Development and use of an in vitro system to study the ripening physiology of strawberry fruit

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Title:
Development and use of an in vitro system to study the ripening physiology of strawberry fruit
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x, 118 leaves : ill. ; 28 cm.
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Perkins-Veazie, Penelope M., 1958-
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Subjects / Keywords:
Strawberries -- Ripening   ( lcsh )
Fruit -- Ripening   ( lcsh )
Fruit -- Physiology   ( lcsh )
Horticultural Science thesis Ph. D
Dissertations, Academic -- Horticultural Science -- UF
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Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references.
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by Penelope M. Perkins-Veazie.
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Typescript.
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Vita.

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University of Florida
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Full Text











DEVELOPMENT AND USE OF AN IN VITRO SYSTEM TO STUDY
THE RIPENING PHYSIOLOGY OF STRAWBERRY FRUIT














By



PENELOPE M. PERKINS-VEAZIE


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


1988




































TO MY HUSBAND, RAYMOND















ACKNOWLEDGMENTS


Drs. D.J. Huber, J.K. Brecht, Earl Albregts,

C.B. Hall and T.E. Humphreys have been an excellent and most

helpful committee. I am most appreciative of the

guidance given by Drs. D.J. Huber and J.K. Brecht,

whose perception and expertise have been exemplary.

Their prompt editing of my manuscripts has been a delightful

experience.

The teamwork displayed by the staff and faculty in

postharvest research have taught me invaluable and

unforgettable lessons. They have always been willing to

provide suggestions, answer questions and help explain

experimental techniques.

I also thank the staff and faculty at the Dover

Agricultural Education and Research Center, who planted and

maintained much of the plant material used in these experiments.

These people have exemplified good field production practices

and teamwork.

Special thanks are given to Dr. Chris Chase, whose loan of

the green chair provided a comfortable seat for the many hours

of contemplation of literature and research.

The help provided by Dean Jack Fry over the past few years

is most deeply appreciated.


iii


















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS........................................ iii

ABSTRACT............ ............................. .. ix

CHAPTERS

I INTRODUCTION.... .................... ........... 1

II LITERATURE REVIEW........ ............. ......... .. 4

Strawberry Fruit Growth and Ripening............ 4
Strawberry Fruit Compositional Characteristics
During Ripening............................. 11
Relationship of Respiration and Ethylene to
Fruit Ripening................................... 17
Ethylene Synthesis........................ ..... 26
Summary ......................................... 34

III DEVELOPMENT AND EVALUATION OF AN IN VITRO SYSTEM
TO STUDY DETACHED STRAWBERRY FRUIT RIPENING...... 37

Materials and Methods......................... 39
Results......................................... 43
Discussion....................................... 55
Conclusions ................................ ..... 59

IV RESPIRATION AND ETHYLENE PRODUCTION IN DEVELOPING
STRAWBERRY FRUIT GROWN IN VITRO................... 61

Materials and Methods............................ 63
Results .......................................... 67
Discussion .................................. ... 79
Conclusions..................... .. ... 83

V REGULATION OF ETHYLENE PRODUCTION IN STRAWBERRY
FRUIT......................................... ... 85

Materials and Methods............................ 87
Results............... ............ .. ........ 91
Discussion.............. .. ............. ... ... 98
Conclusions... .... ... ..... ............ .... .... 100











Page

VI SUMMARY AND CONCLUSIONS.......................... 101

LIST OF REFERENCES........................ .......s. 104

BIOGRAPHICAL SKETCH.................................. 118















LIST OF TABLES


Table Page


3-1 Relationship between the color of in vitro grown
strawberry fruit and HunterColorlab L,a,b values.... 42

3-2 Effect of age on in vitro growth of 'Pajaro'
strawberries harvested and placed in solutions at
6, 12, or 18 days after anthesis.................... 49

3-3 Rates of growth in vitro for primary, secondary
or tertiary fruit order............................. 52

3-4 Comparison of in vitro fruit weight gain and
ripening to field fruit ........... ...................... 53

3-5 Comparison of length and diameter of in vitro
and field-ripened fruit of the same anthesis date... 54

5-1 Comparison of ACC content, EFE activity and
ethylene production from strawberry fruit
harvested from the field at different color stages.. 92

5-2 Effect of STS and ACC addition to vase solutions
with strawberry fruit on ethylene biosynthesis of
fruit grown in vitro................................ 94

5-3 Comparison of ACC content, EFE activity and ethylene
production from green strawberry fruit receptacle
tissue and calyx tissue after incubation in 1 mM ACC
in vase solutions for 2 days......................... 96















LIST OF FIGURES


Figure Page


3-1 Effect of cultivar and sucrose on cumulative
fruit growth in vitro............................... 44

3-2 Effect of age and sucrose on cumulative growth
of 'Pajaro' fruit in vitro.......................... 46

3-3 Effect of fruit age at harvest on cumulative
growth in vitro..................................... 48

3-4 Effect of fruit order on cumulative growth
in vitro............................................ 51

4-1 Changes in respiration and ethylene in strawberry
fruit harvested at selected stages of development... 64

4-2 Influence of propylene on respiration (A) and weight
gain (B) of strawberry fruit harvested 20 days
postanthesis (white)....... ........... ........... 69

4-3 Effect of propylene on respiration (A) and weight
gain (B) of fruit harvested 12 days
postanthesis (green)................................ 70

4-4 Ethylene production (A), respiration (B) and weight
gain (C) of fruit harvested at 20 days postanthesis
(white) and held in vase solutions with 0 or
1 mM ACC............................................ 72

4-5 The effect of ACC on ethylene production (A),
respiration (B) and weight gain (C) of fruit
harvested at 14 days post-anthesis (green) stage
of development and placed in vase solutions......... 74


vii








4-6 Ethylene production of fruit harvested at 14 days
post anthesis (green) and placed in vase solutions
containing 0, 1, or 5 mM ACC........................ 75

4-7 Respiration (A) and weight gain (B) of fruit
harvested at 14 days post anthesis (green) and
placed in vase solutions with 0, 1, or 5 mM ACC...... 77

4-8 The effects of ACC and silver on ethylene production
(A), weight gain and color (B) of fruit harvested at
14 days post anthesis (green) and placed in vase
solutions with 0 or 1 mM ACC and/or STS.............. 78

5-1 Ethylene production from calyxes and whole fruit
harvested at the green stage after calyx removal
and treatment with 1 mM ACC.......................... 97


viii
















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

DEVELOPMENT AND USE OF AN IN VITRO SYSTEM TO STUDY
THE RIPENING PHYSIOLOGY OF STRAWBERRY FRUIT


By

Penelope M. Perkins-Veazie


April 1988



Chairman: Donald J. Huber
Major Department: Horticultural Science (Vegetable Crops)


Attempts to study and modify strawberry (Fragaria X

ananassa) fruit ripening have been limited by the continued

growth of this fruit during ripening and the failure of the

strawberry to continue to ripen once detached. The purpose

of this work was to develop a system which would permit

normal ripening of detached, immature strawberry fruit under

controlled conditions. This system was then implemented to

study the physiological changes involved in ripening.

Normal ripening of detached fruit was achieved by

placing their peduncles in a solution of 200 ul.liter-1

hydroxyquinoline sulfate plus 88 mM sucrose. 'Pajaro'

strawberry fruit harvested at 50% maturity and placed









in vitro consistently gained more weight than did fruit

harvested at 25% or 70% maturity. Fruit placed in vitro

gained less cumulative weight than those in the field, but

both in vitro and field-grown fruit ripened simultaneously.

Ethylene production and respiration trends of fruit

harvested from the field at different stages of color

development were characteristically nonclimacteric. However,

fruit maintained in a 5000 ul.liter-1 propylene atmosphere

or inclusion of 1 mM 1-aminocyclopropane-1-carboxylic acid

(ACC) in vase solutions failed to elicit an elevated

respiration rate, although weight gain and color development

were accelerated. Addition of 0.5 mM silver thiosulfate to vase

solutions suppressed fruit growth but did not delay ripening.

The ACC content of strawberry fruit changed little

throughout ripening, while ethylene-forming enzyme (EFE)

activity and ethylene production decreased as fruit developed

from the green to the white stage. Although no ACC synthase

could be detected in strawberry fruit at any stage of

maturation, the increased levels of ACC found in

silver-treated fruit provided evidence for the presence of

this enzyme. Inclusion of ACC in vase solutions increased

receptacle ACC content, stimulated EFE activity and greatly

increased ethylene production. Both EFE and ACC synthase

activities may control ethylene biosynthesis in strawberry

fruit, but active ACC synthase must be isolated from this

fruit before the mechanisms) responsible for low ethylene

production can be ascertained.














CHAPTER I
INTRODUCTION


Fruits are generally classified into two categories,

depending on their pattern of ripening. Climacteric fruit

such as bananas, avocados, apples and tomatoes exhibit

elevated respiration and ethylene production during ripening.

Other fruit types, such as grapes, watermelons and

strawberries, are nonclimacteric. As these fruit mature and

ripen, there is a downward drift in respiration rate and no

increase in ethylene production. Climacteric fruit have been

intensively studied in attempts to establish a link between

ethylene and other ripening parameters. Limited work has

been conducted with nonclimacteric fruit due to the inability

of these fruit to continue normal ripening once detached, and

the lack of ripening markers such as polygalacturonase

activity or increased ethylene production as found in

climacteric fruit.

Research on strawberry fruit has been extensive but

information on their ripening processes is lacking. The

roles and types of growth regulators in strawberry fruit have

been intensively studied, including changes in abscisic acid

(ABA) and auxin in achenes and receptacles and the action of

auxin in assimilate transport. As yet, the actual





2


involvement of these hormones in the ripening of all fruit

types is unknown. A lack of response to applied ethylene has

been demonstrated in strawberries and the role of endogenous

ethylene is speculative. Sucrose is the major assimilate

transported to the fruit during growth, yet its fate in the

fruit is unknown. Cell wall changes contribute to strawberry

fruit softening but the process is not triggered by

polygalacturonase.

Strawberries have several unique features compared to

other fruit. The true seeds acheness) are located on the

outer surface of the fruit. This morphological

characteristic resulted in the first identification of the

involvement of auxin, from seeds in fruit growth and

development. In addition, strawberries have an extended

peduncle joining the receptacle to the parent plant. The

presence of this extended peduncle suggests the possibility

of using floricultural techniques to study ripening of

detached strawberry fruit.

Strawberry fruit ripening is difficult to study on

detached fruits. Strawberries exhibit a high respiration

rate and continued growth through ripening. When detached at

a preripe stage, the fruit quickly become spongy and exhibit

only limited increases in anthocyanin and soluble solids

contents. Most of the previous studies with strawberries

were done with attached fruit. Using attached fruit for

studies adds a number of potential cultural variables, which

have to be identified, controlled and evaluated in an

analysis of fruit ripening.










Since strawberry fruit fail to continue normal

development once detached, the first objective of this work

was to develop a system which would permit normal ripening of

detached, immature strawberry fruit. Once this system was

developed and optimized, the influence of ethylene on

ripening changes was monitored. Finally, ethylene

biosynthesis enzymes in strawberry fruit were measured and

the relationship of this pathway to fruit ripening was

determined, in the hope that the role of ethylene in

strawberry fruit ripening could be elucidated.














CHAPTER II
LITERATURE REVIEW


The strawberry (Fragaria X ananassa) fruit has a

relatively short life span of 30 to 50 days, depending on

environmental conditions and genotype (Dana, 1980). As the

fruit ripens, its appearance changes dramatically, yet few of

the physiological changes associated with ripening have been

examined in detail. Historically, strawberry fruit have been

used more extensively in studies of parthenocarpy and auxin

action (Coombe, 1976) than in ripening studies.

Strawberry Fruit Growth and Ripening


Fruit Morphology

The strawberry is an aggregate fruit. A number of

ovaries belonging to a single flower adhere as a unit on a

common receptacle (Shoemaker, 1978). Fruitlets, or achenes,

are attached to the outer surface of the swollen receptacle

in spirally arranged rows (Abbott et al., 1970). The achenes

are connected to the receptacle by vascular strands and the

epidermal layer of the receptacle (Avigdori-Avidov, 1986).

The receptacle with achenes is generally referred to as the

fruit.








The receptacle organ consists of pith tissue, forming a

central cylinder, which is surrounded by cortical tissue

containing parenchymal and epidermal cells (Havis, 1943).

Vascular bundles traverse the pith and cortex to the achenes

on the outer surface of the cortex and act as extensions of

the peduncle from the vascular tissue (Lis and Antoszewski,

1982).

Each fruit is attached to the parent plant by a

relatively long peduncle. Each fruit is initiated from a

growth axis terminating in an inflorescence (Dana, 1980). A

primary (10) fruit is initiated at the terminus of the

inflorescence, while secondary (20) and tertiary (30)

fruit are initiated from primordia located between bracts on

the inflorescence below the primary bloom.

Fruit Growth

The growth of the strawberry fruit, measured by changes

in fresh weight, dry weight, fruit length or diameter,

generally is reported to be single sigmoidal (Crane and

Baker, 1953). Single-sigmoidal growth is characterized by an

initially slow growth rate followed by a phase of exponential

growth rate, then a declining growth (Bollard, 1970).

However, unlike other fruits classified as single sigmoidal,

such as muskmelon (Pratt et al., 1977) or apple (Biale and

Young, 1981), strawberries ripen prior to the cessation of

growth (Smith and Heinze, 1958).

There is some evidence which indicates that strawberry

fruit growth may be other than single sigmoidal. Some









researchers have demonstrated double-sigmoidal growth,

depending on cultivar and measurement intervals during

development. Archbold and Dennis (1984) measured fruit of

short-day plants at 3-day intervals throughout development

and reported a double-sigmoidal growth pattern. Stutte and

Darnell (1987) reported single sigmoidal growth for a

day-neutral cultivar. Another day-neutral cultivar was

reported to exhibit apparent single sigmoidicity when

measured every 6 days (Mudge et al., 1981) and, in a later

study, double sigmoidicity when measured every 3 days

(Veluthambi and Pooviah, 1984).

Achene growth. Thompson (1964, 1969) studied both

achene and receptacle growth of strawberry fruit and

concluded that achene development occurs prior to receptacle


enlargement.


Upon


growth becomes rapid

consist of embryo,

(Thompson, 1963). T

bounding the embry

after which time the

fertilized ovules,

after anthesis. The

nutrients to the

(Antoszewski, 1973).

shown that auxin


maturation of the embryo, receptacle

and ripening is initiated. The achenes

endosperm, nucellus and carpel tissue

he endosperm is a free-nuclear layer

o sac until 10 to 14 days after anthesis,

endosperm becomes a cellular layer. In

embryo formation is completed 10 days

vascular bundle of each achene supplies

achene and surrounding parenchyma cells

Studies with radiolabelled auxin have

is translocated basipetally through the


vascular bundles from the achenes to the peduncl

nutrients move acropetally (Antoszewski, 1973).


e, whereas









Receptacle growth. Receptacle development is generally

expressed in terms of changes in color and size. Stages of

development of the receptacle are usually classified as small

green, large green, white, pink or red (ripe), (Huber, 1984;

Culpepper et al., 1935). Fruit are at their maximum size at

the red stage (Huber, 1984). Fruit reach the white stage

approximately 21 days postanthesis and are fully red (ripe)

within 30 to 40 days (Dennis et al., 1970). Time to fruit

ripeness is strongly dependent on cultivar and environmental

conditions, particularly temperature (Dana, 1980).

As with other fleshy fruits, most of the size increase

in strawberry fruit is due to cell expansion. Havis (1943)

measured cell volume in transverse sections of fruit

harvested from the time of pollination through fruit

ripeness. He concluded that cell enlargement accounted for

90% of postanthesis fruit growth. Knee et al. (1977)

reported that cell division ceased in the receptacle tissue

at 7 days after petal fall. Fresh weight increased rapidly

between 14 and 28 days after petal fall and then plateaued

between 28 and 35 days, concomitant with fruit reddening.

Factors in the fruit effecting growth. It has long been

observed that primary fruit are larger than secondary or

tertiary fruit. Sherman and Janick (1966) calculated that

30 and 20 fruit attained 25% and 50% fresh weight,

respectively, of the size of the 10 fruit. These size









differences are believed to be the result of the greater

number of achenes on primary fruit (Abbott et al., 1970).

Webb et al. (1978) observed that a minimum number of achenes

was necessary for fruit development. They speculated that

the number of achenes was set at flower initiation, with

berry size determined at the flower primordia stage.

Although the relationship between the number of achenes and

berry weight is linear, the degree of receptacle tissue

sensitivity to hormones secreted from the achenes also may be

involved (Moore et al., 1970). Large-fruited clones had a

greater relative difference in size between primary and

secondary fruit than did small-fruited cultivars. Webb et

al. (1978) found that the maximum area of receptacle tissue

influenced by achenes was limited to 0.165 cm2. Lis and

Antoszewski (1982) reported that the response of berries

without achenes to exogenously applied auxin was greater in

primary than in secondary fruit. This difference may be

related to other factors, such as vascular bundle size, or

the potential for expansion of parenchyma cells in the

receptacle.

Hormonal Effects on Growth

Auxin. One of the earliest investigations of the

effects of auxin on general fruit development was undertaken

by Nitsch (1950), using strawberry fruit. He found that

removal of achenes arrested receptacle growth whether they

were removed at 4, 7, 14 or 21 days after pollination. When









napthaleneacetic acid (NAA) was

place of achenes, parthenocarpic

occurred, even when the achenes had


pollination.

levels of

was detected

were remove

the receptac

fruit alrea

developed co

Archbol

found high


U


applied to receptacles in

growth of the berries

been removed 4 days after


sing an Avena coleoptile growth bioassay, high


auxin activity were found in

in the receptacle (Nitsch, 1

d 4 days after pollination

les, strawberries reddened,

dy 21-days-old at the time

lor in the absence of auxin.

d and Dennis (1984), using

levels of free indole-3-a


achene tissue and smaller quantities


the achenes but none

952). When achenes

and auxin applied to

whereas only those

achenes were removed



mass spectroscopy,

cetic acid (IAA) in

in the receptacle


tissue. Free IAA was found in achenes 4 days after anthesis

and in the receptacles 11 days after anthesis. Levels in

both tissues peaked at 3 ug/g dry weight by 14 days after

anthesis corresponding to the maximum fruit growth rate. The

IAA level in the receptacle tissue then dropped rapidly,

whereas in the achenes it dropped gradually to a minimum

level of 1 ug/g achene dry weight at 23 days after anthesis

(red receptacle). Esterified-IAA was first detected in

achenes 4 days postanthesis, but did not appear in the

receptacle until 17 days post anthesis (Archbold and Dennis,

1984). High levels of amide-IAA were found in achenes at 11

and again at 23 days after anthesis (Archbold and Dennis,

1984).








Gibberellin and cytokinin. Addition of gibberellic acid

(GA) to strawberry ovaries in culture induced growth only

around the basal portion of the receptacle (Bajaj and

Collins, 1968). No effects were noted from kinetin (CK)

additions. Lis and Antowszewski (1979) removed achenes 14 to

15 days postpollination and treated half of the fruit with

GA, CK, IAA or all 3 in combination. GA and CK had no

influence on the accumulation of labelled 14C02 or 32p

in the receptacle tissue although IAA treatments markedly

increased label accumulation, indicating that auxin evoked a

sink for nutrient assimilation. Additionally, Archbold and

Dennis (1985) reported that GA did not stimulate growth when

applied to fruits without achenes. However, Thompson (1969)

reported normal growth and ripening of parthenocarpic berries

when GA was applied without auxin. Kano and Asahira (1981)

reported that the addition of NAA or CK to tissue cultured

receptacles delayed ripening.

Auxin, CK and GA were detected by bioassay in strawberry

fruit early in development, peaking at 7 days postanthesis

(Lis et al., 1978). The concentrations of all 3 were greater

in the achenes than in the receptacle tissue. After 7 days

postanthesis, the CK level decreased sharply in achenes and

receptacles, but was present at low levels until the fruit

were ripe. The GA levels were high 5 to 6 days

postpollination, decreased, then increased after the fruit

were ripe.








Abscisic acid. Archbold and Dennis (1984) measured

abscisic acid (ABA) levels in achene and receptacle tissues.

Levels of ABA, expressed as ug/fruit or ug/achene, were less

than 0.1 in both tissues at anthesis and then increased with

ripening to 0.45 and 0.25 for achene and receptacle tissue,

respectively. The concentration of ABA was 40 to 50% of

auxin levels. Lis et al. (1978) reported low ABA levels in

unpollinated or 1 day postpollinated receptacles and

achenes; levels increased during ripening, reaching a maximum

in red fruit. Kano and Asahira (1981) found that addition of

10 mg.liter-1 ABA stimulated the growth and ripening of

tissue-cultured strawberry fruit and proposed that ABA was

synthesized actively in the latter half of development after

CK disappeared from the achenes.

Strawberry Fruit Compositional Characteristics
During Ripening
Softening

Softening of strawberry fruit begins at the white stage

of development and increases dramatically as the fruit

progresses through the red stage (Culpepper et al., 1935).

Electron micrographs of cortical parenchyma cells revealed

separation along the the middle lamellar region (Neal, 1965).

A number of studies have been done to determine the mechanism

of softening. Woodward (1972) found very low levels of

water-soluble pectins 14 days after petal fall. These levels

increased at 21 days after petal fall (white fruit) and then

reached 90% of total pectin levels at 42 days (red-ripe).








Despite the observation that water-soluble pectin levels

increase as strawberry fruit ripen, no polygalacturonase (PG)

activity has been detected (Neal, 1965; Barnes and Patchett,

1976; Huber, 1984). However, strawberry cell walls treated

with tomato PG released pectins of similar molecular weight

to those released by tomatoes, indicating the absence of a PG

inhibitor in strawberries (Huber, 1984).

Barnes and Patchett (1976) reported that

pectinmethylesterase (PME) activity increased during

strawberry ripening but Neal (1965) proposed that strawberry

softening was due to increased esterification of cell wall

pectin located in layers of the middle lamella.

Esterification would result in severing of cationic

crosslinks and cause cell separation.

Yields of cell wall per fruit decreased with ripening

(Neal, 1965), although soluble polyuronides as a percentage

of the total extractable polyuronides increased (Huber,

1984). The total polyuronide content remained constant with

ripening, indicating continued synthesis, while neutral

sugars associated with pectin increased (Huber, 1984).

Softening of strawberry fruit is thought to be due

primarily to the increased solubility of polyuronides.

However, hemicellulose degradation may also contribute to

softening. Knee et al. (1977) observed an increase in

xylose, mannose and glucose residues in soluble

polysaccharide fractions during ripening and proposed that








hemicellose polysaccharides were being degraded or released

from interpolymer bonds. Huber (1984) found a shift in

hemicellulose molecular weight during strawberry fruit

ripening.

Cytological Changes

Electron microscopy studies revealed that receptacle

tissue at petal fall consisted of both meristematic and

expanding cells (Knee et al., 1977). Meristematic cells were

no longer observed at 7 days after petal fall. Cells at

petal fall had dense cell walls, small vacuoles, starch

grains in plastids, dictyosomes, ribosomes and endoplasmic

reticuli. Cells of green fruit (7 days postanthesis) had

tubular proliferations of the tonoplast, which became

extensive in ripe fruit. Cell walls were swollen and were

traversed by protoplasmic connections between adjacent cells

(Neal, 1965). At 21 days after petal fall (white), cells

were expanded, vacuolated, plastids had degenerated and most

of the starch had disappeared. The cortical cells, which

were mostly paranchymatous, enlarged and became separated as

the strawberry fruit developed and ripened (Neal, 1965).

During ripening, the cells were connected only by small

projections at the tips of the cells. These junctions were

traversed by protoplasmic connections between the cells.

Cell wall swelling was extreme, resulting in occlusion of the

intercellular spaces by matrix material (Knee et al., 1977).

Mitochondria appeared to be normal in ripe fruit (Knee et

al., 1977).










Pigment Changes

The white stage of strawberry fruit development is

regarded as the phase between the end of

chorophyll/carotenoid synthesis and the beginning of

anthocyanin synthesis (Woodward, 1972). Gross (1982)

detected the presence of residual carotenoids in ripe fruit

which originated from chloroplasts present in green fruit.

Electron microscopy studies revealed that chloroplasts in

ripening strawberry fruit cells disintegrated; evidently

chloroplasts disintegrate during ripening without

transformation to chromoplasts (Knee et al., 1977).

The major pigments synthesized in strawberry fruit are

anthocyanins, primarily as pelargonidin-3-glucoside (Ryan,

1971). Anthocyanin synthesis was very low until 35 days

after petal fall, then reached 75% of the final anthocyanin

concentration in the following 7 days (Woodward, 1972).

Pelargonidin-3-glucoside, formed from the shikimate acid

pathway, is thought to be stored in the vacuole (Rhodes,

1980).

Sugars and Soluble Solids

Total sugars, consisting of reducing sugars and sucrose,

are commonly measured as percent soluble solids. Soluble

solids in strawberry fruit increase steadily during

development (Woodward, 1972). Sugar content increased

logarithmatically with development (Knee et al., 1977).








Soluble solids in ripe fruit vary from mean values of 6 to 9%

depending on cultivar and cultural conditions (Duewar and

Zych, 1967).

Sucrose was shown to be the major assimilate transported

to the strawberry fruit (Lis and Antoszewski, 1979; Forney

and Breen, 1985a). The concentration of glucose and fructose

was higher than sucrose in small green and ripe fruit (Forney

and Breen, 1986). Sucrose was not detected in fruit until 10

days postanthesis; concentrations increased and then

decreased as fruits became red ripe. The rate of sugar

exudation from intact pedicels also increased with fruit size

(Forney and Breen, 1985b). The rate of sucrose transport,

measured with fruit discs, proved to be highest early in

development (9 days postanthesis), quickly falling to 50% of

initial rates by 12-17 days postanthesis, corresponding to

about half of the final growth of the fruit (Forney and

Breen, 1985a).

Starch

The role of starch in strawberry fruit growth has not

been determined. Long (1938) theorized that the

disappearance of starch, which coincided with increased

soluble solid levels, was due to hydrolysis of starch to meet

the sudden high demand for sugar by fruit. Starch has been

found only in the chloroplasts from green receptacle tissue,

but disappeared prior to fruit ripening (Knee et al., 1977).

Degradation of starch in plastids was essentially complete by








21 days after petal fall although starch was still detected

on the basis of iodine stains (Knee et al., 1977). Possibly,

starch is used in early growth of the fruits until the

achenes have developed sufficiently to induce rapid nutrient

assimilation from the parent plant.

Proteins and Amino Acids

Other than the enzymes involved in softening, few

studies have been conducted on protein changes in ripening

strawberry fruit. Veluthambi and Pooviah (1984) found

different polypeptide molecular weights from strawberry

receptacles harvested at 0, 5, 10, 15 and 20 days after

pollination. Removal of achenes resulted in new polypeptide

bands. When auxin was applied, these bands did not appear.

The induced polypeptides were theorized to represent

inhibitors which formed when auxin was not supplied.

Burroughs (1960) and Gallander (1979) reported that 79-87% of

the amino acids in ripe fruit consisted of free aspartic

acid, asparagine, glutamine and glutamic acid.

Titratable Acids

Citric acid is the major organic acid found in

strawberries at all stages of growth (Culpepper et al.,

1935). Acids, mainly citric acid, increased steadily with

fruit development and decreased in overripe fruit (Woodward,

1972). The pH of fruit homogenates were 3.6, 3.3, and 3.7

for green/white, red and overripe fruits, respectively. The

decrease in citric acid occurs at the same time as an

increase in malic acid (Reyes et al., 1982).









Relationship of Respiration and Ethylene to
Fruit Ripening
Introduction

Fruit ripening characteristics include changes in

pigmentation, texture (softening), synthesis of aromatics,

conversion of starch to sugars and abscission (Kader, 1985;

Brady, 1987). In strawberry fruit, ripening is characterized

by enlargement of receptacle tissue, softening, anthocyanin

synthesis and increased levels of soluble sugars.

Fruit ripening has long been associated with changes in

respiration and ethylene production. Ethylene is a plant

hormone that coordinates and unifies ripening in many fruit

(Abeles, 1973). Fleshy fruit are categorized into

nonclimacteric and climacteric classes on the basis of

respiration patterns during maturation and ripening (Yang,

1987). The climacteric fruit undergo a distinct ripening

phase, signalled by a climacteric rise in respiration and

increased ethylene emanation, while nonclimacteric members

generally do not. Although nonclimacteric fruit may have

respiratory rates comparable to climacteric fruit, the lack

of a preclimacteric minimum and climacteric peak in

respiration differentiates them from the climacteric group

(Biale and Young, 1981). Ethylene production from

nonclimacteric fruit is generally much less than from

climacteric fruit. That a fruit is truly nonclimacteric is

sometimes difficult to demonstrate as pathogen infection








(Oslund and Davenport, 1983) or chilling stress (Wang and

Adams, 1982) can induce a respiratory rise and ethylene

production not related to ripening.

The morphological source of fruit tissue is not related

to respiratory behavior (Biale and Young, 1981). The drupe

fruits peach and plum are climacteric whereas the cherry and

olive are nonclimacteric. The fleshy tissues of the apple

climactericc) and strawberry (nonclimacteric) fruit are of

receptacle derivation (Esau, 1977).

Climacteric Fruit

Climacteric fruit are so classified on the basis of a

respiratory upsurge climactericc) coinciding with the

initiation of ripening which generally declines during the

latter period of ripening (Watada et al., 1984). Ethylene

production also increases and this increase may precede,

coincide with or follow the respiratory climacteric,

depending on the species and cultivar. Increased ethylene

production precedes the respiratory climacteric in honeydew

melon (Pratt et al., 1977), coincides with the climacteric in

avocado (Eaks, 1985) and follows the climacteric in chile

pepper (Gross et al., 1986). Other examples of climacteric

fruits include banana, apple, pear, muskmelon, tomato (Biale

and Young, 1981), and blueberry (Ismail and Kender, 1969).

In the blueberry, ethylene production occurs prior to

respiration in the rabbiteye fruit, while ethylene production

and the respiratory climacteric are simultaneous in the

highbush fruit (Shimura et al., 1986).










In climacteric fruit, softening, color development,

hydrolysis of storage polysaccharides, and volatile'formation

are temporally associated with the respiratory climacteric

(Rhodes, 1980). Harvested, immature climacteric fruit may

undergo similar changes if subjected to ethylene treatment,

but the quality is inferior. Ripening is thought to be

initiated by ethylene in many climacteric fruits (Brady,

1987). Increased endogenous ethylene production was related

to an increase in sucrose, color change and softening in

loquat fruit (Hirai, 1980) and in figs (Marei and Crane,

1971). However, Jeffrey et al. (1984) reported that an

increase in soluble solids and a decrease in acidity preceded

the acceleration of ethylene production in tomatoes, although

softening and lycopene synthesis were dependent on the gas.

The magnitude of the respiratory upsurge and ethylene

production varies with the fruit species and cultivar.

Avocados exhibit a very high respiration rate of 225 ml

CO0.kg-1.hr-1 during the climacteric and a

correspondingly high ethylene production rate of 250

ul.kg- .hr-1 (Eaks, 1985). Conversely, the chile pepper

exhibits a much smaller increase in respiration, changing

from 65 to 130 ml.kg-l.hr-1, while the ethylene peaks at

only 0.6 ul.kg-1.hr-1 (Gross et al., 1986). The range of

peak ethylene production in climacteric fruits varies from

less than 0.1 ul.kg-1.hr-1 in the raspberry (Blanpied,








1964) to more than 400 ul.kg 1.hr1 in the mammee apple

(Akamine and Goo, 1978). The degree of the increase in

ethylene production during ripening can range from a doubling

of the preclimacteric level, as in the fig (Marei and Crane,

1971), to a several-hundred fold increase, as in the soursop

(Paull, 1982).

Fruit maturity effects the development of the

climacteric response and ripening. If harvested too

immature, fruit fail to exhibit the same levels of increased

respiration or ethylene production seen in more mature fruit.

Pratt et al. (1977) found that 'Honey Dew' melons failed to

soften or develop color if harvested less than 50% mature.

Ethylene and respiratory climacterics were delayed and

reached only 50% of the peaks from more mature fruit. In the

case of the avocado, mature fruit will develop climacteric

ethylene and respiration only after harvest. This anomaly is

thought to be due to the presence of a ripening inhibitor

exported from the tree to attached fruit (Tingwa and Young,

1975).

The application of ethylene or propylene, an ethylene

analogue, to climacteric fruit will advance the initiation of

ripening without altering the shape or magnitude of the peak

(McMurchie et al., 1972). The magnitude of this effect

depends on fruit maturity and the concentration of ethylene

applied. Once ripening has been initiated, respiration does

not return to preclimacteric levels upon removal of exogenous


M








ethylene. McMurchie et al. (1972) found that application of

500 ul.liter-1 propylene to green bananas initiated the

climacteric within 10 hours after the treatment began and 24

hours before the climacteric in control fruit.

Increasing the concentration of ethylene can advance the

-1
onset of ripening. When 1.0 ul.liter-1 ethylene was

applied to guava (Psidium guajava) fruit at the mature,

preclimacteric stage, the time to the onset of ripening was

decreased by 4 days (Yen and Tzong-shyan, 1986). When 10


ul.1iter-1

advanced by

production

ethylene fa

Often,

sensitivity

ul.liter-1

stimulated

was not s

(McGlasson

individual


was applied, the onset of ripening was further

2 days. In mature fruit, endogenous ethylene

is stimulated. Additional applications of

1 to elicit any further response.

the maturity stage of the fruit determines its

to exogenous ethylene. Application of 1000

xogenous ethylene to immature tomato fruits

respiration, but endogenous ethylene production

imulated until after the start of ripening

et al., 1975). Although the ripening of


fruits was


advanced,


uniform ripening


did not


result. Conversely, application of 1000 ul.liter-1

ethylene to melons only 30% mature stimulated uniform

ripening (Lyons and Pratt, 1964).

Extended application of high levels of ethylene can

result in irreversible suppression of ripening-associated,

endogenous ethylene production. Brecht and Kader (1984a)








found that ethylene production was reduced when nectarines

were treated with 100 ul.liter-1 ethylene for 4 days, compared

to a 2-day treatment. Zauberman and Fuchs (1973) showed a

50% loss in ethylene production when avocados were stored in

100 ul.liter-1 continuous ethylene, compared to 0 or 24

hours of ethylene treatment.

Nonclimacteric Fruit

Nonclimacteric organs lack an upsurge in respiration

during ripening. Furthermore, ripening occurs without an

increase in endogenous ethylene levels. Although the

respiration rate of nonclimacteric fruits is often comparable

to that of climacteric fruit, ethylene production from

nonclimacteric fruit generally remains below 1.0

ul.kg-l.hr-1 (Kader et al., 1985). The pattern of

respiration and ethylene emanation in nonclimacteric fruits

is generally one of higher rates postanthesis, gradually

declining as the fruit mature (Biale and Young, 1981).

Generally, nonclimacteric fruits display a gradual

development of color, softening and increased soluble solids,

not associated with starch breakdown, increased respiration

or ethylene. Some examples of nonclimacteric fruit include

watermelon (Elkashif, 1985), orange, grape, and cucumber

(Biale and Young, 1981). Carambola fruit fail to show an

increase in either respiration or ethylene emanation while

ripening (Oslund and Davenport, 1983). The cherry exhibits a

downward drift in respiration as the fruit ripen, without


1








increased ethylene evolution (Blanpied, 1972). Akamine and

Goo (1979) failed to detect any ethylene production from the

mountain apple (Eugenia malaccensis). On the other hand,

cucumber fruit fail to exhibit a respiratory climacteric

during maturation but do exhibit an increase in ethylene

production in fruit 20 to 30 days after anthesis (Kanellis et

al., 1986).

The unique response of nonclimacteric fruit to exogenous

ethylene has been a useful aid in distinguishing

nonclimacteric from climacteric fruit. Application of

ethylene or propylene to nonclimacteric fruit results in

increased respiration rates while ethylene emanation remains

at low, basal levels (McMurchie et al., 1972). The magnitude

of the respiratory increase depends on the fruit type and on

the concentration of exogenous ethylene applied. Upon

removal of ethylene, respiration returns to previous levels

and reapplication of ethylene will stimulate another

respiratory increase. This technique was first used with

citrus. Vines et al. (1965) applied 50 ul.liter-1

ethylene for 24 hours to Valencia oranges and observed a 70%

increase in respiration. After removal of the ethylene,

respiration returned to near-normal in 5 days. Treatment of
-1
lemons with 1.0 ul.liter- of ethylene increased the

respiration rate by 20%, while application of 100

ul.liter-1 ethylene increased the respiration rate by more

than 100% (McMurchie et al., 1972). One example where








ethylene application has provided evidence of nonclimacteric

behavior is the watermelon. Mizuno and Pratt (1973)

classified the watermelon as a climacteric fruit on the basis

of ethylene production and respiratory rates from fruit

harvested at different stages of development. However, upon

application of 50 ul.liter'1 ethylene, Elkashif (1985)

found a 40% increase in respiration of mature and immature

watermelons. Following removal of ethylene, the respiration

rate decreased to preapplication values after 3 days.

Endogenous ethylene production was not increased and ripening

was not advanced. These results lead to the conclusion that

the watermelon is nonclimacteric.

Generally, the onset of ripening in nonclimacteric fruit

is not hastened by ethylene application. Treatment of detached

cherries with 1000 ul.liter-1 propylene for 24 hours failed

to enhance color formation or endogenous ethylene production

(Reid et al., 1985). Application of 500 ul.liter-1

ethephon to grapes just before the onset of ripening advanced

color formation and softening by 4 to 6 days, but delayed

ripening if applied earlier in growth (Hale et al., 1970).

Fudge (1930) found enhanced color formation in cranberries

treated with 1000 ul.liter-1 but no increase in respiration

rates or total sugars.

Senescence may be advanced by ethylene application.

Elkashif (1985) found that watermelons harvested immature or

mature had accelerated cell wall breakdown, decreased flesh








firmness, and electrolyte leakage after 3 days treatment with

50 ul.liter-1 ethylene. Pathogen infection occurred in

fruit treated 8 days with ethylene. Treatment of carambolas

with an ethephon dip (1000 ul.liter-1) failed to stimulate

ethylene production for more than 2 days but pathogen

infection occurred after 8 days of storage (Lam and Wan,

1987).

Respiration and Ethylene in Strawberry Fruit

Generally, nonclimacteric fruit, exemplified by citrus

and grape, do not exhibit rapid changes in color, softening

or increased soluble sugars (Biale and Young, 1981). One

exception to this generalization is the strawberry fruit,

which softens and ripens within a few days, without

exhibiting an increase in either respiration or ethylene.

The respiration rate of detached ripe 'Geneva' fruit

held at 210C was 56 ml C02.kg-'.hr'1 (Dayawon and

Shutak, 1967) while detached 'Raritan' strawberries harvested

at the white stage and held at 220C respired at 70

ml.kg-1.hr-1 (Janes et al., 1978). The respiration rate

of ripe 'Shasta' fruit decreased with temperature, from 120

to 10 ml.kg-1.hr-1 at 300 and OOC, respectively

(Mitchell et al., 1964). Ethylene production from ripe

strawberry fruit at 200C is generally reported to be less

than 0.1 ul.kg- .hr-1 (Kader et al., 1985). Ethylene

emanation in 'Prizewinner' fruit fell from 2 ul.kg-1.hr-1

at anthesis to 0.08 ul.kg-1.hr-1 at ripeness at 200C








(Knee et al., 1977). Application of 50 ul.liter-1 ethylene

to white fruit failed to increase respiration (Janes et al.,

1978) while 200 ul.liter-1 ethylene applied to detached

green and white fruit failed to initiate color development

(Mason and Jarvis, 1970). Although changes in respiration

and/or ethylene production are associated with ripening in

some fruits, the relationship between respiration, ethylene

production and strawberry fruit ripening remains unknown.

A confounding factor in the above studies has been the

failure of strawberry fruit to develop normally if harvested

prior to the development of color (Mason and Jarvis, 1970).

This complication makes it difficult to assess the validity

of measurements of respiration and ethylene production from

detached fruits and increases the complexity of determining

the true involvement of ethylene in strawberry fruit

ripening.

Ethylene Synthesis

Intermediates in Ethylene Synthesis

The sequence and identification of intermediates

involved in ethylene biogenesis evolved from a series of

experiments with model systems and plant tissues. Initially,

Lieberman et al. (1965) used a model system of linolenic

acid, with copper and ascorbic acid as catalysts to study

ethylene formation. They found that methionine, added as an

inhibitor of free radical reactions, could substitute for

linolenic acid as a substrate and resulted in greatly








increased ethylene production. Subsequently, Lieberman et

al. (1966) demonstrated that addition of methionine to apple

tissue resulted in ethylene formation. The intermediates

involved in the conversion of methionine to ethylene were

subsequently identified as S-adenosylmethionine (SAM) and

1-aminocyclopropane-l-carboxylic acid (ACC) by Adams and Yang

(1979) and Lursen et al. (1979). Lursen et al. (1979)

found that application of ACC to soybean leaf discs resulted

in ethylene formation. They predicted that ethylene

biosynthesis involved the conversion of SAM to ACC. Adams

and Yang (1979), following the conversion of radiolabelled

methionine in apple tissue, found that SAM was converted to

ACC, then ACC was converted to ethylene. All climacteric and

nonclimacteric fruit thus far studied have demonstrated the

ability to convert methionine to ethylene (Yang et al.,

1986).

The content of ACC changes in relation to ethylene

synthesis. Yang et al. (1986) found that mature but unripe

apples contained 0.1 nmole.g fresh weight-1 ACC,

increasing 300-fold during the ethylene climacteric. At the

preclimacteric stage in avocado, banana and tomato fruits,

ACC levels were about 0.1 nmole.g fresh weight-1 in
each of these fruit tissues (Hoffman and Yang, 1980). During

the climacteric, ACC levels increased to 45, 5 and 7

nmole.g fresh weight-1 for avocado, banana and tomato,

respectively.








When aminovinylglycine [2-amino-4-aminoethoxy-trans-3-butenoic

acid] (AVG), a specific inhibitor of ACC synthase, or amino-

oxyacetic acid (AOA), an inhibitor of pyridoxal phosphate

enzymes, was applied to pears or apples prior to the onset

of rapid ACC synthesis, conversion of SAM to ACC was

inhibited and ripening was delayed (Yang, 1985).

Enzymes Involved in Ethylene Synthesis

The enzymes regulating the conversion of methionine to

ethylene were identified as methionine adenosyl transferase,

ACC synthase and ethylene forming enzyme (EFE), respectively

(Yang and Hoffman, 1984). The rate-limiting enzyme in

ethylene synthesis was shown to be ACC synthase. Conversion

of SAM to ACC in mung beans was stimulated by auxin treatment

but the level of SAM did not decrease; thus conversion of

methionine to SAM was not limited (Yu and Yang, 1979).

Furthermore, application of ACC to tissue from several fruits

greatly stimulated ethylene production, indicating that EFE

was constitutive and that the level of ACC, and therefore ACC

synthase, was the rate-limiting step (Cameron et al., 1979).

Yang and Adams (1979) found that AVG inhibited the conversion

of methionine to ACC but not of methionine to SAM or of ACC

to ethylene. They concluded that ACC synthase was a

pyridoxal enzyme. Boiler et al. (1979) isolated ACC

synthase as a soluble enzyme from cell-free extracts of

tomato fruit pericarp. It was inhibited by AVG and had a

molecular weight of about 55000. To date, ACC synthase has








been isolated from tomato pericarp, Cucurbita maxima mesocarp

(Nakajima and Imaseki, 1986), mung bean hypocotyls (Yu and

Yang, 1979), tobacco leaves (Imaseki and Watanabe, 1978),

nectarine (Brecht and Kader, 1984b), citrus peel (Riov and

Yang, 1982a) and cucumbers (Terai and Mizuno, 1985). Acaster

and Kende (1983) reported a molecular weight of 57000 for ACC

synthase isolated from wounded tomato fruit tissue. Privalle

and Graham (1987) reported a molecular weight of about 50000

for the enzyme from wounded, pink tomato pericarp. Nakajima

and Imaseki (1986) estimated the molecular mass of ACC

synthase from winter squash mesocarp to be 160000, made up of

2 subunits, each approximately 84000 molecular weight. They

calculated the specific activity of ACC synthase to be 220

MU/mg protein at 30 C, with 50 uM SAM. ACC synthase is

believed to be located in the cytoplasm (Apelbaum et al.,

1981).

Ethylene forming enzyme, which has not yet been isolated

or fully characterized, is most likely membrane-bound, since

ethylene production is disrupted when plant cells are exposed

to detergents (Lieberman, 1979) or osmotic shock (Imaseki and

Watanabe, 1978). Guy and Kende (1984) suggested that EFE was

located on the tonoplast, and that ACC was sequestered in the

vacuole, since vacuoles isolated from pea protoplasts

produced 80% of the total ethylene. Application of

cycloheximide (CHI), a protein synthesis inhibitor, to

avocado fruit did not inhibit EFE, indicating that EFE

activity is constitutive (Blumenfield et al., 1986).









Regulation of Ethylene Biosynthesis


System I and system II ethylene. The

ene biogenesis during climacteric fruit

red to as autocatalytic ethylene (Biale

Autocatalytic ethylene is defined as

g the increased synthesis of ethylene


exposure


to ethylene (Yang and Hoffman, 1984).


upsurge in

ripening is

and Young,

that produced

triggered by

McMurchie et


al. (1972) proposed that all tissues exhibit a low basal

ethylene system which they referred to as System I.

Climacteric organs have an additional system, which McMurchie

et al. (1972) called System II, thought to be triggered by

increased sensitivity of the tissue to its own System I

ethylene. Yang et al. (1986) proposed that the endogenous

ethylene produced by System I activates ACC synthase,

creating more ACC, which then induced EFE and subsequent

formation of more System II ethylene.

Ethylene levels remain low in nonclimacteric fruits

throughout development and ripening, in contrast to the

many-fold upsurge occurring in climacteric fruits. Ripening

nonclimacteric tissues do not develop System II ethylene

production. This difference may be due to the lack of

development of tissue sensitivity to ethylene or inability to

sustain System II ethylene production (McMurchie et al.,

1972).


ethyl

refer

1981)

duri n








Huber and Sherman (1987) proposed that System I ethylene

may initiate ripening, due to ethylene receptors. These

receptors may be unmasked as inhibitors disappear, or

pre-formed ripening-specific receptors may be modified by

ethylene. The result, over time, is age-accruing

sensitization of the tissue to ethylene.

Climacteric fruit. The regulation of ethylene

production in climacteric fruit is a function of fruit

maturity and species. This regulation has been found to

relate to changes in fruit ACC content. Application of

ethylene to intact, immature tomatoes or cantaloupes greatly

increased the capacity of the tissue to convert ACC to

ethylene, but did not increase the ACC content, indicating

regulation by both ACC synthase and EFE (Liu et al., 1985).

Application of ACC to preclimacteric apples resulted in only

a 5-fold stimulation of ethylene production, compared to a

1000-fold ethylene increase during the climacteric.

Apparently, some EFE must be synthesized after autocatalytic

ethylene production is stimulated. Brecht and Kader (1984b)

compared ACC synthase and EFE activity to ethylene production

and ACC content of nectarine fruit. Detectable EFE activity

preceded the activation of ACC synthase and the rise in

ethylene synthesis. Yang et al. (1986) followed changes in

EFE, ACC and ethylene in apples and found that EFE activity

preceded changes in ACC content or ethylene production.

Blumenfield et al. (1986) studied changes in ACC synthase

and EFE activity, ACC content and ethylene production in








avocados. They propose that attached fruit have low levels

of ACC synthase activity and ACC, which increased upon

harvest. The ACC is then converted to ethylene by EFE; then

the ethylene induces climacteric ethylene production.

Ripening is then promoted by the higher level of ethylene.

Autoinhibition of ethylene synthesis in climacteric

fruit tissue seems to be due to a blockage and decrease of

EFE activity rather than of ACC synthase. In the presence of

100 ul.liter-1 ethylene, EFE activity in nectarines dropped,

although ACC synthase and ACC contents remained at similar

levels relative to endogenous ethylene production (Brecht and

Kader, 1984b).

Nonclimacteric fruit. Few studies have been done on the

regulation of ethylene in ripening nonclimacteric fruit.

Most studies have dealt with the activation of ethylene

production by chilling stress or wounding. Cameron et al.

(1979) applied ACC to a number of nonclimacteric fruits,

including squash and bell pepper, and obtained stimulated

ethylene production in these fruit. The amount of ethylene

produced was dependent on both tissue types and the

concentration of ACC applied. All tissues responded to 1 mM

ACC; thus EFE activity was constitutive in these fruits. The

occurrence of a lag phase between ACC application and

ethylene production in some tissues indicated that de novo

EFE synthesis may be required. To date, the ACC content has

been measured in only 2 nonclimacteric fruit, citrus and








cucumber. In flavedo tissue from intact oranges, the ACC

content remained constant at 0.11 nmole.g fresh

weight-1 (Hyodo and Nishino, 1981), and decreased in whole

cucumber fruit during ripening from 1.3 to 0.5

nmole.g fresh weight-1 (Terai and Mizuno, 1985).

Ethylene biosynthesis in flavedo tissue from mandarin orange

could be stimulated by aging the excised tissue for 30 hours,

which was temporally related to a large increase in ACC

content (Hyodo and Nishino, 1981). Ethylene synthesis could

be stimulated in nonaged tissue when 1 mM ACC was applied.

Cycloheximide (CHI) applied to tissue pretreated with ACC

strongly inhibited the conversion of ACC to ethylene, whereas

treatment with actinomycin, a transcription inhibitor, did

not suppress ethylene synthesis. Apparently, synthesis of

EFE was required, although mRNA synthesis had already taken

place. Aged tissue pretreated with or without ACC was then

treated with AVG, an inhibitor of ACC synthase. Ethylene

from tissue without ACC pretreatment was suppressed, but AVG

did not result in decreased ethylene from tissue with ACC

pretreatment. Evidently, de novo synthesis of ACC synthase

was not stimulated by the increased ethylene production

brought about by ACC application, but de novo synthesis of

this enzyme was required for ethylene production by aged

tissue.

The activity of ACC synthase, ACC content and ethylene

production increased in cucumber fruit chilled at 2.50C








then transferred to 250C (Wang and Adams, 1982). Treatment

of chilled fruit tissue with cycloheximide resulted in

lowered ACC synthase activity, ACC level and ethylene

production, but not with actinomycin or a-amanitin,

indicating de novo synthesis of ACC synthase in response to

chilling. This response was similar to that obtained with

aged mandarin orange tissue.

Inhibitors of Ethylene Action and Synthesis

Several compounds have been found to inhibit or block

ethylene synthesis and action. Pyridoxal enzyme inhibitors,

including the vinylglycine analogs rhizobitoxine and AVG,

irreversibly bind to an active site on ACC synthase,

effectively blocking the conversion of SAM to ACC (Yang and

Hoffman, 1984). Hydroxylamine analogs, such as

aminooxyacetic acid (AOA), react with the pyridoxal phosphate

coenzyme necessary for ACC synthase activity, blocking

formation of more ACC. Neither analog blocks the conversion

of ACC to ethylene, although cobalt and anaerobisis have been

shown to inhibit the conversion of ACC to ethylene (Adams and

Yang, 1979; Yang, 1985). Inhibitors of ethylene action include

2, 5 norbornadiene and silver. Norbornadiene is a volatile,

cyclic olefin that competes with ethylene for a receptor

binding site (Veen, 1985). Beyer (1976) discovered that

silver was a potent inhibitor of ethylene action. Silver has

been found to inhibit System II ethylene synthesis and

lycopene formation in tomato fruit (Hobson et al., 1984;








Saltveit et al., 1978), and prevents senescence in cut

flowers, but the exact mechanism of action is unknown (Veen,

1986).

Summary

Strawberry fruit growth is composed of achene

development and receptacle enlargement. Assimilate transport

from the plant to the developing fruit takes place

through the vascular bundles. Biochemical changes such as

softening and anthocyanin synthesis begin just prior to the

white stage. Respiration and ethylene studies of strawberry

fruit have been done on ripe or nearly-ripe fruit. Ethylene

levels are very low, while respiration rates are high

relative to other fruit.

Fleshy fruit have been classified as climacteric or

nonclimacteric on the basis of respiration patterns during

ripening. Climacteric fruit exhibit a decreased respiration

rate, followed by a respiratory upsurge, just before and

during ripening. Ethylene production exhibits a similar

pattern. When exogenous ethylene is applied to these fruit

at the preclimacteric stage, the onset of ripening is

hastened. Nonclimacteric fruit show no variation in ethylene

production or respiration during growth, other than a general

downward drift from anthesis through ripening. Ripening

changes are associated with ethylene in climacteric fruit,

but researchers have thus far failed to establish the role of

ethylene in the ripening of nonclimacteric fruit.








It is proposed that the difference between climacteric

and nonclimacteric fruit ripening is related to differences

in ethylene production and sensitization. Climacteric fruit

are capable of autocatalytic ethylene production, which may

be the result of increased tissue sensitivity to basal

ethylene levels present throughout growth. Nonclimacteric

fruit either do not become sensitized to basal ethylene

levels or are unable to sustain higher levels of ethylene

production. Nonclimacteric fruit are capable of forming EFE

and ethylene from exogenously applied ACC.

Strawberry fruit exhibit marked changes in growth,

pigmentation, texture and flavor during ripening but the role

of ethylene, if any, in strawberry fruit ripening remains

unknown.















CHAPTER III
DEVELOPMENT AND EVALUATION OF AN IN VITRO SYSTEM
TO STUDY DETACHED STRAWBERRY FRUIT RIPENING


Details on the induction and regulation of ripening have

been largely derived from studies with climacteric fruit

(Biale and Young, 1981). If detached at a mature stage,

these fruit generally will continue to ripen normally,

exhibiting increased respiration and ethylene production

which can be monitored postharvest. Direct study of

nonclimacteric fruit has been limited because normal ripening

generally does not continue following detachment (McGlasson,

1985). Furthermore, respiration and especially ethylene

production do not change dramatically during ripening, making

it difficult to identify the initiation of ripening events.

Strawberries have been classified as nonclimacteric

fruit because of the lack of a respiratory increase during

ripening. Additionally, no rise in ethylene occurs during

ripening (Biale and Young, 1981). Although anthocyanins can

appear in strawberries detached preripe, textural changes are

abnormal and soluble solids do not increase (Austin et al.,

1960).

The ability to harvest strawberry fruit at an early

stage of development and promote normal ripening under








controlled, in vitro conditions would allow experimentation

directed towards elucidating details of the physiology of

ripening of nonclimacteric fruit. Loewus and Kelly (1961)

placed green strawberries having a small portion of the

attached peduncles into vials containing water and

radiolabelled D-galacturonic acid to study uptake and

utilization of this hexuronic acid. Fruit developed to the

pink color stage; the galacturonic acid was incorporated and

used in the receptacle tissue for the synthesis of ascorbic

acid and pectin. Further studies employing this approach hav

not been performed with strawberries. In a study of the

response of cherry fruit to propylene and silver, Reid et al.

(1985) pretreated cherry fruit by immersion of attached

peduncles into a silver thiosulphate solution. Such in vitro

systems have long been employed for the study of cut flower

physiology. Vase solutions in the simplest form contain a

germicide and sucrose and these treatments have resulted in

increased dry weights and the promotion of flower longevity

for carnations (Reid et al., 1980b), roses (Ferreira and

DeSwardt, 1980) and other flowers (Halevy and Mayak, 1979).

The response of cut flowers to vase solutions depends on

species, cultivar and flower maturity at harvest. Based on

the success of vase solutions with cut flowers and the system

used by Loewus and Kelly, it seemed that the morphology of

strawberries might lend themselves to treatment analogous to

cut flowers.








The objectives of this study were to investigate the

conditions necessary to promote growth and ripening of

detached strawberry fruit, and to compare the parameters of

fruit maintained in vitro to those fruit maintained in the

field.

Materials and Methods
Short-day strawberry (Fragaria X anassasa) cultivars

'Douglas' and 'Pajaro' were grown in field plantings in Dover

and Gainesville. Blossoms were tagged at anthesis and fruit

harvested with intact peduncles from 6 to 22 days

postanthesis. Immediately after the fruit with peduncles

were detached, they were placed in plastic bags, sealed, and

placed on ice during transport from the field to the

laboratory. Fruit were stored up to 3 weeks at 10C without

detrimental effects.

In Vitro Growth Experiments

The cultivars 'Douglas' and 'Pajaro' were harvested at

14 days postanthesis, at a time when fruit maturity,

expressed as a percentage based on the total number of days

from anthesis to full ripeness, was approximately 52%. Vase

solution composition was based on that commonly employed for

cut flowers (Halevy and Mayak, 1981) and consisted of

autoclaved distilled water with 200 ug.liter-1

hydroxyquinoline hemi-sulfate (HQS) with or without 3% (88

mM) sucrose (Fisher analytical grade). The pH of each

solution was 4.0. Effects other than antimicrobial have been








reported for HQS (Halevy and Mayek, 1979). However, omission

of this agent resulted in prolific microbial growth and loss

of fruit weight. Twelve fruit were used for each treatment.

Initial fruit fresh weights were matched between treatments.

The average fruit fresh weights were 5.44+1.01g and

5.63+1.10g for 'Pajaro' and 'Douglas', respectively.

After trimming peduncles to a uniform length of 55 mm

with a scalpel, each fruit was placed in an autoclaved, 18.5

ml scintillation vial (Kimble 27 1/4 x 55) filled with 16 ml

of solution and covered with parafilm which was slit to

accommodate the peduncle. The base of each peduncle was

recut to remove 1 to 2 mm every 2 to 3 days during all

experiments. The daily loss in fresh weight due to peduncle

trimming was about 0.1% fresh weight. Fresh weight was

recorded every 1 to 3 days throughout the experiments.

Duplicated experiments were conducted on lab benches under

fluorescent lights (12 hours) at 23 + 30C. Fruit surface

color was measured using a Hunterlab Colorquest color

difference meter. Values were recorded in the L (black to

white); a (green to red); b (blue to yellow) system using a

25 mm aperture calibrated with white (No. 1077, x=81.7

y=86.53 z=93.98) and gray (No. 1077, x=51.37 y=54.64

z=59.20) standards. Values for L, a, b corresponding to

strawberry color stages are listed in Table 3-1.

Influence of Fruit Age at Harvest on In Vitro Development








'Pajaro' fruit were harvested at 14 and 20 days

post-anthesis (approximately 54% and 77% maturities,

respectively). Twelve fruit of each age were placed via

peduncles in solutions containing 200 ug'liter-1 HQS with

or without 88 mM sucrose. Average initial fresh fruit

weights were 2.95+.41 g and 13.65+2.30 g for 14 and 20 day

fruit, respectively. Further experiments were conducted with

'Pajaro' fruit to examine the effects of fruit age on in

vitro response. Fruit at 6, 12 and 18 days postanthesis

(25%, 53% and 78% mature, respectively) were harvested and

placed in solutions consisting of 88 mM sucrose and 200

ug'liter-1 HQS. Solutions and measurements were made as
described above. Receptacle length, defined as the distance

from the calyx to fruit tip, and maximum diameter were

determined with a Vernier caliper to the nearest 0.1 mm.

Influence of Fruit Order on In Vitro Development

Primary, 20 and 30 'Pajaro' fruit of the same

anthesis date were harvested from the field at 14 days

postanthesis (58% mature). Twelve fruit per order were

placed in the 88 mM sucrose with 200 ug'liter-1 HQS

solutions. Measurements were conducted as described above.

Average initial weights were 3.47+.75 g, 2.68+.36 g, and

1.14+.11 g for 10, 20 and 30 fruits, respectively.

Comparison of Field to In Vitro-Ripened Fruit

'Pajaro' fruit tagged on the same date of anthesis were

harvested from the field at 12 and 19 days postanthesis and

placed into in vitro sucrose solutions after harvest as












Table 3-1. Relationship between the color of in vitro
grown strawberry fruit and HunterCoTorlab
L, a, b values.

Value
Fruit Colorz L a by

Green (G) 50+4 -7+1 17+2

White (W) 61+5 -4+2 16+1

Pink" (P") 52+4 -1+2 14+2

Pink (P) 45+4 9+3 14+2

Red" (R") 37+3 14+2 14+2

Red (R) 31+2 20+3 11+3

Red+ (R+) 22+4 30+5 10+2



ZPink=1less thin 1/4 pink on fruit; red"=
light red; red =deep red. Each value represents the mean
of 24 measurements, + SD.

YL=value (black to white), a=hue (green to red),
b=chroma (blue to yellow).








described above. Length and diameter measurements of twelve

tagged fruit left attached to the plants were taken at 2 to 3

day intervals from 12 days postanthesis until harvest at 24

days (fully ripe). Final weights and color values of fruit

harvested at 24 days postanthesis were compared to those

ripened in vitro.

Results

In Vitro Growth Experiments

Figure 3-1 illustrates the cumulative growth of

strawberry fruit harvested 14 days postanthesis and placed

into vase solutions. 'Pajaro' fruit consistently showed

significantly more weight gain than did 'Douglas' fruit,

regardless of the presence of sucrose (Fig. 3-1). 'Pajaro'

fruit in solutions without sucrose gained 30% more weight

than 'Douglas' fruit in solutions with sucrose. This

difference increased to 300% when 'Pajaro' fruit were grown

in solutions containing sucrose. 'Douglas' fruit grown

without sucrose failed to gain weight.

Color development in 'Pajaro' fruit advanced more than

in 'Douglas' fruit. Although 'Douglas' fruit grown in the

presence of sucrose reached the white color stage at the same

time as 'Pajaro' fruit grown in sucrose, further color

development in 'Douglas' fruit was suppressed. 'Pajaro'

fruit grown with sucrose developed to a full red color while

still gaining weight, whereas 'Douglas' fruit grown with

sucrose began to lose weight after reaching the pink color











140-
0
120-



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N,
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AJRACO


I I I I I I I2 3 10 1
0 2 3 4 5 6 7 8 9 10 11 12 13 14


DAYS IN VITRO


















Figure 3-1. Effect of cultivar and sucrose on cumulative
fruit growth in vitro. 'Pajaro' or 'Douglas'" 1
fruit were placed in water with 200 ug.liter
HQS, with or without 88 mM sucrose. Day of
color change is indicated by W (white), P (pink)
or R (red). Vertical bars represent LSD between
cultivars and sucrose levels for each day.


SUCROSE *-*
SUCROSE o---
DOUGLAS
SUCROSE U-*
SUCROSE 0---0


R


RP




P-
w

R
10


N W
---a 0-








stage. Color formation in 'Pajaro' fruit grown without

sucrose was delayed by 4 days relative to 'Pajaro' fruit

grown with sucrose and was nonexistent for 'Douglas' fruit

grown without sucrose. Because of the poor in vitro

performance of the 'Douglas' fruit, only 'Pajaro' fruit were

used for subsequent experiments. Despite its poor growth in

vitro, 'Douglas' fruit in the field were larger than 'Pajaro'

fruit and developed color normally.

Influence of Age on Fruit Growth and Ripening In Vitro

'Pajaro' fruit harvested at 20 days (white; 77% mature)

or 14 days (green; 54% mature) postanthesis responded

differently to the presence of sucrose (Fig. 3-2). White

fruit provided with sucrose showed less weight gain but had

accelerated color development compared to white fruit held in

solutions not containing sucrose (Fig. 3-2A). Color

formation in white fruit grown without sucrose was delayed by

3 days compared to fruit grown with sucrose. Once color

formation was initiated, the rates of color development were

the same for white fruit grown with or without sucrose. The

cumulative weight gain of white fruit grown either in the

presence or absence of sucrose did not exceed 0.40 g/initial

g fruit fresh weight.

Fruit harvested at the green stage gained 400% more

weight when grown with sucrose (Fig. 3-2B). Fruit grown

with sucrose exhibited the greatest weight gain concomitant

with ripening and continued to gain weight as color






















B
-100 o
1 *SUCROSE.--*R
-SUCROSE o- -o

S80-
I *- R-
I-_

O 60-
0: p
w P-
P40-
j W
I P P
S20



0 1 2 3 4 5 6 7 8 9 10 11 12 13
DAYS IN VITRO











Figure 3-2. Effect of age and sucrose on cumulative growth
of 'Pajaro' fruit in vitro. Fruit were
harvested 20 days post-anthesis (white) (A)
or 14 days post-anthesis (green) (B) and placed
in vials with or without 88 mM sucrose. Day of
color change is indicated by W (white) or R
(red). Vertical bars represent standard error
of the mean (SE).








intensified. Fruit grown without sucrose exhibited a

decrease in weight as fruit ripened.

Fruit placed in vitro as early as 6 days postanthesis

(25% mature) had total weight gains similar to fruit

harvested 12 days postanthesis (53% mature) (Fig. 3-3).

Fruit placed in vitro at 18 days postanthesis (78% mature)

gained relatively less cumulative weight than fruit placed in

vase solutions at either 6 or 12 days postanthesis. During

the first day in vitro, the weight gain was 0.10 g/initial g

for fruit at all ages. After 6 days in vitro, fruit

harvested at 18 days postanthesis were red and fruit

harvested 12 day postanthesis had gained 60% more weight

relative to fruit harvested 6 days postanthesis. Fruit

harvested 18 days postanthesis gained less than 0.4 g/initial

g in vitro. Fruit placed in vitro at 12 days postanthesis

exhibited a large increase in growth rate after 8 days in

vitro, concomitant with the start of color development. The

weight gain of these fruit at this time was 0.7 g/initial g.

During ripening in the following 6 days, these fruit gained

an additional 0.5 g/initial g. Fruit placed in solutions at

6 days post-anthesis ripened after 18 days in vitro. After

13 days in vitro, these fruit had gained 0.6 g/initial g,

then gained another 0.6 g/initial g as they developed color

in the following 6 days. Unlike the fruit placed in vase

solutions at 12 days postanthesis, fruit placed in vitro at 6

days postanthesis did not exhibit a large increase in growth

rate concomitant with ripening.














4- DAYS RO
S0 6 R*
0 6o--o R
X120 12R
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=100p /




I-
P P-







-40

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 18 17 18 19 20
DAYS IN VITRO




























Figure 3-3. Effect of.fruit age on cumulative growth
in vitro. Fruit were harvested at 6 days, 12
days or 18 days post-anthesis and placed
in vials containing 88 mM sucrose. Day of
color change is indicated by W (white), or R
(red). Vertical bars represent SE of 12 fruit.


1 7 T 1 1 1 r _


I


I








Regardless of the initial fruit age and days in vitro,

all fruit ripened within 24 days from anthesis (Fig. 3-3).

Fruit initiated ripening (white stage) at 20 days post

anthesis and the number of days between white and red stages

was the same for fruit harvested at 6, 12 or 18 days post

anthesis. The rate of growth, calculated as g/initial g/day,

was greatest for fruit placed in vitro at 12 days

postanthesis and least for fruit grown in vitro 18 days

postanthesis (Table 3-2). However, final fruit size was

greatly influenced by initial fruit size. Fruit harvested

after 18 days postanthesis had greater initial and final

fresh weights, lengths and diameters than fruit harvested

after 12 or 6 days postanthesis. Fruit harvested 18 days

postanthesis had the greatest rate of weight gain of 0.329

g/day.

Comparison of In Vitro Response between Fruit Orders

Primary and 20 fruit generally did not differ in daily

or total weight gain (Fig. 3-4). Fruit from each

inflorescence position gained about 0.15 g/initial g during

the first day in the vase solution, and obtained a final

cumulative weight increase of 0.9 to 1.0 g/initial g.

Tertiary fruit ripened at the same time as 10 and 20

fruit, but gained porportionately more weight as they changed

color from white to red. The growth rates in vitro were

similar between 10, 20and 30 fruit until the white























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R
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830 R

01
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w / W


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0 1 2 3 4 5 6 7 8 9 10 11
DAYS IN VITRO




Figure 3-4. Effect of fruit order on cumulative growth
in vitro. Fruit harvested 14 days post-
anthesis. Primary, secondary, and tertiary
fruit turned white (W) or red (R) on the same
day. Vertical bars represent SE of 12 fruit.













Table 3-3. Rates of growth in vitro for primary,
secondary or tertiary fruit order.

Time Fruit Order
Interval Primary Secondary Tertiary


(Days) (g/initial g/day x 102)
0 to 6 12.7+.16 12.1+.14 12.0+.11

6 to 11 5.4+.09 3.9+.06 13.9+.13





























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stage of color development (Table 3-3), then the growth rates

of 10 and 20 fruit slowed, while the 30 growth rate

remained constant.

Comparison of In Vitro Growth to Field Growth

Fruit harvested at 12 days postanthesis and ripened in

vase solutions had lower final fresh or dry weights than

fruit harvested at 19 days and ripened in vase solutions or

fruit ripened in the field (Table 3-4). Fruit placed in

vitro at 12 days postanthesis achieved 55% of the final

weight of fruit ripened in the field, while fruit placed in

vitro at 19 days postanthesis obtained 90% of field fruit

weight. No differences in color values were. found between

fruit ripened in the field or in vitro, regardless of number

of days in vitro. Fruit ripened in the field had greater

length and diameter than fruit harvested at 12 or 19 days

postanthesis and ripened in vitro (Table 3-5). Fruit placed

in vitro at 19 days postanthesis increased very little in

either length or diameter, relative to the field-ripened

fruit.

Discussion

These results demonstrate that floriculture techniques

employed for increased cut flower longevity provide a

feasible and satisfactory method for examining the

characteristics of strawberry fruit growth and ripening.

Strawberry fruit could be ripened successfully in vitro when

grown in vase solutions containing sucrose and HQS. Similar








success has been reported for floral crops harvested and held

in solutions containing sucrose and an antimicrobial agent,

such as HQS. Hydroxyquinoline salts lower the pH of water

and prevent microbial growth in vase solutions (Marousky,

1971) and reduce vascular resistance to water flow (Burdett,

1970). Addition of sucrose to vase solutions promotes the

longevity of roses (Ferreira and DeSwardt, 1980), carnations

(Reid et al., 1980b) and other cut flowers (Halevy and Mayak,

1981). Sucrose was essential for best growth and ripening of

strawberries harvested at the green stage, Sucrose is the

major assimilate translocated to strawberry fruit (Forney and

Breen, 1985b). Studies with roses employing vase solutions

containing radiolabelled sucrose indicate that sugars are

used for protein and carbohydrate synthesis (Paulin, 1986).

Sucrose also was essential for optimum color development

in strawberry fruit grown in vitro. Enhanced anthocyanin

development in the presence of sucrose has been reported for

carnations held in vitro (Lee et al., 1980) and for

strawberry leaf discs (Creasy et al., 1965). In strawberry

leaves, most of the anthocyanin precursors are derived from

the shikimate acid pathway and develop in response to

ultraviolet light (Rhodes, 1980), as is pelargonidim, the

predominant anthocyanin in strawberry fruit (Fuleki, 1969).

The in vitro growth performance of strawberry fruit was

greatly dependent on cultivar and fruit age. The failure of

'Douglas' fruit to gain weight or ripen in vitro could have


L









been due to several factors. The achenes blackened after 5

days in vitro, whereas the achenes on 'Pajaro' fruit remained

green. Viable achenes provide auxin, which stimulates

enlargement of receptacle tissue (Archbold and Dennis, 1985;

Nitsch, 1950). The failure of 'Douglas' fruit to exhibit

significant weight gains may also have been due to problems

with microbial stem blockage or the physiological composition

of the peduncles. Microorganism blockage of vascular tissue

has been shown to greatly reduce the vase life of cut

carnation flowers, while the effect of these microorganisms

is species-specific (Zagory and Reid, 1986). Van Meeteren

(1978) found that stem break in cut gerbera flowers was due

to reduced water uptake caused by bacterial activity, and

that the incidence of stem break was dependent on the

cultivar. Certain rose cultivars are very susceptible to

"bent neck" which is caused by a lack of stem lignification

and results in a lack of water uptake (Zieslin et al., 1978).

Color development in 'Douglas' was consistently initiated at

the proximal end and proceeded towards the tip (distal end).

Fruit exhibited weight losses before full color was obtained.

In 'Pajaro' fruit held in sucrose solutions, initiation of

surface color occurred in a random fashion and weight gain

continued well into color development.

Fruit placed in vase solutions at 12 to 14 days

post-anthesis (50 to 60% mature) exhibited the most growth

and color development when sucrose was provided. Fruit








harvested at 18 to 20 days post-anthesis (75 to 80% mature)

did not respond to added sucrose in terms of either weight

gain or color development but did ripen normally. Evidently,

fruit harvested at this stage had assimilated sufficient

carbohydrate to permit normal anthocyanin development and had

already achieved most of their growth. The less mature green

fruit, which failed to either gain weight or develop red

color when held in solutions without sucrose, apparently

lacked sufficient carbohydrate levels necessary for

anthocyanin formation or sustained growth.

The facts that green strawberry fruit provided with

sucrose gained more weight and that sucrose hastened the

ripening of both green and white fruit is evidence that fruit

were assimilating and metabolizing sucrose in a manner

similar to cut flowers. Both the xylem and phloem of roses

are involved in the translocation of sucrose from vase

solutions (Ho and Nichols, 1975); these tissues may also have

translocated sucrose to strawberry receptacles.

Although fruit harvested at 6 days postanthesis did

ripen, they exhibited a lower growth rate than fruit

harvested at later stages of development. The low weight

gain of these fruit may have been due some disintegration of

the peduncles in the vase solutions.

Competitive differences between fruit on the plant could

be largely overcome by placing fruit in vitro. Tertiary

fruit exhibited a greater growth rate between the white and








red stages of ripening than did 10 or 20 fruit. The 30

fruit initially weighed 60% less than 10 fruit, and may

have required more time in vitro to overcome the suppression

of growth induced by 10 and 20 fruit competition prior to

harvest. Janick and Eggert (1968) found that removal of 10

fruit resulted in increased 20 fruit weight. Fruit of the

same anthesis date placed in vitro ripened simultaneously

regardless of fruit order.

Final fruit size in vitro was ultimately controlled by

the initial size but color development was not. Lower

initial weights resulted in lower final weights within fruit

age. The lower final fruit weights from in vitro-ripened

fruit compared to field-ripened fruit has also been noted in

tissue-cultured strawberries (Bajaj and Collins, 1968).

Detachment of the fruit may have resulted in vascular system

injury and lower subsequent solution uptake. The vase

solutions employed here also lacked other nutrients and

hormones which may enhance growth of attached fruit. Vase

solutions with 1% and 5% sucrose were tried (data not shown),

but 1% sucrose resulted in nonuniform fruit growth while 5%

sucrose resulted in calyx necrosis.

The onset of ripening was not delayed for fruit grown in

vitro, even in fruit harvested as young as 6 days

postanthesis and weighing as little as 0.85 g fresh weight.

Evidently, initiation of ripening was not dependent on the

attainment of a critical fruit weight and use of an in vitro








system did not interfere with the changes required for the

expression of ripening.

Conclusions

Successful ripening of immature strawberry fruit was

accomplished by placing fruit with attached peduncles in vase

solutions composed of HQS and sucrose. Fruit cultivar and

maturation stage are the important variables which must be

considered when using the in vitro system. 'Pajaro' fruit

harvested at 50 to 60% maturity exhibited the greatest and

most uniform weight gain when placed in vase solutions

containing 200 ul.liter-1 and 88 mM sucrose. Although

the final fruit weight of in vitro ripened fruit was less

than that of field-ripened fruit, color developed in vitro at

the same rate and to the same levels as field fruit.















CHAPTER IV
RESPIRATION AND ETHYLENE PRODUCTION IN DEVELOPING STRAWBERRY
FRUIT GROWN IN VITRO


Fleshy fruits are categorized into nonclimacteric and

climacteric classes on the basis of respiration patterns

during maturation and ripening (Yang, 1987). The climacteric

fruit undergo a distinct rise in respiration and ethylene

emanation, whereas nonclimacteric members do not.

Conclusively identifying fruit as nonclimacteric can

sometimes prove difficult. Fruit subjected to pathogen

infection (Oslund and Davenport, 1983), chilling temperatures

(Wang and Adams, 1982) and other forms of stress can exhibit

elevated respiration and ethylene production unrelated to

ripening.

Application of exogenous ethylene has been shown to

induce respiratory and other physiological responses in both

climacteric and nonclimacteric fruit types. McMurchie et al.

(1972) found that application of propylene, an ethylene

analogue, to bananas would advance ripening and the onset of

the respiratory and ethylene climacterics without altering

the magnitude of the respiratory peak. When propylene was

applied to lemons and oranges, respiration was enhanced but

ripening was not advanced and endogenous ethylene production

was not enhanced over the low basal levels.









Beyer (1976) discovered that silver nitrate was a potent

inhibitor of ethylene action. Subsequent studies were made

with silver thiosulfate (STS), which is more readily

transported in plant tissue (Veen, 1983). Silver has been

shown to inhibit wilting of canrnation flowers (Reid et al.,

1980a,b) and to induce male flowering in cucumbers (Den Nijs

and Vissen, 1980). Lettuce roots exposed to ethylene in the

presence of STS continued normal elongation (Abeles and

Wydowski, 1987). Silver applied to intact tomato fruit

inhibited lycopene and polygalacturonase synthesis (Hobson et

al., 1984), but long-term treatment with STS stimulated ACC

synthesis and conversion of ACC to ethylene (Atta-Aly et al.,

1987), but the exact mechanism by which silver blocks

ethylene action remains unknown.

Attempts to advance the ripening of strawberry fruit,

reportedly a nonclimacteric fruit type, with ethylene have

been unsuccessful. Janes et al. (1978) found that treating

detached white strawberry fruit with 50 ul.liter1 for 25

hours failed to enhance color development or stimulate

respiration. Similarly, postharvest application of 200

ul.liter-1 ethylene for 24 hours to detached green or white

fruit did not initiate color development (Mason and Jarvis,

1970). The ability of exogenous ethylene to stimulate

endogenous ethylene production was not studied.

A confounding factor in the above studies has been the

failure of strawberry fruit to develop normally if harvested









prior to ripeness (Mason and Jarvis, 1970). This

complication makes it difficult to determine the true

involvement of ethylene in strawberry fruit ripening.

In the previous section (Chapter III), it was

demonstrated that strawberry fruit detached immature and

placed via peduncles into vase solutions of 3% sucrose were

able to grow and ripen. This system provided a means of

studying the long-term changes in respiration and ethylene

production of strawberry fruits during growth and ripening

under controlled conditions.

The objectives of these experiments were to characterize

respiratory and ethylene changes of developing strawberry

fruit and to study the effects of ethylene application on

ripening of detached strawberry fruit maintained in vitro.

Materials and Methods

Respiration and Ethylene Production Measurements

'Pajaro' strawberry (Fragaria X ananassa Duch.) fruit

were tagged at anthesis and harvested from field plantings in

Gainesville and Dover, Florida. Fruit were harvested at

green, green-white, white, pink and red stages of color

development. Fruit maturation at each developmental stage

was calculated as the percent of days from anthesis to

field-ripeness (Fig. 4-1). Fruit were cut with peduncles

attached, placed in plastic bags and transported on ice to

the laboratory. Fruit not used immediately were stored at

10C. Storage for up to 4 weeks at 1oC did not impair in



















240



200 1.OC



160 C



120 I
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)80



40


.05


U'W ---- I a R E i i ,


0 12
GREEN


14 16
GW
DAYS


18 20 22
WHITE PINK RED
POST-ANTHESIS


24 26 27
RED RED


50 67 75 83 92 100

PERCENT FRUIT MATURATION


Figure 4-1.


Changes in respiration and ethylene
in strawberry fruit harvested at
selected stages of development. Percent
maturation is a function of days from
anthesis (green) to the red stage of color
development. SE bars are within symbols.


I.
-4


C
(

-i


I1 I I I i I I I I I I I
PAJARO (250C)


___-0----I----
' ~^ ~.~1 1 ~ I ..1


.14
0
C

12 c

-4
.10 2


m


-4
6



.4



2


0
'0









vitro fruit growth upon removal from storage. Six fruit per

color stage were selected to measure the capacity of fruit to

produce CO2 and ethylene at different developmental stages.

Fruit were placed via peduncle into individual 16 ml

scintillation vials containing 15 ml sterile distilled water

to alleviate water stress. Each vial with a single fruit was

placed in a 252 ml jar and sealed for 1 hour at 250C

30. Respiration measurements, determined as CO2

production, were made after 0.5 hr by sampling 0.5 ml of

atmosphere with gas-tight syringes and injecting into a gas

chromatograph equipped with a thermal conductivity detector

(Fisher 1200). Levels of ethylene were determined by

sampling 0.5 ml of atmosphere with airtight syringes and

injecting into a gas chromatograph equipped with a

photoionization tube (Photovac 10A10).

Effect of Propylene on In Vitro Growth and Ripening

Fruit were harvested at the green stage (12 days

postanthesis; 50% mature) and at the white stage (20 days

postanthesis; 71% mature) of development and peduncles

trimmed to approximately 55 mm. Fruit fresh weights were

matched between treatments. Average fruit weights were

3.11+.50 g for green fruit and 10.96+1.8 g for white fruit.

Fruit were placed via peduncles into scintillation vials

containing 16 ml of a solution consisting of 88 mM sucrose

and 200 ul.liter-1 hydroxyquinoline hemi-sulfate (HQS).

Four replications were used for each treatment. Each









replication consisted of 4 fruit in 4 vials placed in a 454

ml wide-mouth Mason jar.

Propylene, an analogue of ethylene with approximately 1%

of ethylene activity (Burg and Burg, 1967), was employed to

determine the ethylene responsiveness of developing

strawberry fruit. Air or air and propylene combinations were

mixed at the desired concentrations by means of a

flow-through system equipped with flow meters and regulating

valves (Gull, 1981). A constant flow rate of the different

gas treatments was maintained at 15 ml.min-1. The

propylene gas mixture was administered to the fruit at a

concentration of 5000 ul.liter-1. The relative humidity

within the jars was measured with a psychrometer (Bendix,

Model 566) and ranged between 80 and 90%. The air

temperature was held at 250 + 30C. At intervals, gas

samples were withdrawn directly from the outlet tubes with

gas-tight syringes. Levels of ethylene and C02 were

determined as described above. Fruit weights were measured

daily. Fruit color was measured at white, pink and red

stages with a Hunterlab Colorquest color difference meter.

Effects of ACC and Silver on Fruit Development In Vitro

Green (12 to 14 days postanthesis; 50 to 56% mature) and

white fruit (20 days postanthesis; 71% mature) were harvested

from the field and placed via peduncle into scintillation

vials containing 16 ml of a solution consisting of 88 mM

sucrose, 200 ul.liter-1 HQS and 0, 1, or 5 mM









1-aminocyclopropane-l-carboxylic acid (ACC). Ethylene

production, respiration, fresh weights and color were

monitored as described above.

Strawberry fruit harvested 14 days after anthesis (50%

mature) were placed in vase solutions via attached peduncles

as described above. The vase solutions consisted of 88 mM

sucrose and 200 ul.liter-1 HQS, with or without 1 mM ACC.

Silver thiosulfate (STS) (0.5 mM) was prepared following the

method of Reid et al. (1980a). STS (0.5 mM) was added to

solutions as a pulse treatment from 0 and 4 days in vitro.

Fruit were then transferred to solutions without STS for 4

days, then were placed in fresh solutions with 0.25 mM STS

from 9 to 12 days in vitro. Strawberry fruit were grown in

vitro at 270 + 20C. Ethylene and fresh weights were

monitored as described above.

Results

Respiration and Ethylene Production as a Function
of Stage of Development

Both respiration and ethylene production of strawberry

fruit harvested at selected stages of maturity and held in

water to alleviate water stress decreased with advanced

maturity (Fig. 4-1). At the green stage, the respiration

rate was 240 ml C02'kg-1-hr-1 and this decreased to

55 ml*kg-1-hr-1 for fruit at the green-white stage.

Ethylene production was 0.6 ul*kg-l1hr-1 at the green

stage of development and decreased to less than 0.4

ul*kg-l hr-1 in green-white fruit. Although fresh









weight increased dramatically as fruit changed in color from

white to pink, respiration during this period decreased from

40 ml C02*kg-l'hr-1 to 20 ml*kg-1.hr-1 while

ethylene production stabilized near 0.05 ul'kg-l.hr1.

Response of Strawberry Fruit to Exogenous Propylene and ACC

The response to 5000 ul.liter-1 propylene of fruit

harvested 50 to 70% mature and maintained in vase solutions

is shown in Figures 4-2 and 4-3. The responses observed were

greatly dependent on fruit maturity. Fruit placed in vitro

at the white stage failed to exhibit elevated respiration,

enhanced fresh weight gain or color formation, over a 7 day

period in response to propylene treatment (Fig. 4-2).

Endogenous ethylene production during the propylene treatment

was too low to register above the 0.1 ppm contaminant

ethylene levels in the propylene. Ethylene levels for fruit

treated with continuous air remained below 0.5

ul*kg-1.hr-1 (data not shown).

Fruit harvested 50% mature (green) and held in vase

solutions also failed to exhibit increased respiration when

subjected to propylene (Fig. 4-3A). However, unlike white

fruit, the fresh weight gains of green fruit were accelerated

in response to either intermittent or continuous propylene

treatments (Fig. 4-3B). Color development was also enhanced

in response to propylene. These effects were related to the

length of fruit exposure to propylene. After 4 days in

vitro, fruit held in atmospheres of continuous (4 days
















10

.X






I-

C

W X
>
P "


<


AIR-* -
CPo--o
60 IP 0-.a



40



20

100
so B
80 B

60

40

20 p

0 ~l)II


0 1 2 3 4 5 6 7 8 90 10

DAYS IN VITRO





















Figure 4-2. Influence of propylene on respiration (A)
and weight gain (B) of strawberry fruit
harvested 20 days postanthesis (white).
Arrows near axis denote times of application
and removal of propylene.











.120: A AIR-
IP 0---0
CPo---o
IP

S100-


S80.


60







1
401I I 1

S200 B

X 180- P P

5 160 -

140 /

120

S100 P

o







0 1 2 3 4 5 6 7 8 9 10
DAYS IN VITRO





Figure 4-3. Effect of propylene on respiration (A) and
weight gain (B) of fruit harvested 12 days
postanthesis (green). Arrows near axis
denote times of application and removal of
propylene.









exposure) or intermittent (2 days exposure) propylene had

reached the white stage 2 days earlier and had gained 20%

more fresh weight than fruit not exposed to propylene (Fig.

4-3B). Fruit placed in vitro at the green stage and

continuously exposed to propylene had gained 50% more weight

than control fruit after 10 days. Fruit treated

intermittently (5 days total) with propylene had gained 30%

more fresh weight than control fruit. At this time, fruit

initially green and exposed to continuous propylene were red,

whereas fruit intermittently exposed were light red and

control fruit were pink.

Effect of ACC on Strawberry Fruit Grown In Vitro

As with the propylene treatments, the response of in

vitro grown strawberry fruit to ACC was greatly dependent on

fruit maturity. Ethylene production more than doubled (0.06

ul.kg-1.hr-1 to 1 ul.kg-1.hr-1) in fruit harvested

70% mature (white) and grown in vase solutions containing 1

mM ACC (Fig. 4-4A). Within 2 days, ethylene production from

fruit held in vase solutions containing 1 mM ACC had peaked

at a level 30 times higher than ethylene production from

fruit without ACC. However, over the entire treatment

period, respiration was unaffected by the endogenously

produced ethylene (Fig. 4-4B). Gains in fresh weight were

the same in both treatments, but color formation was advanced

by 2 days in fruit provided with ACC (Fig. 4-4C).





72





15 I1 ls
0A *
to 10

Ir 5 P 8-



SP P+ 0 mM-
.5 1 mMo--o
0 1J ( 0-
80
U3 R-

S60 A



O 40 m

1 mMo-o


S 100 P

S80

a 0- p 0 -R-
0

S 23 20 M OM .
L I I1m IMo ..o

0 1 2 3 4 5 6 7 8 9 10
DAYS IN VITRO







Figure 4-4. Ethylene production (A), respiration (B) and
weight gain (C) of fruit harvested at 20 days
postanthesis (white) and held in vase solutions
with 0 or 1 mM ACC. SE for weight gain are
within symbols.









Ethylene production in fruit harvested at 50% maturity

(green) and placed in vitro with 1 mM ACC increased from 0.5

to 1 ul.kg-1.hr-1 within 1 day (Fig. 4-5A). Ethylene

production remained at this level until 5 days in vitro.

From 5 to 7 days in vitro, by which time fruit had turned

white, ethylene production increased 5-fold from fruit

provided with ACC. Fruit held without ACC continued to

produce ethylene at a rate of less than 1 ul*kg-1.hr1.

The respiration rate of fruit provided with ACC was lower

than in the control (Fig. 4-5B). Green fruit held in ACC

had gained 30% more in fresh weight after 6 days in vitro and

50% more in relative fresh weight after 10 days in vitro

compared to fruit held in solutions without ACC (Fig. 4-5C).

Color changes were advanced by 2 days in fruit provided with

continuous ACC relative to the control.

In a subsequent study, green fruit were harvested at a

similar stage of development (56% mature) and held in higher

concentrations of ACC (5 mM). After 1 day in 5 mM ACC in

vitro, ethylene production increased 20-fold (0.5

ul*kg-1.hr-1 to 10 ul'kg-1.hr'1). After 5 days

in vitro, fruit held in 5 mM ACC had turned white and

ethylene production increased to 55 ul'kg-1 hr (Fig.

4-6). Fruit held in 1 mM ACC initially produced ethylene at

a rate of 2 ul'kg-1.hr-1, increasing 10-fold (20

ul.kg-1-hr-1) at 5 days in vitro, by which time fruit had

turned white. The ethylene production of the control fruit

decreased 40%, from an initial rate of 0.5 ul'kg-1.hr-'

to 0.3 ul*kg'1.hr-1 after 5 days in vitro.























W


I I I ? I I


DAYS IN VITRO


Figure 4-5.


The effect of ACC on ethylene production (A)
respiration (B) and weight gain (C) of fruit
harvested at 14 days post-anthesis (green)
stage of development and placed in vase
soluti ons.


0

cc
0, x

W
'-.2
MC

-J C


U











60



50



40




330
U,


3 20




10


0 1 2 3 4 5 67 8 9 10

DAYS IN VITRO














Figure 4-6. Ethylene production from fruit harvested at
14 days post anthesis (green) and placed
in vase solutions containing 0, 1,or 5 mM ACC.











Fruit harvested 56% mature (green) and provided with 5

mM ACC in vitro exhibited similar respiration rates as fruit

grown in 0 or 1 mM ACC (Fig. 4-7A). The cumulative fruit

fresh weight gains were 1.70, 1.60 and 1.45 g/initial g for

fruit grown in the presence of 5.0, 1.0 and 0 mM ACC,

respectively (Fig. 4-7B). Fruit in either the 1 or 5 mM ACC

solutions turned white 2 days before the control, and had

greater color development after 10 days in vitro.

Response of Strawberry Fruit Grown in Solutions Containing
Silver

Fruit harvested 50% mature (green) and held in solutions

containing 0.5 mM STS, 1 mM ACC or both exhibited increased

ethylene production (Fig.4-8A). Ethylene production from

fruit held in solutions with STS and without ACC increased

40% over control fruit. After 1 day in vitro, fruit held in

the STS solutions with ACC exhibited a 100% (1

ul*kg'hr-1 to 2 ul*kg'hr-1) increase in ethylene

production compared to fruit held in ACC solutions without

STS, and a 300% increase compared to fruit held without ACC

or STS.

Fruit harvested 50% mature (green) and held in solutions

containing ACC exhibited enhanced growth after 8 days in

vitro (Fig. 4-8B). However, fruit held in solutions

containing STS exhibited greatly suppressed growth, even in

the presence of 1 mM ACC. Fruit in ACC solutions reached the

white and pink color stages 1 day earlier than control fruit.












0 mM *--
1 mM---o
5mMO-o


6 140







20

V-l

20








CI-
0




I 180
C,
a
140



. 100






20
U
o


0 1 2 3 4 5 6 7 8 9 10

DAYS IN VITRO


Figure 4-7.


Respiration (A) and weight gain (B) of fruit
harvested at 14 days post anthesis (green)
and placed in vase solutions with 0, 1, or
5 mM ACC. SE bars for weight gain are within
symbols.


OmM.-.
1 mMo-o
S mUo-a


_ 1 r




















































4 5 6 7 8 9 10 11 12
DAYS IN VITRO


Figure 4-8.


The effects of ACC and silver on ethylene
production (A), weight gain and color (B)
of fruit harvested 14 days post anthesis
(green) and placed in vase solutions with
with 0 or 1 mM ACC and/or STS.


10
CI
*X
*,


0a


0.5

0


- 200

- 180
C
0 160

X 140
I-
120

100

W 80

60

m 40

2 20
C.









Although growth was suppressed in the presence of STS, color

development was not impaired.

Discussion

The respiration and ethylene production patterns of

strawberry fruit harvested at different stages of maturity

and maintained and ripened in vitro or from fruit harvested

from field-grown plants at selected stages during ripening

and held in water for 1 day during ripening indicate that

this fruit is nonclimacteric. Both respiration and ethylene

production rates decreased gradually as fruit ripened.

Similar patterns of respiration and ethylene production have

been reported for cherry (Blanpied, 1972), carambola (Oslund

and Davenport, 1983) and watermelon (Elkashif, 1985) fruit.

The respiration and ethylene production rates from fruit

harvested green or white and ripened in vitro were higher

than those of fruit harvested from the field at selected

stages of development. This discrepancy may be due to the

effects of sucrose and HQS in the vase solutions used with

the in vitro fruit. Roses held in vase solutions containing

sucrose exhibited higher respiration rates than roses held in

vase solutions containing water (Ferreira and De Swardt,

1980). Although the in vitro fruit maintained higher levels

of ethylene production and respiration, the overall trend was

one of decline during ripening, consistent with that from

fruit harvested at different stages of development.









Strawberry fruit harvested from 50 to 70% mature and

provided with a carbohydrate source failed to exhibit

increased respiration rates or stimulation of endogenous

ethylene production when continuously or intermittently

exposed to ethylene over a 10-day period. Janes et al.

(1977) were unable to detect increased respiration in

detached, white strawberry fruit exposed to 50 ul.liter-1

ethylene for 25 hours. Only one other nonclimacteric fruit

type, the cranberry, was reported to exhibit no respiration

response when treated with ethylene (Fudge, 1930).

Application of propylene is a widely employed method for

investigations of the climacteric nature of fruit (McMurchie

et al., 1972). Application of propylene to climacteric

fruit, such as banana, results in stimulation of endogenous

ethylene production, the onset of ripening and the onset of

the respiratory climacteric (McMurchie et al., 1972).

Application of propylene to nonclimacteric fruit generally

results in enhanced respiration but no stimulation of

endogenous ethylene production. The lack of endogenous

ethylene production from strawberries in response to

propylene is further evidence for the nonclimacteric nature

of strawberries. The lack of enhanced respiration obtained

with strawberry fruit is in contrast to other nonclimacteric

fruit exposed to propylene. Elkashif (1985) found that

application of 6500 ul.liter-1 propylene to watermelons

resulted in a 40% increase in the respiration rates.

Saltveit (1977) reported a 38% increase in respiration after









application of 500 ul.liter-1 propylene to green bell

peppers and McMurchie et al. (1972) reported a 100% increase

in respiration of lemons exposed to 500 ul.liter-1

propylene.

Growth and color development of strawberry fruit

harvested green (50% mature) and maintained in vitro was

enhanced in the presence of 50 ul.liter-1 ethylene. Little

stimulation of weight gain or color was evident in fruit

harvested white (70% mature) and treated with ethylene. A

similar change in sensitivity to ethylene with fruit maturity

has been reported for the grape (Hale et al., 1970), a

nonclimacteric fruit, and for the fig (Marei and Crane,

1971), a climacteric fruit. Hale et al. (1970) reported

that application of 20 ul.liter-1 ethylene to grapes on the

vine was not effective after the beginning of veraison, but

advanced the onset of the second rapid renewed growth phase

and ripening if applied just before veraison. Application of

5 ul.liter-1 ethylene to figs on the tree between stage II

and stage III of growth similarly hastened growth and

ripening (Marei and Crane, 1971).

The ability of immature strawberry fruit grown in vitro

to advance in ripening when exposed to ethylene may be

related to the growth pattern of this fruit. Strawberry

fruit continue to grow and assimilate sucrose during ripening

(Forney and Breen, 1985a), without formation of starch (Knee

et al., 1977). The lack of sufficient carbohydrate reserves









may be why Mason and Jarvis (1970) found that strawberries

would not soften, increase in soluble solid levels or color

normally if detached at the green or white stages of

development, despite postharvest treatment with ethylene.

The accelerated growth of the propylene or ethylene

treated strawberries may be due to ethylene-enhanced sucrose

uptake, as has been reported for other tissues. Saftner

(1986) applied ethylene to sugar beet tissue and found a

greater uptake of radiolabelled sucrose, which he proposed to

be due to increased phloem loading. The loquat, a

climacteric fruit, accumulates sucrose within 2 weeks of

maturation from other parts of the plant. A rapid increase

in the fresh weight occurs during ripening (Hirai, 1980).

Hirai (1982) found that ethylene treatment of loquat fruit

accelerated color and sugar accumulation. Since sugar

accumulation in the loquat is not the result of starch

degradation, Hirai (1982) proposed that ethylene triggered an

increase in sink activity or changed assimilate distribution

patterns. Veen (1985) noted that ACC treatment of carnation

buds stimulated the growth of pistils, and the addition of

sucrose to the ACC treatments increased pistil growth by an

additional 40%.

Suppression of strawberry fruit growth but not ripening

in the presence of silver indicates that the processes

controlling growth in this fruit are at least partly

influenced by ethylene. STS blocked the ethylene-stimulated









growth of pistils in carnation buds (Veen, 1985), and the

ripening of banana and tomato tissue slices (Saltveit et al.,

1978). However, application of 2 mM STS to cherries, another

nonclimacteric fruit, at the yellow-pink stage, failed to

prevent ripening (Reid et al., 1985). The ripening of

strawberries, defined as color change, held in STS indicates

that ripening is independent of ethylene action.

Strawberry fruit were able to assimilate and convert ACC

provided in vase solutions into ethylene. A 2-fold increase

in ethylene production occurred soon after fruit were placed

in the ACC solutions. The peak ethylene production from both

white and green fruit was 16 to 30-fold higher than controls.

Ethylene production of bell peppers and cucumbers, considered

to be nonclimacteric fruit types, was stimulated from basal

levels of less than 1 ul.kg-1hr-1 to 30 and 9

ul.kg- 1-hr1 by application of 1 mM ACC to excised tissue

(Cameron et al. 1979).

The highest levels of ethylene production from ACC

occurred after the strawberry fruit turned white but before

they reached the pink stage. The association of increased

ethylene production with the stage of fruit color development

indicates that the activity of ethylene-forming-enzyme (EFE)

is related to fruit maturity. Yang et al. (1986) reported

that the activity of EFE increased 1000-fold in apple fruit,

concurrent with increased ethylene production during

ripening.









Conclusions

Strawberry fruit harvested 50 to 70% mature and

maintained in vitro failed to exhibit a respiratory

climacteric or increased ethylene production during normal

ripening or in response to exogenously applied propylene. In

the presence of ACC, production of endogenous ethylene was

greatly stimulated in both green and white fruit, peaking

after the initiation of ripening. Respiration did not

increase in ACC-treated fruit. Strawberry fruit fresh weight

gain and color were enhanced in fruit harvested green and

maintained in vitro in the presence of ethylene but only

fresh weight gain was inhibited by STS treatment. The

respiration and ethylene production of fruit harvested at

green through red stages of development decreased with

advanced maturity, similar to the decline exhibited by fruit

ripened in vitro. On the basis of these results, the

strawberry can be classified as a nonclimacteric fruit.

Additionally, ethylene appears to be involved in the fresh

weight gain of strawberry fruit.














CHAPTER V
REGULATION OF ETHYLENE PRODUCTION IN STRAWBERRY FRUIT


Historically, the ripening pattern of fleshy fruit has

been categorized as climacteric or nonclimacteric on the

basis of respiration patterns during maturation and ripening

(Biale and Young, 1981). Additionally, McMurchie et al.

(1972) showed that there is a difference in the ethylene

production capacity between climacteric and nonclimacteric

fruit. The climacteric fruit undergo a distinct rise in

respiration and ethylene emanation, whereas nonclimacteric

fruit do not. In climacteric fruit, the increase in ethylene

production is temporally associated with a number of ripening

changes, including softening, pigment synthesis and/or

chlorophyll degradation. Ethylene has been thought to

function as a hormone, initiating and coordinating ripening

events in climacteric fruits (Rhodes, 1980). In the

nonclimacteric fruits studied, such as lemon (McMurchie et

al., 1972), cherry (Reid et al., 1985), carambola (Oslund and

Davenport, 1983) and watermelon (Elkashif, 1985), ripening

was not found to be associated with increased ethylene

production. Since nonclimacteric fruit display no increase

in ethylene production, the involvement of ethylene, if any,

as a ripening initiator in these fruits must relate to an

increased tissue sensitivity to ethylene (McGlasson, 1978).










The intermediates involved in the formation of ethylene

have been identified as methionine, S-adenosylmethionine

(SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) (Adams

and Yang, 1979). This pathway of ethylene biosynthesis has

been characterized as follows:

Methionine----SAM---ACC---ethylene

The pathway of ethylene synthesis has been found without

exception in all higher plant systems thus far studied (Yang

and Hoffman, 1984). Studies of the regulation of ethylene

synthesis in climacteric fruit have shown that the major

controlling enzyme is ACC synthase, which converts SAM to ACC

(Yang, 1987). Ethylene forming enzyme (EFE), which converts

ACC to ethylene, is constitutive but its activity can be

enhanced by the application of ethylene or ACC. The degree

of enhancement is dependent on fruit maturity (Hoffman and

Yang, 1982). Application of ACC to cucumber, pepper or

squash, all nonclimacteric fruits, resulted in a 10 to

30-fold stimulation of ethylene production (Cameron et al.,

1979). In citrus, one of the most intensely studied

nonclimateric fruit, investigations have focused on the wound

response of citrus (Hyodo and Nishino, 1981; Riov and Yang,

1982) rather than regulation during fruit development and

ripening.

Silver, applied as silver thiosulfate (STS), has been

shown to be a potent inhibitor of ethylene action. Silver









thiosulfate inhibited the wilting of carnation flowers (Reid

et al., 1980), blocked the elongation of carnation pistils in

the presence of ACC (Veen, 1985) and inhibited lycopene

synthesis and polygalacturonase activity in tomato fruit

(Hobson et al., 1984). However, the effects of STS on

nonclimacteric fruit ripening are unknown.

There is a lack of information regarding the regulatory

features of ethylene in nonclimacteric fruit. Previously,

the use of an in vitro system allowing the normal development

of strawberry fruit demonstrated clearly that these fruit

exhibit nonclimacteric ripening behavior (Chapter IV). The

objectives of this study were to determine the levels of ACC

and the activities of ACC synthase and EFE during ripening.

Additionally, the effects of exogenously applied silver

thiosulfate (STS), a known inhibitor of ethylene action, and

ACC on ethylene biosynthesis were examined.

Materials and Methods

Plant Material

'Pajaro' strawberry (Fragaria X annassa Duch.) fruit

were tagged at anthesis and harvested from field plantings in

Gainesville and Dover, Florida. Fruit used for measuring

ethylene production capacities in detached fruit were

harvested at the green, white, pink and red stages of color

development with attached peduncles, placed in plastic bags

and transported on ice to the laboratory. Fruit to be

developed in vitro were harvested at the green stage (50%








mature) with intact peduncles, placed in plastic bags and

transported on ice to the laboratory. The maturity of green

fruit was calculated as the percent of days from anthesis to

field ripeness. Fruit not used immediately were stored at

10C and used within 1 week.

Treatment of Strawberry Fruit with STS or ACC

Strawberry fruit were harvested at the green stage (50%

mature) with peduncles attached, weighed and placed

individually via peduncle into 16 ml scintillation vials

containing 15 ml of vase solution (88 mM sucrose and 200

ul.liter-1 hydroxyquinoline sulfate (HQS)), with or without

1 mM ACC. Silver thiosulfate (STS) (0.5 mM) was prepared

following the method of Reid et al. (1980) and used in the

vase solutions from 0 to 4 days. Fruit were then transferred

to solutions without STS for 4 days before being returned to

fresh solutions with 0.25 mM STS for 3 additional days. In

vitro grown fruit were maintained at 270+20C. Four fruit

per treatment were sacrificed after 4 days in vitro for

measuring ACC content, EFE and ACC synthase activities. The

remaining fruit were sacrificed when the fruit were ripe

(red). Determinations of ethylene production, ACC and assays

for EFE and ACC synthase were performed as described below.

Comparison of Ethylene Production Between Receptacle and
Calyx

Green strawberry fruit were harvested, weighed, and

placed in vase solutions containing 1 mM ACC as described

above. After 1 day in vitro, fruit were weighed and calyxes








excised. Ethylene measurements were taken from fruit and

calyxes at intervals from 1 to 6 hours and again after 24

hours as described below. After 2 days in vitro, calyxes

were removed from the remaining fruit and the ACC content and

EFE activity of fruit and calyxes were determined as

described below.

Ethylene measurements. Four replications, consisting of

4 detached fruit with peduncle per color stage or 4 fruit

grown in vitro, with calyxes excised, were placed separately

in 16 ml vials in 252 ml jars. Excised calyxes were placed

in 25 ml Erlenmeyer flasks. Jars containing fruit and flasks

containing calyxes were sealed for 1 and 0.5 hours,

respectively, at 250+20 C. Afterwards, 0.5 ml of

atmosphere was withdrawn using gas-tight syringes and

analyzed using a gas chromatograph equipped with a

photoionization detector (Photovac 10A10) and an activated

alumina column. For fruit grown in vitro with ACC and/or

STS, 4 replications consisting of 4 fruit in individual 16 ml

vials per 454 ml jar were maintained in a flow-through system

with a constant flow rate of 15 ml.min- 1 air. The relative

humidity within the jars ranged between 80 and 90%, and the

air temperature was held at 250+2C. At appropriate

intervals, gas samples were withdrawn directly from the

outlet tubes with gas-tight syringes and levels of ethylene

were determined as described above.









Assay of EFE activity. EFE activity was determined by

measuring the capacity of the strawberry tissue to convert

excess exogenously supplied ACC to ethylene (Hoffman and

Yang, 1982). Tissue plugs (8.5 mm in diameter) were taken

from freshly harvested fruit by insertion of a sterile,

number 5 cork borer through the center of the fruit,

bisecting the cortex, pith and epidermal layers. The

remainder of the receptacle was frozen at -300C for the ACC

assay (see below). Each plug from green or in vitro grown

fruit was sliced into discs 3 mm thick, to aid solution

infiltration. One plug per fruit, intact or sliced into

discs, was vacuum infiltrated for 2 minutes with a solution

of 2% (w/v) KC1 containing 2.5 to 5 mM ACC. Immediately

after infiltration, tissue plugs from white, pink or red

fruit were placed in 25 ml Erlenmeyer flasks and sealed with

serum caps for 1 hour at 250C. Discs from fruit harvested

green or grown in vitro were placed in 50 ml Erlenmeyer

flasks after infiltration, incubated for 3 hours, then sealed

with serum caps for 1 hour. The accumulated ethylene

concentration in the head space was determined as described

above.

Determination of ACC. Fruit used for EFE assays were

frozen at -300 overnight. Five g of frozen receptacle

tissue or 1 g of frozen calyx tissue was then homogenized in

4 ml/g cold 80% ETOH with a Sorvall Omnimixer at maximum

speed for 2 minutes. Homogenates were held on ice for 8

hours, then centrifuged at 10,000 G for 20 minutes. ACC in