanson et al.

1986).









The research presented here describes the natural growth of clonal Ligustrum

japonicum as a basis for understanding how this growth is influenced by water stress,

and ultimately integrate this knowledge to characterize shoot and root growth patterns.

Material and Methods

Growth conditions and experimental design

Twenty-four star-shaped rhizotrons were constructed as described in Chapter 2

(Figure 2-3). Briefly, each rhizotron had four arms and held 0.16 m3 of substrate. They

were 1.76 m across, 0.30 m deep at the end of each arm and 0.35 m deep in the center.

Rhizotrons resided in an open-sided greenhouse with a double polyethylene roof, under

natural light, located in Apopka, FL. Clonal Ligustrumjaponicum plants were selected

from a local nursery (Jon's Nursery, Eustis, FL) to ensure homogeneous size and

health. One plant was transplanted into each rhizotron using a commercial substrate

composed of Canadian sphagnum peat moss, processed pine bark, perlite, vermiculite,

starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc.,

Agawan, MA). Irrigation was supplied using a spray stake (model green 22500-001120,

Netafim Irrigation, Inc., Fresno, CA) at the tip of each arm pointing inward. Two

experiments, conducted consecutively over two years, examined patterns of shoot and

root growth under well-irrigated conditions and water stress applied constantly (2008) or

intermittently (2009).

In the experiment conducted in 2008, substrate moisture was kept constant at

three levels: 32.5% VWC (percent volumetric water content, well-irrigated plants,

Chapter 4, Table 4-1); 17.4% VWC (moderately-stressed plants); and 16.5% VWC

(severely-stressed plants). Plants from 11.4-L containers were transplanted into

rhizotrons in March and grown for 116 days. After a 23 day acclimatization phase,









plants were subjected to three irrigation frequencies (well-irrigated, severely-stressed,

and moderately-stressed), with 12, 5 and 5 replications each, respectively. Substrate

moisture level was managed based on substrate measurements as described in

Chapter 4.

In the experiment conducted in 2009, substrate was irrigated at 2 and 7 day

intervals (well-irrigated and moderately-stressed plants, respectively). These two

treatments resulted in VWC of 22.3% and 19.8% in the driest periods (Chapter 4, Table

4-1). Plants grown in 3.8-L containers were transplanted into each rhizotron in February

and grown for 138 or 159 days. Entire rhizotrons were irrigated daily until saturation for

41 days for plant acclimatization. Thereafter, irrigation was withheld for six days before

the start of irrigation treatments. Treatments consisted of two irrigation frequencies,

once a week for 10 minutes (12 L of water, moderately-stressed plants) and every other

day for 5 minutes (6 L of water, well-irrigated plants), with 12 replications each. Plants

were harvested twice, 138 and 159 DAT (days after transplant). Six plants from each

treatment were harvested at root flush (increasing number of root growing points, RGP)

and six at shoot flush (increasing number of shoot growing points, SGP).

Growth measurements

Growth of roots and shoots were monitored weekly and recorded after

commencement for both experiments. Growth was not disturbed by pruning during

experiments. Number of root growing points visible in the eight rhizotrons' observation

windows, and SGP were counted weekly throughout the experiments. A root growing

point was defined as a root tip with visual characteristics of active growth (Figure 5-1 E,

light color of the root cap, division and elongation zones, and no apparent root hairs). A









shoot growing point was defined as a shoot tip between visual bud break and no

unfolding of new leaves (Figure 5-1D).

In 2009, in addition to RGP and SGP recorded up to 126 and 157 DAT, for root

and shoot flush, respectively, shoot architecture parameters (length, number of leaves,

bud type, and date of bud set for new branches), and number of inflorescences per

plant were recorded weekly up to 100 DAT.

Results and Discussion

Growth under well-irrigated conditions (2 day wetting and drying cycles)

L. japonicum has imbricate buds, buds with more than two scales that overlap one

another (Figure 5-1A-C). At the end of each branch a cluster of buds occurs, that will be

referred to as apex buds (Figure 5-2). These apex buds are composed of one central

terminal bud and two lateral buds on opposite sides. Also part of the apex bud cluster,

there are two accessory buds for each terminal and lateral buds. Apex lateral buds

originate from the last pair of leaves. Leaf Lateral buds refers to lateral buds that

originate from leaves older than the last pair of leaves (Figure 5-2). Each terminal bud

and lateral bud has preformed stem, leaf primordia and lateral buds (Figure 5-3B-D).

L. japonicum is a temperate woody shrub, native to Japan and East Asia (Ishii and

Iwasaki 2008). Preformation, the differentiation of organs in a growing season before

maturation and extension the following growing season, is a common characteristic

among temperate woody species (Meloche and Diggle 2003; Puntieri et al. 2007;

Remphrey and Powell 1984). Neoformation, the simultaneous differentiation and

extension of organs in the same growing season, is considered to be relatively

uncommon among temperate woody plants (Puntieri et al. 2007).









Although under apical dominance, vegetative lateral buds of Rosa hybrida

increased dry mass and developed new leaf primordia with age (Marcelis-Van Acker

1994). Our observations suggest that L. japonicum leaf lateral buds (Figure 5-1 C)

behave as observed for roses, despite preformed lateral bud maturation during the

present growing season, leaf lateral bud located in new branches will burst only in the

next growing season or if apical dormancy is broken. No new leaf lateral bud outgrew

(Table 5-1). However, seven percent of neoformed apex lateral buds expanded to form

second order shoots, while 23.2% of new terminal buds formed a second order shoot

(Table 5-1). This suggests that new leaf lateral buds are preformed and mature in the

present growing season during determinate growth. While new apex lateral buds,

together with new terminal buds, are neoformed. Additionally, neoformed buds appear

less sensitive to dormancy than preformed buds, which need more than one growing

season to naturally outgrow.

Apical dominance is a term generally used to refer to inhibition of growth of lateral

buds by the terminal bud (Little 1970; Napoli et al. 1999). Although a degree of apical

dominance was noted, this species appears to have weak apical dominance. Only

47.6% of total growing points originated from terminal buds (Table 5-2). Additionally, the

first flush of growth was mostly due to leaf lateral bud outgrowth. It is important to note

that leaf lateral bud outgrowth was independent of terminal bud decapitation. Plants

were not pruned. Decapitation causes not only the commonly discussed plant growth

regulators alteration, but also imposes stress by wounding, and causes changes in

xylem fluid flow and in sink strength (Napoli et al. 1999). These dormancy-released

buds were mostly located in the basal zone of the plant. Thus, the first flush of growth,









in the beginning of spring, may be a result of the interaction of ecodormancy release

with positional clues, since the terminal bud in the same branch was not released.

After the first flush, some neoformed apex lateral buds initiated active growth

concurrently with neoformed terminal bud (7% of neoformed apex lateral bud outgrowth,

Table 5-1). Bud formation date did not inhibit neoformed terminal bud to the same

extent, since they produced second and third order shoots in 23.2% of total neoformed

terminal buds (Table 5-1). The outgrowth of neoformed apex lateral bud suggests that

those buds are less sensitive to terminal bud dominance. While some apex lateral bud

outgrew during the experiment, none of the leaf lateral buds outgrew. Any tentative

approach to explain apical dominance or dormancy depth should take bud position and

age into consideration. In Quercus, branching order and age were the most important

influences on bud outgrowth, compared to several manipulative treatments, such as

defoliation and terminal bud removal (Bicl-Sorlin and Bell 2000).

A new branch was defined as a single stem developed from an old bud (bud

expanded in previous growing seasons) and its subsequent second and third order

shoots. A terminal branch was defined as a new branch arising from an old terminal

bud, and a lateral branch as a new branch formed from an old lateral bud. Of the total

number of new terminal branches, 53.7% had two or more flushes of growth (Table 5-

3), while fewer lateral branches (30.1%) had two or more flushes of growth. This

suggests differences in sink strength between branch positions in a plant. Furthermore,

83.8 % of old branches that produced new growth generated only one new branch

(Table 5-3), with the majority of terminal bud and lateral bud outgrowth occurring

isolated (Table 5-2). Isolated outgrowth refers to only a single bud outgrowth from a









branch. In some cases, both lateral buds of the same leaf pair outgrew concurrently. It

has been suggested that lateral bud outgrowth potential is associated with

developmental stage and physiological activity of its dominant terminal bud. In

Arabidopsis, lateral bud is suppressed during vegetative growth. However, after a long

period of vegetative growth, dormancy is released from more distal lateral buds,

resulting in acropetal pattern of growth. After switching to reproductive growth,

dormancy was released from proximal lateral buds, resulting in basipetal pattern

(Vojislava and Anthony 2000). However, only four flower panicles were recorded during

this experiment and branching pattern tended to be basipetal (11.1 leaf lateral branches

per plant versus 7.7 terminal branches per plant, Table 5-3). In roses, shoot apical

meristem of lateral buds remained vegetative unless apical dominance was removed

(Marcelis-Van Acker 1994). The position or age of a bud along the stem can determine

its ability to grow out (Waldie et al. 2010).

Although apex buds occasionally outgrew at the same time, in cases where

terminal bud burst together with one or more other buds, terminal bud always produced

longer shoot stems with a greater number of leaves than a branch from a lateral bud

developing by its side. Branch final size depended on whether growth was

indeterminate or determinate. Indeterminate growth occurred mostly from isolated

lateral branches, which were responsible to 22.6% of all branch length in well-irrigated

plants (data not shown). Stems with apparent determinate growth measured on average

16.1 and 13.9 cm, for terminal and lateral branches respectively, while stems with

indeterminate growth averaged 58.9 and 69.5 cm in length for terminal and lateral

branches, respectively (Table 5-4). For some tree species, neoformation contributed









more to axis formation than preformation (Costes 2003; Guedon et al. 2006; Snowball

1997). Number of leaves per stem and internode length of determinate lateral branches

were smaller than observed for determinate terminal branch and associated second

order branches (Tables 5-6 and 5-4), but there were no differences between terminal

and lateral branches when growth was indeterminate (Tables 5-6 and 5-4). The lower

number of leaves originated by lateral buds than by terminal buds agrees with

observations in buds of apple trees, in which lateral buds had fewer leaf primordia and

bud scales than terminal buds (Costes 2003). The unknown factor that signaled shoot

apical meristem in determinate branches to start differentiating scales and consequently

set a bud was not perceived in indeterminate branches. What arrested growth of

indeterminate branches is unknown. Nutrient availability at the meristematic region may

be involved with bud setting. Reserve carbohydrates are often used for growth of

sprouts and root suckers (Kozlowski 1992). Moreover, mitotic activity and dormancy

were related with carbohydrate levels within the bud of Douglas fir (Owens and Molder

1973). Also, neoformation is considered a plastic response of woody plants to factors

acting at the time of shoot extension (Guedon et al. 2006).

Effect of intermittent water stress on growth

Wetting and drying cycles leading to moderate stress resulted in biomass

reductions of approximately 20% (Chapter 4). Water stress also reduced several other

growth parameters, such as the number of new branches (40% reductions, Table 5-3),

especially lateral branches; total growing points (42% reduction, Table 5-2); and length

of new branches (24% reduction, Table 5-5).

Water stress affected shoot architecture by enhancing apical dominance. Lateral

branching was mainly affected, declining from an average 11.1 new lateral branches









produced by well-irrigated plants to only 5.4 produced by moderately-stressed plants

(Table 5-3). Moreover, water stress affected outgrowth of apex buds by increasing

terminal bud dominance over the two lateral buds located at the apex. The proportion of

apex outgrow (terminal bud, terminal bud+1 lateral bud, terminal bud+2 lateral buds, or

terminal bud+2 lateral buds +1 accessory bud) was distributed differently for

well-irrigated and moderately-stressed plants (51.9, 25.2, 21.4, and 1.6% for

well-irrigated plants, and 74.1, 12.1, 13.8 and 0%, for moderately-stressed plants,

respectively, Table 5-2). Although only terminal bud+1 lateral bud separated statistically

between the two treatments, the tendency towards increased apical dominance in

moderately-stressed plants is clear. Interestingly, moderate water stress did not affect

leaf lateral bud outgrowth in the same way. Both treatments had 86.3% of leaf lateral

buds developing isolated. Still, leaf lateral bud outgrowth of moderately-stressed plants

was 50% less than well-irrigated plants (Table 5-2).

As discussed above, first order terminal branches of well-irrigated plants had

mainly determinate growth, and lateral branching occurred more often than terminal

branching. Water stress restricted new shoot growth to principally determinate terminal

branches but with three times as many indeterminate terminal branches as well-irrigated

plants (Table 5-6). Also, stem length of determinate first order terminal branches in

moderately-stressed plants were considerably longer than those of well-irrigated plants,

while stem length of first order terminal branches decreased for moderately-stressed

plants (Table 5-4). First order terminal branch of moderately-stressed plants had longer

internodes than those of well-irrigated plants (3.7 versus 2.9 cm, Table 5-4), and

approximately one extra pair of leaves (10.5 versus 8.9 leaves per stem, Table 5-6).









The number of new leaves produced by 100, 138, and 159 DAT was similar between

dates within each treatment; however, moderately-stressed plants produced fewer new

leaves than well-irrigated plants at 100 and 159 DAT since there were fewer branches

(Figure 5-4). As discussed on Chapter 4, at 138 DAT (root flush) moderately-stressed

plants had smaller new leaves than well-irrigated plants (11.7 and 13.3 cm2,

respectively), and by 159 DAT (shoot flush) moderately-stressed plants had similar size

new leaves as well-irrigated plants (13.3 and 13.8 cm2, respectively). However, the

number of new leaves did not increase significantly for moderately-stressed plants

(Figure 5-4).

The seven-day wetting and drying cycles changed morphological and

physiological behavior of L. japonicum by potentiating apical dominance. The beginning

of the first shoot flush coincided with the beginning of stress treatment (around 47 DAT).

At the second flush, which occurred later into the stress treatment, old buds burst more

frequently than the newly formed apex lateral buds. Also, as plants began to adapt to

the stress imposed, indeterminate growth was triggered more often in meristematical

regions of terminal buds. Well-irrigated plants tended to set a bud and then invest in the

production of new leaves in a second flush developing second order branches.

Conversely, shoot apical meristem of moderately-stressed plants turned to

indeterminate growth, thus producing neoformed leaves. Towards the end of the

experiment, after a root flush, moderately-stressed plants were established (Chapter 4),

this made possible additional leaf and stem expansion. As a result, moderately-stressed

plants had leaves with similar size as well-irrigated plants.









Root and shoot growth patterns

The initial acclimatization period after transplant into rhizotrons was required to

enable roots to elongate sufficiently to be observed in rhizotron observation windows.

Roots had to grow approximately 20 cm to reach the sides of rhizotron. Root growth

was represented by RGP. This delayed quantification of RGP relative to SGP (Figures

5-5 to 5-8).

Root growth occurred before bud swelling. Longest roots outside the original root

ball, measured the day soil moisture probes were initially installed, was about 10 cm in

the 2008 experiment (23 DAT) and 15 cm in the 2009 experiment (41 DAT). Thus, it is

likely that the initial root flush began soon after transplant into rhizotron. This contrasts

with observations for transplanted Quercus alba and Quercus marilandica seedlings,

which expanded roots only after shoot expansion (Reich et al. 1980), or in Quercus

rubra, which expanded root and shoot concomitantly (Sloan and Jacobs 2008). In

Central Florida, growing root tips have been noted in February, weeks before bud

break, during transplanting of several evergreen and deciduous tree species (Beeson,

per. Comm,). Despite variation on flush size and duration between plants, root growth of

L. japonicum rarely completely stopped during shoot growth.

A flush was defined as the period between increasing and decreasing growing

points until rest (zero value) or before a subsequent increase, which would characterize

the beginning of a new flush. Shoot and root flushes were mostly asynchronous in both

years and in all treatments. L. japonicum is described in literature as an episodical

species (Kuehny and Halbrooks 1993; Kuehny et al. 1997), based on growth monitored

by changes in dry mass. Organ growth is controlled by two processes: cell division and

cell expansion (Kuehny and Halbrooks 1993). However, whole plant growth can be









categorized in two phases: A) cell division and differentiation of organs and tissues in

meristems and; B) expansion and formation of new organs. In shoot tips, bud formation

comprises phase A, which will expand during determinate growth. However, it is phase

B of growth that is usually measured and commonly regarded as growth. Accumulation

of dry mass occurs with growth. However, dry mass accumulation stretches through

organ maturation. In roots, however, the two phases of growth occur concomitantly.

Cockcroft, et al. (2000) concluded that cell division is the main determinant of meristem

activity and overall growth rate. While dry mass accumulation is the most common

method used to categorize growth, and was used previously for L. japonicum, other

species have been categorized using stem or leaf elongation to measure growth

(Borchert 1975; Cockcroft et al. 2000; Schoene and Yeager 2006). Accounting for

growth using growing points was a method to quantify visual growth non-destructively

over time in the same group of plants. Meristems are an important source of plant

growth regulators; thus, counts of growing points are an indirect way to quantify the

contribution of active shoot apical meristem and root apical meristem for the pool of

these compounds.

In general, the first shoot flush coincided with the start of treatments in most

plants, and coincided with the first visible root flush (roots and shoots flushing together).

Well-irrigated plants from 2008 (constant moisture) displayed two distinct growth

patterns (Figure 5-5). The first (A-08) was characterized by two shoot flushes with three

small root flushes and one large root flush towards the end of data collection, and the

second (B-08) by one main shoot flush with two smaller root flushes and one large root

flush again near the end. Plants within each pattern were relatively synchronous. Plants









with pattern A-08 had the first shoot flush starting around 30 DAT, which lasted for

approximately 30 days. The second shoot flush followed the first without a resting

period and lasted from 25 to 50 days. Besides the first root flush, which occurred for all

plants, two root flushes occurred within the second shoot flush, and a fourth large root

flush followed with an obvious decline in SGP for all plants. Shoot growth was delayed

for plants with pattern B-08, as were substantial increases in RGP activity. Shoot

flushes started around 45 DAT and lasted for about 60 days, due to more branches with

indeterminate growth. In this pattern of growth, the second root flush occurred as shoot

flush was finishing, and a third root flush followed when shoots became quiescent.

Episodical growth was not observed in initial stages, but rather in short periods, in which

RGP were decreasing and SGP increasing, or the inverse was occurring. A search of

the literature did not find similar growth patterns to those observed in this study, nor

have growing points of both roots and shoots been used before to distinguish episodical

patterns. Under warm soil and abundant moisture conditions, there was no clear

arresting of RGP during shoot flushes. During periods of low RGP, root mass increase

likely would have been small, and conversely the opposite during periods of high RGP.

Thus, activity of RGP should correspond to traditional dry mass measurements of

episodical growth.

Growth patterns of well-irrigated plants of the 2009 experiment (two day wetting

and drying cycles) also can be divided in two groups (Figure 5-6). The first pattern

(C-09) had three shoot flushes between 38 and 110 DAT, a resting period of

approximately 40 days before new shoot flush, and four root flushes without resting

periods. The second pattern (D-09) had three shoot flushes with a small resting period









between them, and four root flushes without resting periods. Pattern D-09 was similar to

A-08; however, D-09 had smaller peaks, separated by a resting period. Also, no plants

in the 2009 experiment displayed only one shoot flush as in 2008, and roots never

completely came into rest. Although substrate moisture conditions were favorable to

growth, such as in 2008, the variation in moisture affected root tips, such that growth in

part of the roots was always active. Constant high substrate moisture (well-irrigated

plants 2008) stimulated roots to turn quiescent at times, and RGP stayed lower (under

100 tips until around 70 DAT, Figure 5-5) than roots under intermittent irrigation

(well-irrigated plants 2009, over 100 growing tips at 70 DAT, Figure 5-6). In the inverse

situation, resting periods between shoot flushes were a novelty for plants grown in

2009, and these periods were opposed to root flushes, better characterizing an episodic

behavior. This suggests that the natural-observed episodic growth, such as observed

for temperate forests, may depend on environment as well as genetic-dictated

characteristics.

Severe water stress resulted in impaired growth (Chapter 4), which can also be

observed on counts of growing points. During the 2008 experiment, plants under severe

stress that did not receive additional irrigation had only one minor shoot flush and

almost no root growth (lower left 3 repetitions of Figure 5-7). When any additional

irrigation was supplied, SGP increased dramatically, such as on the one occasion of the

plant represented on the top left of Figure 5-7, or on two occasions after irrigation was

supplied for the plant represented by the graph second from the top on the left in Figure

5-7. For both cases, despite large SGP counts, the formed branches did not expand

past a few centimeters and shortly after commencement of visible growth these









branches dehydrated and died. Plants under severe water stress delegated most of

their resources into root growth, rather than shoot growth (46.3 and 41.6% allocation to

roots for severely-stressed and moderately-stressed plants, respectively, Chapter 4).

However, if any irrigation was applied bud burst occurred, thus, water was clearly a

limiting factor for bud outgrowth. Moderately-stressed plants of 2008, had two shoot

flushes and very low counts of RGP up until 100 DAT (Figure 5-7). The majority of

moderately-stressed plants of 2009 displayed growth patterns similar to D-09. However,

growth patterns in these plants were more variable.

Conclusions

Age and position affected L. japonicum's buds capacity for outgrowth. The first

spring flush was mainly due to leaf lateral bud outgrowth, thus resulting in basipetal

branching. Possibly reflecting leaf lateral bud proximity to storage carbohydrates and a

postulated root-to-shoot signal in roots (Beveridge 2000; Waldie et al. 2010) or stored

carbohydrates in the trunk (Salaun and Charpentier 2001). At the second flush of

growth, not only leaf lateral buds, but also neoformed buds were able to outgrow, since

new leaves can generate carbohydrates to support neoformed bud outgrowth. First

order terminal branch had mostly determinate growth, while first order lateral branch

had an increased number of branches with indeterminate growth.

Exposure to intermittent water stress, such as experienced in natural settings,

resulted in stronger apical dominance in L. japonicum. As a result of prolonged

intermittent water stress, bud setting of terminal bud was delayed more often and

indeterminate growth was triggered in these shoot tips. Moreover, in the branches that

stopped growing at the determinate stage and set a bud, the newly formed apex lateral

bud outgrew less often, thus suggesting the increased apical dominance of terminal bud









over apex lateral bud. Further into the treatments, well-irrigated plants tended to invest

in the production of new leaves, developing second order branches in a new shoot

flush. Plants under intermittent water stress continued new leaf production, with

indeterminate growth, instead of setting a bud. These plants had fewer but normal size

leaves by the end of the study because they were able to fully expand leaves after a

root flush, which resulted in the establishment of a normal balance between absorbing

and transpiring surfaces (Chapter 4).

After transplant into rhizotrons, root growth occurred before shoot growth and it

occurred as soon as early March. The temporal variation on moisture caused by wetting

and drying cycles resulted in continuous growth of parts of the root system, while resting

periods were observed for shoot growth. Inversely, continuous high moisture levels

resulted in roots having resting periods for some plants. Classic episodic growth was

not observed as described for this species (Kuehny and Halbrooks 1993; Kuehny et al.

1997) for most of the growing season. However, small ebb and flow cycles of RGP and

SGP were documented.









Table 5-1. Percentages of bud outgrowth and dormancy of neoformed buds on
branches expanded during 100 days of undisturbed growth by L. japonicum
grown in large rhizotrons in 2009.


Treatment


Well-irrigated
plants


Apex bud
outgrow
Apex bud
dormancy


Terminal bud
outgrowth
Terminal bud
dormancy

Apex Lateral bud
outgrow
Apex lateral bud
dormancy

Leaf lateral bud
outgrowth
Leaf lateral bud
dormancy


% of total


Moderately-
stressed plants


Well-irrigated
plants


12.2 az yY 6.9 a y

81.3 a z 47.4 b z


7.4 a y 4.3 b y

23.8 a z 13.8 b z


4.8 a y 2.6 a y

57.6 a z 33.5 b z


0 a y


Moderately-
stressed plants


12.4

87.6


23.2

76.8


7.0

93.0


10.6

89.4


20.5

79.5


94.4


0.0

100.0


0 a y


376.5 a z 268.7 b z


100.0


z Means of 12 replications. Means within rows not followed by the same letter (a, b) are
significant at Ps0.05 (Fisher's Least Significant Difference).
Y Means of 12 replications. Means within columns not followed by the same letter (z, y) are
significant at Ps0.05 (Fisher's Least Significant Difference).









Table 5-2. Number of growing
L. japonicum grown


points recorded during 100 days of undisturbed growth of
in large rhizotrons in 2009.


Treatment


% of total


Well-irrigated
plants


Total growing points
Growing points from:
preformed terminal buds
neoformed terminal buds
preformed leaf lateral buds
neoformed lateral buds

Isolated preformed leaf
lateral bud outgrow
Opposite preformed leaf
lateral bud dormancy

Isolated terminal bud
outgrow
1 terminal bud + 1 lateral
bud outgrow
1 terminal bud + 2 lateral
bud outgrow
1 terminal bud + 2 lateral
bud + 1 accessory bud
outgrow


31.2 az


Moderately-
stressed
plants
18.1 b


7.7
7.4
11.3
4.8


9.5 a z 4.4 b z

1.8 a y 1.1 a y


9.9 a z 7.7 a z

5.3 a y 2.2 b y

4.3 a y 2.8 a y

0.3 a x 0.0 a x


Well-irrigated
plants
100.0


Moderately-
stressed
plants
100.0


24.4
23.2
38.3
14.1


86.3

13.7


51.9

25.2

21.4


31.9
20.5
36.3
11.3


86.3

13.7


74.1

12.1

13.8


z Means of 12 replications. Means within rows not followed by the same letter (a, b) are
significant at P-0.05 (Fisher's Least Significant Difference).
Y Means of 12 replications. Means within columns not followed by the same letter (z, y, x) are
significant at P-0.05 (Fisher's Least Significant Difference).


100









Table 5-3. New branch distribution of L. japonicum grown in large rhizotrons in 2009,
during 100 days of undisturbed growth.
Treatment % of total


New branches produced
Lateral branches
Terminal branches

New branches with
1 flush
>1 flush

Lateral branch with
1 flush
>1 flush

Terminal branch with
1 flush
>1 flush

Old branches with new
growth
Old branches that originated
1 new branch


Well-irrigated
plants
18.8 az
11.1 a zy
7.7 a z


11.3
7.5


7.8 a
3.3 a


Moderately-
stressed
plants
11.2 b
5.4 b z
5.7 a z


Well-irrigated
plants
100.0
58.6
41.4


7.2 b
4.0 b


4.0 b
1.5 a


Moderately-
stressed
plants
100.0
48.5
51.5


60.3
39.7


63.1
36.9


69.9
30.1


66.3
33.7


46.3
53.7


15.7 a


59.9
40.1


9.7 b


13.4 a z 8.4 b z


>1 new branch


a y


83.8
16.2


87.5
12.5


z Means of 12 replications. Means within rows not followed by the same letter (a, b) are
significant at P_0.05 (Fisher's Least Significant Difference).
Y Means of 12 replications. Means within columns not followed by the same letter (z, y) are
significant at P_0.05 (Fisher's Least Significant Difference).


101









Table 5-4. Single stem and internode length (cm) of new branches produced during 100
days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009.
Well-irrigated Moderately-stressed
plants plants
Single stem length


Determinate growth
1st order terminal branch
1st order lateral branch
2nd and 3rd order branch

Indeterminate growth
1st order terminal branch
1st order lateral branch


16.1 az zy
13.9 b y
14.4 a zy


58.9 a
69.5 a


Internode length
Determinate growth
1st order terminal branch 3.2 a
1st order lateral branch 2.9 b
2nd and 3rd order branch 2.6 a


Indeterminate growth
1st order terminal branch 3.9 a z 3.7 a z
1st order lateral branch 3.8 a z 3.8 a z
z Means of 12 replications. Means within rows not followed by the same letter (a, b) are
significant at P_0.05 (Fisher's Least Significant Difference).
Y Means of 12 replications. Means within columns not followed by the same letter (z, y) are
significant at P_0.05 (Fisher's Least Significant Difference).

Table 5-5. Total length (cm) of new branches produced during 100 days of undisturbed
growth of L. japonicum grown in large rhizotrons in 2009.
Treatment % of total


Well-irrigated
plants


Moderately-
stressed
plants


Well-irric
plant


Moderately-
stressed
plants
plants


Total 583.83 az 440.9 b 100.0 10C
1st order
te brah 134.9 a yY 145.8 a z 23.1 3,
terminal branch
1st order
t brah 269.1 a z 192.7 a z 46.1 4,
lateral branch
2nd and and 3rd
d ad a 179.8 a zy 102.5 a z 30.8 2,
order branch
z Means of 12 replications. Means within rows not followed by the same letter (a, b) are
significant at P_0.05 (Fisher's Least Significant Difference).
Y Means of 12 replications. Means within columns not followed by the same letter (z, y) are
significant at P_0.05 (Fisher's Least Significant Difference).


).0
.1

.7

.2


102


13.6
20.1
14.6


73.0
70.0










Table 5-6. Number of stems and leaves per stem per plant produced during 100 days of
undisturbed growth of L. japonicum grown in large rhizotrons in 2009.
Well-irrigated Moderately-stressed
plants plants
Stems per plant
Determinate growth
1st order terminal branch 6.4 az yy 5.5 a z
1st order lateral branch 9.1 a zy 3.8 b z
2nd and 3rd order branch 11.4 a z 7.0 a z

Indeterminate growth
1st order terminal branch 0.3 a y 1.0 a z
1st order lateral branch 2.1 a z 1.6 a z

Leaves per stem
Determinate growth
1st order terminal branch 10.0 a z 9.0 a y
1st order lateral branch 8.9 b y 10.5 a z
2nd and 3rd order branch 10.8 a z 11.2 a z

Indeterminate growth
1st order terminal branch 30.5 a z 39.6 a z
1st order lateral branch 37.1 a z 37.6 a z
z Means of 12 replications. Means within rows not followed by the same letter (a, b) are
significant at P_0.05 (Fisher's Least Significant Difference).
Y Means of 12 replications. Means within columns not followed by the same letter (z, y) are
significant at P_0.05 (Fisher's Least Significant Difference).


103















































Figure 5-1. Organs of L. japonicum at different growth stages. A) Apex buds terminal
bud, TBu; accessory buds, AcB; and lateral buds, LBu. B) Old terminal bud
within apex buds. C) Leaf Lateral bud. D) Left and right panels, respectively,
shows expanding bud at beginning and later on expansion. E) Quiescent
root tip, QR, and growing root tip, GR. F) Neoformed bud at the beginning of
setting, left, and later on bud setting, right.






104













3'" Order Branch



E 2nd Order
SBranch

1 Order
Terminal
Branch .c
Terminal
Bud -\ 2
a Apex 0
L Lateral
Buds .
Lateral I"Order
0 Bud Lateral
Branch

Figure 5-2. Diagram showing branch orders and bud positions of L. japonicum.





















105









































".:7


Figure 5-3. Different organs of L. japonicum. A) Quiescent lateral root with root hairs.
B) Old terminal bud with excised scales to expose leaf primordia, LP.
C) Longitudinal cut of old terminal bud and lateral bud, LBu. D) Fresh
longitudinal cut of apex buds. E) Longitudinal cut of a growing root tip.
F) Fresh longitudinal cut of a growing root tip, left, and quiescent root tip, right.


106











Number of new leaves


S-6000


b
-4000 -
E
2o

-2000 m



0
159 DAT


SWell-irrigated plants

Moderately-stressed


[ Well-irrigated plants

- Moderately-stressed


Figure 5-4. Number of new leaves and leaf area of L. japonicum at variable volumetric
water content (2 day irrigation cycle, well-irrigated plants and 7 day irrigation
cycles, moderately-stressed plants) for 2009 plants. DAT, days after
transplant.


107


600
-



400


0
-



200
E
z


100


-13
138


159 DAT


Leaf area










































1001

I,'
0 1

SGP
A' c-% -
0 20 40 60 80 100 120 0 20 40 60 80 100 120
DAT DAT
Figure 5-5. Growth patterns of L. japonicum grown in rhizotrons at constant high
substrate volumetric water content (well-irrigated plants) in 2008. DAT, days
after transplanting. Vertical axes are RGP (root growing points, broken line)
and SGP (shoot growing points, continuous line). Vertical axes scale is on
the left. A-08 pattern of growth: graphs in the left column and B-08 pattern of
growth: graphs in the right. Each graph represents one plant replication.


108

































RGP100"



0 /- '...



0 20 40 60 80 00 120 140 160 0 20 40 60 BO 100 120 140 160
DAT DAT
Figure 5-6. Growth patterns of L. japonicum grown in rhizotrons at variable volumetric
water content (2 day irrigation cycle, WP) for 2009. DAT, days after
transplanting. Vertical axes are RGP (root growing points, broken line) and
SGP (shoot growing points, continuous line). Vertical axes scale is on the
left. D-09 pattern of growth: graphs in the left column and C-09 pattern of
growth: graphs in the right. The top three graphs in each column are for
plants harvested during root flush. The bottom three graphs in each column
are for plants harvested during shoot flush. Each graph represents one plant
replication.


109



























100
RGP




SGP 10
-oJ..~~~~~~~ -____-___-___-____-___-___- --
0 20 40 60 80 100 120 0 20 40 60 80 100 120
DAT DAT
Figure 5-7. Growth patterns of L. japonicum grown in rhizotrons at severe stress, left
column, and moderate stress, right column (severely-stressed and stressed
plants, respectively) in 2008. DAT, days after transplanting. Vertical axes
are RGP (root growing points, broken line) and SGP (shoot growing points,
continuous line). Vertical axes scale is on the left. Each graph represents
one plant replication.


110















I


9Il


j
-



I''


C, x
'- '4
4 I
I'
I,




I ~*



-
t= / -
'-


0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 16
DAT DAT

Figure 5-8. Growth patterns of L. japonicum grown in rhizotrons at variable volumetric
water content (7 day irrigation cycle, stressed plants) for 2009. DAT, days
after transplanting. Vertical axes are RGP (root growing points, broken line)
and SGP (shoot growing points, continuous line). Vertical axes scale is on
the left. The top three graphs in each column are for plants harvested during
root flush. The bottom three or two graphs in each column, respectively are
for plants harvested during shoot flush. Each graph represents one plant
replication.


0


111


RGP



0


SGP10I
- oI









CHAPTER 6
PATTERNS OF FREE AMINO ACIDS AND NON-STRUCTURAL CARBOHYDRATES
ASSOCIATED WITH EPISODICAL GROWTH OF Ligustrumjaponicum Thunb.

Introduction

The above-ground portion of a plant has the primary function of energy capture,

while below-ground roots' primary purpose is absorption of water and ions. Thus,

equilibrium between the size of shoot and root systems has to be maintained for

optimum growth. In the quest to identify the mechanism that maintains this equilibrium,

Thornley (1972) hypothesized that episodic growth in plants was controlled by the ratio

of total carbon to total nitrogen. Subsequent research has compiled substantial

evidence for this hypothesis (Campagna and Margolis 1989; Ericsson 1995), and the

general concept of carbon-to-nitrogen ratio has been proposed to be refined from total

carbon (C) and total nitrogen (N) to their labile forms (Campagna and Margolis 1989;

Lalonde et al. 2004; Saarinen 1998). Campagna and Margolis (1989) found a strong

correlation (r > 0.90) between total nonstructural carbohydrate to free amino acid ratio

(tnc:faa) and carbon allocation to shoots and roots. When tcn:faa was high, rates of

protein synthesis and other metabolic processes were affected favoring root growth.

When tnc:faa was low, shoot growth was favored. For this reason we appraised C as

total nonstructural carbohydrates (tnc) and N as free amino acids (faa).

Signaling effects are a plant growth regulator-like influence, thus they can be

active in small levels and affect gene expression. These effects can include up or

downregulation of key genes. Much is known about the use of sugars for signaling. A

thorough review of sugar signaling was compiled by Koch (2004). Trouverie et al.

(2003) found a strong correlation between abscisic acid and vacuolar invertase in

response to water stress. Vacuolar invertases are active in most expanding tissues, and


112









are involved in sugar sensing (Koch 2004), activating genes such as for sugar storage

and osmoregulation (Sturm 1999). In animals, amino acid derivates are important

signaling compounds (Forde and Walch-Liu 2009; Lalonde et al. 2004). Possibly plants

have developed similar sensors to assess substrate availability, thus specific receptors

have been investigated (Forde and Walch-Liu 2009; Lam et al. 1998). However, N

signaling is not as well understood as C signaling in plants.

The N-products that act as signals have not been clearly identified. Glutamine

(Gin) and glutamate (Glu) are usually in equilibrium with asparagine (Asn) and aspartate

(Asp). These four amino acids are the major amino acids accumulated and transported

in many plants species and have been hypothesized to function as signals. Glutamate

applied externally to root tips caused inhibition of growth (Sivaguru et al. 2003) and

inhibited lateral root formation and outgrowth (Walch-Liu and Forde 2007). Nitrate

counteracted Glu, thus stimulating root branching and growth of the main root. External

nitrate and Glu were proposed to interact to modulate root growth (Forde and Walch-Liu

2009). However, internal Glu concentrations have not been reported to cause the same

effects. Excessive external concentrations of one amino acid in relation to others can

inhibit growth, with the only exception being Gin, which has the ability to counteract

growth inhibition caused by the excessive concentrations of other amino acid (Singh

and Shaner 1995). An example is Valine-mediated inhibition to growth due to isoleucine

(Iso) starvation (Bonner and Jensen 1997).

In shoots, water stress induced Bermuda grass to increase proline (Pro), Asn, and

Valine (Val), while Glu and alanine (Ala) levels decreased (Barnett and Naylor 1966).

Proline, Val and threonine (Thr) concentration increased in Pisum sativum in response


113









to water stress (Charlton et al. 2008). Proline and amides have been suggested to

function as a storage compound during water stress (Barnett and Naylor 1966; Charlton

et al. 2008). Biosynthesis of branched chain amino acids [Val, leucine (Leu) and Iso]

occurs primarily in young tissues. Expression of the various genes in the branched

chain amino acid pathway may vary in the different organs (Singh and Shaner 1995).

This group of amino acids, especially Val, appears to be related to drought tolerance in

grasses, Cynodon dactylon and Zoysiajaponica (Barnett and Naylor 1966; Carmo-Silva

et al. 2009), wheat, Triticum aestivum (Del Moral et al. 2007), alfafa, Medicago sativa

(Girousse et al. 1996), and kiwifruit, Actinidia deliciosa (Milone et al. 1999).

Intensity of remobilization and use of reserves in woody species may be different

than that observed in herbaceous plants. Furthermore, labile C and N pools in perennial

species are maintained not only by root absorption and shoot production, but also by

break down of reserves as well. Salaun, et al. (2005a) observed that in Ligustrum

ovalifolium, prior to bud break arginine, and then later, glutamine accounted for the

principal components of N mobilization.

Water-stressed leaves tended to maintain soluble sugars at similar levels to those

of non-stressed leaves, despite declining rates of carbon assimilation. This was due to

starch degradation, which drastically reduced starch reserves (Chaves and Oliveira

2003). Roots store carbohydrates, protein and non-protein compounds (Bollmark et al.

1999). Nutrient reserves are important for the first flush of growth of the season. A

better understanding of physiological processes involved in shoot and root growth will

allow more precise and efficient management and application of fertilizer and water.


114









Such information is required for efficient production of woody plants in nurseries and to

promote rapid establishment in a landscape.

There were three main objectives of this research. The first was to elucidate the

relationship between the total nonstructural carbohydrate (tnc), free amino acid (faa),

their ratio (tnc:faa) and the shoot and root growth of L. japonicum. The second objective

was to determine how water stress influenced these variables. The third objective was

to identify, if present, specific amino acids and/or sugars that may function as signals for

growth initiation in roots and shoots as a topic for future research.

Materials and Methods

Growth conditions and experimental design

Twenty-four star-shaped rhizotrons were constructed as described in Chapter 2

(Figure 2-3). Briefly, each rhizotron had four arms and held 0.16 m3 of substrate. They

were 1.76 m across, 0.30 m deep at the end of each arm and 0.35 m deep in the center.

Rhizotrons resided in an open-side greenhouse with a double polyethylene roof, under

natural light, located in Apopka, FL. Clonal Ligustrumjaponicum plants were selected

from a local nursery (Jon's Nursery, Eustis, FL) to ensure homogeneous size and

health. One plant was transplanted into each rhizotron using a commercial substrate

composed of Canadian sphagnum peat moss, processed pine bark, perlite, vermiculite,

starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc.,

Agawan, MA). Irrigation was supplied using a spray stake (model green 22500-001120,

Netafim Irrigation, Inc., Fresno, CA) at the tip of each arm pointing inward.

Plants grown in 3.8-L containers were transplanted into each rhizotron in February

2009 and grown for 138 or 159 days. Entire rhizotrons were irrigated daily until

saturation for 41 days for plant acclimatization. Thereafter, irrigation was withheld for six


115









days before the start of irrigation treatments. Treatments consisted of two irrigation

frequencies, once a week for 10 minutes (12 L of water, moderately-stressed plants)

and every other day for 5 minutes (6 L of water, well-irrigated), with 12 replications

each. These two treatments resulted in volumetric water content (VWC) of 22.33% and

19.8% in the driest periods (Chapter 4, Table 4-1).

Growth measurements

Growth of roots and shoots were monitored weekly for both experiments. Growth

was not disturbed by pruning during experiments. A root growing point was defined as a

root tip with visual characteristics of active growth (Figure 5-1 E, light color of the root

cap, with division and elongation zones, and no visible root hairs). A shoot growing point

was defined as a shoot tip between visual post-quiescence bud break and no new

unfolding of leaf (Figure 5-1D). The cumulative number of root growing points (RGP)

visible at the eight sides of a rhizotron and the cumulative number of total shoot growing

points (SGP) was counted weekly throughout the duration of the experiment.

Plants were harvested twice, at 138 and 159 DAT (days after transplant). Six

plants from each treatment were harvested at root flush (increasing RGP) and six at

shoot flush (increasing SGP). The number of RGP and SGP was recorded up to 126

and 157 DAT, for root flush and shoot flush, respectively.

Biochemical analyses

Just prior to harvest, each plant was sampled for biochemical analyses of five

tissues and xylem fluid was extracted. Tissues sampled were: growing shoot tip (GS, 1

cm, 5 per sample, Figure 5-1D); neoformed bud (NB, 5 per sample, Figure 5-1 F);

dormant terminal bud (DB, 5 per sample, Figure 5-1B); growing root tip (GR, 1 cm, 20

per sample, Figure 5-1E); and quiescent root tip (QR 1 cm, 20 per sample, Figure


116









5-1E). Tissues were immediately sliced in small sections, placed in microcentrifuge

tubes, frozen with dry ice, and then stored at 700 C. Later, samples were freeze-dried

for 5 hours and weighed. Xylem fluid was extracted with a pressure chamber (Plant

Water Status Console, Series 3000, Soil moisture Equipment Corp., Santa Barbara,

CA) following methodology described by Andersen (Andersen et al. 1993), with the

exception that fluid was collected with 0.2 MPa over the balance pressure for 60

seconds. Xylem fluid was extracted between 1100 to 1400 hours EDT. Shoots were

severed at the soil line and roots were cleaned free of substrate using the root separator

described in Chapter 3. Plant components for dry mass measurements were dried at

650 C, until constant dry mass was observed. To calculate root-to-shoot ratio, dry mass

of roots was divided by the dry mass of shoots.

Tissue samples were ground with a homogenizer mixer (SDT-1810 Tissumizer,

Tekmar, Cincinnati, OH) in 10 mL of 80% ethanol (v/v), boiled in water bath for 5 min,

cooled in an ice bath for 5 min, then centrifuged for 15 min at 7000 rpm (IEC clinical

centrifuge, Damon, Needham Heights, MA). Supernatant was decanted and the pellet

was re-extracted twice. Pellets were dried overnight in the oven with forced air at 700 C

for starch analyses. Combined supernatant was concentrated overnight in an oven with

forced air at 350 C to about 1 mL, re-suspended in distilled deionized water, filtered

through a 0.22 pm nitrocellulose membrane, and subdivided for carbohydrate and

amino acid analyses.

Starch was analyzed by an enzymatic method (Beeson and Proebsting 1988). A

pellet was dispersed in 0.1 M acetate buffer, pH 4.8 and reconstituted amyloglucosidase

(1 mg/mL of acetate buffer) was added to each tube for starch digestion at 550 C for 1


117









hr (5 parts of sample/1 part of enzyme). After 5 min of centrifugation, 1mL of

supernatant was mixed with 2 mL of Glucose Reagent (part number 1076-250, Stanbio

Laboratory, Boerne, TX) and incubated at 370 C for 15 min. Absorbance of samples and

standard curve was determined at 500 nm with a Lambda 2 UVNis spectrometer

(Perkin Elmer, Norwalk, CT). Starch content was quantified with a standard curve

derived from (starch solution, 1 wt%, Sigma Aldrich Inc.), from 0 to 2 mg.

Carbohydrates were analyzed by high performance liquid chromatography (HPLC,

Binary LC Pump 250, Perkin Elmer) coupled with a refractive index detector (Series

200a, Perkin Elmer) and a Supelcogel K column (Sigma- Aldrich Inc., Bellefonte, PA).

Carbohydrate separation was accomplished with constant gradient of 15 mmol/L

potassium phosphate, at a flow rate of 0.5 mL/min, and column temperature of 850 C.

The subsample destined for amino acid analysis was diluted with 1 mL of 1 N HCI

and passed through two columns in series. The top column contained 4 cm (after

draining) of polyvinylpyrrolidine (PVPP, Sigma Aldrich Inc.) suspended in deionized

water. The bottom column contained activated Dowex-50W (Sigma Aldrich Inc.). The

Dowex-50W was triple-rinsed with deionized water and activated with 4 volumes of

2.5 N HCI, then rinsed with deionized water to remove excessive HCI. The Dowex was

layered to a 2-cm drained depth in the bottom column. The sample and a 1 volume

rinsate of the 0.1 N HCI was placed on top of the PVPP and eluded with 5 ml of 0.1 N

HCI onto the Dowex column. The PVPP column was removed and the Dowex column

washed with 5 ml of 80% ethanol. Amino acids were then eluted with 4 mL of a 0.2 M

ammonia hydroxide solution and evaporated to dryness under an air stream. The

residue was dissolved in 0.1 N HCI for amino acid derivatization.


118









Free amino acids were analyzed by HPLC (Binary LC Pump 250, Perkin Elmer,

connected to a Series 200 UvNis detector, Perkin Elmer) using pre-column

derivatization with phenylisothiocyanate (PITC, Pierce, Rockford, IL). Separation of

individual amino acids was accomplished with a Nova-Pak C18 reverse phase column (4

ltm, 3.9 x 300 mm, Waters, Milford, MA) at 380 C connected to a Nova-pak C18 Sentry

guard column (Waters, Milford, MA). Tissue extracts were freeze-dried and dissolved in

100 [iL of ethanol:triethanolamine:water (2:2:1, v), then freeze-dried again.

Derivatization was accomplished by adding 30 [iL of ethanol:water:triethanolamine:PITC

(7:1:1:1, v) under a N2 atmosphere in the dark. After 20 min at room temperature,

derivatization was complete and samples were freeze-dried overnight. Derivatized

samples were then diluted to 1 mL in 5.75 mM mono sodium phosphate and 5%

acetonitrile and filtered through a 0.22 pm nitrocellulose membrane prior to injection into

the HPLC system. The injection volume was 20 pL. The mobile phase consisted of a

timed gradient of eluent A and eluent B, with a flow-rate of 1.0 mL/min. Eluent A was

0.14 m sodium acetate, 0.5 mL/L TEA, at pH 6.4. Eluent B was 60% acetonitrile. The

eluent profile began with 93% eluent A, then proceeded as follows, with the time to

change from one concentration to next followed by the beginning and ending

concentrations: 0.5 min, 93-92% eluent A; 0.5 min, 92-87% eluent A; 2.5 min, 87-83%

eluent A; 8 min, 83-59% eluent A; 2 min, 59-54% eluent A; 2.5 min, 54-0% eluent A; 4

min, 0% eluent A; 3 min, 0-93% eluent A; and 4 min, 93% eluent A.

Quantities of soluble sugars and free amino acids were calculated from peak

areas integrated by the TotalChrom software, version 6.3.1 (Perkin Elmer). Calibration

curves for sugars were based on five concentrations of D-(-)-fructose, D-(+)-glucose,


119









sucrose, D-mannitol, stachyose, D-(+)-raffinose (Sigma -Aldrich Inc.), and myoinositol

(Caisson Laboratories, Inc., North Logan, Utah). Calibration curves for amino acids

were based on five concentrations of L-alanine, L-arginine, L-aspartic acid, L-cystine,

L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,

L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine, L-asparagine,

L-glutamine, and L-tryptophan (Sigma- Aldrich Inc.).

A calibration curve was developed for each amino acid and carbohydrate using

regression analysis (Microsoft Office Excel 2007, Microsoft Corp. Redmond, WA) of the

area under each peak. Calibration equations were used to calculate the metabolite

concentration using the area under each peak detected by HPLC. To characterize

effects of water stress, each metabolite concentration was analyzed as a 2 x 2 x 5

factorial, with irrigation frequency, harvest and tissue as treatments, respectively, with

six replications. To distinguish between concentrations at shoot tip tissues, metabolites

were analyzed separately as a 2 x 3 factorial, with harvest and tissue as treatments with

six replications. Root tip tissues metabolite concentrations were analyzed similarly as a

2 x 2 factorial, with harvest and tissue as treatments with six replications. Xylem fluid

metabolite concentrations were analyzed as a 2 x 3 factorial and 2 x 2 factorial, for

shoot and root tissues respectively, with harvest and tissue as treatments with six

replications. All statistical analysis were conducted using SAS (version 9.1, SAS

Institute, Cary, NC), with the exception of correlations between sampled tissue and

concentrations of metabolites, which were calculated using JMP (version 8.0, SAS

Institute, Cary, NC).


120









Results and Discussion

Patterns of faa and tnc under well-irrigated conditions

Plant growth was quantified by the number of growing points. To distinguish

between effects of tnc and faa in shoot versus root growth, plants were harvested in two

episodes: 1) root flush, increasing RGP and decreasing SGP; and 2) shoot flush,

increasing SGP and decreasing RGP. Within each harvest, shoot tips were sampled at

three developmental growth stages, dormant bud, growing shoot tip, and neoformed

buds, and root tips at two developmental growth stages, quiescent root tips and growing

shoot tips.

Total faa concentrations in neoformed buds were much higher than in growing

shoot tips and dormant bud (Figure 6-1). Neoformed buds were sampled at early stages

of bud set, while expansion of younger leaves was still occurring. Upper leaves at this

point were still heterotrophic, thus functioning as sinks was well as neoformed buds.

High sink strength likely was the cause of the high faa. Correlation between RGP and

faa in neoformed buds at root flush was r = -0.90. Thus as plant's growth shifted to root

growth, faa export by roots was greatly reduced and associated with shifts in shoot

apical meristems to the production of bud scales, and thus bud set. Free amino acids in

dormant bud versus RGP at root and shoot flush were also negatively correlated (r = -

0.78 and -0.63, respectively), and associated with inhibition and delay of outgrowth of

this type of bud. Apex buds and leaf lateral buds were responsible for the majority of

branches produced during second flush of L. japonicum (Chapter 5). At shoot flush, faa

increased in growing shoot tips and dormant buds with increases in SGP (r=0.95 and

0.79, respectively), while it decreased in growing root tips (r= -0.73). At the root tip level,

this negative correlation indicates that roots were no longer the main sink.


121









Concentrations of tnc did not vary as much as faa levels in shoot tips (Figure 6-1).

At root flush tnc levels were similar among shoot growth stages. However, at shoot

flush, dormant bud had lower tnc concentrations, indicating they were a weak

carbohydrate sink, unlike the other two tissues at that time. Concentration of tnc in

growing shoot tips and neoformed buds was positively correlated with SGP at shoot

flush (r= 0.65 and 0.57, respectively).

Shoot tips that were still growing during the root flush harvest were closer to

setting a bud than the same tissue at a shoot flush, since harvests occurred at the

beginning of a flush. Similarly, root tips that were still growing during shoot flush harvest

were most probably in a declining growth phase. If the hypothesis that low tnc:faa

stimulates shoot growth is true, than in a comparison between dormant buds, growing

shoot tips and neoformed buds, tnc:faa should be in the order growing shoot tip<

dormant bud< neoformed bud during a shoot flush harvest. This was not the order

observed during shoot flush (Figure 6-1). Growing shoot tips had a significantly higher

tnc:faa than neoformed buds. However, the correlation between tnc:faa and SGP was

negative for growing shoot tips at shoot flush (r = -0.63) and positive for neoformed

buds at root flush (r = 0.57). These two contradictory results observed with tnc:faa

comparison and correlations, together with the strong positive correlation observed

between RGP and faa in neoformed buds at root flush, suggests that faa rather than the

tnc:faa, at the shoot tip regulates growth or quiescence in buds.

Glutamine, Asp and histidine (His) were among the amino acids that varied the

most between dormant and neoformed buds at both harvests (Figure 6-2). The

concentration of amino acids was much higher in neoformed buds compared to other


122









two shoot tip types. These higher faa concentrations, compared with growing shoot tips

were mainly due to increases in His at root flush, and in arginine (Arg), Leu, Tyr,

cysteine (Cys), methionine (Met), and Val during both root and shoot flush. Proline

concentrations were low across shoot tissues and harvests.

Glucose concentration was significantly higher in growing shoot tips at both

harvests than dormant and neoformed buds. Stachyose and raffinose were present in

high concentrations in neoformed buds at root and shoot flush. Growing shoot tips had

higher myoinositol, and dormant bud lower fructose levels than other two growth stages

at shoot flush (Figure 6-2).

For roots, the expansion of the hypothesis infers that a high tnc:faa stimulates root

growth, thus the tnc:faa should be quiescent root tip< growing root tip at a root flush. In

this case, the hypothesis held true (Figure 6-3). Saarinen (1998) concluded that tnc:faa

in roots was a better indicator of internal C to N balance than the carbon-to-nitrogen

ratio in a C4 species, since long-term supply of high levels of N resulted in low tnc:faa

and preferred allocation to shoot. In previous studies, whole organs (leaves, shoots, or

roots) were sampled for biochemical analyses, whereas here, only the meristematic

areas and surrounding tissues were analyzed. Sampling whole organs provides a more

general summation of plant faa and tnc status, while sampling as done here assesses

the influence of compounds available at meristematic sites. Coordination of growth is

ultimately achieved in the root or shoot meristem (Chaves et al. 2003; Clark 2001).

Signals are perceived by meristems, which in turns, coordinates meristem maintenance

and organogenesis (Tax and Durbak 2006).


123









Growing root tips had higher tnc than quiescent root tips at root flush (Figure 6-3).

This increase was due to higher concentrations of all nonstructural carbohydrates,

except mannitol, which did not vary much between root tip types (Figure 6-4).

Interestingly, the less common sugars of stachyose, raffinose and myoinositol,

accumulated in these tissues. Growing root tips at root flush had significantly more

raffinose than at shoot flush. Conversely, myoinositol levels were doubled in quiescent

root tips and growing root tips at shoot flush. Previously labeled myoinositol was

incorporated into cell wall polysaccharides of growing root tips of maize (Harris and

Northcote 1970).

Glutamine, Val, and His were the main faa transported in xylem fluid (Figure 6-5).

Previous work found an increase in Gin in leaf tissues and xylem fluid during shoot flush

of L. ovalifolum (Salaun and Charpentier 2001; Salaun et al. 2005); Val and His were

not measured in those studies. Although high concentrations of Val and His were

detected in neoformed buds, Gin was not among the higher concentrations observed in

shoot tip. This suggests additional amino acid biosynthesis occurred in other tissues

before reaching meristematic sites, possibly in storage tissues such as trunks. An

increasing gradient of Arg and Gin was found in L. ovalifolum from roots to the trunk and

decreasing towards upper parts of stem (Salaun and Charpentier 2001). Free amino

acids were higher in xylem fluid during a shoot flush (Figure 6-6), when faa were

transported to support expanding shoot mass. Consequently, tnc:faa tended to be lower

at this harvest.

High mannitol concentration in xylem fluid indicates translocation of reserves

(Figure 6-5), which were stored or used in tissues other than meristems. Mannitol


124









concentrations were relatively low in buds. Carbohydrate transported in xylem fluid

supports bud break in Juglans regia L. (walnut, Bonhomme et al. 2010). Carbon is

stored as starch in xylem parenchyma cells and cambium of stems. After symplasmic

connections are established in cells around the meristem, starch is converted to soluble

sugars, which are actively transported in to xylem and then move up to support bud

outgrowth. Sucrose and glucose concentration in apical buds of walnut was similar to

those observed herein for L. japonicum at shoot flush.

Influence of water stress in patterns of faa and tnc

At root flush, concentrations of most faa in shoot tips of moderately-stressed

plants (Figures 6-7) were comparable to those present in well-irrigated plants (Figure

6-2), thus in general, faa concentrations were not limited by water stress. Concentration

of most faa in growing shoot tips of moderately-stressed plants were higher than those

of well-irrigated plants, with the most noticeable increases observed in Arg, Val, and

His. Higher concentrations of faa may be a result of the tendency to higher root-to-shoot

ratio at root flush of moderately-stressed plants (Chapter 4). Neoformed buds of

moderately-stressed plants also had significantly more Val. The exceptionally high Val

concentration found in neoformed buds of moderately-stressed plants may be explained

by requirements of surrounding young expanding leaves and bud scales to produce

cuticle as a dehydration defense. Concentrations of faa in dormant bud of

moderately-stressed plants were not affected by water stress. Although water stress-

induced Pro increases were cited in the literature for other species (Barnett and Naylor

1966; Charlton et al. 2008), Pro concentrations were at stable levels across treatments

here.


125









At shoot flush, expanding shoots were the primary sink. As a consequence of sink

strength, growing shoot tips of moderately-stressed plants had similar levels of faa as

growing shoot tips of well-irrigated plants (Figures 6-7 and 6-2). Between treatments,

Asp concentration was somewhat lower in growing shoot tips of water-stressed plants,

but was significantly higher in neoformed buds. Water stress also lowered Val and Arg

concentrations in neoformed buds.

As previously described for leaves (Chaves and Oliveira 2003), shoot and root tips

of moderately-stressed plants maintained similar of tnc levels as well-irrigated plants

(Figure 6-1 x 6-7 and 6-3 x 6-8). Although raffinose family oligosaccharides have been

proposed to counteract dehydration (Bogdan and Zagdanska 2006; Brenac et al. 1997),

neither raffinose nor its precursor myoinositol increased due to water stress in a grass

species (Amiard et al. 2003). Raffinose concentrations did not differ between

moderately-stressed and well-irrigated treatments in L. japonicum. As observed for

well-irrigated plants, growing root tips at root flush had significantly more raffinose than

at shoot flush in moderately-stressed treatment. This trend also occurred for stachyose

in growing root tips of moderately-stressed plants, which had greater concentrations of

this oligosaccharide at root flush. Moreover, myoinositol levels were also higher in

quiescent root tips and growing root tips at shoot flush for moderately-stressed plants as

well as for well-irrigated plants. Carbohydrates can be stored in trunks and also in roots,

which could then support post-transplant root growth, before shoot growth, if soil

temperature is favorable. Storage carbohydrates can also be important for water-

stressed plants, since photosynthesis rates are diminished by drought due to stomatal

closure over the long term. Previous studies found that leaves continue to export


126









C-assimilated after shoot elongation stops (Dickson et al. 2000a; Sloan and Jacobs

2008).

Conclusions

After a shoot flush, as plant growth shifted to root growth, a strong correlation was

found between RGP and faa concentration in neoformed buds, thus suggesting smaller

faa concentration at the shoot tip caused shoot apical meristem to set a bud. While

after a root flush, with the switch to mainly shoot growth, faa concentration in growing

shoot tips and dormant buds increased with increases of SGP, while in growing root tips

it decreased with increases in SGP, thus suggesting higher faa concentrations on

growing shoot tips and dormant bud stimulated growth. Concentrations of tnc in shoot

tips, did not vary as much as faa concentrations. Moreover, contrary to expectations,

growing shoot tips had a significantly higher tnc:faa than neoformed buds earlier in the

shoot flush. These observations lead to the conclusion that faa levels at the shoot tip

are more decisive to meristem growth/quiescence control than the tnc:faa or tnc level by

itself. In roots, tnc:faa observed was as predicted (growing root tips having a higher

tnc:faa than quiescent root tips). This is in agreement with previous observations that

this indicator at the root level better predicted plant growth than when measured in

shoots (Saarinen 1998). Of these, Gin is suggested as the principal candidate for N

signaling for initiation of bud break. It was transported in the greatest quantity and

varied the most between shoot and root flush (Figure 6-3). Additionally, concentrations

measured at the beginning of shoot flush were proportional to the amount of bud break

for well-irrigated and moderately-stressed treatments. While Gin was principal

transported faa concentrations in shoot tips were not high, suggesting it was rapidly

metabolized. Mannitol was also found in large quantities in xylem fluid, indicating


127









translocation of reserves. Yet since bud break was hypothesized due more to faa

concentration than tnc:faa, the importance of high mannitol quantities is questionable.

Water stress resulted in increases in the concentration of some amino acids in

growing shoot tips, such as Arg, Val, and His. Neoformed buds of moderately-stressed

plants had significantly more Val than neoformed buds of well-irrigated plants. The

exceptionally high Val concentration found in neoformed buds of moderately-stressed

plants may be explained by requirements of surrounding young expanding leaves and

bud scales to produce cuticle as a dehydration defense. Valine is among the precursors

of coenzyme A, which is a precursor of leaf cuticular components (Weng et al. 2010).

As observed for well-irrigated plants, growing root tips of moderately-stressed plants at

root flush had higher concentrations of raffinose, stachyose and myoinositol than

quiescent root tips, and in much higher levels than shoot tip tissues. Carbohydrate

storage in roots can be an important source of C-skeletons especially in water stressed

plants, to support root growth after transplant. Precise management and application of

fertilizers and water prior to transplant into a landscape could be essential to shortening

establishment time and improving survival rates.


128











total nostructural
carbohydrates


free amino acids


3 E


2
E


total nostructural
carbohydrate-to-free
amino acid ratio


800

600

400

200


3 800

600
2
400


Well- Moderately- Well- Moderately-
Irrigate sed stressed rr stressed
plants plants plants plants

* Dormant Bud [] Growing shoot


200

0
Well- Moderatell
Irrigated I stressed
plants plants

* Neoformed Bud


Figure 6-1. Total nonstructural carbohydrates, free amino acids, and total nonstructural
carbohydrate-to-free amino acid ratio of L. japonicum shoot tissues of plants
grown in rhizotrons at variable volumetric water content (2 or 7 day irrigation
cycles, well-irrigated and moderately-stressed plants, respectively).
(*) indicates significant difference among tissues (P Significant Difference).


129


E 300


%200
o)
E
100







Well-irrigated plants: shoot tissues


It,
I
111 ilJ.I


jrl j.L


ili.4F


a


I ~


-] _fi *


Asp Glu Gin His Arg Pro Tyr Val Met Cys Leu Suc Man Gluc Fruc Stac Raff Inos


I Dormant bud D Growing shoot U Neoformed bud
Figure 6-2. Free amino acids and nonstructural carbohydrates of L. japonicum shoot
tissues of plants grown in rhizotrons at variable volumetric water content (2
day irrigation cycles, well-irrigated plants). Amino acids which did not vary
between tissues or treatments, and were in low levels were omitted for figure
clarity. Standard abbreviations used for free amino acids. Nonstructural
carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc;
stachiose, stac; raffino, raff; and myoinositol, inos. (*) indicates significant
difference among tissues (P50.05, Fisher's Least Significant Difference).


130


ill ,il










total nosructural
carbohydrates


free amino acids


,rio
E

0
^UiI


800

600

400

200

0


3 800

600
2
400
1
200


total nostructural
carbohydrate-to-free
amino acid ratio







EL


plants


Quiescent root F Growing root

Figure 6-3. Total nonstructural carbohydrates, free amino acids, and total nonstructural
carbohydrate-to-free amino acid ratio of L. japonicum root tissues of plants
grown in rhizotrons at variable volumetric water content (2 or 7 day irrigation
cycles, well-irrigated and moderately-stressed plants, respectively).
(*) indicates significant difference among tissues (P Significant Difference).


131


E 300

j,200
E
10


0
300










Well-irrigated plants: root tissues


Asp Glu Gn His Arg Pro yr Va Met Leu
Asp Glu Gin His Arg Pro Tyr Val Met Cys Leu


100
80
60
-40
- 20
0


Suc Man Gluc Fruc Stac Raff Inos


U Quiescent root [ Growing root
Figure 6-4. Free amino acids and nonstructural carbohydrates of L. japonicum root
tissues of plants grown in rhizotrons at variable volumetric water content (2
day irrigation cycles, well-irrigated plants). Amino acids which did not vary
between tissues or treatments, and were in low levels were omitted for figure
clarity. Standard abbreviations used for free amino acids. Nonstructural
carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc;
stachiose, stac; raffino, raff; and myoinositol, inos. (*) indicates significant
difference among tissues (P0.05, Fisher's Least Significant Difference).


132


S0.5-
m-5
0.4
E OA
u -
0.3
0 o -
E


100 o


60
so80 E


40 o
E
20
0


In, lnnrh












50 -0.8
E 45 E
S 40 .6
S35 0.6
35
CU (n 30
' C 25 0.4
. 20
CL 15
0.2
10 05
10 .J A I 0 ..-_ .l II 0.2
50 0.8
"g 45
U 40 1
f0.6
3 35
% a 30
25 0.4
20
11 0.2
a 10
V 5 rn-
0 0.0
Asp Glu Gin Gly His Arg Pro Tyr Val Met Cys Leu Suc Man Gluc Fruc Stac Raff Inos

Root Flush [] Shoot flush

Figure 6-5. Free amino acids and nonstructural carbohydrates of L. japonicum xylem
fluid extracted from plants grown in rhizotrons at variable volumetric water
content (2 or 7 day irrigation cycles, well-irrigated plants or moderately-
stressed plants, respectively). Amino acids which did not vary between
tissues or treatments, and were in low levels were omitted for figure clarity.
Standard abbreviations used for free amino acids. Nonstructural
carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc;
stachiose, stac; raffino, raff; and myoinositol, inos. (*) indicates significant
difference among harvests (P

133














1.5 100, 0.05
E E
SI so
.080

0.03
40
C 0.5
20

0.0 0 0.00
1.5 100 0.05

ED 1.0

003 -

C. 0.5 40
020
0.0 0.00
total nostructural free amino total nostructural
carbohydrates acids carbohydrate-to-
free amino acid
Root flush ] Shoot flush

Figure 6-6. Total nonstructural carbohydrates, free amino acids, and total nonstructural
carbohydrate-to-free amino acid ratio of L. japonicum xylem fluid extracted
from plants grown in rhizotrons at variable volumetric water content (2 or 7
day irrigation cycles, well-irrigated plants and moderately-stressed plants,
respectively). (*) indicates significant difference among harvests (P<0.05,
Fisher's Least Significant Difference).


134










Moderately-stressed plants: shoot tissues
: 1.0
E 0.9
u 0.5 100 m
O A 8 E
0.3 6 so
S 0.2- 40 c
O E






0 0.2 40
0
CO 0-1 I20
0.0 0- --
Asp Glu Gin His Arg Pro Tyr Val Met Cys Leu Suc Man Gluc Fruc Stac Raff Inos Star

U Dormant bud EI Growing shoot U Neoformed bud
Figure 6-7. Free amino acids and nonstructural carbohydrates of L. japonicum shoot
tissues grown in rhizotrons at variable volumetric water content (7 day
irrigation cycles, moderately-stressed plants). Amino acids which did not
vary between tissues or treatments, and were in low levels were omitted for
figure clarity. Standard abbreviations used for free amino acids.
Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc;
fructose, fruc; stachiose, stac; raffino, raff; and myoinositol, inos.
(*) indicates significant difference among tissues (P<0.05, Fisher's Least
Significant Difference).


135










Moderately-stressed plants: root tissues


S0.5
SE A

: ( 0.3
0S D 0.1



0.5
E O.4

0.3
= D 02

5 o-
0
W O 0.1
0.0
Asp Glu Gin His Arg Pro Tyr Val Met Cys Leu


*









Su* Man Glu Fc Stac Raf nos

suc M~an Gluc Fruc Stac: Raft lInca


0 Quiescent root [ Growing root

Figure 6-8. Free amino acids and nonstructural carbohydrates of L. japonicum root
tissues grown in rhizotrons at variable volumetric water content (7 day
irrigation cycles, moderately-stressed plants). Amino acids which did not
vary between tissues or treatments, and were in low levels were omitted for
figure clarity. Standard abbreviations used for free amino acids.
Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc;
fructose, fruc; stachiose, stac; raffino, raff; and myoinositol, inos. (*)
indicates significant difference among tissues (P<0.05, Fisher's Least
Significant Difference).


136


100 E
80 E
60 v
40 oc
E
20
0
100
80
60
40
20
0










CHAPTER 7
SUMMARY AND CONCLUSIONS

To conduct this research, a system was required that would enable control of

substrate moisture, provide easy root observation and sampling, and be of sufficient

size such that root growth was not impeded. Several designs for a star-shaped rhizotron

were developed and evaluated with the three-part goal of: 1) adjusting volume and

shape for minimal physical restriction and use with mature woody plants; 2) developing

a drainage system comparable to natural soils; and 3) facilitating ease, accuracy and

duration of data acquisition. The final design allowed exceptional root observation, used

a wick-type drainage system to provide a near-uniform profile of soil moisture, and was

easily manageable for precise long-term data acquisition. This rhizotron had eight

independent viewing/sampling windows and held 0.16 m3 of soil. An associated camera

positioning frame developed especially for these rhizotrons facilitated digital

photographs of the soil profiles for time series assessment of morphological and

architectural parameters. The camera positioning frame was compact, light and

effective in small spaces.

After months of growth, plants were harvested for shoot and root mass. Isolation of

plant roots from soil or substrate for biomass measurement is time consuming and can

be a limiting factor influencing experimental designs especially with mature woody

plants. An electric powered root separator was developed that sped sample preparation

for root dry mass determination with a capacity of 40 L of container substrate or 32 kg of

sandy soil. No water was required and a four-fold reduction in total processing time was

achieved. Extent of root recovery was quantified by processing five woody plant species

grown in two different substrates and in soil, resulting in a minimum yield of 98%.


137









To evaluate effects of water stress on growth and identify probable biochemical

components that actuate these water stress effects, Ligustrumjaponicum plants of

landscape size were exposed to varying regimes of substrate water availability: 1)

constant high levels of plant available water, 2) severe stress, 3) constant low levels of

plant available water, 4) constant non-moisture limiting, 5) simulated natural wetting and

drying cycles of two days, and 6) simulated natural wetting and drying cycles of seven

days. This provided insight into how this woody species adjusts to its environment when

exposed to various degrees and types of water stress. Excessive water availability

resulted in marked reductions of carbon allocation towards roots, consequently luxury

shoot growth was observed. Constant or intermittent moderate water stress resulted in

total biomass reductions of approximately 20%, which disproportionally reduced shoot

mass, particularly diminishing leaf growth (leaf number and size). Prolonged, severe

water stress caused a 40% reduction on total biomass. This reduction was also

disproportionally greater for shoot biomass production, with relatively smaller reductions

in root biomass production. Plant responses to water stress differed with time of

exposure and degree of stress. Low correlation of root-to-shoot ratio and irrigation

frequency suggested that root-to-shoot ratio may not be the best indicator of water

status during the growing period of the plant.

Bud outgrowth dynamics and its implications to plant architecture were examined

along with the influence of relative growth of roots and shoots. Results indicated that

neoformed buds had limited sensitivity to dormancy, while preformed buds needed

more than one growing season to naturally outgrow. Following transplant into

rhizotrons, the first flush of shoot growth was mostly due to leaf lateral bud outgrowth.


138









Water stress influenced shoot architecture by enhancing apical dominance. Lateral

branching was diminished 51% in water stressed plants compared with those well-

irrigated. As plants adapted to the stress imposed, indeterminate growth was triggered

more often in meristematic regions of terminal buds. At the second flush, which

occurred later in the stress treatment, old buds burst more frequently than the newly

formed apex lateral bud. After transplant into rhizotrons, root growth began before shoot

growth. Temporal variations of moisture caused by wetting and drying cycles resulted in

continuous growth for portions of the root system, with quiescent periods observed for

shoot growth. Conversely, continuous high moisture levels resulted in roots exhibiting

quiescent periods in some plants. Large cycles of episodic growth were not observed

for most of the experimental period. However, for most surviving plants, trends of

increasing root and decreasing shoot growth were evident near the end as plants

neared balance between roots and shoots before harvest. Patterns of shoot and root

growth varied considerably between these clonal plants, which may be an important

consideration on analyses of populations of woody plants.

Episodical growth has been proposed to be controlled by changes in the carbon to

nitrogen ratio in plants. This ratio was refined in terms of the quantities of free amino

acids (faa) and total non-structural carbohydrates (tnc) in plants and plant parts. The

relationship of tnc and faa concentrations and its ratio in tissues of meristematic regions

were studied with a whole plant growth approach, including roots and shoots at different

growth stages. Additionally, the influence of water stress on these relationships was

considered. The results observed indicates that faa levels at the shoot tip are more

decisive to meristem growth/quiescence control than the tnc:faa or tnc level by itself. In


139









roots, tnc:faa observed was as good predictor of root growth (growing root tips having a

higher tnc:faa than quiescent root tips). Valine, leucine, tyrosine, cystein, metionine, and

arginine increased significantly with bud set in neoformed buds, compared with growing

shoot tips. Root tips contained abundant fructose, stachyose, and myoinositol. Mannitol

was the major transport sugar and glutamine, valine and histidine were the main faa

transported in xylem fluid. Water stress resulted in increases in the concentration of

some amino acids in growing shoot tips, such as Arg, Val, and His, especially valine in

neoformed buds at root flush.


140










APPENDIX
DIFFERENCES BETWEEN METABOLITES ANALYZED IN CHAPTER 6

Table A-1. Differences between metabolites analyzed as 2 x 2 x 5 factorial, with irrigation frequency, harvest and tissue as
treatments described in Chapter 6.


trt Tissue


Dormant bud

Growing shoot

Neoformed bud

Quiescent root

Growing root

Dormant bud

Growing shoot

Neoformed bud

Quiescent root

Growing root


Harvest
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush


Alanine
0.007 bcz
0.006 bc
0.013 bc
0.003 c
0.020 abc
0.014 bc
0.007 bc
0.010 bc
0.013 bc
0.011 bc
0.010 bc
0.005 c
0.009 bc
0.004 c
0.023 ab
0.023 ab
0.011 bc
0.008 bc
0.034 a
0.014 bc


0.C
0.
0.C
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.C
0.


Arginine Asparagine
039 defg 0.001 c
038 defg 0.001 c
090 bcde 0.000 c
008 g 0.002 c
204 a 0.000 c
117 b 0.000 c
047 cdefg 0.024 bc
061 bcdefg 0.009 bc
084 bcde 0.021 bc
105 bc 0.106 a
044 defg 0.003 c
030 efg 0.001 c
014 fg 0.000 c
011 g 0.001 c
193 a 0.000 c
211 a 0.000 c
076 bcde 0.021 bc
060 bcdefg 0.021 bc
093 bcd 0.072 ab
073 bcdef 0.040 abc


Aspartate


0.029
0.033
0.239
0.036
0.334
0.281
0.048
0.037
0.123
0.090
0.041
0.024
0.114
0.071
0.246
0.223
0.046
0.052
0.165
0.098


f
ef
abc
ef
a
ab
ef
ef
cdef
def
ef
ef
cdef
ef
abc
abcd
f
ef
bcde
def


Cysteine Glutamine Glutamate


0.048
0.040
0.120
0.019
0.268
0.209
0.049
0.072
0.073
0.105
0.068
0.031
0.052
0.026
0.280
0.238
0.084
0.066
0.096
0.084


bcde 0.004
cde 0.001
b 0.004
e 0.009
a 0.051
a 0.050
bcde 0.043
bcde 0.053
bcde 0.059
cb 0.083
bcde 0.008
cde 0.005
bcde 0.020
de 0.011
a 0.073
a 0.094
bcde 0.073
bcde 0.045
cde 0.179
bcde 0.058


z Means within columns not followed by the same letter are significant at Ps0.05 (Fisher's Least Significant Difference).


0.077
0.077
0.200
0.033
0.427
0.306
0.062
0.082
0.102
0.155
0.089
0.056
0.113
0.052
0.425
0.367
0.099
0.061
0.127
0.081










Table A-1. Continued
trt Tissue

. Dormant bud

o Growing shoot
U,
U,
Neoformed bud
U:
c Quiescent root

0
-o
o
2 Growing root

Dormant bud
w ------
co Growing shoot
o
o Neoformed bud

j Quiescent root


Growing root


Harest
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush


Glycine
0.000 a
0.001 a
0.000 a
0.001 a
0.000 a
0.000 a
0.000 a
0.001 a
0.004 a
0.000 a
0.001 a
0.000 a
0.000 a
0.000 a
0.000 a
0.000 a
0.000 a
0.000 a
0.005 a
0.005 a


Histidine
0.006 d
0.017 cd
0.064 bc
0.018 cd
0.141 a
0.094 ab
0.000 d
0.027 cd
0.000 d
0.000 d
0.022 cd
0.018 cd
0.006 d
0.024 cd
0.121 a
0.141 a
0.000 d
0.018 cd
0.007 d
0.000 d


Leucine Methionine
0.075 efg 0.032 efghi
0.080 efg 0.025 efghi
0.055 fg 0.049 cdef
0.008 g 0.006 i
0.249 ab 0.094 ab
0.222 abcd 0.074 abc
0.126 def 0.041 defg
0.169 bcde 0.073 abc
0.083 efg 0.053 cde
0.125 ef 0.053 cde
0.100 efg 0.039 defgh
0.059 fg 0.017 ghi
0.024 g 0.020 fghi
0.010 g 0.008 ih
0.295 a 0.105 a
0.235 abc 0.081 abc
0.160 bcde 0.071 bcd
0.146 cdef 0.054 cde
0.124 ef 0.049 cdefg
0.094 efg 0.056 cde


Proline
0.005 f
0.004 f
0.021 bcde
0.013 cdef
0.004 ef
0.001 f
0.016 cdef
0.023 bcd
0.065 a
0.078 a
0.008 cdef
0.006 def
0.014 cdef
0.018 bcdef
0.000 f
0.011 cdef
0.022 bcde
0.026 bc
0.079 a
0.036 b


142


Serine
0.000 b
0.000 b
0.000 b
0.001 a
0.000 b
0.000 b
0.000 b
0.000 b
0.000 b
0.000 b
0.001 a
0.000 b
0.000 b
0.001 ab
0.000 b
0.000 b
0.000 b
0.000 b
0.000 b
0.000 b










Table A-1. Continued
trt Tissue

3 Dormant bud

-o Growing shoot






0)
U,
U Neoformed bud
QU
r Quiescent root
o
o
r Growing root

Dormant bud
w -- ----
c Growing shoot
o
o Neoformed bud

-j Quiescent root


Growing root


Harvest
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush


Threonine


0.008
0.005
0.019
0.003
0.025
0.020
0.007
0.004
0.000
0.004
0.009
0.006
0.011
0.005
0.030
0.042
0.004
0.002
0.000
0.000


def
f
bcde
f
bc
bcd
def
f
f
f
def
ef
cdef
f
ab
a
f
f
f
f


Tyrosine


0.025
0.015
0.022
0.006
0.080
0.059
0.062
0.072
0.037
0.049
0.030
0.012
0.013
0.006
0.081
0.061
0.081
0.054
0.067
0.050


defgh
ghf
efgh
h
a
bcde
abcd
ab
bcdefgh
bcdefg
defgh
hg
gh
h
a
abcde
a
abcdef
abc
bcdefg


Valine
0.113 cdef
0.116 cdef
0.325 bcde
0.017 f
0.951 a
0.439 bc
0.232 bcdef
0.365 def
0.092 f
0.205 bcdef
0.139 cdef
0.060 ef
0.087 bc
0.019 f
0.403 bc
0.483 b
0.235 bcdef
0.231 bcdef
0.248 bcdef
0.229 bcdef


Fructose
2.85 gh
1.67 gh
0.00 h
3.49 efg
4.61 defg
1.92 gh
5.80 def
6.86 def
21.01 c
27.73 a
1.90 gh
1.61 gh
0.00 h
3.77 efg
3.65 efg
4.25 defg
7.27 d
7.29 d
24.10 bc
25.79 ab


Glucose
66.98 defg
33.58 j
89.70 abc
104.09 ab
81.86 cde
54.67 ghi
37.15 ij
39.34 ij
65.02 efg
62.96 efgh
43.40 hij
44.17 hij
84.91 bcd
105.84 a
75.13 defg
64.79 efg
37.73 ij
40.98 ij
67.91 defg
60.97 fgh


143










Table A-1. Continued
trt Tissue

3 Dormant bud
c:
0'--
-o Growing shoot






0)
U,
U Neoformed bud
QU
r Quiescent root
o
o
r Growing root

Dormant bud
w -- ----
c Growing shoot
o
o Neoformed bud

-j Quiescent root


Growing root


Har
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush
Root flush
Shoot flush


Inositol
0.53 ghi
0.07 i
0.00 i
2.08 fghi
0.51 ghi
0.00 i
2.73 defg
4.53 de
9.55 c
17.21 b
0.25 hi
0.36 hi
0.00 i
2.33 efgh
0.57 ghi
0.00 i
3.87 def
4.60 d
10.39 c
19.49 a


Mannitol
17.65 defg
10.65 g
24.09 d
20.96 de
23.55 d
17.96 defg
35.06 c
37.71 c
38.59 c
51.31 a
12.62 fg
13.51 efg
18.41 defg
20.29 def
21.73 d
20.07 def
32.89 c
36.21 c
38.90 bc
46.59 ab


Raffinose
3.08 ef
2.37 f
0.00 f
1.38 f
7.97 cde
8.83 cd
2.15 f
4.47 def
35.68 a
23.13 b
2.50 f
2.81 ef
0.00 f
1.01 f
10.63 c
9.49 cd
2.23 f
5.01 def
37.04 a
21.00 b


Stachyose
1.64 efgh
1.24 h
0.00 h
0.27 h
3.52 defgh
6.04 cdef
6.29 cde
7.35 cd
19.82 b
18.40 b
1.31 gh
1.57 fgh
0.00 h
0.00 h
4.58 defgh
5.99 cdefg
6.38 cd
10.30 c
25.08 a
16.61 b


Sucrose
7.91 def
4.76 fgh
17.16 a
6.67 efg
13.12 b
9.46 cde
2.04 hij
4.15 ghi
0.00 j
2.20 hij
6.48 efg
6.34 efg
12.58 bc
7.40 defg
12.25 cb
10.41 bcd
2.31 hij
4.71 fgh
0.67 ij
1.51 hij


Starch
0.25 cd
0.48 cd
0.00 d
1.84 a
0.00 d
0.00 d
0.15 cd
0.14 cd
0.88 bc
0.44 cd
0.47 cd
0.19 cd
0.72 bcd
1.81 a
0.00 d
0.00 d
0.12 cd
0.19 cd
1.29 ab
0.35 cd










Table
trt




-o
CU






0
C,
C,

CU


o







"o


A-1. Continued
Tissue

Dormant bud

Growing shoot

Neoformed bud

Quiescent root

Growing root

Dormant bud


Growing shoot

Neoformed bud

Quiescent root

Growing root


Free amino
Harvest
acids
Root flush 0.47 fghi
Shoot flush 0.46 fghi
Root flush 1.22 cde
Shoot flush 0.18 i
Root flush 2.85 a
Shoot flush 1.89 bc
Root flush 0.76 defghi
Shoot flush 1.06 defg
Root flush 0.81 defghi
Shoot flush 1.17 cdef
Root flush 0.61 defghi
Shoot flush 0.33 gh
Root flush 0.50 efghi
Shoot flush 0.27 hi
Root flush 2.28 ab
Shoot flush 2.21 ab
Root flush 0.98 defgh
Shoot flush 0.84 defghi
Root flush 1.34 cd
Shoot flush 0.92 defghi


Nonstructural
carbohydrates
100.88 cdefg
54.83 h
130.95 bcd
140.78 b
135.14 bc
98.88 defg
91.37 efg
104.56 cdef
190.56 a
203.39 a
68.94 gh
70.55 fgh
116.62 bcde
142.45 b
128.55 bcd
115.00 bcde
92.82 efg
109.30 bcde
205.38 a
192.31 a


145


Nonstructural carbohydrate-
to-free amino acid ratio
223.60 cdef
147.91 defg
113.09 efg
749.45 a
57.31 g
52.43 g
132.33 fge
112.20 fge
258.98 dc
215.15 cdef
130.56 efg
231.96 cde
228.95 cde
538.13 b
66.94 g
51.64 g
99.75 fg
133.52 efg
176.81 defg
314.22 c









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BIOGRAPHICAL SKETCH

Dilma Daniela Silva was born in Minas Gerais, Brazil. In 2001 she received her

bachelor's degree from the Universidade Federal de Vigosa, Vigosa, Minas Gerais,

Brazil. She worked as a research assistant in the Plant Physiology Laboratory of the

Department of Biological Science at the North Dakota State University in conjunction

with the Northern Crop Science Laboratory, USDA, where she worked with protein

determination, isolation of mitochondria and peroxisomes, tissue respiration rates and

other plant biochemistry analyses. In 2004 she received a Master of Science degree in

plant science from the Universidade Federal de Vigosa, Vigosa, Minas Gerais, Brazil,

with a thesis title of "Ethylene sensitivity of two varieties of geranium and 1-MCP

treatment".


161





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INFLUENCE OF WATER STRESS, NONSTRUCTURAL CARBOHYDRATES AND FREE AMINO ACIDS ON CONTROL OF ROOT AND SHOOT GROWTH OF Ligustrum japonicum Thunb. By DILMA DANIELA SILVA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1

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2010 Dilma Daniela Silva 2

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To my mom, Guiomar, and to my son, Nicolas 3

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ACKNOWLEDGMENTS Successful completion of this work would not have been possible without the support, encouragement and assist ance of many people. First and foremost, I would like to take the opportunity to convey my sincere appreciation to Dr. Richard C Beeson Jr, my advisor and committee chair, for believing on my potential and allowing me the opportunity to pursue a dream. His many hours of guidance are appreciated, especially for the many hours Dr Beeson endured reading and editing this dissertation. Second, I would like to thank my committe e members Drs. Karen E Koch, Michael E Kane, Peter C Andersen, and Timothy A Martin for their guidance, expertise, constructive suggestions, and review of t he manuscripts. All are greatly appreciated. Each members contributions were unique, teaching me the importance of utilizing diverse perspectives, all of which substantially improved the finished result. I am grateful to Christopher Fooshee and Heidi Savage for their thoughts, contributions and reviews to this dissertatio n. Special thanks also goes to those who shared some of the many hour s I spent building rhizotrons planting, collecting data, washing roots or HPLC-fighting: Brian Pearson, Donald Cox, Edward Tillman, Fabieli Lanes, and Julie Brooks. I will always have tender memories of my ti me at MREC. I would also like to thank Ana Willianms, Angelica Cretu, Eliandro Costa, Gregory Alex ander, Mary Risner, and Patricia Ramos for their friendship and encouragement. 4

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TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 7LIST OF FI GURES .......................................................................................................... 9ABSTRACT ................................................................................................................... 12 CHAPTERS 1 INTRODUC TION .................................................................................................... 16Partitioning of P hotosynthat es ................................................................................ 16Episodic Gr owth ...................................................................................................... 18Induction and Control of S hoot and Root Growth ................................................... 20Root-to-Shoot Ratio ................................................................................................ 21Signaling Syst ems in Plants .................................................................................... 242 AN ELEVATED LARGE-VOLUME RHIZOTRON FOR EVALUATING ROOT GROWTH UNDER NATURAL SOIL MOISTURE COND ITIONS. ........................... 28Introducti on ............................................................................................................. 28Material and Methods ............................................................................................. 29Stage 1 Designs for containm ent and observati on ports ............................... 29Stage 2 Designs for drainage systems allowing natural-so il profiles ............. 31Results and Discussion ........................................................................................... 333 DEVELOPMENT AND EVALUATION OF A ROTARY ROOT SEPARATOR ......... 46Introducti on ............................................................................................................. 46Material and Methods ............................................................................................. 47Results and Discussion ........................................................................................... 494 EFFECTS OF WATER STRESS ON PLANT GROWTH OF Ligustrum japonicum Thunb. ................................................................................................... 53Introducti on ............................................................................................................. 53Material and Methods ............................................................................................. 55Experiment 1 .................................................................................................... 55Experiment 2 .................................................................................................... 57Experiment 3 .................................................................................................... 58Results and Discussion ........................................................................................... 60Soil moisture management ............................................................................... 60Experiment 1 .................................................................................................... 62 5

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Experiment 2 .................................................................................................... 63Experiment 3 .................................................................................................... 665 DEVELOPMENT OF SHOOT ARCHITECTURE OF Ligustrum japonicum Thunb. IN RESPONSE TO SOIL MOIS TURE. ....................................................... 82Introducti on ............................................................................................................. 82Material and Methods ............................................................................................. 84Growth conditions and ex perimental design ..................................................... 84Growth meas urement s ..................................................................................... 85Results and Discussion ........................................................................................... 86Growth under well-irrigated conditions (2 day wetting and drying cycles) ........ 86Effect of intermittent water stress on growth ..................................................... 90Root and shoot grow th patte rns ....................................................................... 93Conclusi ons ............................................................................................................ 976 PATERNS OF FREE AMINO ACIDS AND NON-STRUCTURAL CARBOHYDRATES ASSOCIATED WI TH EPISODICAL GROWTH OF Ligustrum japonicum Thunb. ................................................................................. 112Introducti on ........................................................................................................... 112Materials and Methods .......................................................................................... 115Growth conditions and expe rimental design ................................................... 115Growth measur ements ................................................................................... 116Biochemical anal yses ..................................................................................... 116Results and Discussion ......................................................................................... 121Patterns of faa and tnc under we ll-irrigated condi tions ................................... 121Influence of water stress in pa tterns of faa and tnc ........................................ 125Conclusion s .......................................................................................................... 1277 SUMMARY AND CO NCLUSION S ........................................................................ 137APPENDIX DIFFERENCES BETWEEN METABOLITES ANALYZED IN CHAPTER 6 .......................................................................................................... 141LIST OF REFE RENCES ............................................................................................. 146BIOGRAPHICAL SK ETCH .......................................................................................... 161 6

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LIST OF TABLES Table page 3-1 Root ball volume and substrate/soil of root samples. ......................................... 513-2 Dry mass of root samples and yield achi eved using the rotary root separator. .. 514-1 Mean percent volumetric water content (%VWC) achieved in Experiments 1, 2 and 3 determined after recalculati on using equations based on prolonged dual measurement of VWC using Digital TDT and ECH2O probes. .................... 734-2 Experiment 1. Dry ma ss (g) of components of L. japonicum grown at different constant volumetric water contents in rhizotrons. Moisture probes remained near original roots ball for the durat ion of the experiment. Masses between treatments were pooled within har vest. .............................................................. 734-3 Experiment 1. Leaf area, allome tric relationships and percentage of allocation in L. japonicum grown in rhizotrons at constant volumetric water contents. Results between treatment s were pooled within harvest. ................... 744-4 Experiment 2. Dry ma ss (g) of components of L. japonicum grown in rhizotrons at constant volu metric water contents. ............................................... 744-5 Experiment 2. Leaf area, allome tric relationships and percentage of allocation in L. japonicum grown in rhizotrons at constant volumetric water contents. ............................................................................................................. 754-6 Experiment 3. Dry ma ss (g) of components of L. japonicum grown in rhizotrons at variable vo lumetric wate r cont ent................................................... 764-7 Experiment 3. Leaf area, allome tric relationships and percentage of allocation in L. japonicum grown in rhizotrons at variable volumetric water content. ............................................................................................................... 775-1 Percentages of bud outgrowth and dormancy of neoformed buds on branches expanded during 100 days of undisturbed growth by L. japonicum grown in large rhizotrons in 2009. ....................................................................... 995-2 Number of growing po ints recorded during 100 days of undisturbed growth of L. japonicum grown in large rhiz otrons in 2009. ............................................... 1005-3 New branch distribution of L. japonicum grown in large rhizotrons in 2009, during 100 days of undisturbed growth. ............................................................ 1015-4 Single stem and internode length (cm) of new branches produced during 100 days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009. 102 7

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5-5 Total length (cm) of new branches produced during 100 days of undisturbed growth of L. japonicum grown in large rhiz otrons in 2009. ................................ 1025-6 Number of stems and leaves per stem per plant produced during 100 days of undisturbed growth of L. japonicum grown in large rhizot rons in 2009. ............ 103A-1 Differences between metabolites analyzed as 2 x 2 x 5 factorial, with irrigation frequency, harvest and tissue as treatments described in Chapter 6. 141 8

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LIST OF FIGURES Figure page 2-1 Prototype rhizotron with Ligustrum japonicum 2 months after transplanting into the rhiz otron. ................................................................................................ 402-2 Drainage systems te sted on rhizot rons, ............................................................. 412-3 Schematic diagram of a rhizot ron. ...................................................................... 422-4 One-year-old Ligustrum japonicum transplanted in a rhizotron, window open just for demons tration. ........................................................................................ 432-5 Temperature of air and substrat e immediately next to single PVC door (substrate: average of four thermo couples, and air: average of two thermocouples). .................................................................................................. 432-6 Camera positioning frame used to ta ke pictures mounted on rhizotr on. ............. 442-7 Percent volumetric water content (% VWC) observed during drainage trial (average of actual values for 3 repetit ions). ........................................................ 453-1 A) Root separator. B) Detail sho wing internal basket. C) Detail showing reducing transmission and motor. ....................................................................... 524-1 Percent contribution of each plant component to the total dry mass of L. japonicum grown under different substr ate moisture levels. ........................... 784-2 Daily stomatal conductance, Dgs (A) and shoot water potential, T (B) of L. japonicum grown under irrigation varying between saturation and 19.8 and 22.3% VWC for moderartely-stressed and well-irrigated plants respectively on the day of minimum and maximum water stress in Exp. 3. ............................ 794-3 Correlation among root and shoot dry mass. ..................................................... 804-4 Correlation for Exp. 2 among %VWC (differential between saturation and the triggering volumetric water content ) and (A) root dry mass, (B) shoot dry mass, (C) leaf dry mass, and (D) leaf area. ........................................................ 815-1 Organs of L. japonicum at different growth stages. ......................................... 1045-2 Diagram showing branch orders and bud positions of L. japonicum ................ 1055-3 Different organs of L. japonicum. ...................................................................... 106 9

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5-4 Number of new l eaves and leaf area of L. japonicum at variable volumetric water content (2 day irrigation cycle, well-irrigated plants and 7 day irrigation cycles, moderately-stressed pl ants) for 2009 plants. ........................................ 1075-5 Growth patterns of L. japonicum grown in rhizotrons at constant high substrate volumetric water content (well-irrigated plant s) in 2008. ................... 1085-6 Growth patterns of L. japonicum grown in rhizotrons at variable volumetric water content (2 day irrigat ion cycle, WP) for 2009. ......................................... 1095-7 Growth patterns of L. japonicum grown in rhizotrons at severe stress, left column, and moderate stress, right colu mn (severely-stressed and stressed plants, respectively) in 2008. ............................................................................ 1105-8 Growth patterns of L. japonicum grown in rhizotrons at variable volumetric water content (7 day irrigation cycl e, stressed plants) for 2009. ....................... 1116-1 Total nonstructural carbohydrates, free amino acids, and total nonstructural carbohydrate-to-free amino acid ratio of L. japonicum shoot tissues of plants grown in rhizotrons at variable volumetr ic water content (2 or 7 day irrigation cycles, well-irrigated and moderately-stressed plants, respec tively). ............... 1296-2 Free amino acids and nonstr uctural carbohydrates of L. japonicum shoot tissues of plants grown in rhizotrons at variable volumetric water content (2 day irrigation cycles, well -irrigated plant s). ....................................................... 1306-3 Total nonstructural carbohydrates, free amino acids, and total nonstructural carbohydrate-to-free amino acid ratio of L. japonicum root tissues of plants grown in rhizotrons at variable volumetr ic water content (2 or 7 day irrigation cycles, well-irrigated and moderately-stressed plants, respec tively). ............... 1316-4 Free amino acids and nons tructural carbohydrates of L. japonicum root tissues of plants grown in rhizotrons at variable volumetric water content (2 day irrigation cycles, well -irrigated plant s). ....................................................... 1326-5 Free amino acids and nonstr uctural carbohydrates of L. japonicum xylem fluid extracted from plants grown in rh izotrons at variable volumetric water content (2 or 7 day irrigation cycles, well-irrigated plants or moderatelystressed plants, respectively). .......................................................................... 1336-6 Total nonstructural carbohydrates, free amino acids, and total nonstructural carbohydrate-to-free amino acid ratio of L. japonicum xylem fluid extracted from plants grown in rhizotrons at variable volumetric water content (2 or 7 day irrigation cycles, well-irrigated pl ants and moderately-stressed plants, respectively ). .................................................................................................... 134 10

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6-7 Free amino acids and nonstr uctural carbohydrates of L. japonicum shoot tissues grown in rhizotrons at vari able volumetric water content (7 day irrigation cycles, moderately-stressed pl ants). .................................................. 1356-8 Free amino acids and nons tructural carbohydrates of L. japonicum root tissues grown in rhizotrons at vari able volumetric water content (7 day irrigation cycles, moderately-stressed pl ants). .................................................. 136 11

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy INFLUENCE OF WATER STRESS, NONSTRUCTURAL CARBOHYDRATES AND FREE AMINO ACIDS ON CONTROL OF ROOT AND SHOOT GROWTH OF Ligustrum japonicum Thunb. By Dilma Daniela Silva August 2010 Chair: Richard C. Beeson, Jr. Major: Horticultural Science Environmental Horticulture A more profound knowledge of the factors affecting root and shoot post-transplant growth would enable more conscienti ous decisions on plant management and on practices to be adopted. The experimental system required to impose precise drought treatments, and to make possible obs ervation of natural growth of Ligustrum japonicum Thunb. under favorable conditions was developed. Influence of different intensities and duration of water stress on plant growth was tested. Plant architecture was described for undisturbed conditions with an d without moderate water stress. Interactions between labile forms of carbon and ni trogen within different merist em tissues, and control of growth initiation or cessation was studied in a whole plant approach. The final rhizotron designed allowed exce ptional root observation, provided a near-uniform profile of soil moisture, and was easily manageable for precise long-term data acquisition. This rhizotron had eight independent viewing/sampling windows and held 0.16 m3 of soil. An electric powered ro ot separator was developed that sped sample preparation for root dry mass determinat ion with a capacity of 40 L of container 12

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substrate or 32 kg of sandy soil. No water was required and a four-fold reduction in total processing time was achieved with a >98% root mass recovery. Excessive water availability resulted in marked reductions of carbon allocation towards roots, consequently luxury shoot grow th was observed. Constant or intermittent moderate water stress resulted in total bi omass reductions of approximately 20%, which disproportionally reduced shoot mass, particu larly diminishing leaf number and size. Prolonged, severe water stress resulted in a 40% reduction of total biomass. This reduction was also disproportionally greater for shoot biomass production, with smaller reductions in root biomass production. Plant responses to water stress differed with time of exposure and degree of stress. Low correlati on of root-to-shoot ratio and irrigation frequency suggested that root-toshoot ratio may not be the best indicator of water status during the plant growing period. Following transplant into rhizotrons, r oot growth began before shoot growth and the first flush of shoot growth was most ly basipetal. Water stress altered shoot architecture by enhancing apical dominance. As plants adapted to the stress imposed, indeterminate growth was trigger ed more often in meristematic regions of terminal buds. At the second flush, which occurred later in the stress treatment, old buds expanded more frequently than the newly formed apex lateral bud. Temporal variations of moisture caused by wetting and drying cycles resulted in continuous growth for portions of the root system, with quie scent periods observed for shoot growth. Conversely, continuous high moisture levels resulted in roots exhibiting quiescent periods in some plants. Patterns of shoot and root growth varied considerably between these clonal 13

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plants, which may be an important consider ation for analyses of populations of woody plants. Free amino acid levels at the shoot tip were more decisive for initiation of meristem growth or quiescence than the quantit y of total nonstructural carbohydrates or nonstructural carbohydrate-to-free amino acid ratio. In roots, this ratio was a good predictor of root growth. Valine, leuci ne, tyrosine, cysteine, metionine, and arginine increased significantly with bud set, compared with growing shoot tips. Root tips contained abundant fructose, stachyose, and myoinositol. Mannitol was the major transport sugar and glutamine, valine and histidine were the main free amino acids transported in xylem fluid. Water stress resu lted in increases in the concentration of some amino acids in growing shoo tips, such as arginine, valine, and histidine, and especially valine in deve loping buds at the begi nning of root flush. The results observed from these experim ents suggest important considerations for woody plant management. Since it appears t hat water stress can increase apical dominance; the necessity for pruning may increas e if the plant is to be used as a shrub rather than as a small tree. C onversely, well-irrigated plants show basipetal branching in the beginning of the growing season, which can also affect pruning decisions. Wetting and drying cycles result in continuous grow th of portions of t he root system; thus, establishment can benefit by temporal variati ons of soil moisture. Fertilization during the growing period prior to transplant into a lands cape is an important factor for building up internal nutrient reserves to support init ial root growth and bud expansion at the beginning of the growing season. These observations suggest that woody plant 14

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15 management can benefit from appropriate decision making of pruning, frequency of irrigation, and timing of fertilization and transplant.

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CHAPTER 1 INTRODUCTION Transplanted in-ground trees undergo a seve re physiological shock because their capacity for water absorption is greatly decreased. This is the result of injury to roots, loss of small absorbing roots, and disruption of the previously established contact of the root system with a large volume of soil (K ozlowski and Pallardy 2002). Furthermore, textural differences between the substrate used for container grown plants and surrounding soil after transplant diminish water availability for root absorption. Shrubs and trees usually do not have a root growth rate high enough to rapidly replace lost or damaged roots, or to gener ate new root growth immediatel y after transplanting into new soil. As a result, plants normally experienc e water stress and establishment is delayed or not achieved in extreme cases. This requi res additional water, fertilizer and extended care. During the establishment period, roots are expanding out into the landscape soil, and shoots and trunk grow more slowly t han before transplanting (Gilman 2002) or discontinue growth until the root system is establis hed and capable of supplying sufficient amounts of water to abov e-ground organs (Ongaro and Leyser 2008). Partitioning of Photosynthates Partitioning of photosynthates can be infl uenced by environmental stimuli (Engels 1994; Rogers et al. 1996). Earlier views, as described by Thornley (1972), considered growth and partitioning to depend on two plant processes, transport and utilization. However, more recent models have added substrate supply (Campagna and Margolis 1989); substrate sources (Thornley 1998); wate r stress (Chaves and Oliveira 2004) and nutrient availability (Coleman et al. 2004) Campagna and Margolis (1989) stated that allocation is likely to result from combined influences of substrate supply, transport and 16

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utilization. As a plant grow s, equilibrium is maintained between root and shoot growth (Campagna and Margolis 1989). Bloom et al. (1985) describe this mechanism well, stating that when a particular below-ground re source, such as nitrogen, limits growth more than other resources such as light, pl ants that optimize growth would allocate more carbon to the roots to absorb the lim iting resource. Woody plants that grow on poor sites generally allocate a greater propor tion of photosynthates to root production than do those that grow on good sites (Kozlowski and Pallardy 20 02). The resultant changes have obvious impacts on water-absorbing capacity in comparison with transpiration by above-ground structures (K ozlowski and Pallardy 2002). Decreasing availability of water, nitrogen, or both, r educes the leaf/root carbon partitioning ratio (Running and Gower 1991). Factors affecting phot osynthesis can also strongly affect water and nutrient acquisition (E issenstat and Van-Rees 1994). Nitrogen plays a central role in plant metabolism, as a constituent of the chlorophyll molecule and as a key com ponent of enzymes used in photosynthesis, respiration and growth process (Margo lis and Waring 1986). The balance between nitrogen and carbon availability is important in determining the relative allocation of dry matter production between roots and shoots. Ericsson (1995) concluded that the internal balance between labile (easily converted) nitrogen and carbon in roots and in the shoots determines how dry matter is parti tioned in a plant. Ericssons work showed that an increased allocation of dry matter to below-ground parts was associated with a greater amount of starch in the tissues. D epletion of stored ca rbohydrates occurred under all conditions in which root development was inhibited. Three different dry matter allocation patterns were observed depending on which specific mineral nutrients were 17

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withheld in each instance. Reduc tion in atmospheric carbon dioxide concentration led to a strong decrease in root growth. However CO2 enrichment had no effect on dry matter partitioning. Campagna and Margolis (1989) used mani pulation of liquid fertilizer and CO2 enrichment of the air during greenhouse production of black spruce seedlings. They found a strong correlation (r2 > 0.90) between tnc:faa and carbon allocation to shoots and roots. When the relative balance between biochemically -available forms of carbon was high in relation to the biochemically-a vailable form of nitrogen, rates of protein synthesis and other metabolic processes were affected favoring root growth. When tnc:faa was low, shoot growth was favored. The authors also stated that while the ratio of carbon to nitrogen has been known to affect root-to-shoot ratio over relatively long periods of time, the carbon-to-nitrogen rati o may also influence short-term episodic growth patterns. Furthermore, it was sugge sted that the carbon (C) to nitrogen (N) balance offers a partial explanat ion of why root growth predomin ates in the later part of growing season. When shoot growth either sl ows down or stops, th e tnc:faa increases because the carbohydrate sink in leaves declines, corresponding to a predominance of root growth. Episodic Growth Many woody species display an episodic growth habit by fluctuating between periods of rapid shoot growth and slow r oot growth versus periods with the inverse pattern. Relatively little is known about the changing carbon allocation patterns in species with episodic growth cycl es (Dickson et al. 2000a). Kuehny, et al (1997) observed alternate flushes of root and s hoot growth of approximately 20 days in Ligustrum japonicum 18

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The control of vegetative growth invo lves close interdependence between roots and shoots (Kozlowski and Pallardy 1997). It has been hypothesized that episodic shoot and root growth is controlled by hereditary factors (Morrow 1950), and is influenced by several non-hereditary factors including t he carbon-to-nitrogen ratio within a plant (Campagna and Margolis 1989) ; competition for carbohy drates among the various tissues (Eissenstat and Van-Rees 1994; Kozl owski and Pallardy 1997; Kuehny et al. 1997); N fertilization (Huche-Thelier et al. 2006; Kuehny et al. 1997); changes in soil water content (Kozlowski and Pallardy 2002) and temperature fluctuations (Lyr and Garbe 1995; Reich et al. 1980). Plant growth can be characterized by cell proliferation, organ elongation and mass accumulation. Meristematic regions can be ac tive, but not exhibit visible growth for a period of time. Growth measurement method may influence perception of organ growth. For instance, length measurement may not be followed by proportional dry mass increase, which occurs with maturation. Thus differences in measurement method can also be a factor in observation of epis odic growth and cause disparities between experimental results. In northern red oak ( Quercus rubra L.), an episodic species, upper leaves of a branch allocated most 14C-photosynthate upward during l eaf and shoot growth, while lower leaves supplied more 14C to lower stem and roots (Dickson et al. 2000a). Starch and sugar storage in leaves, stems, and ro ots during lag and bud growth stages indicate a feedback response to decreasing sink strength and temporary storage of both starch and sugar in these plant tissues (Dickson et al. 2000b). Kuehny and Halbrooks (1993) determined episodic growth of Ligustrum japonicum plants by measuring total 19

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shoot and root fresh weight nondestructive ly. Kuehny et al. (1997) concluded that changes in endogenous N concentrations and allocation patterns in L. japonicum were linked to the control of episodic shoot and root growth. Induction and Control of Shoot and Root Growth Mechanisms by which plants modulate their growth rate, cell division, and sink strength in response to environmental and de velopmental conditions are unknown, but are presumed to involve meristems (Kuehny et al. 1997). A meristem is a group of undifferentiated, actively dividing plant cells. They include formation of roots and shoots. Individual meristematic cells are dividing, expanding and differentiating. These highly dynamic cells are somehow coordinated in r oot or shoot meristems (Grandjean et al. 2004). The result can affect developmental patterning (Clark 2001) or developmental programs (Lough and Lucas 2006) in plants. Cockcroft, et al (2000) concluded that cell division is the main determinant of meristem activity and overall growth rate. They proposed that control of plant growth rate is achieved through r egulation of the cell phase G1. Tax and Durbak (2006) described the structure of the shoot apical meristem and summarized experimental and molecular data for signaling. Pl ant meristems can also be influenced by cytokinins (Werner et al. 2001). Francis and Halford (2006) reviewed the literature on nut rient sensing in plant meristems and gave special emphasis to the components of sugar sensing/signalin g pathways. Woody plants growth and development ar e regulated by interactions of their heredity and environment as they influence the availability of resources (carbohydrates, hormones, water and mineral nutrients) at meri stematic sites (Kozlowski and Pallardy 1997). Shoot growth largely is determined by genetic factors and environmental factors 20

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such as light, water, temperature, miner al supply, composition of above and belowground atmospheres, physical and chemical soil properties, insects, other plants, and animals (Calfee 2003). Specific shoot acti vity depends upon environmental factors such as photosynthetic photon flux and carbon dioxide concentration. Similarly, specific root activity depends upon factors such as root tem perature, concentration of nutrients in the soil and water uptake (Engels 1994; Thornley 1972). Salaun, et al. (2005) observed that N was translocated in the xylem fluid of Ligustrum ovalifolium L. predominantly in the form of amino acids. Prior to bud break argi nine and, later, glutamine accounted for the principal components of nitrogen mobilization. However, of the 20 common amino acids only five were analyzed in this study. Also working with Ligustrum ovalifolium Salaun and Charpentier (2001) found that in Dece mber, arginine fo llowed an increasing gradient from the root s to the trunk and decreased from t he lower to the upper parts of stems. The more fertilizati on the plants received, the higher the arginine content. Storage content in trunks amounted to 45% of total plant arginine. A high carbon-to-nitrogen ratio promotes r oot development, and, conversely, a low carbon-to-nitrogen ratio promotes shoot dev elopment (Koch 1997). Previous research by Beeson indicated that high N availability fr om biocompost incorporated into the soil at transplanting reduced root grow th into landscape soil, while stimulating shoot growth. Conversely low N in landscape soils of unfer tilized control plants stimulated root growth, while severely restricting new shoot growth (Beeson, unpublished data). Root-to-Shoot Ratio The root-to-shoot ratio, defined as dry mass of roots divided by dry mass of shoots, depends upon partitioning of photosynt hates (Engels 1994; Rogers et al. 1996). 21

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The proportion of above-ground and below-ground mass can be altered by responses of genes that affect uptake, assi milation and storage (Koch 1997). The balance between root and shoot growth is expressed by k, the variation of dry mass of shoots divided by the variation of dr y mass of roots over a given period of time. In other words, k represents the relative partitioning of growth over a specific period of time. In 1972, Thornley published a model which describes root-to-shoot ratio in vegetative plants and how they respond to changes in root and shoot activity. In this model a plant has only two components, r oots and shoots, and growth is dependent upon the supply of carbon (by shoots) and ni trogen (by roots). Later Thornley (1998) refined this model to accommodate infl uence of ontogenesis, scaling, within-plant transport resistances, hormones and active transport. Water deficits and mineral nutrient deficienc ies promote greater relative allocation of photosynthates to root growth. This ultima tely results in plants that have higher rootto-shoot ratio and greater capacity to absorb wa ter and minerals relative to shoots that must be supported (Kozlowski and Pallardy 2002). One of the adaptative mechanisms found in plants to avoid water stress is to pr oduce high root-to-shoot ratio, which permits better exploitation of water reserves to replace transpirational losses. Many plants respond to water stress with an in crease in root-to-shoot ratio, usually attributed to an decrease in shoot growth (Bachelard 1986; Sharp and Davies 1979; Steinberg et al. 1990). However some authors a ttribute the change in root-to-shoot ratio under water shortage mainly to an adaptativ e improvement (drought tolerance) genetically inherited a nd argue that leaf growth slow s down, causing a decrease in foliage area and intercepted solar radiation (F arrell et al. 1996; Osorio et al. 1998). 22

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Ultimately this relative decrease of foliage could represent a decrease in tnc:faa and invert the relationship observed by Cam pagna and Margolis (1989) in well-irrigated plants. In response to drought, or limited soil mois ture availability over extended periods of time, plant water status was proposed to be more strongly influenced by changes in root-to-shoot partitioning and root density rat her than the interaction of soil moisture content with stomatal conduc tance (Thornley 1996). Indeed, this has been verified through many experiments in t he past 15 years relating to physiological responses of woody ornamentals transplanted into lands cape situations (Beeson, pers. comm.). Johnson et al. (1991) developed a water submodel th at includes root-to-shoot message control of stomatal conductance for incorporat ion into mechanistic plant growth models. However, McMillin and Wagner (1995) observ ed that the influence of water stress in root-to-shoot ratio are depend ent on stage of development, thus age may influence establishment as well. Studies with C14 and C13 indicate a significant difference between the amounts of C transported to different plant organs. Fifty percent of assimilates produced in young cereal plants was rapidly transported below -ground, of which 50% was respired. Less than 5% of the fixed ca rbon went to root exudates. When subjected to water stress, the allocation of C13 to roots increased so that at fl owering 38% of shoot C was belowground compared to 31% in well-irrigated plants. CO2 enrichment increased the proportion of root to total ma ss by 55%. While increasing air temperature by a mean of 3 C decreased the proportion of roots from 0.093 in the cool treatment to 0.055 in the warm treatment (Engels 1994). Campagna and Mar golis (1989) reported similar results 23

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in black spruce seedlings. As tnc:faa increased, k decreased during seedling development. Keyes and Grier (1981) reported that in low-productivity sites Pseudotsuga menziesii trees partitioned 36% of total net primary production to fine roots but only 8% when in high-productivity sites. Kuehny, et al. (1997) found that when N is limiting whole-plant growth, more car bohydrates are transloca ted to below-ground, thus facilitating root growth, and also stored in the shoots, where it would aid future episodes of growth. Cheng and Fuchigami (2002) working with a pple trees observed that about 50% of tree N content was remobilized to support new shoot and leaf growth. They concluded that the amount of reserve N remobilized fo r new growth in spring was proportional to tree N status, and was unaffected by current N supply in the soil. Woody plants transplanted into landscapes normally extend substantial roots into the soil before shoot growth returns to pre-tr ansplant levels. This pre-dominance of root growth overrides normal cycles of episodical shoot and root growth. Signaling Systems in Plants Higher plants are sessile organisms, thus the ability to adapt to their environment is a key feature for survival. Environmental stimuli, such as light, water or nutrient availability can be sensed in one organ of a plant and the information can be transmitted to other organs or cells through the use of chemical signals that trigger plant responses. The evolutionary development of a long-distance communication network in higher plants reflects the need to communicate environmental inputs, sensed by mature organs, to meristematic regions of the plant (Sack and Holbrook 2006). Signaling pathways utilize a complex network of interactions to orchestrate biochemical and physiological responses such as flowering, fruit ripening, germination, photosynthetic 24

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regulation, and shoot or root development (Mulligan et al. 1997), and allows an optimal response to environmental conditions (Genoud et al. 2001). Recent studies have identified new molecular components required for proper shoot meristem activity, and they have revealed that complex, interce llular communication pathways play important roles in coordinating meristem function (Fletcher and Meyerowitz 2000; Haecker and Laux 2001). Kepinski (2006) addressed the mechanistic links between hormone signaling and developmental processes. Thum et al. (2003) reported interaction of carbon methabolism with blue, red, and far-r ed-light signaling. Sugar and especially amino acid signaling in plants is in the early stages of research, with the majority of it being done with herbaceous rat her than woody species. Sugar-regulated expression provides a mechanism for control of resource distribution among tissues and organs (Koch 1996). Hartig and Beck (2006) discussed crosstalk between auxin, cytokinins, and sugars in the plant cell cycle and point out the degree of tissue and cell specificity that exists for signal interactions, even in physiologically similar processes like cell divi sion. Gene responses to sugars in a sink organ can depend on the pathway used for sucr ose import into a given tissue (Koch 2004; Koch and Zeng 2002). In plants, diffe rent sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues (Rolland et al. 2006). Sucrose cleavage in cells can be catalyzed by two enzymes invertase (forming fructose + glucose) or the reversible en zyme, sucrose synthase (forming fructose + UDP-glucose). Thus, action of invertase generates twice as much substrate for hexosebased sensing (Koch 2004). ABA accumulation during water stress may often function to help maintain shoot as well as root grow th, rather than to inhibit growth as is 25

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commonly believed (Sharp and LeNoble 2002). The activity of acid vacuolar invertase was highly correlated with xylem fluid ABA concentration (Trouverie et al. 2003) and glucose directly affects ABA biosynthesis (Chaves and Oliveira 2004) by induction of transcription of genes for this hormone biosynthesis (Cheng et al. 2002). Intensity of remobilization and use of reserves in woody species may be different than that observed in herbaceous plants. Furthermore, labile C and N pools in perennial species are maintained not only by root abs orption and shoot produc tion, but by break down of reserves as well. Changes in plant carbohydrate status can lead to a wide array of responses at the whole-plant or gene-expression levels. Certain genes are downregulated, and others upr egulated by sugars affecting the balance between above and below ground processes, including st orage, utilization, photosynthesis, remobilization, export, relative source/sin k activity and growth (Koch 1996). Cell differentiation and the cell cycle can also be st rongly affected by sugar availability (Koch 1996). Nitric oxide acts as a signaling molecule, in particular by mediating the effects of hormones and other primary signaling molecule s in response to environmental stimuli (Chaves and Oliveira 2004). Lough and Luc as (2006) discussed phloem-mediated transport of macromolecules as component s of an integrated long-distance signaling network, with emphasis on proteins and RNA species. Crosstalk between signals for availabili ty of C and N was discussed by Koch (1997) who concluded that interactive effe cts on gene expression ar e possible, although the points of interface are unclear. The same author also stated that the form of both N and C can also be important in the response of photosynthetic genes to C to N balance, 26

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27 particularly since sugars and amino acids can have markedly different effects than unassimilated CO2, NO3, or even metabolically generated NH4 and NO3. Amino acid signaling research in plants is only in its ear ly stages. Glutamate applied externally to root tips caused inhi bition of growth (Sivaguru et al. 2003) and inhibited lateral root formation and out growth (Walch-Liu and Forde 2007). Nitrate counteracted Glu, thus stimul ate root branching and main root growth. External nitrate and Glu were proposed to interact to modulat e root growth (Forde and Walch-Liu 2009). However, internal Glu concentrations have not been cited to cause the same effects. Excessive external concentrati ons of one amino acid in relation to others can inhibit growth, with the only exception being Gln, wh ich has the ability to counteract growth inhibition caused by the excessive amino ac id (Singh and Shaner 1995). An example is Val-mediated inhibition to growth due to isoleucine (Iso) starvation (Bonner and Jensen 1997).

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CHAPTER 2 AN ELEVATED LARGE-VOLUME RHIZOTR ON FOR EVALUATING ROOT GROWTH UNDER NATURAL SOIL MOISTURE CONDITIONS. Introduction Although root growth is cent ral to overall plant perform ance, the study of natural root development has remained a challen ge due to the difficult y of observation. Attempts to observe roots ov er time date back to at least the early 1900s (McDougall 1916). However, most methods used to stud y root development are extremely time consuming and tedious (Calfee 2003). A rhizotron is a device for observing plant roots non-destructively over time (Garrigues et al. 2006). Root observation fac ilities described by Soileau et al. (1974), Karnok (1982), and Taylor and Bohm (1976) consisted of underground laboratories with transparent windows. Root observation windo ws installed in nativ e soil (Gallandt et al. 1990; McDougall 1916; Metcalfe et al. 2007) were less elaborate, but also below-soil level. A different approach was the use of transparent tubes in the greenhouse by Schoene and Yeager (2006; 2007), or more elaborated tubes developed by Bland (1990). The majority of research on root development has relied on narrow observation boxes usually made with plexiglass (Boukcim et al. 2001; Busch et al. 2006; Devienne-Barret et al. 2006; Garrigues et al. 2006; James et al. 1985; Misra 1999; Stepniewski et al. 1991; Ugoji and Laing 20 08; Wiese et al. 2005). Although these boxes and tubes enabled easier greenhouse study of root growth, the volume of soil exploited by roots was severely reduced, narro wing applicability to seedlings or cuttings. Moreover, normal root architecture was biased in these types of chambers because roots were forced to grow in narrow spac es, forming an artificial root arrangement. Wright and Wright (2004) developed a star -shaped rhizotron with eight glass panels, 28

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suitable for greenhouse or field use that over came many of the biases of the past. However, it did not fully mimic in-ground cond itions. A large rhizotron, designed around concepts of enhanced root observation and tracking strategies would enable researchers to study growth of whole mature root systems as they develop. Understanding the effects of env ironmental factors and cultural practices on root growth of mature plants would be of great benefit for applied and fundam ental goals ranging from ecology to agriculture, landscaping, and forestry. The objective of this study was to design a nd test several alternative strategies for above-ground rhizotrons that could enhance observation and recording of undisturbed, natural root growth of woody plants. A primary goal was to mimic in-ground conditions, including minimum physical restrictions and enhanced drainage (the latter being especially valuable for testing effects of soil moisture deficits on root growth). Material and Methods Rhizotron designs tested here proceeded in sequence, each modified based on results of the one immediately preceding it. Stage 1 Designs for containment and observation ports A prototype rhizotron was c onstructed to evaluate diffe rent materials and designs for root observation and sampling. The shape wa s that of a star with four-arms radiating from a central rectangular box (Figure 2-1). This star-shaped rhiz otron enclosed 0.18 m3 of soil, measured 2.1 m acro ss, and was 0.31 m deep at the ti p of the arms. The central box was 0.25 x 0.25 x 0.37 m tall. The bo ttom of each arm was sloped towards the center box to facilitate drainage and to r educe perched water tables common in flat bottom containers (Bilderback and Fonteno 1987; Spomer 1980). The bottom of the center box consisted of a plastic mesh (12.5 mm square) over which woven ground 29

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cover (Lumite Inc., Gainesville, GA) was pl aced to support the substrate. The frame of this prototype rhizotron was welded from angle-iron strips (L-shaped cross-sections [18 x18 mm] of 3mm iron), with narrower width ( 12.5 x 12.5 mm) at the top of the box to facilitate transplanting root balls (typically 11.4L from st andard nursery containers). To facilitate observation and drainag e, the rhizotron was supported by four legs (60 cm), at the tip of each arm. Each lateral (side of the arm of the rhizotron) was used to evaluate different systems for non-disruptive visual ization of roots while holding substrate in place. Four different door designs were evaluated: 1) a single door the length of a lateral held by two brass hinges (2-1A); 2) a single sliding pa nel the length of a lateral (Figure 2-1B); 3) two independent doors each held by two bra ss hinges (Figure 2-1C); and 4) a side panel with two smaller doors attached to panel (Figure 2-1D). For some configurations, 1.25 cm square wire mesh was placed in t he lateral to hold substrate in place. Two materials were tested for doors: 2.2 mm clear plexiglass and 8 mm Thinwall polycarbonate (Lexan, General Elet ric, Fairfield, CT). In ad dition, different methods to seal doors were tested for avoidance of to avoid moisture loss and capacity to remain secured. Rhizotron arms were covered with opaque woven ground cover (Lumite Inc.) to maintain darkness of the root environment. In May 2006, a Ligustrum japonicum Thunb. from an 11.4-L container was transplanted into the rhizotron and grown fo r five months. Irrigation was supplied daily with three bubbler emitters per arm (Model Shrubbler 360o; Antelco, Longwood, FL). Ground cover was removed from the arms only for root recording and monitoring. Roots were traced using permanent markers on the pl exiglass door models. For polycarbonate 30

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doors, transparency sheets (20 x 25 cm) were placed on the side of a soil profile for tracing roots. Stage 2 Designs for drainage syst ems allowing natural-soil profiles Two additional drainage systems were nex t compared to the original design for their capacity to reduce or eliminate ex cess irrigation. Results from Stage 1 (Figure 2-2A) indicated that excess water percolates downward until an impervious surface or perched water tabl e is encountered (as typical in containers and in natural soil profiles). In the elevated rhizotrons te sted here, the 0.6-m air gap between the plastic mesh and the ground below formed an im pervious surface, resulting in perched water tables within the rhizotrons. To simulate in-ground soil moisture characteristics, these perched water tables had to be eliminated. Two additional drainage systems were t hus developed, with the goal of better simulating in-ground moisture characteri stics. The hanging water column system (Figure 2-2B) was intended to remove excess wa ter from the substrate to a set tension level. For this, a 24-cm diameter polyeth ylene funnel was installed and sealed with silicone caulk underneath the center box, and fill ed with medium texture sand of a finer texture than the substrate above. A 60-cm length of Tygon R-3603 laboratory tubing (6.3 mm internal diameter) was attached to the bottom of the fu nnel. The lower end was elevated 7 cm to establish a hanging water column. The third was a wick system (Figure 2-2C), which consisted of a super absorbent felt, covered with perforated black polyethylene landscape fabric (WeedBlock, Easy Gardner Products, Inc., Waco, TX) installed under the substrate. The felt was the bottom la yer of the capillary system Aquamat (Soleno Textiles Inc., Laval, Quebec, Canada) and was hypothesized to wick excess water from the substrate. Resulting fluid was envisi oned to drip and evaporate 31

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from the mat hanging below the rhizotr on center. A WeedBlock layer was added to prevent roots from growi ng into the felt below. The three drainage systems were evaluated for 12 days (four irrigation cycles at three day intervals), with three replications (each a separate rhizotron). Rhizotrons were filled with a commercial substrate composed of Canadian sphagnum peat moss, processed pine bark, perlite, vermiculite, starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc., Agaw am, MA). Soil moisture sensors, EC-5 ECH2O probes (Decagon Devices Inc., Pullman, WA) were used to characterize soil moisture profiles. Sensors were calibrated fo r the substrate prior to use. Calibration was performed by placing sensors at a uniform depth in 0.03 m3 of substrate in a white vinyl cylinder (50-cm diameter). A time domain tr ansmissometry sensor (Digital TDT, model ACC-SEN-TDT, Acclima, Inc., Meridian, ID) was included for measurement of volumetric water content (VWC). The cylinder was filled with an excessive volume of water, and then allowed to dr ain and dry naturally while pos itioned on top of native, sandy soil. Millivolts were recorded for each senor concurrent with the volumetric water content measured by the TDT every 15 minutes for 6 days using a datalogger and multiplexer (CR10X and a 1632 multiplexier, respectively, Campbell Scientific, Inc., Logan, UT). Calibration equations were quadratic for each sensor when including moisture ranges above field capacity (Micros oft Office 2007, Micr osoft Corp. Redmond, WA). Each rhizotron re plication had three ECH2O probes installed to monitor substrate moisture, at the center of one arm, center of the center box, and bottom of the center box. Two thermocouples monitored air temperature. Four additional thermocouples were installed to monitor substrate temper ature 1 cm away from the door material. 32

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Volumetric water content was analyzed as a 3 x 3 factorial, wit h drainage system and probe position as treatments with three replications us ing SAS (version 9.1, SAS Institute, Cary, NC). Results and Discussion After five months of growth in the prototype rhizotron, the L. japonicum plant had expanded its roots only 75% of the length of a rhizotrons arm. It was concluded that the arms could be shortened to reduce cost and overall floor space while still achieving research goals. Final rhizotrons central box was 0.25 x 0.25 x 0.35 m tall, and laterals were decreased from 0.93 m to 0.78 m in lengt h. This reduced the overall width of a rhizotron from 2.1 m to 1.76 m. The slope of the bottom of each arm increased slightly (6.4% to 6.6%). Framework of the final rhizotron was constructed with galvanized sheet metal (Figure 2-3 5 x 3.8 x 5 cm U-shape, 5 x 5 cm V-shape, and 5 x 2 cm L-shaped). Only the top and bottom squares of the center box (25 x 25 cm) were made of 2-cm flat iron strips (Figure 2-3). The pieces we re welded and painted to avoid corrosion. Expanded white PVC board (6 mm, Kommerling Inc., Huntsville, AL) was used in the bottom of the arms, which were sealed on the corners with silicon chalk. Rhizotrons were raised 0.6 m above the ground by wooden legs. The final rhizotron design (Figure 2-4) enclosed 0.16 m3 of soil, measured 1.76 m across, 0.30 m deep in the tip of the arms and 0.35 m deep in the center. Of the door types evaluated, a single door (F igure 2-1A), attached with two hinges proved most effective. It allowed easiest access for root observation, recording, and measurement of roots. Once wet, the substrate was stabl e and remained in place when the door was open. The wire mesh on the si de of the arm was found to be unnecessary. Although offering good stability, sliding panels (Figure 2-1B) were not practical. When 33

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slid open, friction crumbled t he substrate into the tracks causing subsequent mechanical problems. The center post used to help seal double doors (Figure 2-1C) obscured roots and made recording root growth by digita l images troublesome. Moreover, opening and closing two doors for root observations wa s more time consuming. Likewise, side panels with two doors (Figure 2-1D) were more difficult to operate and close and offered no advantages. Investigation of materials for door cons truction indicated several advantages for white 6-mm expanded PVC board (Kommerli ng Inc.). This was opaque, waterproof, insulating, and sufficiently rigid to retain lateral shape of rhizotrons arms when filled with the substrate. These were attached at t heir basal edge with two 5-cm brass hinges. Doors were held in closed position by two 7-cm wooden buttons attached to the frame with machine bolts. Both other materials evaluated in the prototype doors were rigid enough for use, however, roots were observed with much more detail with open doors, thus negating the need for transparency. Even though plexiglass has been the material of choice for many researchers, moistu re accumulation inside the panels can make observation through them difficult. An additional problem associated with clear plexiglass was glare, which makes digital p hotographs of the roots unfeasible. Thinwall polycarbonate was light weight and readily accessible. However, it did not allow root observation through doors, and permitted light penetration to roots unless the system was covered. A better solution for the rhiz otron doors was the ex panded PVC board. It was inexpensive, light weight, sturdy, easy to cut and manipulate, and had the added advantage of being opaque. This eliminated the need for covers The white color of the board also reflected radiation, thus aiding heat management. The difference in 34

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temperature between substrat e immediately next to PVC door and air varied between -4.5o C and 4o C during the course of the day. Substrate temperature varied less than air temperature (Figure 2-5 cooler dur ing the day and warmer during the night). To limit evaporation from t he substrate surface, a per forated black polyethylene landscape fabric (WeedBlock) was cut to fit and placed on top of the substrate. Irrigation was provided by placing a spray stake (mo del green 22500-001120, Ne tafim Irrigation, Inc., Fresno, CA) at the tip of each a rm pointing inward. T he angle created by the irrigation spray approximated the internal angle on the arms. The four sprays of water slightly overlapped at the center providing a uniform coverage. Although a number of authors report the use of permanent markers for root tracing (Boukcim et al. 2001; Metcalfe et al 2007; Misra 1999; Schoene and Yeager 2006; 2007; Stepniewski et al. 1991), we found that for accurate recording of so many roots they were impractical. Each rhizotron o ffered eight surfaces where roots could be observed. Digital photography, followed by software analysis, provided a much more precise account of root system developmen t. Serial photography can also allow for further analyses of morphological characteristics, various r oot orders, and root geometry (Wiese et al. 2005). However, for accurate analysis, serial photographs must be taken at exactly the same location and distance each time. Additionally, t he camera that was used for all digital photography (DSC-W170, S ony Corp. of America, Montvale, NJ) would not cover the entire rhizotron surfac e at the widest lens setting. Moving the camera further away was not an option due to close spacing between rhizotrons, and loss of detail from increasing the lens-to-subject distance. To address this challenge, 35

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two overlapping images were obtained for eac h panel using a device that would assure consistent positioning. To achieve this, a camera positioning fram e (Figure 2-6) was developed to hold the camera at a constant distance and location for time-series digital recording of root growth. The positioning frame was construct ed using a 2.8 x 2.8 x 61 cm magnetic bar (Ningbo Ketian Magnet Co., Z hejiang Province, China) th at could be centered on the top edge of each lateral of an arm of the rhizotr on. From the center of this magnetic bar, an aluminum bar (2.5 x 0.5 cm) was bent into a C-shape with 90 turns at the distal ends so that it braced the frame against the open PVC door. T he distance of the arm to the vertical leg of the C was 0.63 m. A 12-mm square aluminum bar 16 cm long was attached perpendicularly to the vertical porti on of the C using a single screw within a 10-cm section of 1.8-cm aluminum c-channel so that it would pivo t from one side of the vertical leg to the other within the c-channel. The vertical position of the pivot arm was such that the optical center of the camera would point directly at the vertical face of the rhizotron. The camera was connected to this perpendicular bar using one screw enabling the camera to face the rhizotron from the left or right. Up to two pictures were taken of each profile, with each accounting fo r a little more than half, and overlapping at the center of the prof ile. This permitted digital fusion of the pictures. A mark was placed on the rhizotron frame at the center of each of the four sides of the profile and a red mark was placed exactly at the profile cent er to facilitate fusion of pictures when necessary. This flexibility of locations for the camera enabled a shor ter distance from the soil profile to the camera, and re sulted in high resolutions and close spacing between 36

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rhizotrons. Initially, only a single image wa s required to encompass the early root growth. The second picture per pr ofile (from the distal half of the profile) was necessary only on later stages, after roots had grown past the halfway mark. Rhizotron facilities usually devise met hods for drainage (Huck and Taylor 1982; Karnok and Kucharski 1982; Soileau et al. 1974), but persistence of perched water tables has not been appraised or addressed in previous studies of root chamber models (James et al. 1985; Ugoji and Laing 2008; Wright and Wr ight 2004). Nonetheless, unimpeded drainage, especially removal of perched water tables, was a critical objective for the rhizotrons described here. Soil moisture sensors were installed to evaluate the effectiveness of the evaluated methods for removing perched water. The simple mesh drainage system (Figur e 2-2A) was unable to remove excess water efficiently. In this system, the subs trate at the bottom of the rhizotron was consistently at a significantly higher mois ture level than the other two locations, indicating impeded drainage. For example, the bottom, arm and center positions averaged 52.7, 39.4 and 27.9 %VWC, respectively, two hours before irrigation (Figure 2-7A). Moreover, substrate located in the a rm remained at considerably higher moisture levels than that in the cent er of the rhizotron. The hangi ng water column system (Figure 2-2B) worked relatively well at draining wate r (Figure 2-7B). This system however was not able to remove as much water from arm and bottom positions as from substrate located at the center (bottom, arm and center had an average of 37.2, 36.7 and 26.8 %VWC 2 hours before irrigation, respectively). Substrate moisture also appears to have established equilibrium more quickly, and at somewhat lower VWC with the hanging 37

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water column (approximately 12 hours after i rrigation) compared to the mesh system at the same locations (arm and center did not reach equilibrium). Best results were obtained with the wick system (Figure 2-2C). This system came into equilibrium more slowly than the hangi ng water column system (18 and 12 hours, respectively), but maintained substrate mo isture levels more uniformly between all positions than did the other two systems (F igure 2-7C difference of %VWC between bottom and center of 27.8, 9.9 and 4.7, for simple mesh, hanging water column and wick systems respectively). Also, volumetric water content was significantly lower at equilibrium and less variable (30-35 %VWC) t han was that of the other two systems (26-37 %VWC for hanging water column and 28-53 %VWC for simple mesh), thus was considered more characteristic of landscape soils. The majority of root observation devices reported in the literature allow limited observation (one to two ports only), regardle ss of whether these rhizotrons force root growth through narrow spaces or are installed in natural settings. The rhizotron described here minimizes physical restriction to normal root growth, development and distribution, and offers eight viewing profiles, greatly enhanc ing the study of the root system as a whole. The st ructure is sturdy enough to support the large substrate volume, and can be increased in size to accommodate larger r oot balls or for experiments of longer duration. With the large rhizotron si ze, the root system developed by L. japonicum after 5 months of growth showed no ro ot restriction. Roots visible in the window had good spatial separati on, improving root analyses by digital photography or visual observation. Root sampling for biochemical assay or root recording was fast, efficient and minimally disturbing sinc e each door could be opened independently 38

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With removal of the perched water table, the system provides a good simulation of in-ground soil environment. Easy adaptations to this model can enable it to be used in split root trials or as a lysimeter. The rh izotron can be divided in the center box by the same expanded PVC board used on the door s and provide up to four different quadrants for split root studies. These rhizot rons can thus be used to investigate the effect of diverse variables on root growth. Examples could include partial root drying, soil texture (Bengough et al. 2006), fertilizer s (Boukcim et al. 2006; Drew 1975) or agricultural chemicals (Tsakaldimi and Ganatsas 2006). 39

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Figure 2-1. Protot ype rhizotron with Ligustrum japonicum 2 months after transplanting into the rhizotron. Doors of rhizotron open only for demonstration. A) Single door. B) Sliding panel. C) Double doors. D) Side panel with two doors. 40

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Figure 2-2. Drainage system s tested on rhizotrons, i: top view and ii: bottom view. A) Simple mesh. B) Hanging wa ter column. C) Wick system. 41

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A B Figure 2-3. Schematic diagr am of a rhizotron. A) Si de view of open and closed doors (left and right, respectively). B) Top view with details of materials used on construction (left of rhizotron: galvani zed sheet metal and bottom: flat iron). 42

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Figure 2-4. One-year-old Ligustrum japonicum transplanted in a rhizotron, window open just for demonstration. Windows enable r oot visualization and easy sampling. Figure 2-5. Temperature of air and substr ate immediately next to single PVC door (substrate: average of four thermo couples, and air: average of two thermocouples). 43

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Figure 2-6. Camera positioning frame used to take pictures mounted on rhizotron. 44

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45 Figure 2-7. Percent volumetric water cont ent (% VWC) observed during drainage trial (average of actual values for 3 repetitions). A) Simple mesh. B) Hanging water column. C) Wick system.

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CHAPTER 3 DEVELOPMENT AND EVALUATION OF A ROTARY ROOT SEPARATOR Introduction Quantitative studies of plant roots have remained a consistent challenge. Extraction of roots from so il and debris of large samples for biomass quantification is time consuming and tedious (Calfee 2003). Th is tends to limit research to small experiments and small sample sizes, especia lly for woody plants with extensive root systems. Analysis of entire r oot systems from the plants is particularly important, because woody plants allocate a large portion of their resources to root development (up to 69%,Vogt et al. 1995). Still, most k nowledge of woody plants roots is based on partial root extraction from soil cores, and subsequent estimation of the entire root system. Roots from a single plant can be dist ributed differently in the surrounding soil, growing more in wet pockets of soil or with higher levels of fertility (Michelakis et al. 1993; Stevens and Douglas 1994; Wilkin et al. 2006). Larger sample sizes or samples of entire root systems would represent a more accurate account of a plants root system. The most common method used for root isolation has been hand picking and washing with jets of water over a sieve (Prathapar et al 1989). Machines for isolating roots with an elutriation system have also been reported (Benjamin and Nielsen 2004; Brown and Thilenius 1976; Ca rlson and Donald 1986; Fribourg 1953; Smucker et al. 1982). Unfortunately, these approaches were designed for small sample sizes, especially core soil samples. They also requi red large volumes of water. A drawback of elutriation systems is that fi nal separation of roots from debris is made more difficult by adhesion of particles to themselves and roots. Additionally, changes in color make root 46

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identification and separation more difficult during final hand separ ation. Benjamin and Nielsen reported that up to 20 hours was necessary to clean roots from samples of 993 cm3 soil after machine washing. The objective of the research report ed here was to develop a fast, mechanical system to separate roots from large sample volumes, with maximal root recovery, minimal root damage, and little to no water use. Material and Methods A root separator was developed to speed r oot isolation for dry mass determination (Figure 3-1). Mechanical separ ation is provided by a rota ting cylinder that removes small particles. The root separator consists of an external cylinder with four 38 x 20 cm openings at the lower end. The cylinder was m ade of aluminum that was closed on one end (79 cm tall and 46 cm in diameter) wher e it was attached to a right angle gear reducer (1:20, model 13-175-20-R, Worldwi de Electric, Rochester, NY). This was belt-driven by a small electric motor (m odel SKH 47KR383 GS, 110 VAC, 0.75 hp, 1720 rpm, General Electric, Milwaukee, WI, Figur e 3-C). The motor was shielded by a sheet metal box (46 x 30 x 20 cm). A 1.5 x 1.5 cm wire mesh was placed on the outside of the cylinder to serve as a secondary sieve. T he upper end of the cylinder was supported by two rubber tires (A tube type, 25.4 cm tall x 7.6 cm wide, World Caster & Equipment Manufacturing, Inc, Lilburn, GA) and hub assemblies attached to the heavy metallic base for stability. The cylinder was set at a 20o angle from the base. Samples, up to 40 L, were placed into a removable, internal basket (50 cm tall and 45 cm in diameter) of 6 x 6 mm wire mesh. Cylinder rotation speed was adjustable. After preliminary trials, the rotational speed was established at 20 rpm to minimize root damage. During machine use, particles smaller than wire mesh openings dropped through both the basket and 47

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the openings at the bottom of the cylinder, then were channeled aside by a plexiglass ramp (100 x 40 cm). After rotation, the basket was removed from the cylinder, and the remaining material was transferred to a sieve (3 x 3 mm) where roots were hand-separated from remaining la rge pieces of substrate and debris. Roots were rinsed only after isolation, thus significa ntly reducing the use of water. This root separator was used to isolate roots of individual Ligustrum japonicum Thum. grown in rhizotrons (Chapter 2) holding 0.16 m3 of substrate (Mix #4, Conrad Fafard Inc., Agawam, MA). Twenty four pl ants were grown between May and October of 2007, and 24 more between March and June of 2008. Entire root systems were cleaned utilizing the washing and hand-picking me thod in the 2007. In 2008, mechanical isolation using the rotary separator pr eceded hand-picking. For the washing and handpicking method used in 2007, rhizotron contents after shoot removal were placed on top of 3 x 3 mm sieves and roots were wash ed with 0.35 MPa water and hand-picked. For mechanical separation, rhizotron contents were separated in to four parts (ca. 40 L each), and then rotated for 5 to 10 min eac h, depending on substrate moisture level estimated visually (drier substrate needed le ss time). After ro tation, roots were removed and placed on the sieves used for handpicking. They were rinsed with water, and then oven dried at 65o C until constant dry mass was obtained. To calculate the percentage of recovery (yield) from the r oot separator, root samples from five woody plant species were isolated using the machine. Entire root balls of container grown plants of Viburnum odoratissimum Ker Gawl., L. japonicum Illicium parviflorum Michx., and Magnolia grandiflora L. Little Gem (Table 3-1) were sampled for root isolation. Additionally, plants of Ilex cornuta Lindl. & Paxt. transplanted 48

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into sandy soil, were also sampled. Of these plants, one fourth of the roots that extended beyond the original root ball were used for root isolation. Rotation time was varied based on root ball size and substrate moisture level. Rotation was stopped when roots where visibly free of most small par ticles and remaining roots were cleaned as described above for L. japonicum grown in rhizotrons. To determine total root mass, substrate that fell from the cylinder was placed on the sieve and any additional root pieces were collected by hand. Yield was ca lculated as percentage of root mass dry recovered from the basket compar ed to the total dry root mass. Root yield from the five plant species were compared using a completely randomized design, with 3 replications. Percent yield data were arcsi n-square-root-transforme d for data analysis. Data analysis was accomplished using SAS (version 9.1, SAS Institute, Cary, NC). Results and Discussion Manual root washing of a 0.16 m3 sample required up to 16 man-hours (data not shown). In contrast, mechanical isolation followed by hand-picking was completed in approximately 4 man-hours. The time requir ed for each sample to be processed with the root separator was adj usted depending on substrate moisture content. Root systems of L. japonicum from 24 rhizotrons were proce ssed using mechanical isolation, followed by hand-picking. This saved an estimated 288 manhours (96 man-hours versus 384 man-hours for hand washing in 2008 and 2007 experiments, respectively, estimation based on 16 man-hours for hand pi cking and 4 man-hours for mechanical isolation). The substrate medium selected for the rhizotr on experiment was chosen to minimize root cleaning time. However, test s of the container root balls included substrates principally compos ed of composted pine bark (< 25 mm). Results from these and the sandy soil suggested that total cleani ng time can be diminished independently 49

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of the root matrix compos ition. Samples processed fo r yield determination included plants produced in substrates with differ ent particle sizes and components. The substrate for magnolia, anise, and viburnum r oot balls was composed mainly of large pine bark pieces (up to 2.5 cm), which make s root separation much more difficult. The sandy soil had a much finer texture than the rh izotron substrate. Plants selected had different root morphology (fine versus coar se roots). Viburnum roots were coarse and most resistant to breakage. L. japonicum roots were smaller in diameter than viburnum roots, but larger than anise, magnolia or holly roots. Holly roots had the finest texture. Roots of anise were the most tender of t he species tested, and easily broken, yet yield of anise roots was very high (Table 3-2). Yiel d of all species were high (Table 3-2), with viburnum representing the lowest yield at 98.4% of tota l root dry mass. The root isolation process worked best w hen the substrate or soil had low water content. Time required for isolation depended on so il particle size, moisture level, root density and morphology. To avoi d root breakage, especially fi ne lateral roots, attention was directed to minimize dur ation of rotation to no longer than necessary. Fine root recovery was good for all species tested, and observationally better than root washing by hand. High pressure water can break fine r oots which are lost through the sieve. 50

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Table 3-1. Root ball volume and s ubstrate/soil of root samples. Common name Scientific name Plant age (year) Root ball volume (L) Substrate or soil type Viburnum Viburnum odoratissimum 2.0 11.4 commercial substrate z Ligustrum Ligustrum japonicum 3.0 11.4 commercial substrate y Holly Ilex cornuta Burfordii 1.5 40.0 soil x Anise Illicium parviflorum 1.0 1.5 commercial substrate z Magnolia Magnolia grandiflora Little Gem 1.0 1.5 commercial substrate z z Pine bark fine, Florida sedge peat, sand (Florida Potting Soil, Inc., Apopka, FL). y Peat moss, pine bark, perlite, and vermiculit e (Mix 4, Conrad Fafard Inc., Agawam, MA) x Tavares-Millhopper fine sand Table 3-2. Dry mass of root samples and yiel d achieved using the rotary root separator. Common Root ball Rotation Time hand Dry mass Yield name volume (L) time (min) Picking (min) roots (g) (%) Viburnum 11.4 2 45 281.0 az 98.46 cz Ligustrum 11.4 2 60 194.0 b 99.58 b Holly 40 1 15 23.9 c 99.86 ab Anise 1.5 0.25 5 5.8 c 99.85 ab Magnolia 1.5 0.25 5 6.5 c 99.90 a z Means of 3 repetitions. Means within columns not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). 51

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52 Figure 3-1. A) Root separat or. B) Detail showing internal basket. C) Detail showing reducing transmission and motor.

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CHAPTER 4 EFFECTS OF WATER STRESS ON PLANT GROWTH OF Ligustrum japonicum THUNB. Introduction Plant responses to water shortage vary with severity as well as with duration of the stress imposed (Farooq et al. 2009) The balance between dehydration and photosynthetic activity is enabled by adaptation, acclimation and short-term physiological regulation (Beikircher and Ma yr 2009). Water stress can cause changes from a subcellular to a whole plant level (Maseda and Fernandez 2006); however, the major contributor to dehydration avoidance for a given species will vary depending on the stress magnitude and dur ation. Plant adjustments to wa ter availability may include changes in aquaporin activity (Tyerman et al. 1999); abscisic acid (ABA) mediated stomatal closure (Schraut et al. 2004) ; changes in morphological and anatomical features such as change in leaf angle, depo sition of cuticle, shedding of leaves, and shifts in allocation of resources between roots and shoots (Abrams 1990; Kozlowski and Pallardy 2002; Maseda and Fernandez 2006; Mishio 1992); deposition of dehydrins (Pelah et al. 1997; Wisniewski et al. 2006) and osmotic adjustment (Kozlowski and Pallardy 2002). Constant or slowly imposed water st ress can inhibit photosynthesis due to stomatal closure (Saccardy et al. 1996), and reduced plant size (height, biomass, leaf area, Maseda and Fernandez 2006) Water equilibration by whole organs can be on the order of hours or even days. However, under conditions which result in very large and rapid changes in extracellular water potentia l, such as moistening of soil following a prolonged drought, aquaporins in root cells may play an important role in rapid water absorption (Tyerman et al. 1999). 53

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Cell enlargement is the cellular process most sensitive to drought stress. Plants subjected to long-term water stress tend to slow shoot growth more than root growth. Increased drought tolerance occurs with the onset of osmotic adjustments, following wetting and drying cycles (Kozlowski and Pallardy 2002). Abiscisic acid is a prominent contributor to maintenance of capacity for expansion of cells behind root apices under severe water stress (Sharp et al. 2004). This enables root growth towards unexploited soil. Over time, a larger root system can expl oit a greater volume of soil, thus increasing water absorption potential. Moreover, this switch in root-to-shoot ratio helps avoid water stress because, proportionally, the volume of shoots that transpire will be smaller than the volume of roots for water absorption. McMillin and Wagner (1995) observed that the influence of water stress on root-toshoot ratio is dependent on stage of developmen t. In addition, root-to-shoot ratio is modified by natural growth patterns. Many woody species, such as L. japonicum show an episodic growth habit by fluctuating betwe en periods of rapid shoot growth and slow root growth, versus periods with the inverse pattern. Relatively li ttle is known about the changing carbon allocation patterns in species with episodic growth cycles (Dickson et al. 2000a). The objective of this research was to characterize and compare the effects of water stress on dry matter partitioning and growth of L. japonicum exposed to: 1) constant non-moisture-limiting conditions; 2) se vere water stress; 3) constant low levels of plant available water; 4) constant high le vels of plant available water; 5) simulated natural wetting and drying cycles of two da ys; and 6) simulated natural wetting and drying cycles of seven days. Comparisons of plant growth among the experiments 54

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described here provide an opportunity to view plant growth responses to water stress in similar settings, therefore avoiding mi sleading comparisons between species, developmental stages and growth conditions. Material and Methods Twenty-four star-shaped rhizotrons were c onstructed as described in Chapter 2 (Figure 2-3). Briefly, each rhizotron had four arms and held 0.16 m3 of substrate. They were 1.76 m across, 0.30 m deep at the end of each arm and 0.35 m deep in the center. Rhizotrons resided in an open-side greenhouse with a double polyethylene roof, under natural light, located in Apopka, FL. Clonal Ligustrum japonicum plants were selected from a local nursery (Jons Nursery, Eu stis, FL) to ensure homogeneous size and health. One plant was transplanted into each rhizotron using a commercial substrate composed of Canadian sphagnum peat moss, proc essed pine bark, perlite, vermiculite, starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc., Agawan, MA). Irrigation was supplied using a spray stake (model green 22500-001120, Netafim Irrigation, Inc., Fresno, CA) at the tip of each arm pointing inward. Three experiments, conducted consecutively over three years, exam ined shoot and root growth under well-irrigated conditions and seve ral different scenarios of water stress. Experiment 1 Rhizotrons were used without the wick dr ainage system described in Chapter 2, and thus had poor drainage. Plan ts from 11.4-L containers were transplanted into rhizotrons in May 2007 and grown for 186 or 217 days. Substrate moisture was managed based on measurements made with a soil moisture sensor (EC-5 ECH2O probe, Decagon Devices Inc., Pullman, WA). Five ECH2O probes were used to read a series of 10 moisture levels determined gr avimetrically based on a mass of substrate 55

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oven dried at 70 C and a single calibrati on curve was developed based on average readings (Nemali et al. 2007). One probe per rhizotron was installed at time of transplant, adjacent to the original root system and half way down in the soil column, and remained in place throughout the ex periment. Irrigation was managed using a datalogger (CR10X, Campbell Scientific Inc., Logan, UT) connected to a AM16-32 multiplexer and two SDM-CD16AC remote relay controls (Campbell Scientific, Inc.). The datalogger was programmed to query each probe every 60 minutes. If the volumetric water content (VWC) at the probe was below the set point established for each rhizotron, irrigation would be activated fo r 30 seconds. Times were established based on trial and error to allow for substrate re-hydration around a probe. Plant available water (PAW) was calculated for each indivi dual rhizotron as the drained saturation volume measured by the ECH2O probes minus available wate r of air dried substrate. Drained saturation was determined by pouring 1 L of water around the newly inserted probe, and then averaging VWC m easured beginning from five until seven hours after drenching. Treatments cons isted of applying irrigati on when PAW dropped below 30% (moderately-stressed plants) and 70% (well-irrigated plants), with two harvest times as the blocks, with 4 repetitions per block. Stomatal conductance (gs) of three sun-ex posed, fully expanded leaves from the first growth flush of the cu rrent growing season of each plant was measured at 169 days after transplant (DAT), bet ween 1000 and 1400 hours with a steady-state porometer (LI-1600; LI-COR, Lincoln, NE). Leaf area, dry mass of transplanted r oot ball, new roots, leaves and stems were measured at final harvest. Entire root systems were cleaned 56

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utilizing the washing and hand-picking method (P rathapar et al. 1989). Plant material was oven dried at 65o C until constant dr y mass was obtained. Experiment 2 The wick drainage system was installed on rh izotrons as described in Chapter 2 prior to initiating this experiment, t hus rhizotrons had good drainage. Plants from 11.4-L containers were transplanted into rhizotr ons in March 2008 and grown for 116 days. After a 23-day acclimatization phase, plants were subjected to two treatments of constant moisture levels (30% and 70 % PAW), with 12 replications each. Substrate moisture level was managed based on substrat e measurements as de scribed for Exp. 1. Prior to initiation, sensors were re-c alibrated against a time domain transmissometry sensor (Digital TDT, model ACC-SEN-TDT, A cclima, Inc., Meridian, ID). The Digital TDT was placed horizontally in the center of a 0.6-m polyvinyl ri ng made from landfill liner material which was placed directly on porous ground cloth covering a native sand soil base. The ring was 30-cm tall with the white side facing outward. Prior to placement of the TDT, about 8 cm of the rhizotron substrate was placed in the bottom of the ring and lightly packed. ECH2O probes were placed horizontally around the TDT, separated from the TDT and other probes by at least 5 cm. Additional substrate was placed on top of the sensors and lightly compacted to appr oximately 5-cm depth. Water from an onsite well (electrical conductivity <0.2 ds/cm) wa s gently added to the s ubstrate to a depth of about 10 cm. Data was then collected from probes every 30 minutes for 13 days until VWC had declined to approximately 20%. Data from each probe was analyzed by regression (Microsoft Office Excel 2007, Mi crosoft Corp. Redmond, WA) against the VWC recorded by the TDT to fit the best line (regression line for most probes was quadratic). 57

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Initially, ECH2O probes were placed close to the edge of a root ball and half way down the soil column as in Exp. 1. S ubsequently, probes were moved at 35-day intervals to the edge of newly extended root tips along one arm of a rhizotron for all rhizotrons. Plant available water was calc ulated as in Exp. 1 each time a probe was relocated. Irrigation was managed as described for Exp. 1, but ran for 1.5 minutes each event. Leaf area and dry mass of roots, leaves and stems were measured at final harvest. The mechanical root separator described in Chapter 3 was used to isolate roots for the entire root ball. Pl ant material was oven dried at 65o C until constant dry mass was obtained. Experiment 3 Plants grown in 3.8-L containers were transplanted into each rhizotron in February 2009 and grown for 138 or 159 days. Entire rhizotrons were irrigated daily until saturation for 41 days for plant acclimatization. Thereafter, irrigation was withheld for six days before the start of irri gation treatments. Treatments consisted of two irrigation frequencies, once a week for 10 minutes (12 L of water, moderat ely-stressed plants) and every other day for 5 minutes (6 L of water, well-irrigated plants), with 12 replications each. Irrigation was initiated at 1930 hours each time. To characterize variations of substrate moisture imposed by treatments, %VWC was measured every 5 min by an ECH2O probe and averages were recorded every 30 minutes. Probes were re-calibrated against the Digital TDT before placement as described in Exp. 2. ECH2O probes were placed close to the root system and half way down in the soil column, 18 DAT, and then relocated at 41 and 84 DAT to the edge of newly-extended root tips. At 84 DAT, a second sensor, a Digital TDT, was in stalled in each rhizotron at the middle of 58

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the arm, 20 cm from the ECH2O probe, to also monitor substrate moisture content. ECH2O probe measurements from the entire experiment were corrected using equations developed from regressi ons made of TDT versus ECH2O probe measurements for the time bot h sensors were installed simultaneously. Saturation was calculated as the average of measurement s recorded from 5 to 7 hours after an irrigation event (2 hours, 4 readings). The lowest achieved %VWC was the average of the 2 hours before an irrigation event. Stomatal conductance was measured with a steady-state porometer (LI-1600) at 99, 101, and 105 DAT. Measurements were taken between sunrise and sunset, in intervals of 2.25 hours, on three sun-expo sed, fully-expanded leaves from the first growth flush of the current growing season chosen at random on each plant replication (5 leaves per treatment). Stomatal conduc tance was measured for well-irrigated plants at 1 and 2 days after irrigation (DAI), and fo r moderately-stressed pl ants at 1, 3 and 7 DAI. Daily stomatal conductance (Dgs) was developed as previously described for daily accumulative water stress (Beeson 1992a). Dail y stomatal conductance for each plant was integrated each day by calculating the area under a gs curve of each repetition. This parameter permitted simple comparison between the treatments, which represented the potential quantit y of water transpired during the day on a leaf area basis. Shoot water potentials were determined in six plants of each treatment 143 and 144 DAT at predawn and midday with a pressu re chamber (Model 3000; Soil Moisture Equipment Corp., Santa Bar bara, CA) using compressed nitrogen. Measurement days corresponded to one day before and one day afte r irrigation. Measurements were made 59

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on individual twigs (ca. 10 cm long). Leaf area, number of leaves, and dry mass of roots, leaves and stems of old and new growth were measured at final harvest. Plants were harvested twice (138 and 159 DAT). Six plants from each treatment were harvested at root flush (increas ing root growing points, Chapter 6) and six at shoot flush (increasing shoot growing points) at the te rmination of the exper iment. The mechanical root separator described in Chapter 3 was used to isolate roots for the entire root ball. Plant material was oven dried at 65o C, until constant dr y mass was obtained. Results and Discussion Soil moisture management Over the course of the three experiment s described here, ECH2O probe measurements were found to vary up to f our-fold among probes. For this reason, a Digital TDT was used in conjunction with each of the ECH2O probe on Exp. 3 to develop individual calibration equations for each ECH2O probe. TDT measurements of VWC are similar to TDR (Blonquist et al. 2005; Burke et al. 2005; Harlow et al. 2003). TDR sensors are considered the most accurate measurement of so il VWC, and the standard by which other soil moisture sensors are evaluated (Blonquist et al. 2005). The calibration equations represented a 30 day period comprising several irrigation cycles. Calibration equations (r2 = 0.72 to 0.96) were used to re-calibrate ECH2O probe measurements for all three experiments. Each experiment was intended to provide a moisture deficient condition in one treatment (30% PAW) and a well-irrigated c ondition in the other (70% PAW). The irrigation control system used for Experim ents 1 and 2 was developed to impose a set VWC, and maintain substrate moisture leve ls close to the set-point throughout the duration of the experiment. The system compensated for plant size and microclimate 60

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effects on water content of t he substrate. Nonetheless, water availability experienced by the plants should be viewed in the light that probes used to control irrigation produced highly variable values among rhizotrons. Thus, substrate moisture readings of each experiment were reevaluated to provide more precise definition of the treatments for comparisons and correct data interpretation. Percent VWC in Experiments 1 and 2, and drained saturation and lowest achieved %VWC in Exp. 3 were analyzed as a split-plo t design (version 9.1, SAS Institute, Cary, NC), with repeated measurements over time. Mean recalcul ated %VWC which occurred during the three experiments were still different between treatments (Table 4-1). Although measurements from Exp. 1 resulted in signifi cant differences, 21.2 and 31.3 %VWC for moderately-stressed and well-irrigated plants respectively, poor drainage from rhizotrons resulted in abundant water availability for plant growth for both treatments. Insufficient remova l of excess irrigation from t he rhizotron resulted in the occurrence of a perched water tabl e at the bottom, which the ECH2O probe, installed at one arm, was not able to detect (Chapter 2). The ECH2O probe-measured saturation VWC wa s above the average and high in some Exp. 2 rhizotrons. When this occurred in rhizotrons designated for 70% PAW, the effect was negligible for most parameters measured; only leaf area was significantly different between rhizotrons with dissimilar le vels of saturation VWC. However, when this occurred in rhizotrons designated fo r an irrigation trigger VWC of 30% PAW (moderately-stressed plants), differentials between saturation (mean 46.1% VWC) and the triggering VWC (mean 23.0% VWC) were 1.8 times higher for some moderately-stressed plant replic ations than others. This re sulted in irrigation occurring 61

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only once or not at all in some moderately-st ressed treatment rhizotrons after the initial acclimation period. Additionally, each time a sensor was reloca ted; it was watered-in to ensure close contact with the substrate to prevent erroneous measurements. This created a localized increase in substrate moisture around the probe, thus further delaying irrigation. The resu lt of the differentiated i rrigation was an unintended, but unique subgroup of the moder ately-stressed treatment wh ich died near the end of the experiment. These plants were designated as severely-stressed plants and analyzed as an independent th ird treatment. Experiment 1 Although averaged half-hour %VWC recorded by ECH2O probes during the experiment was higher ( P <0.05) for the well-i rrigated plants than for moderately-stressed plants (31. 30 and 21.17%, respectively, T able 4-1), total plant dry mass was not significantly different ( P <0.05) between the tw o treatments at each harvest. Also, as discussed above, all plants were exposed to excessive moisture due to the perched water table discovered in rh izotrons (Chapter 2). Data of the two treatments is presented polled (Table 4-2 and 3). All plant s directed over 70% of mass allocation to shoots (Figure 4-1, Table 4-3). Root growth was limited due to ample water availability for root absorption even in the moderately-stressed treatment. Biomass accumulation of all plant components wa s similar between treatments within each harvest (data not shown) although %VWC was di fferent at the sensor site (Table 4-1). Allometric relationships were not affected in the treatment moderatestress. Mean gs at midday was similar ( P >0.05) 138.5 and 132.8 mmol m-2 sec-1 for well-irrigated and moderately-stressed treat ments, respectively. 62

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Stationary single capacitance probe measurements did not represent PAW throughout the entire growing period in a large soil volume, especially for the low VWC treatment. As roots elongated into unexplored substrate, they could exploit areas of high VWC, since the entire substrate was moistened at each irrigation event. Furthermore, water depletion in the soil region around the probe increased with time due to accumulation of roots around it, which incr eased irrigation frequency (data not shown). Additionally, later tests rev ealed the presence of a perched water table as discussed in Chapter 2. These results sugges ted that irrigation control us ing single probes in large soil volumes should be more accurate if the probe was relocated as new roots expanded into unexplored substrate. Variat ion on water distribution within the soil exploited by the root system should also be considered. Hedley and Yule (2009) concluded that the accuracy of predicting VW C in a large native so il area was limited by spatial variability of the soil moisture pattern within the area. Experiment 2 Moderate constant stress (moderately-stressed plants) resulted in significantly smaller biomass production in all plant components (Table 4-4). After 100 days under severe stress conditions, all plants were dead. Dry mass of severely-stressed plants was lower than for moderately-stressed and we ll-irrigated plants (Table 4-4). Allocation to roots and root-to-shoot ra tio (Table 4-5) was higher in severely-stressed plants than in plants of the other two tr eatments. The high root-to-shoot ratio obtained in Exp. 2 noticeably contrasts with results from Exp. 1 (Figure 4-1). The average ratio was 0.36 between treatments and harvests of Exp. 1, where water availability was at excess levels, versus 0.87, 0.75, and 0.72 for se verely-stressed, moderately-stressed and well-irrigated plants respectively in Exp. 2. Stabler and Martin (2006) studying 63

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Caesalpiinia pulcherrima found that frequent irrigation (every 2 days) decreased rootto-shoot ratio by ca. 40% compared with m oderate irrigation (every 5 days), and by approximately 48% compared with in frequent irrigation (every 10 days) Here root-toshoot ratio was 21% less for well-irrigated plan ts compared to severely-stressed plants. Root-to-shoot ratio appears to be very speciesand perhaps situation-dependent. Irrigation cycles of 2 days resulted in root-to-shoot ratio of 0.25 for Pittosporum tobira and 0.49 for Viburnum odorissimum (Scheiber et al. 2007); 0.80 and 1.0 for Citrus sinensis grown in sand and peat, respectively (Sanchez and Syvertsen 2009); 0.53 for Caesalpinia pulcherrima, and 0.43 for Cercidium floridum (Stabler and Martin 2006). Yeager et al. (1990) testing growth responses of L. japonicum to vesicular-arbuscular mycorrhizae inoculation obser ved root-to-shoot ratio of 0.95 and 1.25 in containergrown plants inoculated and noninoculated respectively. Differences between the first two expe riments are due to: 1) the success in creating a more natural percolation of wate r through the entire substrate column; and 2) relocation of moisture probes to compens ate for root elongation into very moist substrate. Average %VWC was significantly different for all three treatments in Exp. 2 (Table 4-1). Dissimilarities between results observed in Experiments 1 and 2 demonstrate the preferential allocation to r oot growth by plants under chronic water stress, and preferential allocation toward s shoots under luxurious water conditions. Plants in the severely-stressed treat ment overcame the imbalance between transpiration and water absorption through changes in allometric relationships (Maseda and Fernandez 2006; Reddy et al. 1998). Specific leaf area was diminished by 31.5% in severely-stressed plants compared with moderately-stressed plants (Table 4-5), 64

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indicating these plants produced smaller and thi cker leaves to reduce transpiration by the canopy, as reported for other species (Abrams 1990; Fernandez et al. 2002; Montague et al. 2000; Zwieniecki et al. 2002). The ratio of leaf area to root dry mass (leaf area-to-root dry mass) a comparison between the transpiring surface and the absorption mass, was also decreased by 45. 8% compared with well-irrigated plants (Table 4-5). Water stress reduced whole plant biomass, but affected root growth to a lesser extent than leaf growth (Table 4-4). Severe stress caused a reducti on of 33.7% in root biomass production compared to well-irrigat ed plants, while chronic water stress provoked by moderately-stressed treatment resulted in a 26.4% reduction. In contrast total leaf area was severely decreased by severe water stress, 64.2% and 26.4% reductions for severelyand moderately-str essed plants (respectively) compared to well-irrigated plants. Death of severely-stressed plants was t he result of insurmountable high water stress. Plant water loss cannot exceed the maximum supply through roots indefinitely (Jackson et al. 2000). Root growth occurred during the early perio d of the experiment through the first relocation of the moisture probe (as evidenced by the count of root growing points, Chapter 5). Root growth was limited after relocation of the probes. With increased water stress, turgor pressures required for root growth apparently were insufficient to expand root tips of these plant s through dry substrate to reach into moist substrate only centimeters away. High wa ter stress was associ ated with slow root growth in Quercus virginiana (Beeson 1992b). Moisture dist ribution in the substrate profile was observed to be uneven at 100 DAT. Substrate located close to root systems 65

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was visually dry, while substrate located to wards the tips of each arm of a rhizotron were wet. As observed in other species, plants that were able to explore further into the substrate were able to survive, even under low VWC, such as observed in the moderately-stressed treatment (Table 4-1). Volaire et al (1998) concluded that an important trait associated with survival of perennial grasses under prolonged drought was the ability to develop deep root systems. Deep root systems enabled Quercus spp. to maintain relatively high predawn wate r potentials during drought (Abrams 1990). Hibiscus glaber avoided severe stress by developi ng a dense deep root system (Mishio 1992). Experiment 3 When L. japonicum plants were subjected to wetting and drying cycles, the moderately-stressed treatment predominately affected abov eground components of a plant, particularly by diminished leaf growth Compared to the well-irrigated treatment, the moderately-stressed treatm ent resulted in reductions of 28.9% and 32.4% in dry mass of new leaves harvested at root flush and at shoot flush respectively (Table 4-6); 30.7% in the number of new leaves when in shoot flush (Table 4-7); and 31.8% and 30.3% in leaf area of plants in root flush and shoot flush, respectively (Table 4-7). Reduced leaf growth was the main contribut or to reductions of 27.3% and 29.4% in the dry mass of new shoots produced by plants on root and shoot flush, respectively (Table 4-6). Biomass reductions caused by water stress are well documented. For example, grasses responded to drought with a 60% biom ass reduction (Fernandez et al. 2002), Caesalpinia pulcherrima responded with up to 36% shoot biomass reduction (Stabler and Martin 2006), and Pittosporum tobira responded with up to 34% shoot biomass reductions (Scheiber et al. 2007). In exper iments described here, the biggest decrease 66

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in shoot biomass was observed in severely -stressed plants of Exp. 2, which exposed plants to low VWC for a prolonged period ( 40.4% compared with well-irrigated plants, Table 4-4). Comparison of biomass shri nkage suffered by the moderately-stressed treatment in Experiments 2 and 3 (constant exposure to 16.5% VWC versus intermittent exposure to 19.8% VWC) exhibits similar levels (ca. 20% compared with respective well-irrigated plants). Partitioning of resources in the stressed and well-irrigated treatments were similar (averaged allocation to shoots of 59.1% on r oot flush and 56.1% on shoot flush, Table 4-7), and occurred in similar levels of those observed in Exp. 2 (averaged 57.7% allocation to shoots, Table 4-5), but shoot allocation was much lower than observed in Exp. 1 (averaged 73.7%, Figure 4-1). In Exp. 1, dry mass distribution between aboveand below-ground portions of the plant is clea rly toward promoting shoot growth, while in Exp. 2 and 3 it is more balanced (Fi gure 4-1). Kozlowski and Pallardy (2002) reviewed evidence that drought promotes relati ve allocation of photosynthates to root growth. In our experiments, how ever, only severe stress resulted in preferred allocation to roots. Specific leaf area of plants of Exp. 3 was consistent between treatments (Table 47), and was similar to the levels observ ed in moderately-stressed and well-irrigated treatments of Exp. 2 (Table 4-5). Stress treatment not only diminished dry mass, but also leaf size (Table 4-6). Old leaves of plants in this experiment had an average leaf area of 8.53 cm2 (leaf area /number of leaves, T able 4-7). The average size for new leaves at root flush was 11.73 cm2 for moderately-stressed plants and 13.34 cm2 for well-irrigated plants; however, by t he second harvest, new leaves of 67

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moderately-stressed plants had increased to similar size of those growing in well-irrigated plants (13.34 and 13.81 cm2 for moderately-stressed and well-irrigated plants, respectively). Similarly, despite lo wer leaf area:root caus ed by moderate stress treatment at the root flush harvest, by the second harvest this difference was no longer significant. This comes as evidence that mo derately-stressed plants were close to, or had established a balance between transpiring ar ea and absorbing mass, which if in a landscape, the plants would hav e been considered established. Ilex cornuta, irrigated every 7 days, took 24 weeks to reach similar levels of those observed in plants irrigated every 2 days of cumulative water stress, thus being considered established (Scheiber et al. 2007). Daily stomatal conductance (Dgs) of m oderately-stressed plants was lower than well-irrigated plants at the day of maximum water stress during Exp. 3 (7 and 2 DAI for moderately-stressed and well-irrigated plants, respectively, Figure 4-2A). However, Dgs values were similar between irrigation tr eatments at 3 DAI fo r moderately-stressed plants and 2 DAI for well-irrigated plants (5,983 and 6,344 mol m-2 day-1, respectively). Thus, there was sufficient water available ne ar the end of the expe riment to moderatelystressed plants for normal transpiration for at least the first three days. This explains some of the differences in dry mass. Within flushes, which were separated by 21 days, there were no differences in root dry mass between treatm ents (Table 4-6). However, there were differences in shoot dry mass, with more mass during shoot flush for the well-irrigated plants than for moderately-str essed plants due to new shoot growth, with new leaves contributing the most. 68

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Predawn shoot water potentials were mo re negative for moderately-stressed than well-irrigated plants, whether on the day befor e irrigation (maximum water stress) or on the day after irrigation (minimum water stress, 10.5 hours after irrigation, Figure 4-2B). Midday water potentials were similar bet ween treatments each day, but were significantly different between days. More negative water potentials of moderately-stressed plants would result in smaller increases in shoot elongation (Beeson 1992a) compared with r oot elongation, and could explain why there were few differences in dry mass of plant components at root flush, yet signifi cant differences in new and total shoot growth, and new and total l eaf mass at shoot flush (Table 4-6). In Pinus pinaster increased root growth was attribut ed to cell wall loosening at moderate water stress (0.15 MPa), with inhibition of root growth attributed to severe water stress (0.45 and 0.66 MPa) due to the inability of the plants to mainta in turgor (Triboulot et al. 1995). Exp. 3 subjected plants to wetting and dryi ng cycles, which are more common in a natural landscape setting. A plant under high water availability has the opportunity to expand growing tissues, and impl ement strategies to tolera te drought that plants under constant stress do not. Severely-stressed plants mainly directed resources towards root growth, and had a severe impairment of sh oot expansion, thus their photosynthate production required to sustain r oot growth was in turn limit ed. Additionally, long-term stress experienced by severely-stressed pl ants likely impaired net photosynthesis due to stomata closure. Saccardy et al. (1996) concluded that the drop in photosynthesis rate caused by water stress was mainly due to stomatal closure when a maize plant dehydrates slowly, such as severelyand moderately-stressed plants of Exp. 2, while it 69

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was mainly due to inhibition of non-stomatal processes when a plant is rapidly dehydrated, such as in Exp. 3. Severely-s tressed plants experienc ed slow dehydration, with the initial high %VWC in these rhiz otrons, 35 days were generally required to decrease the %VWC to ca. 17%. Thus, inhibi tion of photosynthesis due to water stress was likely constant in severely-stressed plant s due to stomatal closure. Kanechi et al. (1996) reported 65% of the limitation of photosynthesis in coffee after rapid dehydration was due to non-stomatal processes (water pot ential declined from -1.0 to -4.0 over the course of one week). The moderately-stre ssed plants in the Exp. 3 likely experienced rather rapid dehydration since the %VWC declined to an average of 19.8% within a week after irrigation. Recently it has been reported that transpl anted woody species undergo a severe water stress unless they re ceive irrigation every 2 days (Scheiber et al. 2007). Correlations between root dry mass and shoot dry mass were consistently high in all three experiments (Fi gure 4-3). In Exp. 2, %VWC (differential between saturation and the triggering volumetric wa ter content) was what ultimate ly dictated frequency of irrigation, and in Exp. 3, it represented the frequency of irrigati on. Although measured biomass had strong correlations with %VWC in Exp. 2 (Figure 4-4), root-to-shoot ratio did not correlate well with %VWC (r = 0.56, data not show n). Correlation of root-toshoot ratio and %VWC in Exp. 3 was also only moderate (r = 0.6). Overall, lower relationships observed for root-to-shoot ratio and %VWC suggests that root-to-shoot ratio may not be the best indicator of water status during the growing period of the plant. Root-to-shoot ratios calculated from Exp. 2 and 3 were comparatively similar and higher relative to those observed in Exp. 1. Act ual percent contributions of roots to total 70

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biomass for moderately-stressed and well-i rrigated plants of Exp. 2 and 3 were generally around 42%, and varied marginally bet ween these two water moisture levels (Figure 4-1), yet there were substantial differences in root and shoot dry mass quantities between treatments in Exp. 2 (F igure 4-3 and 4-4, Table 4-4). This contrasts with suggested relationships of root-to-shoot ratio in the literature. Johnson et al. (1991) developed a water subm odel that included root-to-shoot message control of gs for incorporation into mechanistic plant growth models. Kozlowski and Pallardy (2002) suggested that wa ter deficits ultimately result in plants that have higher root-to-shoot ratio and greater capacity to absorb water and minerals relative to shoots that must be supported. Thornley (1996) proposed that in response to limited soil moisture availability over extended periods of time, plant water status is more strongly influenced by changes in root-to-shoot partiti oning and root density than the interaction of soil moisture content with gs. The increase in root-to-shoot ratio is an adaptive response to water stress (Chaves et al. 2003; Jackson et al. 2000), howev er, our results indicate that a severe stress is needed to induce this response in L. japonicum plants. The balance between root dry mass and shoot dry ma ss, namely root-to-shoot ratio, was influenced by water availability in our results; however, it should not be viewed as a sole indicator of plant water availability during the growth period. As previously observed for C4 species (Sharp et al. 2004), root growth is less s ensitive to water stress than shoot growth. L. japonicum (C3) maintained root growth even with slow dehydration to ultimately fatal levels, even though shoot growth was severely impaired. Constant, moderate levels of water stress also reduced plant growth as whole; however, shoot growth, though 71

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restricted, still occurred. Th is enabled these plants to survive since photosynthates required for root growth could be supplied. In termittent exposure to moderate levels of water stress reduced plant biomass by simila r amounts to that achieved by continuous exposure. However, the wetting and drying cycles enabled plants under stress to achieve greater carbon assimilation than plan ts under constant water stress, which led to establishing a normal balance between absorbing and transpiring surfaces by the end of the study. Accurately imposing and measuring water stress in mature woody plants in a large scale, while keeping other variables as const ant as possible, is not an easy task. Soil moisture measurements can be deceiving, thus a system has to be evaluated as a whole for best performance. The final ex perimental system described here appears to have achieved this goal. Future research delvi ng into the study of water stress or other growth variables of perennial plan ts may find this system valuable. 72

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Table 4-1. Mean percent volumetric water c ontent (%VWC) achieved in Experiments 1, 2 and 3 determined after recalculati on using equations based on prolonged dual measurement of VWC using Digital TDT and ECH2O probes. Severely-stressed plants Moderately-stressed plants Well-irrigated plants Exp. 1 21.17bz 31.30 a Exp. 2 16.48c 17.44b 32.55 a Exp. 3 Drained saturation 25.03a 25.06 a Low achieved %VWC 19.80b 22.33 a Differential 5.22a 2.68 b z Means within rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). Table 4-2. Experiment 1. Dr y mass (g) of components of L. japonicum grown at different constant volumetric water contents in rhizotrons. Moisture probes remained near original roots ball for the durat ion of the experiment. Masses between treatments were pool ed within harvest. Harvest 1 Harvest 2 186 DATz 217 DAT Shoot 958.5 by 1461.1a Leaves 529.5 b 868.6a Stem 429.0 b 592.6a Total roots 331.5 b 539.4a Old rootsx 201.6 b 274.1a New rootsw 129.9 b 265.3a Plant 1290.0 b 2000.6a z Days after transplant. y Means representative of 4 replications. Means within rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). x Roots within the transplanted root ball. w Roots grown during the duration of experiment. 73

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Table 4-3. Experiment 1. Leaf area, a llometric relationships and percentage of allocation in L. japonicum grown in rhizotrons at constant volumetric water contents. Results between treatm ents were pooled within harvest. Harvest 1 Harvest 2 186 DATz 217 DAT Leaf area (cm2) 43,587ay 55,073 a Specific leaf area (cm2 g-1) 82.92a 62.22 b Root-to-shoot ratio (g g-1) 0.34a 0.37 a Root dry mass-to-leaf dry mass ratio (g g-1)0.57a 0.63 a Leaf area-to-root dry mass ratio (cm2 g-1) 132.03a 101.25 b % Allocation to shoots 74.44a 73.05 a % Allocation to roots 25.56a 26.95 a z Days after transplant. y Means representative of 4 replications. Means wi thin rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). Table 4-4. Experiment 2. Dr y mass (g) of components of L. japonicum grown in rhizotrons at constant vo lumetric water contents. Severely-stressed plants Moderately-stressed plants Well-irrigated plants 100 DATz 116 DAT 116 DAT Shoot 217.24 cy 308.52b 396.01 a Leaves 116.88 c 165.44b 215.38 a Stem 100.36 c 143.08b 180.63 a Roots 187.40 b 233.72b 282.49 a Plant 404.64 c 542.24b 678.50 a z Days after transplant. y Means of 5, 5, and 12 replications for severely-stressed, moderately-stressed, and well-irrigated plants, respectively. Means within rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). 74

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Table 4-5. Experiment 2. Leaf area, a llometric relationships and percentage of allocation in L. japonicum grown in rhizotrons at constant volumetric water contents. Severely-stressed plants Moderately-stressed plants Well-irrigated plants 100 DATz 116 DAT 116 DAT Leaf area (cm2) 4,205cy 8,649b 11,750a Specific leaf area (cm2 g-1) 35.86b 52.37a 54.44a Root-to-shoot ratio (g g-1) 0.87a 0.75b 0.72b Root dry mass-to-leaf dry mass ratio (g g-1) 1.61a 1.40b 1.32b Leaf area-to-root dry mass ratio (cm2 g-1) 22.70b 37.73a 41.94a % Allocation to shoots 53.69b 57.09a 58.37a % Allocation to roots 46.31a 42.91b 41.63b z Days after transplant. y Means of 5, 5, and 12 replications for severely-stressed, moderately-stressed, and well-irrigated plants, respectively. Means within rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). 75

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Table 4-6. Experiment 3. Dr y mass (g) of components of L. japonicum grown in rhizotrons at variable volumetric water content. Moderately-stressed plants Well-irrigated plants 138 DATz 159 DAT Root flush Shoot flush Root flush Shoot flush Leaves Old 53.5 ay 58.1 a 57.5a 58.1a New 53.6 c 67.8 bc 75.4b 100.2a Total 107.0 b 125.9 b 132.8ab 158.2a Stem Old 46.3 b 55.2 ab 48.5b 69.7a New 19.8 b 25.4 ab 25.6ab 31.8a Total 66.1 b 80.7 ab 74.1b 101.4a Shoot Old 99.7 b 113.4 ab 106.0ab 127.7a New 73.4 c 93.2 bc 101.0b 131.9a Total 173.1 b 206.6 b 207.0b 259.7a Root Old 82.8 a 96.1 a 84.3a 98.6a New 44.1 b 79.8 a 47.4b 87.4a Total 126.9 b 175.9 a 131.7b 186.1a Plant 300.0 c 382.5 ab 338.7bc 445.7a z Days after transplant. y Means of 6 replications. Means within rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). 76

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Table 4-7. Experiment 3. Leaf area, a llometric relationships and percentage of allocation in L. japonicum grown in rhizotrons at variable volumetric water content. Moderately-stressed plants Well-irrigated plants 138 DATz 159 DAT Root flush Shoot flush Root flush Shoot flush Old leaves Number of leaves 288.2ay 317.8a 322.0 a 323.2a Leaf area (cm2) 2,570a 2,734a 2,528 a 2,824a Specific leaf area (cm2 g-1) 48.3a 47.1a 44.4 a 48.5a New leaves Number of leaves 288.8b 284.6b 372.3 ab 410.7a Leaf area (cm2) 3,386c 3,949bc 4,967 ab 5,669a Specific leaf area (cm2 g-1) 63.2ab 59.1ab 66.4 a 56.7b Total leaves Number of leaves 577.0c 602.4bc 694.3 ab 733.8a Leaf area (cm2) 5,957c 6,683bc 7,495 ab 8,493a Specific leaf area (cm2 g-1) 55.8a 53.4a 56.9 a 53.6a Allometric relationships Root-to-shoot ratio (g g-1) 0.75ab 0.85a 0.64 b 0.72b Root dry mass-to-leaf dry mass ratio (g g-1) 1.21ab 1.39a 0.99 c 1.18bc Leaf area-to-root dry mass ratio (cm2 g-1) 47.30b 38.76b 57.77 a 46.10b % Allocation to shoots 57.43ab 54.08b 61.25 a 58.28ab to roots 42.57ab 45.92a 38.75 b 41.72ab z Days after transplant. y Means of 6 replications. Means within rows not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). 77

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Figure 4-1. Percent contribution of each pl ant component to the total dry mass of L. japonicum grown under different substrate moisture levels. Experiment 1: moisture probes remained near original roots ball for t he duration of the experiment. Experiment 2: constant VW C due to consistent relocation of moisture probes. Experiment 3: irrigation varied between saturation and 19.8 and 22.3% VWC for moderately-st ressed and well-irrigated plants respectively. Plants harvested during root and shoot flush. 78

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Figure 4-2. Daily stomatal conductance, Dgs (A) and shoot water potential, T (B) of L. japonicum grown under irrigation varying between saturation and 19.8 and 22.3% VWC for moderartely-stressed and we ll-irrigated plants respectively on the day of minimum and maximum water stress in Exp. 3. Each bar represents the mean of fifteen daily curves of Dgs (5 plants x 3 leaves) and 6 replications of T. Bars not followed by the same letter within each time are significant at P 0.05 (Fishers Least Significant Difference). Bars followed by within each treatment and ti me are significant at P 0.05 (Fishers Least Si g nificant Difference ) 79

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Figure 4-3. Correlation am ong root and shoot dry mass. (A) Ex periment 1, (B) Experiment 2 and (C) Experiment 3. The correlation coefficient (r) is included for each relationship (all samples pooled). 80

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81 Figure 4-4. Correlation for Exp. 2 among %VWC (differential between saturation and the triggering volumetric water content ) and (A) root dry mass, (B) shoot dry mass, (C) leaf dry mass, and (D) leaf area. The co rrelation coefficient (r) is included for each relationship (all samples pooled).

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CHAPTER 5 DEVELOPMENT OF SHOOT ARCHITECTURE OF Ligustrum japonicum Thunb. IN RESPONSE TO S OIL MOISTURE. Introduction The process of shoot branching is an impor tant determinant of a plants shape. Shoot branching is the process by which ax illary buds, located on the axil of a leaf, develop and form new flowers or branc hes. Bud outgrowth is regulated by the interaction of environmental and endogenous signals, such as plant growth regulators. These interacting factors have a major effect on shoot system ar chitecture (Ongaro and Leyser 2008). Depth of bud dormancy has been proposed to be related to abscisic acid (ABA) levels (Tamura et al. 1993) and water status of the bud (A rora et al. 2003). Prolonged exposure to drought can trigger ABA responses in shoot meristems, such as inhibition of leaf production, growth and development (Kuang et al. 1990; Sauter et al. 2001). Growth restrictions result in meristems lo sing relatively little water during water stress (Arora et al. 2003). Frugis et al. (2001) dem onstrated that overexpression of a gene in lettuce associated with accumulation of specif ic types of cytokinins, caused alterations to plant architecture, which changed from determinate to indeterminate leaf growth. Overall plant form is achieved by regulati on of initiation and outgr owth of axillary meristems (Kerstetter and Ha ke 1997). The tendency of a dormant lateral bud to outgrow is regulated by a network of vari ables (Waldie et al. 2010). Among the most important variables are age of the bud relative to its initiation, and the zone of the stem in which the bud develops (basal versus aeria l branching, Napoli et al. 1999). Roles of cytokinin as promoter and au xin as an inhibitor of lateral bud outgrowth have long been known and studied in branches with decapita ted terminal bud. However, it has been 82

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suggested that these two plant growth regulators may not be the only factors in lateral bud breakage of dormancy durin g undisturbed growth. A net work of shoot and root signals that regulates branching has been proposed (Thomas and Hay 2009). A branching signal has been postulated to exist, wh ich moves in the direction of root-toshoot (Beveridge 2000). The i dentity of this signal is st ill unknown, although a strong candidate has recently em erged (Waldie et al. 2010). Borchert (1975) observed variations of shoot growth patterns between young and mature plants of Quercus palustris Muenchh. Patterns ranged from a series of flushes characterized by determinate growth to l onger flushes characterized by indeterminate growth. Branch position or geometry alone was not sufficient to account for the various growth patterns existing between species; other factors, such as timing of shoot growth, also affected shoot architectura l pattern (Napoli et al. 1999). Napoli et al. (1999) noted that research on dormancy and shoot growth has mainly focused on herbaceous plants; thus, all conc epts are not necessarily applicable to woody species. Developing a clearer understanding of bud dynamics and their typespecific contributions under undisturbed conditions, is a necessary prerequisite for predicting their responses under distur bed conditions (Zhang et al. 2009). Many researchers have studied shoot architecture, but have given exclusive attention to shoot growth by itself. Recently a root signal wa s proposed (Waldie et al. 2010). However, the influence of relative growth of roots and s hoots are still obscure. Relationships between physiological processes and environmental c onditions or plant development are often confounded by variation on geneti c, environmental or ontogenet ic factors (Hanson et al. 1986). 83

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The research presented here describes the natural growth of clonal Ligustrum japonicum as a basis for understanding how this growth is influenced by water stress, and ultimately integrate this knowledge to c haracterize shoot and root growth patterns. Material and Methods Growth conditions and experimental design Twenty-four star-shaped rhizotrons were c onstructed as described in Chapter 2 (Figure 2-3). Briefly, each rhizotron had four arms and held 0.16 m3 of substrate. They were 1.76 m across, 0.30 m deep at the end of each arm and 0.35 m deep in the center. Rhizotrons resided in an open-sided greenhouse with a double polyethylene roof, under natural light, located in Apopka, FL. Clonal Ligustrum japonicum plants were selected from a local nursery (Jons Nursery, Eu stis, FL) to ensure homogeneous size and health. One plant was transplanted into each rhizotron using a commercial substrate composed of Canadian sphagnum peat moss, pr ocessed pine bark, perlite, vermiculite, starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc., Agawan, MA). Irrigation was supplied using a spray stake (model green 22500-001120, Netafim Irrigation, Inc., Fresno, CA) at the tip of each arm pointing inward. Two experiments, conducted consecutively over two years, examined patterns of shoot and root growth under well-irrigated conditions and water stress applied constantly (2008) or intermittently (2009). In the experiment conducted in 2008, substrate moisture was kept constant at three levels: 32.5% VWC (percent volumetr ic water content, well-irrigated plants, Chapter 4, Table 4-1); 17. 4% VWC (moderately-stressed plants); and 16.5% VWC (severely-stressed plants). Plants from 11.4-L containers were transplanted into rhizotrons in March and grown for 116 days. After a 23 day acclimatization phase, 84

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plants were subjected to three irrigation fr equencies (well-irrigated, severely-stressed, and moderately-stressed), with 12, 5 and 5 rep lications each, respectively. Substrate moisture level was managed based on substr ate measurements as described in Chapter 4. In the experiment conducted in 2009, substrate was irrigated at 2 and 7 day intervals (well-irrigated and moderately-stressed plants, respectively). These two treatments resulted in VWC of 22.3% and 19.8% in the drie st periods (Chapter 4, Table 4-1). Plants grown in 3.8-L containers were transplant ed into each rhizotron in February and grown for 138 or 159 days. Entire rhizotr ons were irrigated daily until saturation for 41 days for plant acclimatization. Thereafter, irrigation was withheld for six days before the start of irrigation treatm ents. Treatments cons isted of two irrigation frequencies, once a week for 10 minutes (12 L of water, moderately-stressed pl ants) and every other day for 5 minutes (6 L of water, well-irrigat ed plants), with 12 replications each. Plants were harvested twice, 138 and 15 9 DAT (days after transplant). Six plants from each treatment were harvested at root flush (increasing number of root growing points, RGP) and six at shoot flush (increasing num ber of shoot grow ing points, SGP). Growth measurements Growth of roots and shoots were m onitored weekly and recorded after commencement for both experiments. Growth was not disturbed by pruning during experiments. Number of root growing points visible in the eight rhizotrons observation windows, and SGP were counted weekly thr oughout the experiments. A root growing point was defined as a root tip with visual char acteristics of active growth (Figure 5-1E, light color of the root c ap, division and elongation zones and no apparent root hairs). A 85

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shoot growing point was defined as a shoot tip between visual bud break and no unfolding of new leaves (Figure 5-1D). In 2009, in addition to RGP and SGP re corded up to 126 and 157 DAT, for root and shoot flush, respectively, shoot architecture parameters (length, number of leaves, bud type, and date of bud set for new branches), and number of inflorescences per plant were recorded weekly up to 100 DAT. Results and Discussion Growth under well-irrigated conditions (2 day wetting and drying cycles) L. japonicum has imbricate buds, buds with more than two scales that overlap one another (Figure 5-1A-C). At the end of each branch a cluster of buds occurs, that will be referred to as apex buds (Figure 5-2). These apex buds are composed of one central terminal bud and two lateral buds on opposite sides Also part of the apex bud cluster, there are two accessory buds for each termi nal and lateral buds. Apex lateral buds originate from the last pair of leaves. Leaf Lateral buds refers to lateral buds that originate from leaves older than the last pair of leaves (Figure 5-2). Each terminal bud and lateral bud has preformed stem, leaf prim ordia and lateral buds (Figure 5-3B-D). L. japonicum is a temperate woody shrub, native to Japan and East Asia (Ishii and Iwasaki 2008). Preformation, the differentiati on of organs in a growing season before maturation and extension the following growi ng season, is a common characteristic among temperate woody species (Meloche and Diggle 2003; Puntieri et al. 2007; Remphrey and Powell 1984). Neoformation, the simultaneous differentiation and extension of organs in the same growing season, is considered to be relatively uncommon among temperate woody plants (Puntieri et al. 2007). 86

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Although under apical dominance, vegetative lateral buds of Rosa hybrida increased dry mass and developed new leaf primordia with age (Marcelis-Van Acker 1994). Our observations suggest that L. japonicum leaf lateral buds (Figure 5-1C) behave as observed for roses, despite pr eformed lateral bud maturation during the present growing season, leaf lateral bud loca ted in new branches will burst only in the next growing season or if apical dormancy is broken. No new leaf lateral bud outgrew (Table 5-1). However, seven percent of neof ormed apex lateral buds expanded to form second order shoots, while 23.2% of new terminal buds formed a second order shoot (Table 5-1). This suggests that new leaf lateral buds are preformed and mature in the present growing season during determinat e growth. While new apex lateral buds, together with new terminal buds, are neofor med. Additionally, neoformed buds appear less sensitive to dormancy than preform ed buds, which need more than one growing season to naturally outgrow. Apical dominance is a term generally used to re fer to inhibition of growth of lateral buds by the terminal bud (Little 1970; Napoli et al. 1999). Although a degree of apical dominance was noted, this species appears to have weak apical dominance. Only 47.6% of total growing points originated from terminal buds (Table 5-2). Additionally, the first flush of growth was mostly due to leaf lateral bud outgrowth. It is important to note that leaf lateral bud outgrowth was independent of terminal bud decapitation. Plants were not pruned. Decapitation causes not only the commonly discussed plant growth regulators alteration, but also imposes st ress by wounding, and causes changes in xylem fluid flow and in sink strength (N apoli et al. 1999). These dormancy-released buds were mostly located in the basal zone of the plant. Thus, the fi rst flush of growth, 87

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in the beginning of spring, may be a result of the interaction of ecodormancy release with positional clues, since the terminal bud in the same branch was not released. After the first flush, some neoformed apex lateral buds initiated active growth concurrently with neoformed terminal bud (7% of neoformed apex lateral bud outgrowth, Table 5-1). Bud formation date did not i nhibit neoformed terminal bud to the same extent, since they produced second and third order shoots in 23.2% of total neoformed terminal buds (Table 5-1). The outgrowth of neoformed apex latera l bud suggests that those buds are less sensitive to terminal bud dominance. While some apex lateral bud outgrew during the experiment, none of the leaf lateral buds outgrew. Any tentative approach to explain apical dominance or do rmancy depth should take bud position and age into consideration. In Quercus, branching order and age were the most important influences on bud outgrowth, compared to seve ral manipulative tr eatments, such as defoliation and terminal bud removal (Bicl-Sorlin and Bell 2000). A new branch was defined as a single stem developed from an old bud (bud expanded in previous growing seasons) and its subsequent second and third order shoots. A terminal branch was defined as a new branch arising from an old terminal bud, and a lateral branch as a new branch fo rmed from an old lateral bud. Of the total number of new terminal branche s, 53.7% had two or more flushes of growth (Table 53), while fewer lateral branches (30.1%) had two or more flushes of growth. This suggests differences in sink strength betw een branch positions in a plant. Furthermore, 83.8 % of old branches t hat produced new growth generated only one new branch (Table 5-3), with the majority of termi nal bud and lateral bud outgrowth occurring isolated (Table 5-2). Isolated outgrowth refe rs to only a single bud outgrowth from a 88

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branch. In some cases, both lateral buds of the same leaf pair outgr ew concurrently. It has been suggested that lateral bud outgr owth potential is associated with developmental stage and physiological activity of its dominant terminal bud. In Arabidopsis, lateral bud is suppressed during vegetat ive growth. However, after a long period of vegetative growth, dormancy is re leased from more distal lateral buds, resulting in acropetal patte rn of growth. After switch ing to reproductive growth, dormancy was released from proximal late ral buds, resulting in basipetal pattern (Vojislava and Anthony 2000). Ho wever, only four flower panicles were recorded during this experiment and branching pattern tended to be basipetal (11.1 leaf lateral branches per plant versus 7.7 terminal branches per plant, Table 5-3). In roses, shoot apical meristem of lateral buds remained vegetative unless apical dominance was removed (Marcelis-Van Acker 1994). The position or age of a bud along the stem can determine its ability to grow out (Waldie et al. 2010). Although apex buds occasiona lly outgrew at the same time, in cases where terminal bud burst together with one or more other buds, terminal bud always produced longer shoot stems with a greater number of leaves than a branch from a lateral bud developing by its side. Branch fina l size depended on whether growth was indeterminate or determinate. Indeterminat e growth occurred mostly from isolated lateral branches, which were responsible to 22.6% of all branch length in well-irrigated plants (data not shown). St ems with apparent determinate growth measured on average 16.1 and 13.9 cm, for terminal and lateral br anches respectively, while stems with indeterminate growth averaged 58.9 and 69.5 cm in length for terminal and lateral branches, respectively (Table 5-4). For some tree species, neoformation contributed 89

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more to axis formation t han preformation (Costes 2003; Guedon et al. 2006; Snowball 1997). Number of leaves per stem and internode length of determinate lateral branches were smaller than observed for determi nate terminal branch and associated second order branches (Tables 5-6 and 5-4), but th ere were no differences between terminal and lateral branches when growth was indete rminate (Tables 5-6 and 5-4). The lower number of leaves originated by latera l buds than by terminal buds agrees with observations in buds of apple trees, in which lateral buds had fewer leaf primordia and bud scales than terminal buds (Costes 2003). T he unknown factor that signaled shoot apical meristem in determinate branches to start differentiating scales and consequently set a bud was not perceived in indeterminat e branches. What arrested growth of indeterminate branches is unknown. Nutrient availability at the me ristematic region may be involved with bud setting. Reserve carbohy drates are often used for growth of sprouts and root suckers (Kozlowski 1992). Moreover, mitotic activity and dormancy were related with carbohydrat e levels within the bud of Douglas fir (Owens and Molder 1973). Also, neoformation is considered a plas tic response of woody plants to factors acting at the time of shoot extension (Guedon et al. 2006). Effect of intermittent water stress on growth Wetting and drying cycles leading to moderate stress resulted in biomass reductions of approximately 20% (Chapter 4). Water stress also reduced several other growth parameters, such as the number of new branches (40% reductions, Table 5-3), especially lateral branches; total growing po ints (42% reduction, Table 5-2); and length of new branches (24% reduction, Table 5-5). Water stress affected shoot architecture by enhancing apical dominance. Lateral branching was mainly affected, declining fr om an average 11.1 new lateral branches 90

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produced by well-irrigated plants to only 5. 4 produced by moderately-stressed plants (Table 5-3). Moreover, water stress affect ed outgrowth of apex buds by increasing terminal bud dominance over the two lateral buds located at the apex. The proportion of apex outgrow (terminal bud, terminal bud+1 la teral bud, terminal bud+2 lateral buds, or terminal bud+2 lateral buds +1 accessory bud) was distributed differently for well-irrigated and moderately-stressed plant s (51.9, 25.2, 21.4, and 1.6% for well-irrigated plants, and 74.1, 12.1, 13.8 and 0%, for moderately-stressed plants, respectively, Table 5-2). Although only termi nal bud+1 lateral bud s eparated statistically between the two treatments, the tendency towards incr eased apical dominance in moderately-stressed plants is clear. Interest ingly, moderate water stress did not affect leaf lateral bud outgrowth in the same way. Both treatments had 86. 3% of leaf lateral buds developing isolated. Still, leaf lateral bud outgrowth of moderat ely-stressed plants was 50% less than well-irri gated plants (Table 5-2). As discussed above, first order termi nal branches of well-irrigated plants had mainly determinate growth, and lateral branc hing occurred more often than terminal branching. Water stress restri cted new shoot growth to pr incipally determinate terminal branches but with three times as many indet erminate terminal branches as well-irrigated plants (Table 5-6). Also, stem length of det erminate first order terminal branches in moderately-stressed plants were considerably longer than thos e of well-irrigated plants, while stem length of first order terminal branches dec reased for moderately-stressed plants (Table 5-4). First order terminal branch of moderate ly-stressed plants had longer internodes than those of well-irrigated pl ants (3.7 versus 2.9 cm, Table 5-4), and approximately one extra pair of le aves (10.5 versus 8.9 leav es per stem, Table 5-6). 91

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The number of new leaves produced by 100, 138, and 159 DAT was similar between dates within each treatment; however, moderately-stressed plants produced fewer new leaves than well-irrigated plants at 100 and 159 DAT since there were fewer branches (Figure 5-4). As discussed on Chapter 4, at 138 DAT (root flush) moderately-stressed plants had smaller new leaves than well-irrigated plants (11.7 and 13.3 cm2, respectively), and by 159 DAT (shoot flush) moderately-stressed plants had similar size new leaves as well-irrigated plants (13.3 and 13.8 cm2, respectively). However, the number of new leaves did not increase signi ficantly for moderately-stressed plants (Figure 5-4). The seven-day wetting and drying cycles changed morphological and physiological behavior of L. japonicum by potentiating apical dominance. The beginning of the first shoot flush coin cided with the beginning of stress treatment (around 47 DAT). At the second flush, which occurred later into the stress treatment, old buds burst more frequently than the newly formed apex latera l buds. Also, as plants began to adapt to the stress imposed, indeterminate growth was triggered more often in meristematical regions of terminal buds. Well-irrigated plant s tended to set a bud and then invest in the production of new leaves in a second flush developing second order branches. Conversely, shoot apical meristem of moderately-stressed plants turned to indeterminate growth, thus producing neoformed leaves. Towards the end of the experiment, after a root flush, moderately-stressed plants were established (Chapter 4), this made possible additional l eaf and stem expansion. As a result, moderately-stressed plants had leaves with similar si ze as well-irrigated plants. 92

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Root and shoot growth patterns The initial acclimatiz ation period after transplant into rhizotrons was required to enable roots to elongate sufficiently to be observed in rhizotron observation windows. Roots had to grow approximately 20 cm to re ach the sides of rhizotron. Root growth was represented by RGP. This delayed quantifi cation of RGP relative to SGP (Figures 5-5 to 5-8). Root growth occurred befor e bud swelling. Longest roots outside the original root ball, measured the day soil moisture probes we re initially installed, was about 10 cm in the 2008 experiment (23 DAT) and 15 cm in the 2009 experiment (41 DAT). Thus, it is likely that the initial root fl ush began soon after transplant into rhizotron. This contrasts with observations for transplanted Quercus alba and Quercus marilandica seedlings, which expanded roots only after shoot ex pansion (Reich et al. 1980), or in Quercus rubra which expanded root and shoot concomitantly (Sloan and Jacobs 2008). In Central Florida, growing root tips have been noted in February, weeks before bud break, during transplanting of several ever green and deciduous tree species (Beeson, per. Comm,). Despite variation on flush size and duration between plants, root growth of L. japonicum rarely completely stopped during shoot growth. A flush was defined as the period betw een increasing and decreasing growing points until rest (zero value) or before a subsequent increase, which would characterize the beginning of a new flush. Shoot and root flushes were mo stly asynchronous in both years and in all treatments. L. japonicum is described in literature as an episodical species (Kuehny and Halbrooks 1993; Kuehny et al. 1997), based on growth monitored by changes in dry mass. Organ growth is controlled by two processes: cell division and cell expansion (Kuehny and Halbrooks 1993). However, whole plant growth can be 93

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categorized in two phases: A) cell division a nd differentiation of organs and tissues in meristems and; B) expansion and formation of new organs. In shoot tips, bud formation comprises phase A, which will expand during det erminate growth. However, it is phase B of growth that is usually measured and commonly regarded as growth. Accumulation of dry mass occurs with growth. However, dry mass accumulation stretches through organ maturation. In roots, however, the two phases of gr owth occur concomitantly. Cockcroft, et al. (2000) conclude d that cell division is the main determinant of meristem activity and overall growth rate. While dry mass accumulation is the most common method used to categorize growth, and was used previously for L. japonicum other species have been categorized using stem or leaf elongation to measure growth (Borchert 1975; Cockcroft et al. 2000; Schoene and Yeager 2006). Accounting for growth using growing points was a method to quantify visual growth non-destructively over time in the same group of plants. Me ristems are an important source of plant growth regulators; thus, counts of growing points are an indirect way to quantify the contribution of active shoot apical meristem and root apical meristem for the pool of these compounds. In general, the first shoot flush coincid ed with the start of treatments in most plants, and coincided with the first visible root flush (roots and shoots flushing together). Well-irrigated plants from 2008 (constant moisture) displayed two distinct growth patterns (Figure 5-5). The first (A-08) was c haracterized by two shoot flushes with three small root flushes and one large root flush towards the end of data collection, and the second (B-08) by one main shoot flush with tw o smaller root flushes and one large root flush again near the end. Plants within each pa ttern were relatively synchronous. Plants 94

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with pattern A-08 had the first shoot flus h starting around 30 DAT, which lasted for approximately 30 days. The second shoot flush followed the first without a resting period and lasted from 25 to 50 days. Besides the first root flush, which occurred for all plants, two root flushes occu rred within the second shoot flus h, and a fourth large root flush followed with an obvious decline in SG P for all plants. Shoot growth was delayed for plants with pattern B-08, as were subs tantial increases in RGP activity. Shoot flushes started around 45 DAT and lasted for about 60 days, due to more branches with indeterminate growth. In this pattern of growth, the second root flush occurred as shoot flush was finishing, and a third root fl ush followed when shoots became quiescent. Episodical growth was not observed in initial stages, but rather in short periods, in which RGP were decreasing and SGP increasing, or the inverse was occurring. A search of the literature did not find similar growth pa tterns to those observed in this study, nor have growing points of both roots and shoots been used before to distinguish episodical patterns. Under warm soil and abundant mois ture conditions, there was no clear arresting of RGP during shoot flushes. During periods of low RGP, root mass increase likely would have been small, and conversely the opposite during periods of high RGP. Thus, activity of RGP should correspond to traditional dry mass measurements of episodical growth. Growth patterns of well-irrigated plants of the 2009 experiment (two day wetting and drying cycles) also can be divided in two groups (Figure 5-6). The first pattern (C-09) had three shoot flushes between 38 and 110 DAT, a resting period of approximately 40 days before new shoot flush, and four root flushes without resting periods. The second pattern (D-09) had three shoot flushes with a small resting period 95

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between them, and four root fl ushes without resting periods. Pattern D-09 was similar to A-08; however, D-09 had smaller peaks, separ ated by a resting period. Also, no plants in the 2009 experiment disp layed only one shoot flush as in 2008, and roots never completely came into rest. Although substrat e moisture conditions were favorable to growth, such as in 2008, the variation in moistu re affected root tips, such that growth in part of the roots was always active. Constant high substrate moisture (well-irrigated plants 2008) stimulated roots to turn quiescent at times, and RGP stayed lower (under 100 tips until around 70 DAT, Figure 5-5) than roots under intermittent irrigation (well-irrigated plants 2009, over 100 growing tips at 70 DAT, Figure 56). In the inverse situation, resting periods between shoot fl ushes were a novelty for plants grown in 2009, and these periods were opposed to root flushes, better charac terizing an episodic behavior. This suggests that the natural-observed episodic growth, such as observed for temperate forests, may depend on environment as well as genetic-dictated characteristics. Severe water stress resulted in impaired growth (Chapter 4), which can also be observed on counts of growing points. Duri ng the 2008 experiment, plants under severe stress that did not receive additional irrigation had only one minor shoot flush and almost no root growth (lower left 3 repetitions of Figur e 5-7). When any additional irrigation was supplied, SGP increased dramatically, such as on the one occasion of the plant represented on the top left of Figure 5-7, or on two o ccasions after irrigation was supplied for the plant represent ed by the graph second from the top on the left in Figure 5-7. For both cases, despite large SGP counts, the formed br anches did not expand past a few centimeters and shortly after commencement of visi ble growth these 96

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branches dehydrated and died. Plants under se vere water stress delegated most of their resources into root growth, rather than shoot growth (46.3 and 41.6% allocation to roots for severely-stressed and moderately-st ressed plants, respectively, Chapter 4). However, if any irrigation was applied bud burst occurred, thus, water was clearly a limiting factor for bud outgr owth. Moderately-stressed pl ants of 2008, had two shoot flushes and very low counts of RGP up until 100 DAT (Figure 5-7). The majority of moderately-stressed plants of 2009 displayed growth patterns similar to D-09. However, growth patterns in these plants were more variable. Conclusions Age and position affected L. japonicum s buds capacity for outgrowth. The first spring flush was mainly due to leaf lateral bud outgrowth, thus resu lting in basipetal branching. Possibly reflecting leaf lateral bud proximity to storage carbohydrates and a postulated root-to-shoot signal in roots (Beveridge 2000; Waldie et al. 2010) or stored carbohydrates in the trunk (Salaun and Charpentier 2001). At the second flush of growth, not only leaf lateral buds, but also neoformed buds were able to outgrow, since new leaves can generate carbohydrates to support neoformed bud outgrowth. First order terminal branch had mostly determinate growth, while first order lateral branch had an increased number of branches with indeterminate growth. Exposure to intermittent water stress, su ch as experienced in natural settings, resulted in stronger apical dominance in L. japonicum As a result of prolonged intermittent water stress, bud setting of terminal bud was delayed more often and indeterminate growth was triggered in these shoot tips. Moreover, in the branches that stopped growing at the determi nate stage and set a bud, the newly formed apex lateral bud outgrew less often, thus suggesting the increased apical dominance of terminal bud 97

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over apex lateral bud. Further into the treatments, well-irrigated plants tended to invest in the production of new leaves, developi ng second order branches in a new shoot flush. Plants under intermittent water st ress continued new leaf production, with indeterminate growth, instead of setting a bud. These plants had fewer but normal size leaves by the end of the study because they were able to fully expand leaves after a root flush, which resulted in the establishment of a normal balance between absorbing and transpiring surfaces (Chapter 4). After transplant into rhizotrons, root gr owth occurred before shoot growth and it occurred as soon as early March. The temporal variation on moisture caused by wetting and drying cycles resulted in continuous growth of parts of the root system, while resting periods were observed for shoot growth. Inve rsely, continuous high moisture levels resulted in roots having resting periods for some plants. Classic episodic growth was not observed as described for this species (Kuehny and Halbrooks 1993; Kuehny et al. 1997) for most of the growing season. Howe ver, small ebb and flow cycles of RGP and SGP were documented. 98

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Table 5-1. Percentages of bud outgrow th and dormancy of neoformed buds on branches expanded during 100 days of undisturbed growth by L. japonicum grown in large rhizotrons in 2009. Treatment % of total Well-irrigated plants Moderatelystressed plants Well-irrigated plants Moderatelystressed plants Apex bud outgrow 12.2 az yy 6.9ay 12.4 10.6 Apex bud dormancy 81.3 a z 47.4bz 87.6 89.4 Terminal bud outgrowth 7.4 a y 4.3by 23.2 20.5 Terminal bud dormancy 23.8 a z 13.8bz 76.8 79.5 Apex Lateral bud outgrow 4.8 a y 2.6ay 7.0 5.6 Apex lateral bud dormancy 57.6 a z 33.5bz 93.0 94.4 Leaf lateral bud outgrowth 0 a y 0ay 0.0 0.0 Leaf lateral bud dormancy 376.5 a z 268.7bz 100.0 100.0 z Means of 12 replications. Means within rows not followed by the same letter (a, b) are significant at P 0.05 (Fishers Least Significant Difference). y Means of 12 replications. Means within columns not followed by the same letter (z, y) are significant at P 0.05 (Fishers Least Significant Difference). 99

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Table 5-2. Number of growing points recorded during 100 days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009. Treatment % of total Well-irrigated plants Moderatelystressed plants Well-irrigated plants Moderatelystressed plants Total growing points 31.2az 18.1b 100.0 100.0 Growing points from: preformed terminal buds 7.7a yy 5.7az 24.4 31.9 neoformed terminal buds 7.4a y 4.3bzy 23.2 20.5 preformed leaf lateral buds 11.3a z 5.5bz 38.3 36.3 neoformed lateral buds 4.8a y 2.6ay 14.1 11.3 Isolated preformed leaf lateral bud outgrow 9.5a z 4.4bz 86.3 86.3 Opposite preformed leaf lateral bud dormancy 1.8a y 1.1ay 13.7 13.7 Isolated terminal bud outgrow 9.9a z 7.7az 51.9 74.1 1 terminal bud + 1 lateral bud outgrow 5.3a y 2.2by 25.2 12.1 1 terminal bud + 2 lateral bud outgrow 4.3a y 2.8ay 21.4 13.8 1 terminal bud + 2 lateral bud + 1 accessory bud outgrow 0.3a x 0.0ax 1.6 0.0 z Means of 12 replications. Means within rows not followed by the same letter (a, b) are significant at P 0.05 (Fishers Least Significant Difference). y Means of 12 replications. Means within columns not followed by the same letter (z, y, x) are significant at P 0.05 (Fishers Least Significant Difference). 100

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Table 5-3. New branc h distribution of L. japonicum grown in large rhizotrons in 2009, during 100 days of undisturbed growth. Treatment % of total Well-irrigated plants Moderatelystressed plants Well-irrigated plants Moderatelystressed plants New branches produced 18.8az 11.2b 100.0 100.0 Lateral branches 11.1a zy 5.4bz58.6 48.5 Terminal branches 7.7a z 5.7az41.4 51.5 New branches with 1 flush 11.3a z 7.2bz60.3 63.1 >1 flush 7.5a y 4.0bz39.7 36.9 Lateral branch with 1 flush 7.8a z 4.0bz69.9 66.3 >1 flush 3.3a y 1.5ay30.1 33.7 Terminal branch with 1 flush 3.5a z 3.2az46.3 59.9 >1 flush 4.2a z 2.5az53.7 40.1 Old branches with new growth 15.7a 9.7b Old branches that originated 1 new branch 13.4a z 8.4b z83.8 87.5 >1 new branch 2.3a y 1.4ay16.2 12.5 z Means of 12 replications. Means within rows not followed by the same letter (a, b) are significant at P 0.05 (Fishers Least Significant Difference). y Means of 12 replications. Means within columns not followed by the same letter (z, y) are significant at P 0.05 (Fishers Least Significant Difference). 101

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Table 5-4. Single stem and internode length (cm) of new branches produced during 100 days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009. Well-irrigated plants Moderately-stressed plants Single stem length Determinate growth 1st order terminal branch 16.1az zy 13.6b y 1st order lateral branch 13.9b y 20.1a z 2nd and 3rd order branch 14.4a zy 14.6a y Indeterminate growth 1st order terminal branch 58.9a z 73.0a z 1st order lateral branch 69.5a z 70.0a z Internode length Determinate growth 1st order terminal branch 3.2a z 3.0a y 1st order lateral branch 2.9b y 3.7a z 2nd and 3rd order branch 2.6a x 2.5a x Indeterminate growth 1st order terminal branch 3.9a z 3.7a z 1st order lateral branch 3.8a z 3.8a z z Means of 12 replications. Means within rows not followed by the same letter (a, b) are significant at P 0.05 (Fishers Least Significant Difference). y Means of 12 replications. Means within columns not followed by the same letter (z, y) are significant at P 0.05 (Fishers Least Significant Difference). Table 5-5. Total length (cm) of new branc hes produced during 100 days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009. Treatment % of total Well-irrigated plants Moderatelystressed plants Well-irrigated plants Moderatelystressed plants Total 583.83 az 440.9b 100.0 100.0 1s t order terminal branch 134.9 a yy 145.8az23.1 33.1 1s t order lateral branch 269.1 a z 192.7az46.1 43.7 2n d and and 3r d order branch 179.8 a zy102.5az30.8 23.2 z Means of 12 replications. Means within rows not followed by the same letter (a, b) are significant at P 0.05 (Fishers Least Significant Difference). y Means of 12 replications. Means within columns not followed by the same letter (z, y) are significant at P 0.05 (Fishers Least Significant Difference). 102

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Table 5-6. Number of stem s and leaves per stem per pl ant produced during 100 days of undisturbed growth of L. japonicum grown in large rhizotrons in 2009. Well-irrigated plants Moderately-stressed plants Stems per plant Determinate growth 1st order terminal branch 6.4 azyy5.5az 1st order lateral branch 9.1 a zy 3.8 b z 2nd and 3rd order branch 11.4 a z 7.0 a z Indeterminate growth 1st order terminal branch 0.3 a y 1.0 a z 1st order lateral branch 2.1 a z 1.6 a z Leaves per stem Determinate growth 1st order terminal branch 10.0 a z 9.0 a y 1st order lateral branch 8.9 b y 10.5 a z 2nd and 3rd order branch 10.8 a z 11.2 a z Indeterminate growth 1st order terminal branch 30.5 a z 39.6 a z 1st order lateral branch 37.1 a z 37.6 a z z Means of 12 replications. Means within rows not followed by the same letter (a, b) are significant at P 0.05 (Fishers Least Significant Difference). y Means of 12 replications. Means within columns not followed by the same letter (z, y) are significant at P 0.05 (Fishers Least Significant Difference). 103

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Figure 5-1. Organs of L. japonicum at different growth stages. A) Apex buds terminal bud, TBu; accessory buds, AcB; and lateral buds, LBu. B) Old terminal bud within apex buds. C) Leaf Lateral bud. D) Left and right panels, respectively, shows expanding bud at beginning and later on expansion. E) Quiescent root tip, QR, and growing r oot tip, GR. F) Neoform ed bud at the beginning of setting, left, and later on bud setting, right. 104

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Figure 5-2. Diagram showing br anch orders and bud positions of L. japonicum 105

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Figure 5-3. Different organs of L. japonicum. A) Quiescent lateral root with root hairs. B) Old terminal bud with excised scales to expose leaf primordia, LP. C) Longitudinal cut of old terminal bud and lateral bud, LBu. D) Fresh longitudinal cut of apex buds. E) Longitu dinal cut of a growing root tip. F) Fresh longitudinal cut of a growing root tip, left, and quiesce nt root tip, right. 106

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Figure 5-4. Number of new leaves and leaf area of L. japonicum at variable volumetric water content (2 day irrigation cycle, well-irrigated plants and 7 day irrigation cycles, moderately-stressed plants) for 2009 plants. DAT, days after transplant. 107

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Figure 5-5. Growth patterns of L. japonicum grown in rhizotrons at constant high substrate volumetric water content (well-irrigated plants) in 2008. DAT, days after transplanting. Vertical axes are RGP (root growing points, broken line) and SGP (shoot growing points, continuous line). Vertical axes scale is on the left. A-08 pattern of growth: graphs in the left column and B-08 pattern of growth: graphs in the right. Each graph represents one plant replication. 108

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Figure 5-6. Growth patterns of L. japonicum grown in rhizotrons at variable volumetric water content (2 day irrigation cycle, WP) for 2009. DAT, days after transplanting. Vertical axes are RGP (root growing points, broken line) and SGP (shoot growing points, continuous li ne). Vertical axes scale is on the left. D-09 pattern of growth: graphs in the left column and C-09 pattern of growth: graphs in the right. The top th ree graphs in each column are for plants harvested during root flush. The bottom three graphs in each column are for plants harvested during shoot flush. Each graph represents one plant replication. 109

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Figure 5-7. Growth patterns of L. japonicum grown in rhizotrons at severe stress, left column, and moderate stress, right colu mn (severely-stressed and stressed plants, respectively) in 2008. DAT, days after tr ansplanting. Vertical axes are RGP (root growing points, broken line) and SGP (shoot growing points, continuous line). Vertical axes scale is on the left. Each graph represents one plant replication. 110

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111 Figure 5-8. Growth patterns of L. japonicum grown in rhizotrons at variable volumetric water content (7 day irrigation cycle, stressed plants) for 2009. DAT, days after transplanting. Vertical axes are RGP (root growing points, broken line) and SGP (shoot growing points, continuous line). Vertical axes scale is on the left. The top three graphs in each column are for plants harvested during root flush. The bottom three or two graphs in each column, respectively are for plants harvested during shoot flus h. Each graph represents one plant replication.

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CHAPTER 6 PATERNS OF FREE AMINO ACIDS AND NON-STRUCTURAL CARBOHYDRATES ASSOCIATED WITH EPISODICAL GROWTH OF Ligustrum japonicum Thunb Introduction The above-ground portion of a plant has the primary function of energy capture, while below-ground roots primary purpose is absorption of water and ions. Thus, equilibrium between the size of shoot and root systems has to be maintained for optimum growth. In the quest to identify the mechanism that maintains this equilibrium, Thornley (1972) hypothesized that episodic growth in plants was controlled by the ratio of total carbon to total nitrogen. Subseque nt research has compiled substantial evidence for this hypothesis (Campagna and Margolis 1989; Ericsson 1995), and the general concept of carbon-to-nitrogen ratio has been proposed to be refined from total carbon (C) and total nitrogen (N) to their labile forms (Campagna and Margolis 1989; Lalonde et al. 2004; Saarinen 1998). Ca mpagna and Margolis (1989) found a strong correlation (r > 0.90) between total nonstructura l carbohydrate to free amino acid ratio (tnc:faa) and carbon allocation to shoots and roots. When tcn:faa was high, rates of protein synthesis and other metabolic proce sses were affected fa voring root growth. When tnc:faa was low, shoot growth was fa vored. For this reason we appraised C as total nonstructural carbohydrates (tnc ) and N as free ami no acids (faa). Signaling effects are a plant growth regulator-like infl uence, thus they can be active in small levels and affect gene expr ession. These effects can include up or downregulation of key genes. Much is known about the use of sugars for signaling. A thorough review of sugar signaling was comp iled by Koch (2004). Trouverie et al. (2003) found a strong correlation between absci sic acid and vacuolar invertase in response to water stress. Vacuolar invertases are active in most expanding tissues, and 112

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are involved in sugar sensing (Koch 2004), activating genes such as for sugar storage and osmoregulation (Sturm 1999). In animals, amino acid derivates are important signaling compounds (Forde and Walch-Liu 2009; Lalonde et al. 2004). Possibly plants have developed similar sensors to assess substrat e availability, thus specific receptors have been investigated (Forde and Walch-Li u 2009; Lam et al. 1998). However, N signaling is not as well understood as C signaling in plants. The N-products that act as signals have not been clearly indent ified. Glutamine (Gln) and glutamate (Glu) are usually in equ ilibrium with asparagine (Asn) and aspartate (Asp). These four amino acids are the ma jor amino acids accumulated and transported in many plants species and have been hypothesized to function as signals. Glutamate applied externally to root tips caused inhi bition of growth (Sivaguru et al. 2003) and inhibited lateral root formation and out growth (Walch-Liu and Forde 2007). Nitrate counteracted Glu, thus stimulating root branching and growth of the main root. External nitrate and Glu were proposed to interact to modulate root growth (Forde and Walch-Liu 2009). However, internal Glu concentrations have not been reported to cause the same effects. Excessive external concentrations of one amino acid in relation to others can inhibit growth, with the only exception being Gln, which has the ability to counteract growth inhibition caused by the excessive c oncentrations of other amino acid (Singh and Shaner 1995). An example is Valine-mediated inhibition to growth due to isoleucine (Iso) starvation (Bonner and Jensen 1997). In shoots, water stress induced Bermuda gra ss to increase proline (Pro), Asn, and Valine (Val), while Glu and alanine (Ala) levels decreased (Barnett and Naylor 1966). Proline, Val and threonine (Thr) concentration increased in Pisum sativum in response 113

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to water stress (Charlton et al. 2008). Proline and amides have been suggested to function as a storage compound during water stress (Barnett and Naylor 1966; Charlton et al. 2008). Biosynthesis of branched chain amino acids [Val, leucine (Leu) and Iso] occurs primarily in young tissues. Expre ssion of the various genes in the branched chain amino acid pathway may vary in the different organs (Sin gh and Shaner 1995). This group of amino acids, especially Val, appears to be related to drought tolerance in grasses, Cynodon dactylon and Zoysia japonica (Barnett and Naylor 1966; Carmo-Silva et al. 2009), wheat, Triticum aestivum (Del Moral et al. 2007), alfafa, Medicago sativa (Girousse et al. 1996), and kiwifruit, Actinidia deliciosa (Milone et al. 1999). Intensity of remobilization and use of reserves in woody species may be different than that observed in herbaceous plants. Furthermore, labile C and N pools in perennial species are maintained not only by root abs orption and shoot production, but also by break down of reserves as well. Sala un, et al. (2005a) observed that in Ligustrum ovalifolium, prior to bud break arginine, and then later, glutamine accounted for the principal components of N mobilization. Water-stressed leaves tended to maintain solu ble sugars at similar levels to those of non-stressed leaves, despite declining rates of carbon assimilation. This was due to starch degradation, which drastically reduced starch reserves (Chaves and Oliveira 2003). Roots store carbohydrat es, protein and non-protein co mpounds (Bollmark et al. 1999). Nutrient reserves are important for t he first flush of growth of the season. A better understanding of physiological processes involved in shoot and root growth will allow more precise and efficient managemen t and application of fertilizer and water. 114

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Such information is required for efficient pr oduction of woody plants in nurseries and to promote rapid establis hment in a landscape. There were three main objectives of this research. The first was to elucidate the relationship between the total nonstructural ca rbohydrate (tnc), free amino acid (faa), their ratio (tnc:faa) and the shoot and root growth of L. japonicum The second objective was to determine how water stress influenced these variables. The third objective was to identify, if present, specific amino acids and/or sugars t hat may function as signals for growth initiation in roots and shoots as a topic for future research. Materials and Methods Growth conditions and experimental design Twenty-four star-shaped rhizotrons were c onstructed as described in Chapter 2 (Figure 2-3). Briefly, each rhizotron had four arms and held 0.16 m3 of substrate. They were 1.76 m across, 0.30 m deep at the end of each arm and 0.35 m deep in the center. Rhizotrons resided in an open-side greenho use with a double polyethylene roof, under natural light, located in Apopka, FL. Clonal Ligustrum japonicum plants were selected from a local nursery (Jons Nursery, Eu stis, FL) to ensure homogeneous size and health. One plant was transplanted into each rhizotron using a commercial substrate composed of Canadian sphagnum peat moss, pr ocessed pine bark, perlite, vermiculite, starter nutrients, wetting agents, and dolomitic limestone (Mix #4, Conrad Fafard Inc., Agawan, MA). Irrigation was supplied using a spray stake (model green 22500-001120, Netafim Irrigation, Inc., Fresno, CA) at the tip of each arm pointing inward. Plants grown in 3.8-L containers were transplanted into each rhizotron in February 2009 and grown for 138 or 159 days. Entire rhizotrons were irrigated daily until saturation for 41 days for plant acclimatization. Thereafter, irrigation was withheld for six 115

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days before the start of irri gation treatments. Treatments consisted of two irrigation frequencies, once a week for 10 minutes (12 L of water, moderat ely-stressed plants) and every other day for 5 minutes (6 L of water, well-irrigated), with 12 replications each. These two treatments resulted in volume tric water content (VWC) of 22.33% and 19.8% in the driest periods (Chapter 4, Table 4-1). Growth measurements Growth of roots and shoots were monitor ed weekly for both experiments. Growth was not disturbed by pruning duri ng experiments. A root growing point was defined as a root tip with visual characteristics of active growth (Figure 5-1E, lig ht color of the root cap, with division and elongati on zones, and no visible root hai rs). A shoot growing point was defined as a shoot tip between visual post-quiescence bud break and no new unfolding of leaf (Figure 5-1D). The cumula tive number of root growing points (RGP) visible at the eight sides of a rhizotron and the cumulative number of total shoot growing points (SGP) was counted weekly throughout the duration of the experiment. Plants were harvested twice, at 138 and 159 DAT (da ys after transplant). Six plants from each treatment we re harvested at root flush (increasing RGP) and six at shoot flush (increasing SGP). The number of RGP and SGP was recorded up to 126 and 157 DAT, for root flush and shoot flush, respectively. Biochemical analyses Just prior to harvest, each plant was sa mpled for biochemical analyses of five tissues and xylem fluid was extracted. Tiss ues sampled were: growing shoot tip (GS, 1 cm, 5 per sample, Figure 5-1D); neoform ed bud (NB, 5 per sample, Figure 5-1F); dormant terminal bud (DB, 5 per sample, Figure 5-1B); growing root tip (GR, 1 cm, 20 per sample, Figure 5-1E); and quiescent r oot tip (QR 1 cm, 20 per sample, Figure 116

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5-1E). Tissues were immediately sliced in small sections, placed in microcentrifuge tubes, frozen with dry ice, and then stored at 70o C. Later, samples were freeze-dried for 5 hours and weighed. Xylem fluid was ex tracted with a pressu re chamber (Plant Water Status Console, Series 3000, Soil mo isture Equipment Corp., Santa Barbara, CA) following methodology described by An dersen (Andersen et al. 1993), with the exception that fluid was collected with 0.2 MPa over the balance pressure for 60 seconds. Xylem fluid was extracted between 1100 to 1400 hours EDT. Shoots were severed at the soil line and root s were cleaned free of substrat e using the root separator described in Chapter 3. Plant components for dry mass measurements were dried at 65o C, until constant dry mass was observed. To calculate root-to-shoot ratio, dry mass of roots was divided by t he dry mass of shoots. Tissue samples were ground with a homogen izer mixer (SDT-1810 Tissumizer, Tekmar, Cincinnati, OH) in 10 mL of 80% ethanol (v/v), boiled in wa ter bath for 5 min, cooled in an ice bath for 5 min, then centri fuged for 15 min at 7000 rpm (IEC clinical centrifuge, Damon, Needham He ights, MA). Supernatant was decanted and the pellet was re-extracted twice. Pellets were dried overnight in the oven with forced air at 70o C for starch analyses. Combined supernatant was concentrated overnight in an oven with forced air at 35o C to about 1 mL, re-suspended in di stilled deionized water, filtered through a 0.22 m nitrocellulose membrane, and subdivided for carbohydrate and amino acid analyses. Starch was analyzed by an enzymatic method (Beeson and Proebsting 1988). A pellet was dispersed in 0.1 M acetate buffer, pH 4.8 and reconstituted amyloglucosidase (1 mg/mL of acetate buffer) was added to each tube for starch digestion at 55o C for 1 117

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hr (5 parts of sample/1 part of enzyme) After 5 min of cent rifugation, 1mL of supernatant was mixed with 2 mL of Glucose Reagent (part number 1076-250, Stanbio Laboratory, Boerne, TX ) and incubated at 37o C for 15 min. Absorbance of samples and standard curve was determined at 500 nm with a Lambda 2 UV/Vis spectrometer (Perkin Elmer, Norwalk, CT). Starch content was quantified with a standard curve derived from (starch solution, 1 wt%, Sigm a Aldrich Inc.), from 0 to 2 mg. Carbohydrates were analyzed by high per formance liquid chromatography (HPLC, Binary LC Pump 250, Perkin Elmer) coupled with a refractive index detector (Series 200a, Perkin Elmer) and a Supelcogel K column (Sigma Aldrich Inc., Bellefonte, PA). Carbohydrate separation was accomplish ed with constant gradient of 15 mmol/L potassium phosphate, at a flow rate of 0. 5 mL/min, and column temperature of 85o C. The subsample destined for amino acid analysis was diluted with 1 mL of 1 N HCl and passed through two columns in series. The top column contained 4 cm (after draining) of polyvinylpyrrolidine (PVPP, Si gma Aldrich Inc.) suspended in deionized water. The bottom column contained activat ed Dowex-50W (Sigma Aldrich Inc.). The Dowex-50W was triple-rinsed with deionized water and activated with 4 volumes of 2.5 N HCl, then rinsed with deionized water to remove excessive HCl. The Dowex was layered to a 2-cm drained depth in the bo ttom column. The sample and a 1 volume rinsate of the 0.1 N HCl was placed on top of the PVPP and eluded with 5 ml of 0.1 N HCl onto the Dowex column. The PVPP column was removed and the Dowex column washed with 5 ml of 80% ethanol. Amino acid s were then eluted with 4 mL of a 0.2 M ammonia hydroxide soluti on and evaporated to drynes s under an air stream. The residue was dissolved in 0.1 N HCl for amino acid derivatization. 118

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Free amino acids were analyzed by HPLC (Binary LC Pump 250, Perkin Elmer, connected to a Series 200 Uv/Vis detecto r, Perkin Elmer) using pre-column derivatization with phenylisothi ocyanate (PITC, Pierce, Rockford, IL). Separation of individual amino acids was accomplished with a Nova-Pak C18 reverse phase column (4 m, 3.9 x 300 mm, Waters, Milford, MA) at 38o C connected to a Nova-pak C18 Sentry guard column (Waters, Milford, MA). Tissue extracts were freeze-dried and dissolved in 100 L of ethanol:triethanolamine:water (2:2 :1, v), then freeze-dried again. Derivatization was accomplished by adding 30 L of ethanol:water:t riethanolamine:PITC (7:1:1:1, v) under a N2 atmosphere in the dark. After 20 min at room temperature, derivatization was complete and samples were freeze-dried overnight. Derivatized samples were then diluted to 1 mL in 5.75 mM mono sodium phosphate and 5% acetonitrile and filt ered through a 0.22 m nitrocellulose membrane prior to injection into the HPLC system. The injection volume was 20 L. The mobile phase consisted of a timed gradient of eluent A and eluent B, with a flow-rate of 1.0 mL/min. Eluent A was 0.14 m sodium acetate, 0.5 mL/L TEA, at pH 6.4. Eluent B was 60% acetonitrile. The eluent profile began with 93% eluent A, then proceeded as follows, with the time to change from one concentration to next fo llowed by the beginning and ending concentrations: 0.5 min, 93-92% eluent A; 0. 5 min, 92-87% eluent A; 2.5 min, 87-83% eluent A; 8 min, 83-59% eluent A; 2 min, 5954% eluent A; 2.5 min, 54-0% eluent A; 4 min, 0% eluent A; 3 min, 0-93% eluent A; and 4 min, 93% eluent A. Quantities of soluble sugars and free amino acids were calculated from peak areas integrated by the TotalChrom software, version 6.3.1 (Perkin Elmer). Calibration curves for sugars were based on five concent rations of D-(-)-fruct ose, D-(+)-glucose, 119

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sucrose, D-mannitol, stachyose, D-(+)-raffi nose (Sigma Aldrich Inc.), and myoinositol (Caisson Laboratories, Inc., North Logan, Ut ah). Calibration curves for amino acids were based on five concentrations of L-alani ne, L-arginine, L-aspar tic acid, L-cystine, L-glutamic acid, glycine, L-histidine, L-isol eucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-prolin e, L-serine, L-threonine, L-ty rosine, L-valine, L-asparagine, L-glutamine, and L-tryptophan (Sigma Aldrich Inc.). A calibration curve was developed for eac h amino acid and carbohydrate using regression analysis (Microsoft Office Excel 2007, Microsoft Corp. Redmond, WA) of the area under each peak. Calibration equations were used to calculate the metabolite concentration using the area under each peak detected by HPLC. To characterize effects of water stress, each metabolite concentration was analyzed as a 2 x 2 x 5 factorial, with irrigation frequency, harvest and tissue as treatments, respectively, with six replications. To distinguish between conc entrations at shoot tip tissues, metabolites were analyzed separately as a 2 x 3 factoria l, with harvest and tissue as treatments with six replications. Root tip tissues metabolit e concentrations were analyzed similarly as a 2 x 2 factorial, with harvest and tissue as tr eatments with six replic ations. Xylem fluid metabolite concentrations were analyzed as a 2 x 3 factorial and 2 x 2 factorial, for shoot and root tissues respectively, with harvest and tissue as treatments with six replications. All statistical analysis we re conducted using SAS (version 9.1, SAS Institute, Cary, NC), with the exception of correlations between sampled tissue and concentrations of metabolites, which were calculated using JMP (version 8.0, SAS Institute, Cary, NC). 120

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Results and Discussion Patterns of faa and tnc under well-irrigated conditions Plant growth was quantified by the number of growing points. To distinguish between effects of tnc and faa in shoot versus root growth, plants were harvested in two episodes: 1) root flush, increasing RG P and decreasing SGP; and 2) shoot flush, increasing SGP and decreasing RG P. Within each harvest, shoot tips were sampled at three developmental growth stages, dormant bud, growing shoot tip, and neoformed buds, and root tips at two devel opmental growth stages, quiesc ent root tips and growing shoot tips. Total faa concentrations in neoformed buds were much higher than in growing shoot tips and dormant bud (Figure 6-1). Neof ormed buds were sampled at early stages of bud set, while expansion of younger leaves was still occurring. Upper leaves at this point were still heterotrophic, thus functi oning as sinks was well as neoformed buds. High sink strength likely was the cause of the high faa. Correlation between RGP and faa in neoformed buds at root flush was r = -0. 90. Thus as plants growth shifted to root growth, faa export by roots was greatly reduced and associated with shifts in shoot apical meristems to the production of bud sc ales, and thus bud set. Free amino acids in dormant bud versus RGP at r oot and shoot flush were also negatively correlated (r = 0.78 and -0.63, respectively), and associated with inhibition and delay of outgrowth of this type of bud. Apex buds and leaf lateral buds were responsible for the majority of branches produced during second flush of L. japonicum (Chapter 5). At shoot flush, faa increased in growing shoot tips and dormant buds with increases in SGP (r=0.95 and 0.79, respectively), while it decreased in growing root tips (r= -0.73). At the root tip level, this negative correlation indicates that roots were no longer the main sink. 121

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Concentrations of tnc did not vary as much as faa levels in shoot tips (Figure 6-1). At root flush tnc levels were similar among shoot growth stages. However, at shoot flush, dormant bud had lower tnc concentrations, indicating they were a weak carbohydrate sink, unlike the other two tissues at that time. Concentration of tnc in growing shoot tips and neoformed buds was positively correlated with SGP at shoot flush (r= 0.65 and 0.57, respectively). Shoot tips that were still growing during the root flush harvest were closer to setting a bud than the same tissue at a shoot flush, since harvests occurred at the beginning of a flush. Similarly, root tips that were still growing during shoot flush harvest were most probably in a declining growth phas e. If the hypothesis that low tnc:faa stimulates shoot growth is true, than in a comparison between dormant buds, growing shoot tips and neoformed buds, tnc:faa shoul d be in the order growing shoot tip< dormant bud< neoformed bud during a shoot flush harvest. This was not the order observed during shoot flush (Figure 6-1). Gro wing shoot tips had a significantly higher tnc:faa than neoformed buds. However, t he correlation between tnc:faa and SGP was negative for growing shoot tips at shoot fl ush (r = -0.63) and pos itive for neoformed buds at root flush (r = 0.57). These two contradictory results observed with tnc:faa comparison and correlations, together with the strong positive correlation observed between RGP and faa in neoformed buds at root flush, suggests that faa rather than the tnc:faa, at the shoot tip regulat es growth or quiescence in buds. Glutamine, Asp and histidine (His) were among the amino acids that varied the most between dormant and neoformed buds at both harvests (Figure 6-2). The concentration of amino acids was much hi gher in neoformed buds compared to other 122

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two shoot tip types. These higher faa concent rations, compared with growing shoot tips were mainly due to increases in His at root flush, and in arginine (Arg), Leu, Tyr, cysteine (Cys), methionine (Met), and Val duri ng both root and shoot flush. Proline concentrations were low across shoot tissues and harvests. Glucose concentration was significantly hi gher in growing shoot tips at both harvests than dormant and neoformed buds. St achyose and raffinose were present in high concentrations in neoformed buds at root and shoot flush. Growing shoot tips had higher myoinositol, and dormant bud lower fructo se levels than other two growth stages at shoot flush (Figure 6-2). For roots, the expansion of the hypothesis in fers that a high tnc:faa stimulates root growth, thus the tnc:faa should be quiescent root tip< growing root tip at a root flush. In this case, the hypothesis held true (Figure 63). Saarinen (1998) concluded that tnc:faa in roots was a better indicator of inter nal C to N balance than the carbon-to-nitrogen ratio in a C4 species, since long-term supply of high levels of N resulted in low tnc:faa and preferred allocation to shoot. In previous studies, whole organs (leaves, shoots, or roots) were sampled for biochemical analy ses, whereas here, only the meristematic areas and surrounding tissues were analyzed. Sampling whole organs provides a more general summation of plant faa and tnc status while sampling as done here assesses the influence of compounds available at merist ematic sites. Coordination of growth is ultimately achieved in the root or shoot meristem (Chaves et al. 2003; Clark 2001). Signals are perceived by meristems, which in turns, coordinates meristem maintenance and organogenesis (Tax and Durbak 2006). 123

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Growing root tips had higher tnc than quiescent root tips at root flush (Figure 6-3). This increase was due to higher concentra tions of all nonstructural carbohydrates, except mannitol, which did not vary mu ch between root tip types (Figure 6-4). Interestingly, the less common sugars of stachyose, raffinose and myoinositol, accumulated in these tissues. Growing root tips at root flush had significantly more raffinose than at shoot flush. Conversely, my oinositol levels were doubled in quiescent root tips and growing root tips at shoot flush. Previously labeled myoinositol was incorporated into cell wall polysaccharides of growing root tips of maize (Harris and Northcote 1970). Glutamine, Val, and His were the main f aa transported in xylem fluid (Figure 6-5). Previous work found an increase in Gln in leaf tissues and xylem fluid during shoot flush of L. ovalifolum (Salaun and Charpentier 2001; Sal aun et al. 2005); Val and His were not measured in those studies. Although high concentrations of Val and His were detected in neoformed buds, Gln was not among the higher concentrations observed in shoot tip. This suggests additional amino acid biosynthesis occurred in other tissues before reaching meristematic sites, possibly in storage tissues such as trunks. An increasing gradient of Arg and Gln was found in L. ovalifolum from roots to the trunk and decreasing towards upper parts of stem (Salaun and Charpentier 2001). Free amino acids were higher in xylem fluid during a shoot flush (Figure 6-6), when faa were transported to support expand ing shoot mass. Consequently tnc:faa tended to be lower at this harvest. High mannitol concentration in xylem flui d indicates translocation of reserves (Figure 6-5), which were stored or used in tissues other than me ristems. Mannitol 124

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concentrations were relatively low in buds Carbohydrate transported in xylem fluid supports bud break in Juglans regia L. (walnut, Bonhomme et al. 2010). Carbon is stored as starch in xylem parenchyma cells and cambium of stems. After symplasmic connections are established in cells around the meristem, starch is converted to soluble sugars, which are actively transported in to xylem and then move up to support bud outgrowth. Sucrose and glucose concentration in apical buds of walnut was similar to those observed herein for L. japonicum at shoot flush. Influence of water stress in patterns of faa and tnc At root flush, concentrations of most faa in shoot tips of moderately-stressed plants (Figures 6-7) were comparable to t hose present in well-irrigated plants (Figure 6-2), thus in general, faa concentrations were not limited by water stress. Concentration of most faa in growing shoot tips of moder ately-stressed plants were higher than those of well-irrigated plants, with the most not iceable increases observed in Arg, Val, and His. Higher concentrations of faa may be a resu lt of the tendency to higher root-to-shoot ratio at root flush of moderately-stress ed plants (Chapter 4). Neoformed buds of moderately-stressed plants also had significantly more Val. The exceptionally high Val concentration found in neoformed buds of moder ately-stressed plants may be explained by requirements of surrounding young exp anding leaves and bud scales to produce cuticle as a dehydration defense. Conc entrations of faa in dormant bud of moderately-stressed plants were not affected by water st ress. Although water stressinduced Pro increases were cited in the liter ature for other species (Barnett and Naylor 1966; Charlton et al. 2008), Pro concentration s were at stable levels across treatments here. 125

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At shoot flush, expanding shoots were the primary sink. As a consequence of sink strength, growing shoot tips of moderately-stressed plants had similar levels of faa as growing shoot tips of well -irrigated plants (Figures 6-7 and 6-2). Between treatments, Asp concentration was somewhat lower in gr owing shoot tips of water-stressed plants, but was significantly higher in neoformed buds. Water stress also lowered Val and Arg concentrations in neoformed buds. As previously described for leaves (Chaves and Oliveira 2003), shoot and root tips of moderately-stressed plants maintained similar of tnc levels as well-irrigated plants (Figure 6-1 x 6-7 and 6-3 x 68). Although raffinose family oligosaccharides have been proposed to counteract dehydration (Bogdan and Zagdanska 2006; Brenac et al. 1997), neither raffinose nor its precursor myoinositol increased due to water stress in a grass species (Amiard et al. 2003). Raffinose concentrations did not differ between moderately-stressed and we ll-irrigated treatments in L. japonicum As observed for well-irrigated plants, growing root tips at root flush had significantly more raffinose than at shoot flush in moderately-stressed treatment. This trend also occurred for stachyose in growing root tips of m oderately-stressed plants, which had greater concentrations of this oligosaccharide at root flush. Moreover, myoinositol le vels were also higher in quiescent root tips and growing root tips at shoot flush for moderately-stressed plants as well as for well-irrigated plants. Carbohydrates can be stored in trunks and also in roots, which could then support post-transplant root growth, before shoot growth, if soil temperature is favorable. Storage carbohy drates can also be important for waterstressed plants, since photosynthesis rates are diminished by dr ought due to stomatal closure over the long term. Previous studi es found that leaves continue to export 126

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C-assimilated after shoot elongation stops (Dickson et al. 2000a; Sloan and Jacobs 2008). Conclusions After a shoot flush, as plant growth shi fted to root growth, a strong correlation was found between RGP and faa concentration in neoformed buds, thus suggesting smaller faa concentration at the shoot tip caused shoot apical meri stem to set a bud. While after a root flush, with the switch to main ly shoot growth, faa concentration in growing shoot tips and dormant buds increased with increases of SGP, while in growing root tips it decreased with increases in SGP, thus suggesting higher faa concentrations on growing shoot tips and dormant bud stimulated growth. Conc entrations of tnc in shoot tips, did not vary as much as faa concentra tions. Moreover, contrary to expectations, growing shoot tips had a significantly highe r tnc:faa than neoformed buds earlier in the shoot flush. These observations lead to the c onclusion that faa levels at the shoot tip are more decisive to meristem growth/quiescence control than the tnc:faa or tnc level by itself. In roots, tnc:faa obs erved was as predicted (growing root tips having a higher tnc:faa than quiescent root tips ). This is in agreement wit h previous observations that this indicator at the root level better predicted plant growth than when measured in shoots (Saarinen 1998). Of thes e, Gln is suggested as the principal candidate for N signaling for initiation of bud break. It wa s transported in t he greatest quantity and varied the most between shoot and root flush (Figure 6-3). Additionally, concentrations measured at the beginning of shoot flush were proportional to the am ount of bud break for well-irrigated and moderately-stressed treatments. While Gln was principal transported faa concentrations in shoot tips were not high, suggesting it was rapidly metabolized. Mannitol was also found in la rge quantities in xylem fluid, indicating 127

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128 translocation of reserves. Yet since bud break was hypothesized due more to faa concentration than tnc:faa, t he importance of high mannitol quantities is questionable. Water stress resulted in increases in the concentration of some amino acids in growing shoot tips, such as Arg, Val, and His. Neoformed buds of moderately-stressed plants had significantly more Val than neofo rmed buds of well-irrigated plants. The exceptionally high Val conc entration found in neoformed buds of moderately-stressed plants may be explained by requirements of surrounding young expanding leaves and bud scales to produce cuticle as a dehydration defense. Valine is among the precursors of coenzyme A, which is a precursor of leaf cuticular components (Weng et al. 2010). As observed for well-irrigated plants, growing root tips of moderat ely-stressed plants at root flush had higher concentrations of ra ffinose, stachyose and myoinositol than quiescent root tips, and in much higher le vels than shoot tip tissues. Carbohydrate storage in roots can be an important source of C-skeletons especially in water stressed plants, to support root grow th after transplant. Precise management and application of fertilizers and water prior to transplant into a landscape could be essential to shortening establishment time and improving survival rates.

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Figure 6-1. Total nonstructural carbohydrates, free amino acids, and total nonstructural carbohydrate-to-free amino acid ratio of L. japonicum shoot tissues of plants grown in rhizotrons at variable volumetr ic water content (2 or 7 day irrigation cycles, well-irrigated and moderately-st ressed plants, respectively). ( ) indicates significant difference among tissues ( P 0.05, Fishers Least Significant Difference). 129

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Figure 6-2. Free amino acids and nonstructural carbohydrates of L. japonicum shoot tissues of plants grown in rhizotrons at variable volumetric water content (2 day irrigation cycles, well-irrigated plants). Amino acids which did not vary between tissues or treatments, and were in low levels were omitted for figure clarity. Standard abbreviations used fo r free amino acids. Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc; stachiose, stac; raffino, raff; and myoinositol, inos. ( ) indicates significant difference among tissues ( P 0.05, Fishers Least Significant Difference). 130

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Figure 6-3. Total nonstructural carbohydrates, free amino acids, and total nonstructural carbohydrate-to-free amino acid ratio of L. japonicum root tissues of plants grown in rhizotrons at variable volumetr ic water content (2 or 7 day irrigation cycles, well-irrigated and moderately-st ressed plants, respectively). ( ) indicates significant difference among tissues ( P 0.05, Fishers Least Significant Difference). 131

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Figure 6-4. Free amino acids and nonstructural carbohydrates of L. japonicum root tissues of plants grown in rhizotrons at variable volumetric water content (2 day irrigation cycles, well-irrigated plants). Amino acids which did not vary between tissues or treatments, and were in low levels were omitted for figure clarity. Standard abbreviations used fo r free amino acids. Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc; stachiose, stac; raffino, raff; and myoinositol, inos. ( ) indicates significant difference among tissues ( P 0.05, Fishers Least Significant Difference). 132

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Figure 6-5. Free amino acids and nonstructural carbohydrates of L. japonicum xylem fluid extracted from plants grown in rh izotrons at variable volumetric water content (2 or 7 day irrigation cycles well-irrigated plants or moderatelystressed plants, respectively). Amino acids which did not vary between tissues or treatments, and were in low le vels were omitted for figure clarity. Standard abbreviations used for free amino acids. Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc; stachiose, stac; raffino, raff; and myoinositol, inos. ( ) indicates significant difference among harvests ( P 0.05, Fishers Least Significant Difference). 133

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Figure 6-6. Total nonstructural carbohydrates, free amino acids, and total nonstructural carbohydrate-to-free amino acid ratio of L. japonicum xylem fluid extracted from plants grown in rhizotrons at vari able volumetric water content (2 or 7 day irrigation cycles, well-irrigated pl ants and moderately-stressed plants, respectively). ( ) indicates significant difference among harvests ( P 0.05, Fishers Least Significant Difference). 134

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135 Figure 6-7. Free amino acids and nonstructural carbohydrates of L. japonicum shoot tissues grown in rhizotrons at vari able volumetric water content (7 day irrigation cycles, moderat ely-stressed plants). Amino acids which did not vary between tissues or treatments, and we re in low levels were omitted for figure clarity. Standard abbreviati ons used for free amino acids. Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc; stachiose, stac; ra ffino, raff; and myoinositol, inos. ( ) indicates significant difference among tissues ( P 0.05, Fishers Least Significant Difference).

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Figure 6-8. Free amino acids and nonstructural carbohydrates of L. japonicum root tissues grown in rhizotrons at vari able volumetric water content (7 day irrigation cycles, moderately-stressed plants). Amino acids which did not vary between tissues or treatments, and we re in low levels were omitted for figure clarity. Standard abbreviati ons used for free amino acids. Nonstructural carbohydrates: sucrose, suc; manitol, man; glucose, gluc; fructose, fruc; stachiose, stac; ra ffino, raff; and myoinositol, inos. ( ) indicates significant difference among tissues ( P 0.05, Fishers Least Significant Difference). 136

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CHAPTER 7 SUMMARY AND CONCLUSIONS To conduct this research, a system was required that would enable control of substrate moisture, provide easy root observation and sampling, and be of sufficient size such that root growth was not impeded. Several designs for a star-shaped rhizotron were developed and evaluated with the threepart goal of: 1) adjusting volume and shape for minimal physical restriction and use with mature woody plants; 2) developing a drainage system comparable to natural so ils; and 3) facilitating ease, accuracy and duration of data acquisition. The final design allowed exceptional r oot observation, used a wick-type drainage system to provide a nea r-uniform profile of soil moisture, and was easily manageable for precise long-term dat a acquisition. This rhizotron had eight independent viewing/sampli ng windows and held 0.16 m3 of soil. An associated camera positioning frame developed especially for these rhizotrons facilitated digital photographs of the soil profiles for time series a ssessment of morphological and architectural parameters. The camera positioning frame was compact, light and effective in small spaces. After months of growth, plants were harve sted for shoot and root mass. Isolation of plant roots from soil or subs trate for biomass measuremen t is time consuming and can be a limiting factor influencing experimental designs especially with mature woody plants. An electric powered root separator was developed that sped sample preparation for root dry mass determination with a capacity of 40 L of container substrate or 32 kg of sandy soil. No water was required and a four-f old reduction in total processing time was achieved. Extent of root recovery was quantif ied by processing five woody plant species grown in two different substr ates and in soil, resulting in a minimum yield of 98%. 137

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To evaluate effects of water stress on growth and identify probable biochemical components that actuate thes e water stress effects, Ligustrum japonicum plants of landscape size were exposed to varying regi mes of substrate water availability: 1) constant high levels of plant available water, 2) severe stress, 3) constant low levels of plant available water, 4) constant non-moistu re limiting, 5) simulated natural wetting and drying cycles of two days, and 6) simulat ed natural wetting and drying cycles of seven days. This provided insight into how this woody species adjusts to its environment when exposed to various degrees and types of wate r stress. Excessive water availability resulted in marked reductions of carbon allo cation towards roots, consequently luxury shoot growth was observed. Constant or in termittent moderate wate r stress resulted in total biomass reductions of approximately 20 %, which disproportionally reduced shoot mass, particularly diminishing leaf growth (leaf number and size). Prolonged, severe water stress caused a 40% reduction on to tal biomass. This reduction was also disproportionally greater for shoot biomass pr oduction, with relatively smaller reductions in root biomass production. Plant responses to water stress differed with time of exposure and degree of stress. Low correlati on of root-to-shoot ratio and irrigation frequency suggested that root-toshoot ratio may not be the best indicator of water status during the growi ng period of the plant. Bud outgrowth dynamics and its implications to plant architecture were examined along with the influence of relative growth of roots and shoots. Re sults indicated that neoformed buds had limited sensitivity to dormancy, while preformed buds needed more than one growing season to naturally outgrow. Following transplant into rhizotrons, the first flush of shoot growth was mostly due to leaf lateral bud outgrowth. 138

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Water stress influenced shoot architectu re by enhancing apical dominance. Lateral branching was diminished 51% in water stressed plants compared with those wellirrigated. As plants adapted to the stress im posed, indeterminate growth was triggered more often in meristematic regions of te rminal buds. At the second flush, which occurred later in the stress treatment, old buds burst more frequently than the newly formed apex lateral bud. After transplant into rhizotrons, root growth began before shoot growth. Temporal variations of moisture caused by wetting and drying cycles resulted in continuous growth for portions of the root system with quiescent periods observed for shoot growth. Conversely, continuous high moisture levels resulted in roots exhibiting quiescent periods in some plants. Large cycles of episodi c growth were not observed for most of the experimental period. However, for most surviving plants, trends of increasing root and decreasi ng shoot growth were evident near the end as plants neared balance between roots and s hoots before harvest. Patter ns of shoot and root growth varied considerably between these cl onal plants, which may be an important consideration on analyses of popul ations of woody plants. Episodical growth has been proposed to be controlled by changes in the carbon to nitrogen ratio in plants. This ratio was refined in terms of the quantities of free amino acids (faa) and total non-structural carbohydr ates (tnc) in plants and plant parts. The relationship of tnc and faa concentrations and its ratio in tissues of meristematic regions were studied with a whole plant growth approach, including roots and shoots at different growth stages. Additionally, the influence of water stress on these relationships was considered. The results observed indicates t hat faa levels at the shoot tip are more decisive to meristem growth/qui escence control than the tnc:faa or tnc level by itself. In 139

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140 roots, tnc:faa observed was as good predictor of root grow th (growing root tips having a higher tnc:faa than quiescent root tips). Vali ne, leucine, tyrosine, cystein, metionine, and arginine increased significantly with bud set in neoformed buds, compared with growing shoot tips. Root tips contained abundant fructo se, stachyose, and myoinositol. Mannitol was the major transport sugar and glutamine, valine and histidine were the main faa transported in xylem fluid. Water stress resu lted in increases in the concentration of some amino acids in growing shoot tips, such as Arg, Val, and His, especially valine in neoformed buds at root flush.

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APPENDIX DIFFERENCES BETWEEN METABOLITES ANALYZED IN CHAPTER 6 Table A-1. Differences between metabolites analyzed as 2 x 2 x 5 factorial, with irrigation fr equency, harvest and tissue as treatments described in Chapter 6. trt Tissue Harvest Alanine Arginine Asparagine Aspartate Cysteine Glutamine Glutamate Moderately-stressed plants Dormant bud Root flush 0.007bcz 0.039defg 0.001c 0.029f 0.048bcde0.004b 0.077ef Shoot flush 0.006bc 0.038defg 0.001c 0.033ef 0.040cde 0.001b 0.077ef Growing shoot Root flush 0.013bc 0.090bcde 0.000c 0.239abc 0.120b 0.004b 0.200d Shoot flush 0.003c 0.008g 0.002c 0.036ef 0.019e 0.009b 0.033f Neoformed bud Root flush 0.020abc0.204a 0.000c 0.334a 0.268a 0.051b 0.427a Shoot flush 0.014bc 0.117b 0.000c 0.281ab 0.209a 0.050b 0.306bc Quiescent root Root flush 0.007bc 0.047cdefg 0.024bc 0.048ef 0.049bcde0.043b 0.062ef Shoot flush 0.010bc 0.061bcdefg0.009bc 0.037ef 0.072bcde0.053b 0.082ef Growing root Root flush 0.013bc 0.084bcde 0.021bc 0.123cdef 0.073bcde0.059b 0.102def Shoot flush 0.011bc 0.105bc 0.106a 0.090def 0.105cb 0.083b 0.155de Well irrigated plants Dormant bud Root flush 0.010bc 0.044defg 0.003c 0.041ef 0.068bcde0.008b 0.089ef Shoot flush 0.005c 0.030efg 0.001c 0.024ef 0.031cde 0.005b 0.056ef Growing shoot Root flush 0.009bc 0.014fg 0.000c 0.114cdef 0.052bcde0.020b 0.113def Shoot flush 0.004c 0.011g 0.001c 0.071ef 0.026de 0.011b 0.052ef Neoformed bud Root flush 0.023ab 0.193a 0.000c 0.246abc 0.280a 0.073b 0.425a Shoot flush 0.023ab 0.211a 0.000c 0.223abcd0.238a 0.094ab 0.367ab Quiescent root Root flush 0.011bc 0.076bcde 0.021bc 0.046f 0.084bcde0.073b 0.099def Shoot flush 0.008bc 0.060bcdefg0.021bc 0.052ef 0.066bcde0.045b 0.061ef Growing root Root flush 0.034a 0.093bcd 0.072ab 0.165bcde0.096cde 0.179a 0.127def Shoot flush 0.014bc 0.073bcdef 0.040abc 0.098def 0.084bcde0.058b 0.081ef z Means within columns not followed by the same letter are significant at P 0.05 (Fishers Least Significant Difference). 141

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Table A-1. Continued trt Tissue Harest Glycine Histidine Leucine Methionine Proline Serine Moderately-stressed plants Dormant bud Root flush 0.000a 0.006d 0.075efg 0.032 efghi 0.005f 0.000b Shoot flush 0.001a 0.017cd 0.080efg 0.025 efghi 0.004f 0.000b Growing shoot Root flush 0.000a 0.064bc 0.055fg 0.049 cdef 0.021bcde 0.000b Shoot flush 0.001a 0.018cd 0.008g 0.006 i 0.013cdef 0.001a Neoformed bud Root flush 0.000a 0.141a 0.249ab 0.094 ab 0.004ef 0.000b Shoot flush 0.000a 0.094ab 0.222abcd 0.074 abc 0.001f 0.000b Quiescent root Root flush 0.000a 0.000d 0.126def 0.041 defg 0.016cdef 0.000b Shoot flush 0.001a 0.027cd 0.169bcde 0.073 abc 0.023bcd 0.000b Growing root Root flush 0.004a 0.000d 0.083efg 0.053 cde 0.065a 0.000b Shoot flush 0.000a 0.000d 0.125ef 0.053 cde 0.078a 0.000b Well irrigated plants Dormant bud Root flush 0.001a 0.022cd 0.100efg 0.039 defgh0.008cdef 0.001a Shoot flush 0.000a 0.018cd 0.059fg 0.017 ghi 0.006def 0.000b Growing shoot Root flush 0.000a 0.006d 0.024g 0.020 fghi 0.014cdef 0.000b Shoot flush 0.000a 0.024cd 0.010g 0.008 ih 0.018bcdef0.001ab Neoformed bud Root flush 0.000a 0.121a 0.295a 0.105 a 0.000f 0.000b Shoot flush 0.000a 0.141a 0.235abc 0.081 abc 0.011cdef 0.000b Quiescent root Root flush 0.000a 0.000d 0.160bcde 0.071 bcd 0.022bcde 0.000b Shoot flush 0.000a 0.018cd 0.146cdef 0.054 cde 0.026bc 0.000b Growing root Root flush 0.005a 0.007d 0.124ef 0.049 cdefg 0.079a 0.000b Shoot flush 0.005a 0.000d 0.094efg 0.056 cde 0.036b 0.000b 142

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Table A-1. Continued trt Tissue Harvest Threonine Tyrosine Valine Fructose Glucose Moderately-stressed plants Dormant bud Root flush 0.008def 0.025defgh 0.113 cdef 2.85gh 66.98defg Shoot flush 0.005f 0.015ghf 0.116 cdef 1.67gh 33.58j Growing shoot Root flush 0.019bcde0.022efgh 0.325 bcde 0.00h 89.70abc Shoot flush 0.003f 0.006h 0.017 f 3.49efg 104.09ab Neoformed bud Root flush 0.025bc 0.080a 0.951 a 4.61defg81.86cde Shoot flush 0.020bcd 0.059bcde 0.439 bc 1.92gh 54.67ghi Quiescent root Root flush 0.007def 0.062abcd 0.232 bcdef5.80def 37.15ij Shoot flush 0.004f 0.072ab 0.365 def 6.86def 39.34ij Growing root Root flush 0.000f 0.037bcdefgh 0.092 f 21.01c 65.02efg Shoot flush 0.004f 0.049bcdefg 0.205 bcdef27.73a 62.96efghWell irrigated plants Dormant bud Root flush 0.009def 0.030defgh 0.139 cdef 1.90gh 43.40hij Shoot flush 0.006ef 0.012hg 0.060 ef 1.61gh 44.17hij Growing shoot Root flush 0.011cdef 0.013gh 0.087 bc 0.00h 84.91bcd Shoot flush 0.005f 0.006h 0.019 f 3.77efg 105.84a Neoformed bud Root flush 0.030ab 0.081a 0.403 bc 3.65efg 75.13defg Shoot flush 0.042a 0.061abcde 0.483 b 4.25defg64.79efg Quiescent root Root flush 0.004f 0.081a 0.235 bcdef7.27d 37.73ij Shoot flush 0.002f 0.054abcdef 0.231 bcdef7.29d 40.98ij Growing root Root flush 0.000f 0.067abc 0.248 bcdef24.10bc 67.91defg Shoot flush 0.000f 0.050bcdefg 0.229 bcdef25.79ab 60.97fgh 143

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Table A-1. Continued trt Tissue Har Inositol Mannitol Raffinose Stachyose Sucrose Starch Moderately-stressed plants Dormant bud Root flush 0.53ghi 17.65defg3.08ef 1.64efgh 7.91def 0.25cd Shoot flush 0.07i 10.65g 2.37f 1.24h 4.76fgh 0.48cd Growing shoot Root flush 0.00i 24.09d 0.00f 0.00h 17.16a 0.00d Shoot flush 2.08fghi 20.96de 1.38f 0.27h 6.67efg 1.84a Neoformed bud Root flush 0.51ghi 23.55d 7.97cde 3.52defgh 13.12b 0.00d Shoot flush 0.00i 17.96defg8.83cd 6.04cdef 9.46cde 0.00d Quiescent root Root flush 2.73defg35.06c 2.15f 6.29cde 2.04hij 0.15cd Shoot flush 4.53de 37.71c 4.47def 7.35cd 4.15ghi 0.14cd Growing root Root flush 9.55c 38.59c 35.68a 19.82b 0.00j 0.88bc Shoot flush 17.21b 51.31a 23.13b 18.40b 2.20hij 0.44cd Well irrigated plants Dormant bud Root flush 0.25hi 12.62fg 2.50f 1.31gh 6.48efg 0.47cd Shoot flush 0.36hi 13.51efg 2.81ef 1.57fgh 6.34efg 0.19cd Growing shoot Root flush 0.00i 18.41defg0.00f 0.00h 12.58bc 0.72bcd Shoot flush 2.33efgh20.29def 1.01f 0.00h 7.40defg 1.81a Neoformed bud Root flush 0.57ghi 21.73d 10.63c 4.58defgh 12.25cb 0.00d Shoot flush 0.00i 20.07def 9.49cd 5.99cdefg 10.41bcd 0.00d Quiescent root Root flush 3.87def 32.89c 2.23f 6.38cd 2.31hij 0.12cd Shoot flush 4.60d 36.21c 5.01def 10.30c 4.71fgh 0.19cd Growing root Root flush 10.39c 38.90bc 37.04a 25.08a 0.67ij 1.29ab Shoot flush 19.49a 46.59ab 21.00b 16.61b 1.51hij 0.35cd 144

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145 Table A-1. Continued trt Tissue Harvest Free amino acids Nonstructural carbohydrates Nonstructural carbohydrateto-free amino acid ratio Moderately-stressed plants Dormant bud Root flush 0.47fghi 100.88cdefg223.60cdef Shoot flush 0.46fghi 54.83h 147.91defg Growing shoot Root flush 1.22cde 130.95bcd 113.09efg Shoot flush 0.18i 140.78b 749.45a Neoformed bud Root flush 2.85a 135.14bc 57.31g Shoot flush 1.89bc 98.88defg 52.43g Quiescent root Root flush 0.76defghi 91.37efg 132.33fge Shoot flush 1.06defg 104.56cdef 112.20fge Growing root Root flush 0.81defghi 190.56a 258.98dc Shoot flush 1.17cdef 203.39a 215.15cdef Well irrigated plants Dormant bud Root flush 0.61defghi 68.94gh 130.56efg Shoot flush 0.33gh 70.55fgh 231.96cde Growing shoot Root flush 0.50efghi 116.62bcde 228.95cde Shoot flush 0.27hi 142.45b 538.13b Neoformed bud Root flush 2.28ab 128.55bcd 66.94g Shoot flush 2.21ab 115.00bcde 51.64g Quiescent root Root flush 0.98defgh 92.82efg 99.75fg Shoot flush 0.84defghi 109.30bcde 133.52efg Growing root Root flush 1.34cd 205.38a 176.81defg Shoot flush 0.92defghi 192.31a 314.22c

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161 BIOGRAPHICAL SKETCH Dilma Daniela Silva was born in Minas Gerais, Brazil. In 2001 she received her bachelors degree from the Universidade Feder al de Viosa, Viosa, Minas Gerais, Brazil. She worked as a research assistant in the Plant Physiol ogy Laboratory of the Department of Biological Sci ence at the North Dakota Stat e University in conjunction with the Northern Crop Sci ence Laboratory, USDA, wher e she worked with protein determination, isolation of mitochondria and peroxisomes, tissue respiration rates and other plant biochemistry analyses. In 2004 she received a Master of Science degree in plant science from the Universidade Federal de Viosa, Viosa, Minas Gerais, Brazil, with a thesis title of Ethylene sensitivit y of two varieties of geranium and 1-MCP treatment.