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Identification and characterization of cDNA clones for chromoplast-associated proteins in tomato fruit

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Identification and characterization of cDNA clones for chromoplast-associated proteins in tomato fruit
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Lawrence, Susan D
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English
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vi, 153 leaves : ill. ; 29 cm.

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Carotenoids ( jstor )
Chloroplasts ( jstor )
Chromoplasts ( jstor )
Complementary DNA ( jstor )
Enzymes ( jstor )
Imports ( jstor )
Plastids ( jstor )
Ripening ( jstor )
RNA ( jstor )
Tomatoes ( jstor )
Tomatoes -- Genetics ( lcsh )
Tomatoes -- Ripening ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 136-152).
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Susan D. Lawrence.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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IDENTIFICATION AND CHARACTERIZATION OF cDNA CLONES FOR
CHROMOPLAST-ASSOCIATED PROTEINS
IN TOMATO FRUIT











By

SUSAN D. LAWRENCE


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


1993














ACKNOWLEDGEMENTS


I wish to thank Drs. Donald Grierson and Wolfgang Schuch for providing us

with clones from their ripening tomato fruit cDNA library. I would also like to take

this opportunity to thank the members of my committee Drs. Gloria Moore, Ken

Cline, Eduardo Vallejos, Don Huber and Bill Gurley for their advice and support.

I am especially indebted to Eduardo Vallejos for extensive years of committee

service. This dissertation is dedicated to Mike McCaffery for his tireless computing

assistance and to Ralph Henry for his photographic skills. Finally, I would like to

express my appreciation to all my Fifield Hall lab pals, past and present, for making

this an experience I will fondly recollect.















TABLE OF CONTENTS


ACKNOWLEDGMENTS ...................................... ii

ABSTRACT ................................................ v

CHAPTERS

1 INTRODUCTION ................................. 1
2 LITERATURE REVIEW ............................ 3
Morphological Changes .............................. 5
Biochemistry and Molecular Biology ..................... 6
Changes in Plastid Structure .......................... 15
Regulation of Photosynthetic Proteins ................... 16
The Role of Ethylene in Tomato Fruit Ripening ........... 18
Transcriptional Regulation of Fruit Ripening .............. 19
Processes Associated with Chromoplast
Development ..................................... 20
Heat Shock Proteins and Oxidative
Stress .......................................... 40
Conclusions ...................................... 51

3 CHROMOPLAST-TARGETED PROTEINS
IN TOMATO FRUIT .............................. 54
Introduction ..................................... 54
Materials and Methods .............................. 55
Results and Discussion .............................. 58

4 CHARACTERIZATION OF TWO cDNAS FOR
CHROMOPLAST-TARGETED PROTEINS ............. 74

Introduction ..................................... 74
Materials and Methods .............................. 75
R results ......................................... 82
D discussion ...................................... 105

5 CONCLUSIONS ................................. 118








APPENDIX CONSTRUCTION AND EXPRESSION OF ANTISENSE
pTOM 41 AND Ptom 111 PLASMIDS IN TOBACCO ..... 120

REFERENCES ............................................ 136

BIOGRAPHICAL SKETCH .................................. 153













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

IDENTIFICATION AND CHARACTERIZATION OF cDNA CLONES FOR
CHROMOPLAST-ASSOCIATED PROTEINS
IN TOMATO FRUIT

By

Susan D. Lawrence

December 1993

Chairman: Dr. Gloria A. Moore
Major Program: Plant Molecular and Cellular Biology

The chloroplast to chromoplast transition during tomato fruit ripening is

characterized by a dramatic change in plastid structure and function. The thylakoid

membranes of photosynthetically competent chloroplasts are broken down and the

carotenoid pigment lycopene is deposited within the developing chromoplast. A goal

of this investigation was to determine whether this process is mediated by an increase

in the steady-state level of RNA for plastid-targeted proteins. Assays for import of

radiolabeled translation products into isolated pea chloroplasts were used to monitor

levels of chromoplast-targeted proteins at four stages of tomato fruit development.

During development, striking increases in levels of translatable RNA for two such

proteins were found. Additionally, the import of in vitro translation products was

examined for seven individual cDNA clones known to encode RNA that increases








during fruit ripening. Three of these clones produced in vitro translation products

that imported into pea chloroplasts. One clone, pTOM5, has recently been shown by

others to encode the enzyme responsible for the first committed step in the synthesis

of lycopene.

Subsequent sequence analysis of another cDNA, pTOM 111, revealed that it

encodes a plastid-localized low MW heat shock protein. Not only does this transcript

increase during development at normal temperatures, but it is increasingly inducible

by elevated temperatures as ripening progresses. This is the first report of the

developmental induction of a plastid-localized low MW heat shock protein.

Identification of the induction of a heat stress protein during this transition suggests

that either the plastid is subjected to an increasingly stressful environment or, as is

consistent for the developmental induction of other heat stress proteins, a massive

structural reprogramming is underway.

These studies imply that there is synthesis and import of new proteins during

the transition from chloroplast to chromoplast, and that the plastid conversion is an

active developmental program rather than a simple decline in synthesis of the

photosynthetic apparatus. Furthermore, these results demonstrate the utility of this

method for identification of structural genes involved in plastid morphogenesis.














CHAPTER 1
INTRODUCTION



Ripening of tomato fruit involves a wide variety of developmental changes

that include but are not limited to changes in cell wall structure, in metabolism, in

ultrastructure, and an especially dramatic change in the form and function of the

plastid. Biochemical and molecular studies suggest that most or all of these changes

are mediated by the enhanced expression of ripening specific genes. I have chosen

this system as a means to investigate plastid development and interconversion.

We are interested in determining how the transition from chloroplast to

chromoplast is regulated, e.g., does a subset of plastid proteins increase during this

process? In an initial examination of this question, we have asked whether an

increase in the accumulation of RNA for a subset of plastid proteins correlates with

the chloroplast to chromoplast transition. Since transcription and/or translation of

the plastid genome seems to diminish during chromoplast formation (Kobayashi,

1991), chromoplast-specific proteins are probably encoded in the nucleus. Nuclear-

encoded plastid proteins are synthesized in the cytosol and posttranslationally

imported into plastids. The import of proteins into plastids is a highly specific

process (Keegstra, 1989). The proteins are made as precursors with transient amino

terminal "transit peptides," which are both necessary and sufficient for the correct








2
targeting into this organelle. It is this latter observation that makes the import into

plastids a potentially diagnostic assay for the identification of chromoplast associated

proteins.

The approach described within this dissertation has been to monitor

chloroplast import of in vitro translation products from RNA of tomato fruit isolated

at different ripening stages. We have also studied translation products programmed

with transcripts from cDNAs (Slater et al., 1985) whose steady state RNA level

increases during ripening. Since individual cDNAs were analyzed, subsequent

sequence analysis allowed further characterization of the proteins that mediate this

transition. Eventually the role of chromoplast-associated proteins in chromoplast

development can be assessed directly via antisense technology. The final outcome of

such a study should be an understanding of the role new protein synthesis plays in

this process and the isolation, identification and characterization of genes that

mediate this transition.













CHAPTER 2
REVIEW OF LITERATURE



The manner in which the transition from chloroplast to chromoplast is

regulated is largely unknown. Electron micrographic analysis has shown that a

massive reordering of plastid structure occurs during the conversion (Thelander et

al., 1986). In tomato fruit, this is characterized by the overall breakdown of

chloroplast specific components, e.g. the thylakoid membrane system and starch, with

the concomitant accumulation of membrane-associated deposits of the carotenoid

lycopene (Thelander et al., 1986). It has not been determined whether the

breakdown of chloroplast structures is caused simply by the decrease in synthesis of

photosynthetic proteins or if increases in degradative enzymes actually enhance this

process. An examination of nuclear-encoded, plastid-targeted, photosynthetic proteins

has shown that their transcription stops 5 to 10 days before the fruit reaches full size

(Piechulla et al., 1986). Analysis of plastid-encoded transcripts has demonstrated that

some are greatly reduced, whereas others persist, and one appears to increase during

chromoplast formation (Kobayashi, 1991; Richards et al., 1991). Similarly, little has

been ascertained about the function or expression of genes involved in the synthesis

of chromoplast structures as few clones specific to this developmental stage have

been available for analysis (for example, Kuntz et al., 1992).








4
In considering what aspects of fruit ripening directly affect the proteins that

are targeted to the plastid, it quickly became apparent that two types of functions

could cause increases in proteins directed to the developing chromoplast. First,

proteins involved in the degradation of the photosynthetic apparatus or the build up

in carotenoids are excellent candidates for chromoplast-targeting and enhanced

synthesis during fruit ripening. A broad description of plastid senescence will be

considered, since that transition also involves the degradation of chloroplast

structures and because an examination of RNA from senescing leaves has shown that

many of the same transcripts are also present. Secondly, other aspects of maturation

may have indirect effects on the environment in which the developing plastid resides.

For example, the autocatalytic production of ethylene, the respiratory climacteric or

an increase in oxidative stress during ripening could secondarily create increases in

plastid-targeted proteins. Since the plastid is responsible for a number of basic

biosynthetic functions, such as the synthesis of aromatic amino acids, fatty acids and

8-aminolevulinic acid these changes outside of the plastid could create a need for

substrates synthesized within this organelle.

A consequence of the work described in this dissertation was the

characterization of a cDNA that encodes a plastid-localized low molecular weight

(MW) heat shock protein (hsp). Because transcript for this hsp increases during

ripening at normal temperatures, it was necessary to assess how this unexpected

finding fit into chromoplast development or fruit ripening. Since oxidative stress has

been associated with senescence and hsps have been induced by oxidative stress in








5
animal tissues, a consideration of heat stress proteins and oxidative stress during fruit

ripening will also be examined.



Morphological Changes

The overall changes that occur during the ripening of tomato fruit are as

familiar to ones mother as they are to any post-harvest physiologist. Full sized green

fruit harvested from the garden and brought into the kitchen will ripen normally. The

color changes observed during the ripening of tomato fruit are due to the breakdown

of chlorophyll and the deposition of lycopene within the fruit plastids. The breaker

stage is identified by the appearance of external color at the blossom end. The exact

fruit color that then develops is dependent on variety and consequently, the fruit may

turn yellow, pink, or orange-red. As the fruit becomes uniformly red, the cell walls

begin to soften. The characteristic flavor of ripe tomatoes is created by a

combination of glucose, fructose, citric and glutamic acids, which accumulate in the

vacuoles, along with the synthesis of hundreds of volatile compounds (Davies and

Hobson 1981; Grierson, 1986).

The young fruit is green because it contains chlorophyll in photosynthetically

functional chloroplasts. It is considered immature green (IMG) until it reaches its

full size. Growth is complete at mature green (MG). A green tomato may appear

morphologically quiescent; however, it is undergoing important ripening changes. Its

locular tissue transforms from a solid to a gelatinous substance, it can respond

rapidly to exogenous ethylene and the endogenous ethylene level also increases at








6
this time. By late MG, carotenoids begin to appear in the locules. Thus the color

change begins in the interior of the fruit (Kader and Morris, 1976). After the fruit

has passed through mature green, the transformation to a red ripe soft tomato

proceeds rapidly.



Biochemistry and Molecular Biology

Although the change in fruit color is a convenient method of classifying

tomato fruit ripening, a number of other biochemical changes also occur. For

example, a dramatic rise in respiration and ethylene production takes place. This

phenomenon is known as the climacteric. Climacteric ethylene levels reach about one

half of their maximum by the breaker stage (Picton and Grierson, 1988). The

respiratory peak occurs soon after the rise in ethylene (Grierson, 1986).



Alterations in Basic Metabolism During Fruit Ripening

Changes take place in the type of carbohydrate that accumulates as tomato

fruit ripens. Starch granules transiently appear between 14-28 days postanthesis in

the immature green chloroplasts (Davies and Cocking, 1965). Although these

plastids can fix carbon at up to 35% of the rate attained by leaf plastids, the starch

granules are mostly the product of sucrose imported from the leaves (Thelander et

al., 1986). Since the import of carbohydrate from the leaves stops by MG (Thelander

et al., 1986), fruit at this stage will develop normally if harvested from the plant.

About 28-35 days after anthesis degradation of starch occurs as an increase in soluble








7
sugars takes place. Consequently, little starch remains in the mature green fruit

(Yelle et al., 1988). The rapid degradation of starch precedes the climacteric rise in

respiration (Davies and Cocking, 1965) and this carbohydrate is stored as glucose

and fructose in the vacuole (Grierson, 1986). The degradation of starch seems to

occur via phosphorylase (Robinson et al., 1988), and the further steps of hexose to

triose occur via the pentose phosphate pathway and glycolysis (Thelander et al.,

1986). Therefore, as the tomato fruit matures, a shift of stored carbohydrate occurs

from starch in the plastid to hexose sugars in the vacuole. In fact, the red ripe

tomato contains no measurable starch and 47% hexose by dry weight (Davies and

Hobson, 1981). The changes in stored carbohydrate precede the breakdown of the

thylakoid membranes and consequently, any plastid associated enzymes responsible

for starch degradation are not expected to increase during chromoplast development.

It is unclear what causes the increase in CO, during ripening and,

consequently, the molecular regulation of this increase is also unknown (Solomos,

1988). Fluctuations in enzymes involved in the tricarboxylic acid (TCA) cycle and

glycolysis occur but it has not been determined whether these changes are due to a

variation in the level of enzymatic effectors or are caused by alterations in the

amount of de novo synthesis of these enzymes during ripening. Solomos (1988)

reviewed the literature on respiration in ripening fruit and concluded that the

respiratory burst measured is probably a result of an increase in substrate level

phosphorylation caused by enhanced glycolysis. An increase in the activities of

cytosolic NADP linked malic enzyme (Goodenough et al., 1986) and the glycolytic








8
enzyme phosphofructokinase (PFK) have been observed (Grierson, 1986). PFK is a

control point in glycolysis and an increase in this enzyme activity probably results in

an increase in carbon flow through this pathway (Solomos, 1988). Indeed, an increase

in the accumulation of ATP is generally noted during the respiratory rise (Solomos,

1988). Investigation of mitochondrial electron transport in ripening fruit suggested

that it remains unchanged (Solomos, 1988). However, the TCA cycle enzymes citrate

synthase, malate dehydrogenase, NAD-linked malic enzyme, and isocitrate

dehydrogenase decrease in ripening tomato fruit (Grierson, 1986; Jeffery et al.,1986).

Overall, these data do not explain why an increase in respiration is measured during

ripening. Since these changes are underway during the plastid conversion while

photosynthesis is declining, it is unclear how this may effect the synthesis of proteins

targeted to the plastid.



Other Changes Associated with Fruit Ripening

Apparently, the level of the anoxia-responsive enzyme, alcohol dehydrogenase

(ADH), increases during ripening in tomato fruit (Longhurst et al., 1990; Van Der

Straeten et al., 1991). Longhurst et al. (1990) concluded that the increase in ADH

during ripening may be caused by the cytoplasmic increase in pH. It has been

hypothesized that low oxygen levels could result within such a bulky organ, but by

exposing ripening fruit to different levels of 02 and CO2 these authors surmised that

the change in pH leads to an induction of this enzyme.








9
As fruit matures, the cell walls soften and an increase in soluble pectin occurs

(Grierson, 1986). Characterization of the regulation of the enzymes involved in cell

wall degradation has begun. These data suggest that the pattern of expression for

enzymes involved in cell wall degradation is complex and can even involve more than

one isozyme (DellaPenna et al., 1986; Grierson et al., 1986; Fisher and Bennett,

1991; Harriman et al., 1991). For example, antisense mutagenesis of the cell wall

softening enzyme, polygalacturonase (PG), has shown that although this enzyme is

dramatically induced during fruit ripening, loss of this enzymatic function has little

overall effect on maturation (Sheehy et al., 1988; Smith et al., 1988 and 1990).

The synthesis of hundreds of volatile compounds occurs as tomato fruit ripens

(Davies and Hobson, 1981). However, little is known about how these compounds

are synthesized during maturation.



Oxidative Stress and Fruit Ripening

Since a chromoplast-associated low MW heat shock protein was identified in

this work, it is important to understand the role this stress protein may play in fruit

development. Oxidative stress occurs when the cell's ability to respond is exceeded

by the level of reactive oxygen species present (Barja de Quiroga, 1992). The

induction of hsps by compounds that cause oxidative stress has been shown in animal

tissues (Drummond and Steinhardt, 1987; Cajone and Bernemmi-Zazzera, 1988;

Courgeon et al., 1988). In addition, oxidative stress has often been associated with

ripening and senescence. One theory postulates that a buildup in the deleterious








10

effects of free radicals results in the physiological manifestations of aging (Harman,

1992).

Free radical production during normal cellular metabolism

Reactive oxygen compounds can cause damage to proteins and especially

membrane components of the cell. A number of normal cellular functions result in

the production of these species, which can be a side effect of reactions that reduce

molecular oxygen. For example, at least one percent of the oxygen utilized during

respiratory electron transport is incompletely reduced (Fleming et al., 1992).

Furthermore, an increase in basal metabolism can result in an increase in the

amount of reactive oxygen generated even without invoking a breakdown in oxidative

phosphorylation (Barja de Quiroga, 1992). Photosynthesis is another normal cellular

function during which a large amount of free radicals can form (Scandalios, 1993).

In addition, some enzymatic reactions directly produce reactive molecules. Examples

of this latter group include xanthine oxidase (an ATP degrading enzyme) and beta

oxidation of fatty acids (Scandalios, 1993). Finally, certain environmental stresses can

enhance the level of reactive molecules. For example, radiation, herbicides,

pathogens, hyperoxia, and temperature fluctuations can induce free radical

production (Scandalios, 1993). Perhaps a buildup of these deleterious compounds

occurs during the ripening of tomato fruit. Since any cellular function that involves

the reduction of oxygen causes production of reactive oxygen, either autocatalytic

ethylene synthesis or the respiratory climacteric would be excellent sources of free

radicals.








11
One source of reactive oxygen is lipoxygenase activity, which increases during

ripening (Thompson et al., 1987). This enzyme incorporates oxygen into

polyunsaturated fatty acids (Hatanaka et al., 1992) and leads to a build up of the

reactive oxygen compounds hydroperoxide or superoxide. Recently, Bowsher et al.

(1992) have purified a membrane associated lipoxygenase from breaker stage tomato

fruit. They find that the activity fractionates with the thylakoid membrane.

Consequently, this could result in increases in the amount of reactive oxygen species

in the developing chromoplast.

The change in membrane integrity during fruit ripening

A decrease in membrane fluidity, which may be created by oxidative stress,

occurs during ripening and senescence along with a progressive loss of specialized

membrane function (Thompson et al., 1987). Many aspects of membrane structure

can affect the level of membrane viscosity. For example, the length or degree of

unsaturation of the hydrocarbons and the level of sterols all can play a part.

Although a decrease in membrane fluidity occurs in the plasma membrane of tomato

fruit (Legge et al., 1986) and pepper microsomal membranes (Whitaker, 1991), no

change in plastid membrane fluidity occurs in either tomato or pepper chromplasts.

Neither fluorescent membrane probes, which directly assess the structure within

specific environments of the membrane used by Legge et al. (1986), nor

measurements of the free sterol or total sterol to phospholipid ratios determined by

Whitaker (1991) could detect a change in the developing chromoplast membrane.

Considering the increase in carotenoid biosynthesis during ripening, which is








12
associated with plastid envelope membranes, it is appropriate that the integrity of

this membrane persists.

Free radical scavengers and oxidative stress

Initially, an increase in free radicals results in an increase in enzymes or

antioxidants that neutralize these reactive species. For example, a family of

superoxide dismutases (SOD) acts to scavenge superoxide and create oxygen and

hydrogen peroxide. Five isozymes of this enzyme have been described in tomatoes.

Two are cytoplasmic, one is mitochondrial and two occur in chloroplasts (Perl-Treves

and Galun, 1991). Also, ascorbic acid and a-tocopherol can act as antioxidants

(Leshem, 1988). A number of enzymes are also involved in reducing the levels of

hydrogen peroxide. Catalase, peroxidase and glutathione peroxidase all act in this

manner (Leshem, 1988). Additionally, glutathione reductase is required to regenerate

glutathione lost by interaction with hydrogen peroxide (Leshem, 1988).

Consequently,the increase in these enzymes is correlated with an increase in reactive

oxygen molecules. Any decrease in these enzymes with senescence, therefore, could

contribute to degeneration of membranes and disturbance of cellular function.

Increases in basal metabolism may induce SODs in plant tissue. Bowler et al.

(1989) concluded that the mitochondrial SOD may respond to changes in the activity

of respiratory electron transport, because the induction of this enzyme in tobacco

correlated with increased activity for cytochrome oxidase.

In addition, oxidative stress in one compartment can lead to the induction of

free radical scavengers in another part of the cell. This was shown when paraquat,








13
which is thought to act mainly on chloroplasts, was applied to tobacco and caused

a strong induction of SOD transcript in the mitochondria, cytoplasm and chloroplast

(Tsang et al., 1991).

The level to which SOD is induced within a cellular compartment may depend

on the amount of SOD that is currently present. This was the explanation of Perl-

Treves and Galun (1991) when they found that the addition of paraquat resulted in

a greater induction of the cytoplasmic SOD in comparison to the plastid message in

tomato leaves. The level of plastidial enzyme activity in mature leaves is already high

in comparison with transcript level. They suggested that induction of plastidial SOD

transcript may therefore be unnecessary.

Ethylene has been shown to induce cytoplasmic and plastidial SOD transcripts

and enzyme activity in tomato leaves (Perl-Treves and Galun, 1991) and

mitochondrial SOD message in tobacco leaves (Bowler et al., 1989). In response to

ethylene, therefore, an increase in reactive oxygen species has been noted and

enzymes within all three cellular compartments can be affected.

Free radical scavengers in ripening fruit

The role of free radical scavengers during fruit ripening, however, is somewhat

confusing. Peroxidase has been shown to decrease during ripening (Rabinowitch et

al., 1982) or peak during the pink stage in tomato fruit (Rothan and Nicolas, 1989).

Perhaps the techniques used by Rabinowith et al. (1982) were not sensitive enough

to detect this peak of activity. The transient increase in basic peroxidase activity

paralleled the increase in ethylene (Rothan and Nicolas, 1989). Since the conversion








14
of 1-aminocyclopropane-l-carboxylate (ACC) to ethylene requires oxygen, this may

lead to a transient increase in reactive oxygen molecules and an increase in this free

radical scavenger.

Changes in SOD have been reported during tomato fruit ripening but results

were contradictory. SOD activity has been found to peak at the pink or orange phase

(Rabinowitch et al., 1982; Livine and Gepstein, 1988). However, no change in the

two plastidial SOD isozymes was reported by Perl-Treves and Galun (1991), nor was

a peak of the cytoplasmic or plastidial SOD message noted during ripening. In fact,

they found that the plastid-associated message actually decreased with maturation.

They explain these contradictory results by suggesting that the enzyme could be quite

stable and turnover very slowly. It is hard to explain all of the varied data reported

by these three groups. More work needs to be done before a conclusion can be made

about free radical scavengers and ripening in tomato fruit. Since the messages for the

additional plastidial and cytoplasmic SODs have not been examined, perhaps these

additional data will help reconcile these dissimilar observations.

Pepper chromoplasts do appear to respond to an oxidative stress during fruit

maturation (Romer et al., 1992). The antioxidant producing enzyme cysteine synthase

was purified and a cDNA was isolated. They found not only did this enzyme increase

during ripening but the activity of glutathione reductase, glutathione synthetase and

glutathione content within chromoplasts also increased during their development.

Since all these compounds protect organisms from oxidative damage and an increase

in a-tocopherol also occurs during ripening, they concluded that these increases in








15
antioxidants and peroxide scavenging compounds may allow protection of the

overaccumulated carotenoids and other membrane components from oxidative stress.

Since the results from tomato fruit on the induction of free radical scavengers

is contradictory, information from other systems may help predict what occurs in

tomato fruit chromoplasts. Leaf tissue responds to an increases in basal metabolism

and the addition of ethylene by inducing SOD (Bowler et al., 1989; Perl-Treves and

Galun, 1991). In addition, a compound expected to affect predominantly one

compartment can induce SOD in other portions of the cell (Tsang et al., 1991).

Finally, the level of induction may be dependent on current levels of enzyme activity

(Perl-Treves and Galun, 1991). Perhaps the results from pepper chromoplasts where

increases in a number of these free radical scavengers has been measured, explains

how the chromoplast, unlike the plasma membrane, avoids oxidative damage and

maintains membrane integrity (Whitaker, 1991; Romer et al., 1992). One might

predict that the plastid can respond to an environment that contains an increasing

amount of reactive oxygen and consequently it is able to maintain chromoplast

membrane integrity during ripening. These data also suggest that the production of

ethylene and/or the respiratory climacteric may induce the production of

chromoplast-associated proteins involved in free radical scavenging.



Changes in Plastid Structure

Massive changes take place in plastid ultrastructure during the chloroplast to

chromoplast transition. The thylakoid membranes are dismantled and chlorophyll








16
degrades (Thelander et al., 1986). Concomitantly, lycopene begins to accumulate in

the stroma. The green flesh mutant, which is inhibited in chlorophyll degradation

during ripening, is helpful in the determination of how pigment accumulates in the

developing chromoplast. Electron micrographs show that the granal stacks persist in

this mutant even as pigment is deposited (Goodwin, 1980), and suggests that

lycopene builds up around the disintegrating thylakoids. By the time a normal tomato

is fully ripe, the chromoplasts contain little else but large stores of lycopene, which

accumulate as lipophilic globules and crystals of pigment (Harris and Spurr, 1969).

The type of chromoplast that develops may be dependent on the type of

carotenoids that accumulate. The 'r' mutant, for example, with its low level of both

lycopene and B-carotene, contains only plastoglobules (Goodwin, 1980).



Regulation of Photosynthetic Proteins

Transcripts for the nuclear-encoded components of photosynthesis decrease

long before the breakdown of the thylakoid membranes. The mRNA for rbcS and

cab (Rubisco small subunit and chlorophyll a/b binding protein respectively) are not

detected after 30 days post anthesis (Piechulla et al., 1985, 1986). This loss of nuclear

transcripts occurs 5 to 10 days before MG, and precedes the end of chlorophyll

synthesis and the disassembly of the thylakoid membranes by 2-3 weeks.

The plastid encoded messages rbcL and psbA (Rubisco large subunit and the

32kD photosystem II protein), however, persist throughout development (Piechulla

et al., 1986). Most of the other plastid transcribed proteins, such as the photosystem








17
I and II proteins (psaA psbB psbC or psbD), all diminish in message levels

throughout development.

Once ripening has progressed to an intermediate stage of development beyond

mature green, levels of photosynthetic activity have greatly diminished and this is

reflected in decreased amounts of photosynthetic proteins. For example, by the

yellow stage of development, the amount of LHCP protein has declined to 10% of

the level present in IMG fruit (Livine and Gepstein, 1988).

At the yellow-orange stage, the level of Rubisco activity has diminished to 2%

of the level present at MG (Piechulla et al, 1987) while the amount of measurable

protein for the large subunit has also decreased. The level of photosynthetic electron

transport has decreased to 13% of what was observed at MG (Piechulla et al., 1987).

Only plastocyanin and the 32kD protein were detected by this stage of development.

These data underscore the dramatic change in the protein components of the

plastid that occurs between mature green and this intermediate stage of ripening. It

is unclear from this type of analysis whether the loss of photosynthetic proteins is

simply due to a decrease in synthesis or is also caused by an enhanced production

of degradative enzymes.

Examination of plastid gene expression during chromoplast development has

shown a reduction in the level of plastid transcript during this process. It is uncertain

whether this is the result of posttranscriptional regulation (Gruissem et al., 1989;

Marano and Carillo, 1991), or whether the rate of transcription is decreased due to

methylation of the plastid genome (Ngernprasirtsiri et al., 1988; Kobayashi et al.,








18

1990). The level of translation is also dramatically reduced in Capsicum plastids

(Kuntz et al., 1989) where ultrastructural analysis has shown that red fruit contain

no ribosomes or rRNA (Carde et al., 1988). Richards et al. (1991) have identified

a plastid transcript that is more abundant in chromoplast RNA than chloroplast

RNA of tomato fruit. However, given the lack of functional translation in pepper

chromoplasts and the decreased amount of transcripts present in tomato

chromoplasts, it is expected that most proteins that are increasingly present in

chromoplasts will be nuclear-encoded.



The Role of Ethylene in Tomato Fruit Ripening

The rise in ethylene during the climacteric is mediated by de novo synthesis

of at least the final two enzymes in the biosynthetic pathway (Holdsworth et al. 1987;

OIson et al., 1991; Rottmann et al., 1991). To date, only the genes for the enzymes

that catalyze these final two steps in ethylene biosynthesis have been isolated.

Ethylene is synthesized from methionine via S-adenosyl-L-methionine and 1-

aminocyclopropane-1-carboxylic acid (ACC). The enzyme that synthesizes ACC

(ACC synthase) is thought to be a pyridoxal 5'-phosphate utilizing enzyme (Yip et

al., 1990). The final step in the synthesis of ethylene is mediated by ethylene oxidase

(EFE), requires oxygen, and can be inhibited by the addition of free radical

quenchers (Matoo and Aharoni, 1988). The synthesis of ACC is considered the rate

limiting step in ethylene biosynthesis. However, EFE is also increasingly synthesized








19
during ripening in tomato and avocado fruit, (Holdsworth et al., 1987, McGarvey et

al., 1992) and during the senescence of carnation petals (Wang and Woodson, 1991).

Production of transgenic tomato plants that contain antisense transcripts for

the enzyme ACC synthase has shown that ripening involves both ethylene dependent

and independent pathways (Theologis, 1992). A number of laboratories have

produced transgenic tomato plants with reduced levels of ethylene (Hamilton et al.,

1990, Klee et al., 1991, Oeller et al., 1991). The most dramatic decrease occurred in

a transgenic plant carrying ten antisense copies of part of a gene for ACC synthase.

The authors reported that the plant has a 99.5% reduction in the level of ethylene

(Oeller et al., 1991) and produces fruit that remain firm and green until exogenous

ethylene is applied. If no ethylene is added, the mutant fruit still express RNA for

the ripening induced cell wall softening enzyme, polygalacturonase (PG) (Oeller et

al., 1991). This result was unexpected because ethylene is required for the expression

of PG. This suggests that the regulation of ripening by ethylene is more complex than

just simple transcriptional control. While the transcription of PG is ethylene

independent, the expression of the enzyme is dependent on the continued presence

of this hormone.



Transcriptional Regulation of Fruit Ripening


A molecular analysis of fruit ripening in tomato has led to the conclusion that

the de novo synthesis of ripening specific proteins occurs and is responsible for many

of the changes associated with maturation. Several researchers have characterized








20

changes in the profile of in vitro translation products that occur during development

of tomato fruit (Rattanapanone et al., 1978; Speirs et al., 1984; Biggs et al., 1986;

Lincoln et al., 1987). A number of groups have constructed cDNA libraries enriched

in ripening specific sequences (Mannsson et al., 1985; and Slater et al., 1985; Lincoln

et al., 1987). Generally, the onset of ripening in tomato fruit is mediated by an

increase in a specific subset of transcripts, including those for PG, EFE and ACC

synthase (DellaPenna et al., 1986; Grierson et al., 1986; Holdsworth et al., 1987;

Olson et al., 1991; Rottman et al., 1991).

Examination of the clones isolated by Grierson's group has shown that, for

most of their cDNAs, RNA fails to accumulate when the fruit is held at high

temperature (Picton and Grierson, 1988). High temperature (35C) has an inhibitory

effect on fruit ripening, depressing the synthesis of lycopene, PG, and ethylene

(Picton and Grierson, 1988). Perhaps this effect of high temperature represents a

heat shock response (Picton and Grierson, 1988). This was not an overall effect for

all clones, however, since RNA increased initially, for some clones, in response to

an increase in temperature. Unfortunately, many of these clones have yet to be

assigned a function.



Processes Associated with Chromoplast Development

Upon review of the work characterizing the molecular changes that occur

during the chloroplast to chromoplast transition, it is notable that mainly

photosynthetic proteins have been examined during this phase (Piechulla et al., 1985,








21
1986, 1987; Livine and Gepstein, 1988). To undertake the investigation of proteins

that increase during chromoplast development, a method must be found to identify

proteins specific to this process. Isolation of chromoplasts and the subsequent

comparison of proteins on SDS-PAGE from different developmental stages has been

attempted and proteins that increase in amount have been identified (Iwatsuki et al.,

1984, Bathgate et al., 1985, Wrench et al., 1987, Hadjeb et al., 1988). Using this

method, specific chromoplast-associated proteins have been isolated from only

pepper chromoplasts (Hadjeb et al., 1988). Antibody has been purified for two

proteins that increase during Capsicum chromoplast development (Newman et al.,

1989). One of these proteins binds carotenoids and is a major constituent of

Capsicum chromoplast membranes (Cervantes-Cervantes et al., 1990). However, this

protein probably binds capsanthin since the antibody does not react to any protein

present in chloroplasts or tomato fruit chromoplasts (Cervantes-Cervantes et al.,

1990). While this type of approach has resulted in the characterization of a few

proteins involved in chromoplast function, only abundant proteins can be identified

in this manner.

The plastid is involved in the synthesis of many compounds necessary for cell

function. For example, the synthesis of fatty acids, aromatic amino acids and other

substances such as 8-aminolevulinic acid have all been localized to plastids. Many of

these compounds are precursors to a wide array of secondary metabolites, so it is

possible that an increased synthesis of these plastid-synthesized compounds is

required during chromoplast development. A recent review by Hrazdina and Jensen








22
(1992) however documented that in many instances isozymes for the synthesis of

amino acids involved in secondary metabolism may be present in the cytoplasm

rather than in the plastids. More information on the compartmentalization of these

pathways is needed before conclusions about the localization of enzymes for

secondary metabolism can be drawn.



Carotenoids

Enzymes responsible for the synthesis of carotenoids are obviously the most

likely candidates for proteins that may be increasingly produced during chromoplast

development. Therefore, it is necessary to consider what enzymes and cofactors may

be involved in this pathway and how it may be regulated.

Carotenoid function

In the chloroplast, carotenoids are an integral part of the photosynthetic

apparatus, where they act as accessory pigments funneling light energy into the

reaction centers. If light intensity becomes too great, the photosynthetic carotenoids

can also trap excessive light energy. Without these peripheral pigments, the plants

may photobleach (Mayfield et al., 1986). The necessity of carotenoids to forestall this

deleterious event is apparent when synthesis of carotenoids is blocked by inhibitors

or mutation.

Another function of carotenoids, however, is the pigmentation of many

flowers and fruits. Most of the orange, yellow or red pigments found in these tissues

can be attributed to carotenoids, which accumulate in the chromoplasts.










Carotenoid structure

Carotenoids are long chain hydrocarbons. The conjugated double bonds give

them their color. The synthetic pathway produces carotenes, xanthophylls and

abscissic acid. The desaturations that occur in the biosynthesis of carotenoids can

create either a cis or trans configuration. Cyclization at one or both ends can occur

(Goodwin, 1980). A beta, epsilon or gamma ring forms based on the position of the

double bond (Goodwin, 1980). The end groups can be acyclic in the case of lycopene.

If the pigment is a long chain hydrocarbon then it is a carotene. Xanthophylls on the

other hand, are carotenoids that contain oxygen.

A number of different modifications are possible to this basic long

hydrocarbon chain; 500 different structures have been described. The complement

of carotenoids found in green leaves are relatively uniform throughout the higher

plant kingdom (Goodwin and Mercer, 1983). This may be expected considering the

importance these compounds play in photosynthesis and the conservation of structure

required of the proteins and pigments of the thylakoid membranes. However, wide

structural variety occurs in carotenoids found in petals and fruit. The type of

molecule that accumulates is species dependent.

Carotenoid synthesis

The basic scheme for the synthesis of higher plant carotenoids is shown in

Figure 2-1. The production of phytoene represents the first committed step in the

synthesis of carotenoids. Usually, step-wise desaturation of phytoene leads to all-

trans-lycopene. The normal product of phytoene synthase is 15-cis-phytoene. Where








24

does the cis-trans isomerization take place? Beyond zeta-carotene, trans

intermediates are involved. Four stepwise removals of 2 hydrogen molecules occur

from phytoene to lycopene, as oxygen is reduced in this reaction. Proton attack of

the C1 double bond of lycopene is involved in the formation of the ring structures.

Different enzymes are responsible for the different rings that are formed. Little

interconversion of these rings occurs. Xanthophyll formation results from the

addition of hydroxyl groups.

Location of carotenoid biosynthesis

Where are the enzymes of carotenoid synthesis located? All activities after the

synthesis of isopentyl pyrophosphate (IPP) have been shown to occur in the plastids

(Gray, 1987). Compartmentalization of the enzyme activity has been best analyzed

in chloroplasts as opposed to chromoplasts, since they are most amenable to

fractionation. Mayfield (1986) examined the chloroplasts of spinach, and concluded

that phytoene desaturase is located on the inner envelope membrane. Ultrastructural

examination of developing chromoplasts suggests that pigments associate with

membrane as they are produced in the inner envelope, which is followed by

vesiculation of this membrane pigment complex (Camara and Brangeon, 1981). In

addition, examination of the lipid component of these membranes confirms that they

are a product of the inner envelope (Sitte et al., 1980).

Genetic studies have mapped all the known tomato mutants to the nuclear

genome (Tomato Genetic Cooperative Report, 1987), which suggests that the genes

involved in the synthesis of carotenoids are nuclear-encoded. Furthermore, inhibition













Isopenlenyl pyrophosplile

Geranylgeronyl pyrophosphole

Phyloene


Cis-C-orolene

1
Proneursporene

Prolycopene


.'Corolene
v Co*C tene


y -Corolene


*-CaCoroene


Fig. 2-1 Pathway for the Biosynthesis of Carotenoids in Higher Plants








26
of plastid translation has demonstrated that this process is not necessary for

carotenoid accumulation (Camara, 1984). Finally, the enhanced synthesis of phytoene

caused by the addition of CPTA is blocked by the cytoplasmic translation inhibitor

cycloheximide. Therefore, the evidence indicates that the enzymes required for

carotenoid synthesis are imported into the plastid.

Enzymology of carotenoid biosynthesis in tomato fruit

The purification of the carotenoid biosynthetic enzymes is difficult because all

the enzymes in this pathway beyond the synthesis of phytoene are associated with

plastid membranes. Consequently, studies that characterize the number and type of

enzymatic steps responsible for the synthesis of these compounds in higher plants

have relied on adding radioactive precursors to solubilized crude enzyme

preparations. The distribution of radioactivity between the different carotenoids is

then measured. These experiments have shown that when, for example, red tomato

extracts are fed labeled lycopene, cyclic carotenoids are produced (Quershi et al.,

1974). This suggests that the enzymes for the production of these compounds are

present, but do not normally accept the lycopene that is produced in vivo. Perhaps

an inhibitor is present in vivo that has been diluted out in vitro. Alternatively, a

spatial separation between these enzymes may exist in vivo that is disrupted in vitro

(Papastephanou et al., 1973). These in vitro studies involved the addition of

quantities of substrate and cofactors that may drastically differ from the situation in

vivo. These unexpected results demonstrate that the pathway for the synthesis of








27
carotenoids is complicated and a more thorough understanding of the production of

carotenoids requires analysis in less disrupted systems.

Examination of mutants in the synthesis of tomato fruit carotenoids has

predicted that the photosynthetic pigments are produced by different isozymes. A

number of tomato mutants have been studied (for review see Goodwin and Goad,

1970). Many have been mapped, their complement of carotenoids has been measured

and the position of the genetic lesion in the biosynthetic pathway has been predicted

(Goodwin and Goad, 1970; and Khudari, 1972; Tomato Genetic Cooperative Report,

1987). Many of these mutants affect only fruit color, while the photosynthetic

pigments found in the leaves are completely normal (Thelander et al., 1986).

Genetic evidence suggests that fruit carotenoids are produced by two separate

pathways. One pathway produces the abundant amount of lycopene generally

associated with red ripe tomatoes, while the other pathway appears to make a small

amount of B-carotene. Evidence that two pathways exist includes the tomato mutant

apricot, which is deficient in the accumulation of lycopene without affecting the level

of B-carotene in the fruit (Goodwin, 1980). Additional evidence includes the fact that

the production of lycopene is sensitive to high temperatures in normal red tomatoes

(Thelander et al., 1986), while the high f3 mutant has reduced lycopene and enhanced

temperature sensitive synthesis of B-carotene. In other words, the dominant high f.

tomato seems to have used the same temperature sensitive pathway to augment the

levels of B-carotene. Both the existence of the apricot mutant and the residual non-








28

temperature sensitive amount of B-carotene found in high f fruit imply that a second

pathway exists.

The residual, but ever present, level of total carotenoids found in some

mutants, the presence of temperature sensitive B-carotene, and enzymes that function

in vitro but fail to accumulate their product in vivo suggest that a major ripening

enhanced inducible pathway for carotenoid biosynthesis may exist along with a minor,

possibly constitutive, path. If the minor pathway is responsible for the production of

the cyclic carotenoids, this predicts that enzymes for the production of cyclic

carotenoids may not necessarily be enhanced during ripening in normal red

tomatoes.

Characterization of carotenoid biosynthesis in bacteria: a model for higher plant
analysis

The cloning and subsequent complementation of genes for carotenoid

biosynthesis in bacteria demonstrates how powerful such a systematic analysis can be

in the understanding of how the pathway functions. These genes are organized into

clusters in the bacteria Erwinia herbicola and Erwinia uredovora. In addition, the

carotenoids in these species are similar to the pigments that accumulate in higher

plants. A number of investigators have used these genes to transform E. coli,

Agrobacterium and Zymomonas (Misawa et al., 1990; Hundle et al., 1991; Misawa et

al., 1991; Naagawa and Misawa, 1991). Deletion analysis of the cluster transformed

into E. coli allowed an unambiguous ordering of the pathway by analysis of the

carotenoids that accumulated in the resulting mutants (Misawa et al., 1990).








29
The power of the characterization of carotenoid biosynthesis in E. coli is

demonstrated by the analysis of individual genes from different species by Linden et

al. (1991). A plasmid containing the genes responsible for the synthesis of phytoene

in Erwinia uredovora can be transformed into E. coli and sequentially cotransformed

with a plasmid containing a phytoene desaturase (PDS) gene. The type of pigment

that is produced by the specific PDS enzymes can then be monitored. The PDS

cDNAs cloned from Synechococcus, Rhodobacter and Erwinia uredovora were tested.

Analysis of the carotenoids produced in these transgenic E. coli showed that the

different enzymes produced a different complement of products. For example, the

Synechococcus enzyme was able to introduce two double bonds in phytoene while the

Rhodobacter enzyme introduces three double bonds. This analysis also shed some

light on the level of cis-trans isomerizations that accumulate in the various

complemented bacteria. The authors concluded that a gradual shift from cis to trans

carotenoids occurs with an increase in the number of conjugated double bonds

produced by the different enzymes. This suggests that a specific cis-trans isomerase

may not be required for this interconversion. Finally, since the Rhodobacter PDS

adds 3 double bonds, the specificity of the Erwinia lycopene cyclase and beta-

carotene hydroxylase on the asymmetrical molecule formed could be assessed. This

experiment was performed by the introduction of a plasmid containing the entire

Erwinia gene cluster except for a mutant PDS gene. Cotransformation of this plasmid

with theRhodobacter PDS created unique carotenoids. This experiment demonstrated

that the cyclase or the hydroxylase recognizes half of the carotenoid molecule. This








30

type of analysis can allow a careful identification of the number and types of enzymes

required to produce a specific complement of carotenoids. The sequential cloning

and analysis of the different carotenoid biosynthetic genes in transgenic bacteria

demonstrate how powerful such an approach could be for the dissection of this

pathway in higher plants. If the higher plant carotenoid genes were cloned, a similar

series of experiments could be undertaken.

Molecular biology of carotenoid biosynthesis in higher plants

The gene for phytoene synthesis has been isolated from tomato fruit.

Originally, the clone for the phytoene synthase cDNA (pTOM5) was isolated by

Slater et al. (1985) simply because it recognized increasing amounts of RNA as

ripening progressed. Significant homology to the deduced amino acid sequence of the

tomato cDNA, pTOM5, to a clone for phytoene synthase in Rhodobacter was

identified (Armstrong et al., 1990a). Bartley et al. (1992) used this information to

clone a similar cDNA from tomato fruit and provided evidence, by complimenting

a mutant Rhodobacter, that it indeed coded for phytoene synthase. Recently, a

normal red variety of tomato has been transformed with a constituitively expressed

antisense version of pTOM5 (Bird et al., 1991). This resulted in yellow fruit and pale

flowers. Chlorophyll a and b levels were normal in the leaves of the transformed

plants, which suggested that the leaf carotenoids were unaffected by the constitutively

expressed antisense message. These results confirm that pTOM5 codes for a

phytoene synthase that is specific for ripening fruit. A more complete analysis of the

transgenic plants by Bramley et al. (1992) confirmed that antisense pTOM5 alters








31
carotenoid accumulation in the flower and fruit but not the leaves of transgenic

plants.

The gene for geranylgeranyl pyrophosphate (GGPP) synthase has been cloned

from pepper fruit (Kuntz et al., 1992). The enzyme was purified, antibody was

produced, and a cDNA was isolated from a ripe pepper fruit expression library. Both

the mRNA and the protein increased during fruit ripening. Immunological

examination has shown that this stromal enzyme is mainly found concentrated

around chromoplast plastoglobuli (Cheniclet et al., 1992).

The deduced amino acid sequence from the cyanobacterium Synechococcus

PDS allowed the cloning of a cDNA from soybean cotyledon by Scolnik's lab

(Bartley et al., 1991). Similar work by Pecker et al. (1992) produced a PDS from

ripening tomato fruit. An interesting observation resulting from this work is that,

while the cyanobacterium and higher plant PDSs share more than 65% deduced

amino acid identity, no homology beyond a short amino terminal region that contains

a conserved dinucleotide-binding motif exists with the fungal, Rhodobacter or Erwinia

PDS. This suggests that separate PDSs may have evolved for higher plants and green

algae in contrast with other photosynthetic organisms. Following a different

approach, Camara's lab purified a phytoene desaturase from Capsicum and used

antibody to isolate a cDNA clone (Hugueney et al., 1992). The deduced amino acid

sequence of that clone has striking homology to the tomato fruit sequence. Only

slight increases in the steady-state level of RNA that hybridizes to this clone occur

at MG. This is in contrast with the analysis of the protein and the enzyme activity,








32
which increases significantly with ripening. It appears from the evidence provided by

both laboratories (Hugueney et al., 1992; Pecker et al., 1992) that the quantity of this

mRNA is low during ripening. Perhaps during chromoplast development large

increases in mRNA occur only for the early enzymes involved in carotenoid

biosynthesis.

Mayer et al. (1992) have purified an oxidoreductase that is specific for

chromoplasts and crucial for desaturation of carotenoids. The peripheral nature of

the involvement of this enzyme in carotenoid synthesis is supported by the fact that

antibody to this protein does not inhibit the synthesis of carotenoids. However,

oxidized quinones are required for carotenoid desaturation, so it is assumed that this

enzyme acts to affect the redox state of the chromoplast membranes where a large

amount of oxidized quinones and tocopherols found. Perhaps the oxidoreductase and

the increased amount of antioxidants in pepper chromoplasts point to an

environment that contains an escalating amount of reactive oxygen (Romer et al.,

1992). The desaturation of phytoene requires the transfer of electrons to oxygen.

This might cause an increase in reactive oxygen and lead to the induction of enzymes

that generate antioxidants.

Two pathways are predicted for the synthesis of carotenoids in ripening

tomato fruit. None of the genes for the enzymes responsible for steps in the pathway

beyond the synthesis of zeta-carotene have been cloned. It is interesting that only

minor increases in the steady-state level of RNA for PDS occur during fruit ripening.

Since large increases in steady-state levels of RNA accumulate for both phytoene








33
synthase in tomato and GGPP synthase in pepper fruit (Slater et al., 1985; Kuntz et

al., 1992), perhaps only the very early steps of this pathway are transcriptionally

induced during ripening, while transcript for the later steps will be constitutively

expressed at a low level. Conceivably the dominant mutants del+ and b+, which

produce increased amounts of 8 and B carotenoids respectively, will contain increased

levels of RNA for the the enzymes responsible for these later steps in the

biosynthetic pathway.



Chloroplast Senescence

Chloroplast senescence involves the degradation of chlorophyll and the

dismantling of photosynthetic membranes. Considering that the analysis of

chloroplast breakdown during chromoplast formation has relied on ultrastructural

analysis and characterization of mostly the decrease in steady-state levels of

photosynthetic proteins, perhaps an understanding of how this process occurs during

leaf sensecence will allow predictions for the plastid transition in ripening fruit.

Indeed, 7 out of 12 fruit ripening enhanced clones share homology to transcripts that

increase in senescing leaves (Davies and Grierson, 1989). During ripening and

senescence starch, chlorophyll and photosynthetic proteins degrade (Davies and

Grierson, 1989). A burst in ethylene synthesis has been correlated with senescence

in leaf tissue (Davies and Grierson, 1989) and respiration also increases.










Photosynthetic protein synthesis during leaf senescence

A decrease in the synthesis of photosynthetic proteins occurs during

chloroplast senescence (Roberts et al., 1987). This loss of proteins follows a similar

pattern as described for chromoplast development by Piechulla. The synthesis of

protein in different ages of Phaseolus leaves was measured, and the amounts of

cytochrome f, cytochrome b6, a and b subunits of the ATPase, 68kD PSI protein, and

LHCP decline in leaves. However, the 32 kD protein persists throughout senescence.

Ultrastructural analysis of chloroplast senescence

Plastid senescence is a gradual ordered process, which occurs within the

plastid. It was initially assumed that chloroplasts were engulfed and degraded within

vacuoles (reviewed by Gepstein, 1988). These early studies concluded that senescing

leaves actually contained fewer plastids. However, reappraisal of this work suggested

that the isolation of fewer plastids in senescing leaves was due to the increased

difficulty in isolating these fragile organelles (reviewed by Gepstein, 1988).

Additional ultrastructural analysis showed that plastids in senescing leaves undergo

a transition in which thylakoids become unstacked and plastoglobuli accumulate

(Gepstein, 1988). This suggests that the degradation of plastid components during

senescence occurs within the plastid itself (Thayer et al., 1987), and is gradual and

ordered. The plastid envelope maintains its integrity until very late in senescence,

long after the degradation of thylakoid membranes is complete (Thomson and Platt-

Aloia, 1987). Furthermore, this means that if enhanced degradation of plastid








35
components occurs during sensecence then it is possible that new proteins would be

imported into the plastid for this purpose.

The degradation of chlorophyll during chloroplast senescence

The degradation of chlorophyll has been suggested to be the trigger for the

proteolysis of the protein components of the thylakoid membranes. Unfortunately,

the pathway for the catabolism of chlorophyll is not well understood. Work with the

non-yellowing mutant of the temperate grass festuca suggested that an initial step in

thylakoid degradation involves dephytlation of the chlorophyll molecule while it

remains in the pigment-protein complex (Thomas et al., 1989). A polar green

chlorophyll degradation product can be isolated from purified light harvesting

complexes (LHCs) of these mutant plastids. This degradation product is also

produced in normal plastids exposed to anoxic conditions. The buildup of this

intermediate occurs in the mutant plant because a downstream step in the

chlorophyll catabolism pathway is missing. Since the product could be isolated from

purified LHCs the authors concluded that the first step in the degradation of

chlorophyll occurs within the protein-pigment complexes of the thylakoid membrane.

Chlorophyllase has been isolated from chloroplasts and could perform this function

(Matile et al., 1989). However, the protein does not seem to increase during

senescence. Alternatively, the enzyme could remain latent in the plastid and be

activated when necessary. Perhaps the activation of the chlorophyllase requires a

newly synthesized protein?








36
The next step in the catabolism of chlorophyll is probably an oxidation.

However, it is not clear whether this occurs in the plastid or vacuole (Matile et al.,

1989). One theory suggested that a thylakoid associated chlorophyll oxidase is

induced by an increase in linolenic acid during senescence (Luthy et al., 1984), which

triggers the catabolism of chlorophyll and the resulting cascade of degradative

reactions. This idea may require reappraisal in light of the fact that oxidized pyyroles

have been found in vacuoles and not isolated from senescing plastids (Matile et al.,

1989).

Degradation of the thylakoid membranes during chloroplast senescence

The analysis of stay-green mutants in soybean and the previously described

mutant in festuca demonstrates that the degradation of the thylakoid membrane is

mediated by at least two separate pathways and is the result of both nuclear and

plastid-encoded proteins.

For example, the cytoplasmic mutant cytG of soybean specifically retains the

(LHCs) (Guiamet et al., 1991). Clearly the breakdown of the core proteins involves

a separate path from the degradation of the LHCs. Since chlorophyll a and b are still

catabolized in this plant, these data suggest that a plastid-encoded protease is lacking

and is involved in LHC breakdown.

When two homeologous recessive nuclear-encoded genes (did2) of soybean are

present in this tetraploid plant, most of the chlorophyll is retained and all of the

thylakoid proteins appear to be present, in contrast with nonmutant leaves (Guiamet








37

et al., 1991). These data imply that the degradation of the thylakoid membrane in

soybean is also mediated by nuclear-encoded genes.

Evidence from the mutant festuca underscores the complexity of the

degradation of thylakoids. The phenotype of this recessive mutant is a selective

maintenance of components of the photosynthetic membranes. For example, the level

of LHCP is maintained in the mutant plant long after the amount of this protein has

begun to diminish in normal plants (Thomas and Hilditch, 1987). Additionally, the

degradation of the plastid D1 protein, which normally turns over in either the light

or the dark, is inhibited in the dark, but not the light (Thomas and Hilditch, 1987).

The 33Kd protein, however, degrades normally in this mutant (Thomas and Hilditch,

1987). These mutant plants are also able to photobleach if they are subjected to high

light intensities (Thomas and Hilditch, 1987). These data suggest that two separate

protease systems are responsible for the degradation of thylakoid membranes. One

system is light activated and unaffected by this mutation and another functions in the

dark and is inhibited by this genetic lesion.

Degradation of Rubisco

An examination of the degradation of Rubisco suggests how a stromal protein

may be broken down during senescence. The breakdown of Rubisco can be induced

by oxidative stress, in a process that appears to mimic natural senescence (Mehta et

al., 1992). A specific and highly conserved cysteine is oxidized by the addition of

CuSO4 and leads to dimerization of the protein. This is followed by a transient

accumulation of this stromal protein on the membrane, and, finally, degradation. In








38
vitro studies have also demonstrated that the oxidized form of Rubisco is inactive and

more susceptible to proteolytic digestion (Penarrubia and Moreno, 1990). This

suggests that the turnover of stromal protein is selective and may involve membrane

associated proteases. It is uncertain whether this response to oxidative damage is

analogous to senescence, however, it does demonstrate that an ordered system of

stromal protein degradation exists in the plastid. It remains to be seen if natural

senescence involves a build up of oxidized protein that enters into this degradation

pathway, or if it is developmentally manifested in some other manner.

Changes in other components of the chloroplast during senescence

A decrease in the level of galactolipids has been noted in senescing

chloroplasts (Woolhouse, 1984). However, Woolhouse (1984) pointed out that it is

uncertain whether this change in lipid content is caused by decreased biosynthesis or

increased degradation.

The isolation of proteases involved in plastid protein degradation has been

fairly unsuccessful (Dalling, 1987). The majority of proteases are found in the

vacuole, and their presence masks an examination of changes in plastid proteases

during senescence.

Control of chloroplast senescence

Overall, plastid senescence requires continued protein synthesis, since the

process can be inhibited by cycloheximide (reviewed by Gepstein, 1988). Although

plastid senescence can be triggered by a number of environmental stimuli such as

darkness, oxidative compounds, or nitrogen deprivation, it is still unclear whether this








39
process is mediated by an increase in de novo synthesis of specific proteins. In the

case of Rubisco, turnover is slow until a nuclear encoded event (assumed since it is

inhibited by cycloheximide or kinetin) triggers the rapid degradation of this abundant

photosynthetic protein (Thayer et al., 1987).

Perhaps the breakdown of chlorophyll acts as a signal for the degradation of

photosynthetic membrane proteins (Nock et al., 1992). This has been suggested by

the results from studies of the mutant festuca where a mutation in chlorophyll

catabolism inhibits the breakdown of the protein components of the thylakoid

membranes. The production of the dephytlated chlorophyll degradation product

requires protein synthesis (Thomas et al., 1989), which implies that the synthesis of

a cytoplasmic protein is required for induction of chloroplast senescence.

Conclusions about chloroplast senescence

To date, ultrastructural analysis has suggested that the senescence of

chloroplasts is a gradual event within the organelle. The degradation of plastid

proteins is a complex process involving multiple proteases with different properties.

Since the breakdown of chloroplasts is inhibited by cycloheximide, this suggests that

senescence requires continued protein synthesis (Gepstein, 1988). Additionally, the

rapid degradation of Rubisco during senescence implies that this upsurge in protein

loss may require a de novo increase in proteins involved in this degradative process

rather than simply continued constitutive expression.

It is difficult to draw generalizations about what may trigger senescence from

the analysis of the few mutants available. The mutant phenotype could simply result








40

from the accumulation of particular plastid components due to loss of a single

function and may not represent a protein that is developmentally induced during

senescence. It seems safe to assume that the non-yellowingfestuca demonstrates that

although the thylakoid membranes can degrade in response to photooxidative stress,

the more gradual and perhaps dark associated breakdown requires proteases that are

not active until the pigment component has been modified.

It would be extremely helpful if proteins or the genes responsible for the

senescence of chloroplasts could be isolated. An understanding of their regulation

could suggest how senescence is controlled. It is possible that the conversion from

chloroplast to chromoplast may represent an ideal system for the isolation and

identification of cDNA clones for proteins involved in this degradative process since

the chromoplast transition does not take place in a sensecing tissue.



Heat Shock Proteins and Oxidative Stress

The identification of a chromoplast-associated low MW heat shock proteins

(hsp) during this study provoked an investigation into the literature on why this hsp

may have been induced during the chloroplast to chromoplast transition. One

possibility is that the low MW hsp is induced during certain developmental stages.

Historically, the induction of low MW hsps occurs during times when rapid

restructuring of the tissue is underway (Arrigo, 1987). It is also feasible that the hsp

is induced because the tissue is enduring an oxidative stress. Hsp induction by

compounds that create oxidative damage has been described in animal tissues








41
(Drummond and Steinhardt, 1987; Cajone and Bernelli-Zazzera, 1988; Courgeon et

al., 1988). Both the induction of hsps during development and oxidative stress will

be considered.



Introduction to Heat Shock Proteins

Heat shock proteins were first identified by their massive induction upon

increases of about 100C above ambient temperature (recently reviewed for plants by

Vierling, 1991; and generally by Jaattela and Wissing, 1992). Hsps have been found

in a diverse range of organisms from bacteria to mammals. These proteins are

broadly categorized by their molecular weight in SDS-PAGE. An individual organism

may contain a number of spatially and functionally distinct proteins from each size

class. For example, a number of different hsp 70 proteins have been described, some

of which are present constitutively (hsc).

The function of these proteins has been best characterized for the constitutive

members of the hsp60 and 70 classes. The 60 kD protein plays a role in the assembly

of the multisubunit Rubisco enzyme complex (Roy, 1989). Involvement in the

assembly of multimeric proteins in the mitochondria has also been shown (Cheng et

al., 1989). The 70 kD proteins have been implicated in moving newly synthesized

proteins from ribosomes to the cellular compartment in which they function (Weber,

1992). The role of the hsps during heat shock has been inferred from the function

of their constitutive forms. It is thought that the hsps bind to proteins denatured by

superoptimal temperatures and may facilitate refolding (Weber, 1992). A role for








42
hsps in providing thermotolerance has been suggested by experiments that create

protection from severe heat shock by the induction of these proteins with a brief or

moderate increase in temperature (reviewed by Lindquist and Craig, 1988).

The induction of hsp messages is rapid and transient (reviewed by Neumann

et al., 1989). Examination of the steady-state level of RNA for a number of different

hsps in soybean revealed that levels decay more quickly at normal temperatures than

during heat stress and the absolute amount of hsps induced is lower in plants that

have been previously exposed to a small heat stress (Kimpel et al., 1990). This is

reminiscent of the thermotolerance response in that the reaction to heat shock is

reduced by heat pretreatment. The transient nature of this response is apparent in

the fact that the hsp message had already begun to decay while the temperature was

still high, and the message was undetectable within 17 hours after the peak

temperature (Kimpel et al., 1990). The induction of heat shock proteins in field

grown plants has also been demonstrated (Burke et al., 1985; Kimpel and Key, 1985).

Kimpel and Key (1985) found that a transient increase in the message for both low

and high MW hsps occurs under fairly normal Georgia summer field conditions with

soybean. This induction is dramatically enhanced in non-irrigated plants. Burke et

al. (1985) also showed similar responses in material from irrigated versus non-

irrigated fields. However, they compared the canopy temperatures and found that the

non-irrigated field was 100C above the temperature in the irrigated field. This is an

important point since the quantity of hsp message produced increases with increasing

temperature (Chen et al., 1990; Kimpel et al., 1990).










Low MW Heat Shock Proteins

In plants the low MW hsps are generally not present at detectable levels in

leaves under nonstress conditions (Vierling, 1991). The low MW hsps represent a

small multigene family that has been subdivided on the basis of amino acid sequence

similarity (Vierling, 1991). Classes of this family have been found in the cytosol,

plastid, and endoplasmic reticulum. In fact, one class of low MW hsp is induced

during meiosis (Bouchard, 1990). Upon induction, the low MW hsps can accumulate

to massive amounts and form large crystalline deposits within the cytoplasm.



Low MW Heat Shock Proteins and Development

The low MW hsps have been found to increase transiently at different times

during development. The best characterized developmental induction of these

proteins is found during Drosophila embryonic development. Arrigo (1987) suggested

that the period of development in which these proteins are most highly induced

represents a time of massive developmental change when the cell may need carefully

controlled mechanisms for overall transformation. Four low MW hsps (ranging in

size from 22 to 27 kD) follow a complex pattern of expression that appears to be

mediated by the molting hormone ecdysterone. Addition of this hormone to

embryonic cell lines induces these proteins (reviewed in Bond and Schlesinger, 1987).

Deletion analysis of their promoters indicated that regions of the DNA responsive

to hormone and heat induction reside in separate portions of the gene. The same hsp

is, therefore, induced developmentally and by heat stress. Since heat causes








44
destabilization and unfolding of proteins and embryo development suggests rapid

protein biogenesis, the induction of the same group of proteins during both events

implies that they are involved in either stabilizing or degrading incorrectly folded

proteins.

Evidence for a developmental change in low MW hsps has begun to

accumulate in higher plants. The 14 and 40 kD hsps are present in dry embryos of

wheat and their mRNA has also been isolated from quiescent wheat embryos not

subjected to heat stress (Helm and Abernethy, 1990). These authors propose that

these hsps may act to protect the seed early in development from the effects of

excess heat. Since the germinating wheat embryo has fewer mechanisms to control

ambient temperature in comparison to an older seedling, maybe the production of

hsps allows the organism to tolerate this transiently harsh environment (Helms and

Abernethy, 1990).

The wheat embryo hsps also demonstrate how the genes for hsps may respond

differently to heat stress at different stages of development. These proteins are heat

inducible at early times during imbibition. However, they are not induced after as

little as 12 hours of imbibition (Helm et al., 1989). The wheat seed is initially quite

tolerant to heat stress, such that pretreatment with a low but hsp inducing

temperature does not improve this thermotolerance (Abernethy et al., 1989). By 9

hours of imbibition, however, a pretreatment can improve the survival of the seed

to heat stress (Abernethy et al., 1989). Another interpretation of these data from

wheat, however, could be that rather than protecting the young embryo from heat









45
stress perhaps the hsps are present in preparation for development and consequently,

additional induction by heat may be unnecessary.

Messages for low MW hsps have been found in dry embryos in other plant

species. The mRNA to a 17.6 kD hsp is present in dry seed and accumulates during

embryo development of sunflower (Almoguera and Jordano, 1992). This cDNA also

hybridizes to mRNA from 3-day-old seedlings subjected to ABA, mannitol or heat

stress. The mRNA for an alfalfa hsp 18.1 has been found to be present in

microcallus suspension and increases transiently during embryo formation at normal

temperatures (Gyorgyey et al., 1991). This cDNA also hybridizes to RNA from callus

subjected to CdCI2, sucrose and heat stress. These data demonstrate that transcripts

for low MW hsps have been found in plant embryos and appear to be

developmentally produced at nonheat stress temperatures. However, it is unclear

from these data whether the transcript induced by stress is encoded by the same

developmentally induced gene or represents another member of a multigene family.



Plastid-Localized Low MW Heat Shock Proteins

A structural analysis of plastid-localized low MW heat shock proteins may

suggest a possible function. Chen and Vierling (1991) have compared the deduced

amino acid sequences of 5 of these plastid proteins, and have reported three

conserved regions. Two of these regions are also present in the low MW hsps found

in the cytoplasm. A third carboxyl terminal consensus region, which is abundant in

methionine, is unique to the plastid-localized low MW proteins. Computer analysis








46
of the tertiary structure of the protein in this methionine rich region suggested that

an alpha helix with a hydrophobic and hydrophilic face could form (Chen and

Vierling, 1991). This same type of three dimensional structure is predicted for three

such methionine rich regions of the SRP 54 (signal recognition particle), which is

involved in transporting newly synthesized proteins in the endoplasmic reticulum

(Lutke et al., 1992). Careful structure/function analysis of this region in SRP 54 and

its interaction with signal peptides using crosslinking reagents, antibodies produced

against specific fragments of the SRP 54 and deletion analysis of this protein

revealed that binding of signal sequences of nascent proteins occurs here (Lutcke et

al., 1992). Binding experiments with the 7S RNA showed that this same region is

involved in interaction with the RNA. High and Dobberstein (1991) suggested that

the SRP 54 interacts with signal peptide on the methionine rich hydrophobic face

and with RNA on the hydrophilic face. It is tempting to speculate that the low MW

plastid-localized hsp is involved in a similar protein-protein interaction. Perhaps the

need for these hsps is increased during times when excessive incorrectly folded

proteins are present.

The sublocalization of the plastid-localized low MW hsp has been

controversial. Work by Kloppstech and colleagues suggested that this hsp becomes

associated with thylakoid membranes upon heat stress (Kloppstech et al., 1985).

However, this occurs only with plants grown at low light intensities (Glaczinski and

Kloppstech, 1988). Binding of hsp to the membrane occurs most abundantly at a light

intensity below the level at which net carbon is fixed. Considering the transient








47
nature of these proteins, and that their transcription usually requires heat stress, this

combination of circumstances could occur at dusk in summer or on the floor of a

tropical rain forest. Chen et al. (1990) have shown that under heat stress and higher

light levels this hsp remains predominantly in the stroma. They reported that the only

plastid-localized low MW hsp from pea associates into 200 kD stromal complexes.

Clarke and Critchley (1992) showed that one of the two low MW plastid-localized

hsps, the 32 kD form, from heat stressed barley leaves associates into 250 to 265 kD

complexes within the stroma and contains no RNA, DNA or the a-subunit of the

Rubisco binding protein. This protein is reminiscent of an hsp 28 found in soybean

plastids, which also contains two low MW hsps of 22 and 28 kD (Clark and Critchley,

1992).

The idea that the methionine rich region could interact with denatured

proteins during heat stress suggests that these complexes could sequester unfolded

proteins. The hsp 60 also associates into an 800-900 kD oligomer of 14 subunits

(Roy, 1989). Perhaps the role of the plastid-localized low MW hsp will be easier to

resolve if, like the hsp 60 and 70s, a constitutive or developmentally associated form

can be identified. Maybe the chromoplast-associated low MW hsp described within

this work is induced by the degradation of proteins for the photosynthetic apparatus

that occurs between mature green and intermediate stages of fruit ripening.

Induction of Heat Shock Proteins By Other Stresses

Hsps can also be induced by heavy metals, ethanol, amino acid analogues and

glucose starvation (Vierling, 1991). Arsenite and cadmium produced the greatest








48
induction of hsps (Czarnecka et al., 1984; Edelman et al., 1988). At lower

concentrations of these compounds, the response, like the heat stress response, was

also transient. By 12 hours of exposure, the messages had begun to diminish

(Edelman et al., 1988).

Oxidative stress and the induction of hsps

The initial correlation between heat and oxidative stress was made in an

examination of Drosophila salivary glands. Puffs, representing high levels of

transcription, may occur at the same loci along the chromosome when either a heat

or oxidative stress is applied (reviewed by Loven, 1988). A combination of heat

treatment and application of an oxidizing agent can act synergistically to reduce the

deleterious effect of a subsequent heat stress (Issels et al., 1987). This

thermotolerance developed from oxidative stress inducers can be blocked by the

addition of catalase during the induction period (Issels et al., 1987).

Studies in Drosophila cells (Drummond and Steinhardt, 1987; Courgeon et al.,

1988) and rat hepatocytes (Cajone and Bernelli-Zazzera, 1988) show a mild induction

of some hsps by oxidative stress. In addition, different hsps were induced by different

chemicals (Cajone and Bernelli-Zazzera, 1988). Hsps are also induced by oxidative

stress in Drosophila cells (Courgeon et al., 1988) where hsp 70 and hsp 23 were

induced by hydrogen peroxide. These experiments point to the conclusion that

oxidative stress and heat shock, which induce many of the same proteins, do not

provoke an identical response.










Heat stress and the induction of free radical scavenging enzymes

It still is not entirely clear how heat and oxidative stress are related. Heat

shock uncouples oxidative phosphorylation and this creates an increased level of

reactive oxygen species (Loven, 1988). Additionally, a link has been made between

the application of a heat stress and an induction of free radical scavenging enzymes.

A hyperthermia induced increase in SOD, CAT and glutathione levels occurs with

exercise training of skeletal muscle (reviewed in Barja de Quiroga, 1992). The

implication is that these antioxidants are induced by mild heat stress produced during

exercise. These additional antioxidants reduce the long term deleterious effects of

free radicals produced in this tissue from the uncoupling of oxidative

phosphorylation.

The heat induced induction of plant SOD was also noted by Tsang et al.

(1991) in tobacco. The cytosolic form of SOD was induced most dramatically

compared with the mitochondrial or plastid form in tobacco. This infers that plants

also may respond to heat stress by inducing enzymes involved in oxidative stress.

Conclusions about the relationship of heat and oxidative stress

Since both heat and oxidative stresses produce a similar response, a number

of hypotheses have been developed to explain how they are related. Some authors

have suggested that the deleterious effects of heat stress are due solely to oxidative

damage (Loven, 1988). This idea may be a bit extreme in view of the fact that hsps

can be induced under anoxia in Drosophila cells (Drummond and Steinhardt, 1987).

Rather than the induction of antioxidants, therefore, this type of assertion requires








50

that direct oxidative damage be measured, which can be done by examining the

amount of lipid peroxidation. However, few studies have been preformed in which

hyperthermia is directly correlated with oxidative stress (Barja de Quiroga, 1992). A

corollary to this idea was developed in Ame's lab (Lee et al., 1983), in which the

synthesis of bisnucleoside polyphosphates, also termed alarmones, acts as the signal

for oxidative stress induction of hsps. Apparently an increase in these compounds is

observed during both of these stresses. However, it was soon demonstrated that the

synthesis of alarmones is inhibited by heat stress rather than induced. A buildup in

these compounds occurs because their degradation is actually inhibited by heat.

Addition of alarmones to cells was also shown to have no effect on the induction of

hsps and hsps could actually be induced without an increase in the amount of these

compounds (Guedon et al., 1986).

The hypothesis that is currently favored for the induction of hsps by oxidative

stress is that both heat and free radicals create aberrant proteins that lead to the

production of hsps (Neumann et al., 1989). This probably explains why the two stress

responses do not completely overlap. Since hyperthermia creates unstable proteins

and uncouples oxidative phosphorylation, the increase in free radicals and the

resulting response may occur secondarily. Oxidative stress may mainly induce free

radical scavengers, which can forestall the production of large numbers of aberrant

proteins.










Conclusions

The molecular characterization of fruit ripening has shown that the events that

characterize the maturation of tomato fruit involve the de novo synthesis of a subset

of ripening associated transcripts. At this point, however, only a few genes involved

in this process have been identified and their mode of regulation assessed.

Even though the conversion of chloroplasts into chromoplasts involves the

degradation of chlorophyll and the breakdown of thylakoid membranes along with

a rapid synthesis of carotenoids, only a small number of genes have been identified

that encode proteins that may be involved in this transformation. To date, two genes

in tomato fruit and one in pepper that code for proteins involved in carotenoid

biosynthesis have been isolated. In addition, only a single gene for synthesis of

cysteine synthase has been identified in pepper chromoplasts. Therefore, it is unclear

how many proteins may be involved in this process and whether their synthesis

increases during development.

Historically the characterization of carotenoid biosynthesis in higher plants

involved following the incorportaion of radiolabeled precursors into crude enzyme

preparations or the identification of the compliment of carotenoids produced by

mutants in the biosynthetic pathway. This type of examination was complicated by

the presence of multiple pathways and by the complexity inherent in assays of

disrupted tissue. The analysis of carotenoid biosynthesis, as demonstrated in bacteria,

in which individual genes can be cloned and the activity can be assessed by

complementation in E. coli may now be possible in higher plants. In this way, an








52
unambiguous assessment of the role an individual enzyme plays on the synthesis of

carotenoids can finally be evaluated. The determination of how many enzymes are

involved in the biosynthesis of fruit carotenoids can be determined. In addition, the

cloning and characterization of the genes involved in this pathway will allow an

understanding of how the synthesis of carotenoids is controlled during chromoplast

formation.

The senescence of chloroplasts in leaves involves the breakdown of

photosynthetic structures, in a gradual process within the plastid. It is clear from

evidence accumulated from the analysis of stay-green mutants that the degradation

of thylakoid membranes involves at least two different systems of protein

degradation, portions of which are encoded in both the nucleus and the cytoplasm.

However, it is uncertain whether de novo synthesis of new proteins occurs during

chloroplast senescence in the leaves.

During the transition of chloroplast to chromoplast in pepper chromoplasts,

an increase of compounds that scavenge free radicals occurs. Since the induction of

hsps has been observed during oxidative stress, it is possible that the induction of an

hsp during fruit ripening is caused by such an increasingly harsh environment.

Clearly, the ripening-associated hormone ethylene can induce SOD in tomato and

tobacco leaves. However, the evidence for an increase in enzymes involved in

oxidative stress in tomatoes remains contradictory. More information is needed

before conclusions can be drawn about what role oxidative stress plays during tomato

fruit ripening.








53
Clearly the transition within the developing chromoplast involves a rapid

degradation of proteins. The period between mature green and yellow fruit appears

to be one in which a particularly rapid degradation of plastid proteins occurs. Since

an increase in hsps has been associated with developmental stages where a great deal

of structural change occurs, the chloroplast to chromoplast transition may be an ideal

environment for the induction of these proteins.














CHAPTER 3
CHROMOPAST-TARGETED PROTEINS IN TOMATO FRUIT



Introduction

The transformation of chloroplasts into chromoplasts results in a dramatic

alteration of organellar structure. The thylakoid membranes are dismantled in a

ordered process while carotenoids build up within the plastid (Thelander et al.,

1986). The structural changes that occur during this transition have been documented

by electron microscopy (Harris and Spurr, 1969). However, it is unclear how this

event is regulated.

One of the objectives of this project was to determine whether the

development of chromoplasts during tomato fruit ripening is mediated by an increase

in nuclear-encoded transcripts of chromoplast-associated proteins. Since plastid

transcription and translation is greatly reduced during chromoplast development

(Kobayashi, 1991) we have focused on proteins that are nuclear-encoded, translated

in the cytoplasm and imported into the plastid. The import of proteins into plastids

is highly specific and requires an amino-terminal transit peptide, which is necessary

and sufficient for targeting into the organelle (Keegstra, 1989). We have monitored

chloroplast import of in vitro translation products from RNA of tomato fruit isolated

at different ripening stages. We have also examined translation products from








55
transcripts encoded from cDNAs (slater et al, 1985) whose steady-state RNA level

increases during ripening.



Materials and Methods



Plant Material

Tomato plants (Lycopersicon esculentum Mill. cv. Rutgers) were grown in a

greenhouse in Gainesville, Florida. Fruit were harvested at four ripening stages.

MG1 was characterized by full-size green fruit with firm locular tissue; MG3 fruit

had completely developed locular gel; the 20-50% ripe fruit were red over less than

half of the outer pericarp; and >80% ripe fruit were red over 80% of the outer

pericarp. The pericarp was frozen in liquid nitrogen immediately after harvesting.



Preparation and Translation of RNA

Total RNA was extracted by combining the protocols of McCarty (1986) and

Grierson et al.(1985). Briefly, this involved grinding the frozen tissue with a coffee

grinder and homogenizing in extraction buffer with a polytron. The extraction buffer

consisted of 100 mM Tris-HCl pH 9.0, 200 mM NaCI, 5 mM DTT, 1% SDS and 20

mM EDTA. The insoluble material was pelleted and reextracted with 1/2 volume of

extraction buffer. The supernatant was extracted 2 times with phenol/chloroform

(1:1) and one time with chloroform. The final supernatant was brought to 0.1M KCI

and precipitated overnight with 2 volumes of ethanol at 40C. The precipitate was








56
washed several times as described in Grierson et al. (1985) and the soluble material

was applied to a cellulose (Sigmacell type 50) column to remove substances that

interfere with extraction of poly (A)' RNA (Grierson et al., 1985). Poly (A)* RNA

was isolated from total RNA by one passage over oligo(dT)-cellulose (BRL).

Individual pTOM clones pTOM 5, 25, 36, 41, 92, 111, and 114 (Slater et al.,

1985) were subcloned into the in vitro transcription vector pSport (BRL) at the PstI

site. Plasmid DNA was isolated by the alkaline lysis method of Lee and Rasheed

(1990). The plasmid DNA was linearized and transcribed with either SP6 polymerase

or T7 polymerase as described in Cline (1988). The poly (A)+ RNA and the in vitro

transcribed RNA were translated (see Figure legends for specific experiments) with

either a wheat germ system (Cline, 1988) or a rabbit reticulocyte lysate system from

Promega (Madison,Wi.) in the presence of [35S]-methionine.



Chloroplast Isolation and Import Assay

Chloroplasts were isolated from pea (Pisum sativum L. cv Laxton's Progress

9) shoots by a combination of differential centrifugation and Percoll gradient

centrifugation (Cline et al., 1989). Import assays were conducted as described by

Cline (1988) except that translation products were diluted 1:3 with unlabelled

methionine in import buffer; 50 Vl of the diluted translation products were added to

100 pil of chloroplasts (1 mg chlorophyll/ml). Import assays were conducted for 20

min at 250 with white light and 10mM Mg-ATP. After incubation, intact chloroplasts

were reisolated by centrifugation through 35% Percoll with or without a treatment








57

of the protease thermolysin to remove surface bound proteins (Cline, 1988).

Repurified plastids were lysed in 30 pil of 10mM Hepes/KOH pH 8.0. Experiments

testing the requirement of ATP were conducted in foil wrapped tubes to maintain

darkness.



Subfractionation and Treatment of Plastids after Import

In experiments examining import of translation products from total poly (A)+

RNA, the repurified plastids were lysed in 10mM Hepes/KOH pH 8.0 and

subsequently subfractionated into stromal (soluble) and membrane (pellet) fractions

by centrifugation for 10 min at 10,000 x g. In experiments with translation products

from individual cDNA clones, the soluble fraction was isolated from the membranes

by lysing the plastids in 10mM Hepes/KOH pH 8.0 on ice for 5 min and centrifuging

30 min at 37,500 x g. The membranes were washed one time with 1 ml of 10mM

Hepes/KOH pH 8.0 and resuspended in the same solution. Alternatively, the

membrane fraction was washed with 0.1 N NaOH (Cline, 1986). Envelopes were

separated from thylakoid membranes as described in Cline (1986), however, 1.3 ml

of plastids (1 mg chlorophyll/ml) were used for each import reaction.

Sensitivity of the imported proteins to thermolysin was tested as follows: A

standard import assay was carried out and divided into two equal fractions. These

fractions were either resuspended in import buffer or lysed with 10mM Hepes/KOH

pH 8.0. Aliquots of 25 pl (1mg chlorophyll/ml) were incubated with 0 or 20 pg of

thermolysin for 45 min at 40 C. EDTA was added to 5mM and the samples were








58
immediately frozen. An equal volume of 2X-SDS PAGE buffer (Cline, 1986) was

added to freshly thawed samples and the tubes were heated to 980 C for 3 min.



Analysis of Import Products

Import products from total poly (A)+ RNA were examined by SDS-PAGE

and fluorography on 7.5%-20% gradient gels (Cline, 1986). Gel lanes received

equivalent amounts of protein as determined by chlorophyll estimation of the

recovered plastids. Import products from in vitro transcribed RNA were examined

on 12.5% SDS PAGE gels.



Results and Discussion

Profiles of in vitro translation products of poly (A)+ RNA differed

markedly between ripe fruit and mature green tissue (Fig.3-1A). The profiles of the

two later ripening stages closely resembled one another, as did the translation

products from the two samples of mature green tissue. Speirs et al. (1984) reported

a similar result from a more detailed analysis of the polypeptide profiles of total

mRNA from ripening tomato fruit.

Pea chloroplasts were chosen to analyze import into plastids because of their

ease of isolation. The isolation of tomato chromoplasts has been reported by others

(Iwatsuki et al., 1984; Bathgate et al., 1985; Hunt et al., 1986; Wrench et al., 1987),

but we were unable to isolate pure and intact fruit chromoplasts that withstand the

handling required to demonstrate successful import. However, in vitro import of an








59
amyloplast specific protein into chloroplasts (Klosgen et al., 1989) and the in vivo

import of a chloroplast protein into non-green plastids using transgenic plants

(deBoer et al., 1988) implied that chromoplast proteins should also be imported into

pea chloroplasts.

Changes in the profile of imported proteins during chromoplast development

were detected (Fig.3-1B). A number of proteins decreased during this period (Fig.

3-1B). For example, a 30kD and a 14kD soluble fraction protein diminished as

ripening progressed. Two imported translation products dramatically increased in the

later ripening stages. A 26 kD protein fractionated with the soluble plastid proteins,

whereas a 17 kD protein was associated with the membrane fraction. These results

imply that there is synthesis and import of new proteins during the transition from

chloroplast to chromoplast and that the plastid conversion may be an active

developmental program rather than a simple decline in synthesis of the

photosynthetic apparatus.

We next asked whether an import assay could be used to identify individual

clones coding for chromoplast-targeted proteins. A specific cDNA clone, pTOM5,

was selected to evaluate this system. Slater et al. (1985) originally isolated this cDNA

because its RNA becomes more abundant as ripening proceeds. Armstrong et al.

(1990) recognized that the deduced protein sequence shares approximately 25%

identity with the predicted polypeptide of a bacterial prephytoene synthase. Bird et

al. (1991) confirmed the involvement of pTOM5 in carotenoid synthesis by creating


























Figure 3-1. Profiles of in vitro translation products and import products are altered
during tomato fruit ripening. A, In vitro translation of total poly (A)+
RNA with four different ripening stages from tomato fruit pericarp.
1 ug of total poly (A)+ RNA per 50 ul reaction was translated in vitro
in a wheat germ system in the presence of [35S]-methionine.
Translation products were examined by SDS-PAGE and fluorography
on 7.5%-20% gradient gels. (MG1) mature green 1, (MG3) mature
green 3, (20-50%) 20-50% red over the outer pericarp, (>80%)
greater than 80% red over the outer pericarp. B, Import into pea
chloroplasts with in vitro translation products of total poly (A)+ RNA
extracted from tomato fruit pericarp at four different ripening stages.
Import assays were conducted and fractionated as described in
Materials and Methods. The (soluble) fraction was isolated from the
(membranes) of the re-purified lysed plastids by centrifugation for 10
min at 10,000 x g. Equivalent amounts of plastid protein (as
determined by chlorophyll estimation) from the +/- protease treated
plastids were separated on 7.5%-20% SDS-PAGE gradient gels and
fluorographed. ["C]-labelled 10 to 70 kD molecular weight standards
(Sigma) appear as arrows on the left side of the figure. The size of the
two import products were derived from a standard curve and are
designated by arrows on the right side of the figure.




































~DG)


-protease +protease
total total


e e C


+protease
soluble






,P-

4WD


*protease
membrane


~ 5 esA








26
A 17


66
45
36 ,
29 ,


20.1 ,
14.20.


,low 4w








62
an antisense pTOM5 tomato transformant that produces yellow rather than normal

red fruit. The transgenic fruit are blocked in the synthesis of phytoene from its

immediate precursor, implying that pTOM5 encodes phytoene synthase (Bramley et

al., 1992). We selected this clone because enzymological data suggest that synthesis

of this nonpigmented carotenoid occurs in plastids (Lutke-Brinkhaus et al., 1982;

Mayfield et al., 1986; Dogbo et al., 1987;).

The major translation product of the pTOM5 transcript was 46 kD (Fig. 3-2,

lane 1); this is in agreement with the size of the hybrid-selected translation product

for pTOM5 identified by Slater et al. (1985). Minor lower molecular weight

translation products were also produced from this transcript and may represent

commencement of translation from internal methionines because these products are

not imported into plastids. Incubation with pea chloroplasts resulted in the

appearance of a 41 kD polypeptide that fractionated with intact plastids and was

resistant to protease treatment of the chloroplasts (Fig. 3-2, lane 4). Protease

resistance was not due to an inherent property of the 41 kD protein because it was

degraded by thermolysin if the plastids were lysed prior to treatment (data not

shown). This result is expected of a protein produced in the cytosol, imported into

the plastid, protected from protease by the organellar envelope, and cleaved of its

transit peptide.

Additional criteria must also be demonstrated for the confirmation of import

into plastids. Import is an energy dependent process (Keegstra, 1989); white light or

















+ Protease


- ATP


Figure 3-2.


The 41 kD form of pTOMS is protected from protease digestion by the
plastid envelope and its appearance requires the addition of ATP.
Lane 1) In vitro transcribed RNA was translated with a rabbit
reticulocyte lysate system in the presence of ["S]-methionine. Import
assays and post assay treatments were conducted as described in
Materials and Methods. Lanes 2-5) Import of pTOM5 in vitro
translation products was conducted with (lanes 2,4) or without (lanes
3,5) 10mM Mg-ATP in darkness. Intact chloroplasts were re-isolated
with (lanes 4,5) or without (lanes 2,3) protease post-treatment.
Proteins from lysed plastids were separated on 12.5% SDS-PAGE gels
and flurographed.


m .m


+


1 2


+


+


4








64

exogenous ATP must be provided for the successful import of proteins. Fig. 3-2,

lanes 2 and 3 show that processing of the 46 kD protein into the 41 kD peptide

required exogenous ATP. In the absence of ATP, the 46 kD putative precursor

bound to the chloroplasts but was destroyed by the protease (Fig. 3-2, lanes 3 and

5). The import into plastids should also be time dependent; this was found to be the

case for the production of the 41 kD protein (Fig. 3-3). Thus, the successful import

of the pTOM5 translation product verified the utility of import assays for the

identification of genes encoding chromoplast-targeted proteins.

Pea chloroplasts were fractionated into soluble and membrane fractions to

allow analysis of the sublocalization of the 41 kD protein. The protein distributed

between these two compartments (Fig 3-3). Parallel import assays with the thylakoid

localized light-harvesting chlorophyll a/b protein (LHCP) and the stromal small

subunit of Rubisco (SS) were used to assess the quality of our subfractionation.

LHCP was exclusively found in the membrane fraction whereas virtually all of the

SS was recovered in the soluble fraction (data not shown). During preparation of this

manuscript, Bartley et al. (1992) have reported the isolation of a cDNA from a

tomato fruit library by hybridization with a PCR fragment produced with

oligonucleotides derived from the sequence of pTOMS. They found that their clone

differs from pTOM5 in 19 amino acids that reside 5 amino acids away from the

deduced carboxyl terminal end. They also demonstrated that their in vitro translation

product imports via an ATP requiring process and that their import product also

appears in both stromal and membrane fractions of pea chloroplasts.






























Membrane


0 1 2 4 6 8 10 0 1 2 4 6 8 10


Min


t""

;


. ... .':


Figure 3-3. The production of the 41 kD form of pTOM5 was time dependent
This protein associated with both stromal and membrane fractions.
pTOM5 import assays were conducted as described in Materials and
Methods. One half of a standard import assay was immediately repurified
on a 35% Percoll cushion after 0, 1, 2, 4, 6, 8, or 10 minutes of
incubation. Chloroplasts were protease treated, repurified and
subfractionated as described in the Materials and Methods. Samples were
analyzed by SDS-PAGE and fluorography.


Stroma








66
The membranes of pea chloroplasts were further subfractionated into

envelopes and thylakoid membranes (Cline, 1986); upon fractionation of the

membranes after import of pTOM5, virtually all of the mature-sized protein was

found to be associated with the thylakoid fraction (data not shown). A mild NaOH

wash removed the pTOM5 protein from the membrane (data not shown). This

suggested that the protein was only peripherally associated with this fraction (Cline,

1986).

Previous attempts by others to localize the site of phytoene synthesis within

the plastid via enzyme assays have resulted in conflicting conclusions. Lutke-

Brinkhaus et al. (1982) concluded that the production of phytoene is associated with

the plastid envelope. However, Mayfield et al. (1986), and independently, Dogbo et

al. (1987) deduced that phytoene is synthesized in the stroma. The unexpected

association of the protein with thylakoids in import assays and its peripheral

association with the membranes demonstrated by the NaOH extraction may have

provided additional evidence that this protein is functionally associated with the

stroma. Alternatively, perhaps the 41 Kd protein has not folded or localized correctly

in pea chloroplasts. Bartley et al. (1991) reported the import of phytoene desaturase

from soybean leaf into pea chloroplasts. Although this protein imported effectively,

Bartley et al. found that it does not also fractionate into the plastid compartment

expected from biochemical analysis. Immunocytolocalization may clarify these

contradicting results. Therefore, it appears that import assays can be useful for the

identification of chromoplast-targeted proteins; however, it is currently unclear what








67
conclusions can be made from the in vitro organellar sublocalization when using

heterologous systems.

Six additional ripening enhanced pTOM cDNA clones (pTOM 25, 36, 41, 92,

111, 114) of unknown function (Slater et al., 1985) were tested to determine whether

they encode plastid targeted proteins. These particular clones were selected because

a comparison of the published sizes of the cDNA inserts with the size of their

respective mRNA suggested that they might be full length (Slater et al., 1985;

Maunders et al., 1987). In vitro transcription of the cDNAs was carried out in both

directions because the orientation of the clones within the vector was unknown. In

vitro translation products of these transcript pairs failed to import in four of the six

clones. Fig. 3-4, lanes 1 to 7 shows examples of the translation products and lack of

import products produced from these clones. However, since the transit peptide,

which is required for successful import of the protein, is located on the amino

terminal end, and since in vitro translation can begin from any internally situated

methionine, any cDNAs that produced an in vitro translation product but not an

imported product may simply not include the entire coding sequence. Translation

products from pTOM41 and pTOM111 were imported successfully into pea

chloroplasts (Fig. 3-4, lanes 9 and 11). The imported product from both proteins was

smaller than the translation product, which is consistent with processing of the plastid

transit peptide. These proteins were sensitive to protease treatment (see chapter 4),

but resistant after import into plastids (Fig. 3-4). As with pTOMS, import of

pTOM111 and pTOM41 translation products were ATP and time dependent (see



















1 23 4 5 6 7 8 9 1011

AMEN -

OIL..


Figure 3-4.


Translation products from transcripts of additional pTOM clones were
tested for their ability to import into isolated pea chloroplasts. In vitro
translation products from pTOM41 and pTOM111 imported into
isolated pea chloroplasts. In vitro transcribed RNA was translated with
a rabbit reticulocyte lysate system in the presence of [35S]-methionine.
Import assays were conducted as described in Materials and Methods.
Samples were analyzed by SDS-PAGE and fluorography. In vitro
translation with no added RNA (lane 1). pTOM92 transcribed in vitro
with SP6 polymerase, translated in vitro, (lane 2) and imported (lane
3). pTOM92 transcribed in vitro with T7 polymerase, translated in
vitro, (lane 4) and imported (lane 5). pTOM36 transcribed in vitro
with T7 polymerase, translated in vitro, (lane 6) and imported (lane 7).
pTOM41 transcribed in vitro with T7 polymerase, translated in vitro,
(lane 8) and imported (lane 9). pTOM111 transcribed in vitro with T7
polymerase, translated in vitro, (lane 10) and imported (lane 11).








69
chapter 4). These data support the idea that the cDNAs do indeed code for plastid-

targeted genes. We have begun sequence analysis (see chapter 4) and antisense

transformation experiments of these cDNAs (see Appendix) to investigate their role

in chromoplast function.

We wondered whether our initial examination of import from total poly (A)+

translation products (Fig. 3-1B) might underestimate the number of chromoplast-

targeted proteins. Therefore, we asked whether the pTOM import products could be

recognized among the total poly (A)' RNA import products. The pTOM5 protein

comigrated with a band shown to increase in the import products from the

membrane fraction of late stage fruit (Fig. 3-5, lanes 5 and 6). It is interesting that

the 26kD soluble import product (Fig. 3-1B) migrates similarly to the pTOM111

import product (Fig. 3-5). However, analysis of these two proteins on several gels

suggested that the pTOM111 product was slightly larger. Consequently, it is unclear

if pTOM111 represents a different isozyme or a completely dissimilar protein. Since

the import of the in vitro translation product of pTOM41 associates with the stromal

fraction, this was compared with the soluble fraction of total poly A' import

products. No protein appears to increase in the later stages of ripening fruit which

comigrates with this band (Fig. 3-5, lanes 2 and 3). We concluded, therefore, that not

all of the chromoplast-targeted proteins could be detected in our analysis of total

poly (A)+ RNA import products.



















Figure 3-5.


Import products corresponding in size to those for pTOM111 and
pTOM5 were enhanced in late stage tomato fruit. However, no
polypeptide was seen that coincided to the pTOM41 import product.
pTOM41, pTOM5 and pTOM111 were transcribed in vitro with T7
polymerase. RNA was translated with rabbit reticulocyte lysate in the
presence of [35S]-methionine. In vitro translation, import assays and
subfractionation were conducted as described for analysis of individual
translation products in Materials and Methods. Lane 1) Soluble
fraction of the pTOM111 import product. Lane 2) Soluble fraction of
the pTOM41 import product. Lane 3) Soluble fraction of imported in
vitro translation products of total poly (A)+ RNA from >80% ripe
fruit. Lane 4) Soluble fraction from imported, in vitro translation
products of immature green total poly (A)+ RNA. Lane 5) Membrane
fraction of the pTOM5 import product. Lane 6) Membrane fraction
from imported, in vitro translation products of total poly (A)' RNA
from >80% red ripe fruit. Lane 7) Membrane fraction from imported,
in vitro translation products of immature green total poly (A)+ RNA.
















1 2 3 4


5 6 7


amM


--ib
MON&

----








72
It is presently unclear how many plastid targeted proteins increase during the

chloroplast to chromoplast transition. The data presented in Fig. 3-4 and 5 clearly

demonstrate that our original examination of chromoplast targeted proteins shown

in Fig. 3-1 underrepresents the number of proteins that increase during this process.

In fact, this is not surprising when considering the limited sensitivity of import of

total poly (A+) translation products. Therefore, we believe that individual testing of

ripening enhanced cDNA clones, with the inherent increase in the quantity of the

individual protein product, is required to clearly identify all the chromoplast targeted

proteins whose RNA increases during this developmental process.

Little detailed analysis of the regulation of ripening enhanced genes has been

undertaken. Lincoln and Fisher (1988) and DellaPenna et al. (1989) concluded that

increases in transcription rate were mainly responsible for the increases in steady-

state RNA levels for the ripening enhanced genes they have analyzed. However, they

also invoked post-transcriptional mechanisms to explain differences between the rate

of transcription and accumulation. Although we cannot exclude post-transcriptional

mechanisms for the increase in steady-state levels of the RNAs we have analyzed,

the simplest interpretation for our data that is consistent with other ripening

enhanced genes is that the processes involved in the development of the chromoplast

are directed at a transcriptional level in the nucleus.

Utilizing an in vitro assay for the import of proteins into plastids we have

identified two proteins from in vitro translation products of total poly (A)+ RNA that

increase during the ripening of tomato fruit. Additionally, we have shown that the








73
in vitro translation products from three individual ripening enhanced cDNA clones

import into plastids. Therefore, the transition from chloroplast to chromoplast

coincides with an increase in steady-state levels of RNA for proteins destined to this

organelle. This suggests that this process is not a simple decline in the synthesis of

photosynthetic proteins that causes a breakdown of the thylakoid membranes and a

shunting of substrates into the carotenoid pathway, but an active developmental

program.












CHAPTER 4
CHARACTERIZATION OF TWO cDNAS FOR CHROMOPLAST-
TARGETED PROTEINS





Introduction

As tomato fruit ripens, cell walls soften, hexoses are deposited into vacuoles,

and carotenoids accumulate in developing chromoplasts. The transformation of

chloroplasts into chromoplasts is distinguished by the breakdown of the

photosynthetic apparatus and a massive synthesis and deposition of carotenoids.

Little is known about how the catabolism of thylakoid membranes or the synthesis

of carotenoids is regulated. Only two tomato genes for early steps in the carotenoid

biosynthetic pathway have been cloned (Bird et al., 1991; Bartley et al., 1992; Pecker

et al., 1992). Besides proteins involved in the synthesis of carotenoids, only the

plastidial superoxide dismutase, characterized by Livine and Gepstein (1988), has

been shown to increase during tomato fruit ripening. However, Romer et al. (1992)

have also isolated and cloned a cysteine synthase from pepper chromoplasts. These

latter findings suggest, therefore, that the chromoplast may be responding to an

increase in oxidative stress during development.

In order to understand how the transformation from chloroplast to

chromoplast is mediated one objective of the present study was to identify and clone








75
genes for proteins involved in chromoplast development. We have identified plastid

proteins that may be increasingly synthesized during chromoplast development by

examining cDNAs isolated from a ripening enhanced tomato fruit library, translating

their transcripts in vitro and testing whether the resulting proteins can be imported

into isolated pea chloroplasts. This chapter describes the characterization of two

cDNAs that code for transcripts whose steady-state level increases during

chromoplast development and whose proteins can be imported into plastids. The

characterization of these two cDNAs represents a novel use of import assays to

examine chromoplast development. Since this type of analysis is free of functional

bias, the identification of unexpected components of chromoplast development is

possible.



Materials and Methods



Plant Material

Tomato plants (Lycopersicon esculentum Mill cv. Sunbeam) were grown in the

field at the Horticultural Unit of the University of Florida in Gainesville, Florida.

Fruit were harvested at 9:00 am on June 19, 1992 from five different ripening stages.

Mature green 2 (MG2) was characterized by full size green fruit with fully developed

gel in at least one but not all of the locules; mature green 3 (MG3) fruit had

completely developed locular gel; breaker stage fruit were characterized by the

appearance of carotenoids at the blossom end; 20-50% ripe fruit were red over less








76
than half but more than 20% of the outer pericarp; and >80% ripe fruit were red

over 80% of the outer pericarp. Pericarp from fruit of each stage was frozen in liquid

nitrogen immediately after harvesting at -700C for analysis at a later date.

For the examination of ripening under controlled temperature, MG fruit were

placed in jars with a flow through system of humidified air and allowed to ripen at

220C in the dark. This prevented water stress or a buildup of respiratory CO2. Fruit

from three different ripening stages (MG3, breaker, and 20-50% ripe) were selected

and exposed for 6 hours to 360C in this humidified environment. Following the

incubation period, pericarp was frozen in liquid nitrogen.



Preparation of RNA

Total RNA was extracted by combining the methods of Chomczyinski and

Sacchi (1987) and Cathala et al. (1983). Briefly, this involved grinding 30 g of frozen

tissue in a coffee grinder and homogenizing in 60 ml of extraction buffer with three

30 sec bursts of a Brinckman polytron. The extraction buffer contained 4M guanidine

isothiocyanate (Bethesda Research Laboratories), 0.5% sarkosyl, 25 mM sodium

citrate pH 7.0, and 0.1M 2-mercaptoethanol. Sodium acetate pH 4.0 was added to

0.18M, followed by one volume of phenol and 1/10 volume chloroform. The

homogenate was shaken for 10 sec after each addition and then cooled on ice for 15

min. The phases were separated by centrifugation at 10,000 x g for 20 min, the

aqueous phase filtered through 2 layers of Miracloth (Calbiochem), and nucleic acids

precipitated with an equal volume of isopropanol for one hour at -200C. The








77
precipitate was collected by centrifugation for 45 min at 10,000 x g. The pellet was

dissolved in extraction buffer and RNA was precipitated with 5 volumes of 4M LiCI

for 15 hours at 40C. Following centrifugation for 90 min at 10,000 x g, the pellet was

washed with 3M LiCI and recovered by centrifugation for 1 hour. The RNA was

dissolved in 0.1% SDS, extracted with 1 volume of phenol/ chloroform (1:1), then

reextracted with 1 volume chloroform, and precipitated with 0.3M sodium acetate

and 2.5 volumes of ethanol. Precipitated RNA was dissolved in sterile water and

stored at -700C.



Template Preparation, In Vitro Transcription and In Vitro Translation

A cDNA for the hsp 21 from heat stressed pea leaves, pHSP 21, was provided

by E. Vierling (Vierling et al., 1988). The pTOM 111 and pTOM 41 cDNA were

provided by D. Grierson (Slater et al., 1985). The pTOM cDNAs were digested with

Pstl and ligated into pSport 1 (Bethesda Research Laboratories) at the PstI site, and

transformed into E. coli strain TB1. Plasmid DNA was isolated by the alkaline lysis

method of Lee and Rasheed (1990). The pHSP 21 plasmid was linearized with Barn

HI and in vitro transcribed with SP6 polymerase as described in Cline (1988). The

pTOM 41 and pTOM 111 plasmid DNAs were linearized with Eco RI and in vitro

transcribed with T7 polymerase (Bethesda Research Laboratories). The in vitro

transcribed RNA transcripts were translated with a rabbit reticulocyte lysate system

from Promega (Madison, Wi.) in the presence of either [3S]-Met or [3H]-Leu

according to procedures described in the Promega manual.










Northern Hybridization

Total RNA, quantified by spectrophotometric absorbance at A2, was loaded

(15 Ig/lane) onto a formaldehyde denaturing gel (Sambrook et al., 1989). After

electrophoresis the gel was washed in sterile water and stained in ethidium bromide

(l1g/ml) for 30 min and destined in 20X SSC, which contained 0.3 M sodiun citrate

and 3 M sodium chloride, for 45 min. The amount of ribosomal RNA was visually

assessed to ensure that approximately equivalent amounts of RNA were loaded. The

RNA was capillary blotted onto nylon membrane (Amersham Corp.) in 20X SSC

overnight. The blot was washed with 10X SSC and exposed to 1.5 J/cm2 UV light

with a transilluminator (Bios).

For hybridization probes, the entire cDNA insert was excised with PstI,

isolated by gel electrophoresis, and electro-eluted. 100 ng of DNA was radiolabeled

with 75 pCi of a32P-dCTP by random primer labelling with a kit (Boehringer

Mannheim).

Prehybridization and hybridization conditions were as described in Church and

Gilbert (1984). The prehybridization buffer contained 0.5M sodium phosphate pH

7.2, 7% SDS and 1% bovine serum albumin (Sigma, fraction V) at 650C. Blots were

prehybridized in 40 ml of buffer for at least 1 hour at 650C. The labeled probe was

denatured at 1000C for 5 min and added to the blot with 5 ml of prehybridization

solution. This was incubated overnight at 65*C. The blot was washed one time in 1X

SSC and 0.1% SDS at room temperature and then 3 times in 0.2X SSC and 0.1%

SDS for 30 min. X-ray film (Kodak, X-OMAT) was exposed to the blot overnight








79
with two intensifying screens (DuPont) to locate bands. Bands were excised from the

membrane and quantified by liquid scintillation counting.



DNA Sequencing

The PstI excised pTOM 111 cDNA insert was subcloned into pBluescript (SK)

plasmid (Stratagene) at the PstI site. This was restricted with either XbaI and SstI or

KpnI and Eco RV for production of ExoIII/Mungbean nested deletion plasmids

following the protocol described in the manual (Stratagene). The resulting plasmids

were precipitated, resuspended in TE, ligated directly, and transformed into E. coli

strain TB1. Plasmids containing nested deletions from either direction were selected

and sequenced using the T3 or M13-40 primer of the pBluescript vector. Double

stranded plasmid DNA was used as a template and prepared as described by Lee

and Rasheed (1990). DNA sequencing procedures were as described in the

Sequenase sequencing kit manual (US Biochemical).

The pTOM 41 cDNA was blunt end ligated (Sambrook et al., 1989) into

pGEM-4Z (Promega) at the Smal site. Both the pGem constructs described above

and the pSport constructs described in the section on in vitro transcription were used

as template for double stranded DNA sequencing as described above. Initially the

T7 or M13-40 sites of the pSport or pGem vectors were used as primer sites.

Oligomers (15mers) specific to pTOM 41 were synthesized by the UF-ICBR DNA

Synthesis Core and used in subsequent sequencing reactions. Some portions of the

sequence were generated by the ICBR DNA Sequencing Core. Sequences were








80
analyzed with the University of Wisconsin Genetics Computer Group computer

programs (Devereux et al., 1984). Protein sequence comparisons were performed at

the National Center for Biotechnology Information using the GENINFO(R) program

of the Experimental Blast Network Service (Altschul et al., 1990) made available by

the ICBR Biological Computing Facility. The petunia hsp 21 sequence (Chen and

Vierling, 1991) is available as EMBL accession #X54103. The pTOM66 sequence

(Fray et al.,1990) is available as EMBL accession #X56138. The sequence for the

hypothetical 30.9 Kd protein from E. coli (Post et al., 1992) is available in GenBank

accession #M77236.



Chloroplast Isolation and Import Assays

Chloroplasts were isolated from pea (Pisum sativum L. cv Laxton's Progress

9) shoots by a combination of differential centrifugation and Percoll gradient

centrifugation (Cline et al., 1989). Import assays were conducted as described by

Cline (1988) except that translation products were diluted 1:3 with unlabelled

methionine or leucine in import buffer; and that 50 l of diluted translation products

were added to 100 pl of chloroplasts (1 mg chlorophyll/ml) for a 300 pl total assay

volume. Import assays were conducted for 15 minutes at 250C with white light and

10mM Mg-ATP unless indicated otherwise. Experiments testing the requirement of

ATP for import of pTOM 111 protein involved incubation of 200 pl of the

translation mixture with or without 4 units of apyrase (Sigma) for 5 min at 25C. The

import assays were conducted in foil wrapped tubes to maintain darkness at 250C.








81
After incubation, intact chloroplasts were reisolated by centrifugation through 35%

Percoll with or without a treatment of the protease thermolysin (Sigma) to remove

surface bound proteins (Cline, 1988). Plastids were treated with 50 tig thermolysin

for 40 min at 4C unless indicated otherwise. Import products were diluted 1:1 with

2X-SDS PAGE buffer, incubated 8 min at 600C and analyzed by SDS-PAGE and

fluorography (Cline, 1986) on 12.5% gels.

In order to demonstrate that the imported protein was sensitive to

thermolysin, an import assay was performed and the chloroplasts were lysed before

the addition of the protease. One fraction of a standard assay was resuspended in

100 jIl import buffer, the other fraction was lysed with the same amount of 10mM

Hepes/KOH pH 8.0. Thirty pl aliquots (0.5mg chlorophyll/ml) were incubated with

0, 12, and 24 pg of thermolysin for 45 min at 40C. For the pTOM 41 protein, [3H]-

leucine was used in the in vitro translation and 1/2 of a standard import reaction (50

pl of plastids at 1mg chlorophyll/ml) was resuspended in 25 pl 10mM Hepes/KOH

pH 8.0. This sample was divided in half and 5 ig of thermolysin was added to an

aliquot. After 40 min incubation at 40C, EDTA was added to a final concentration

of 5mM and the samples were immediately frozen. An equal volume of 2X-SDS

PAGE buffer (Cline, 1986) was added to freshly thawed samples, the tubes were

heated at 980C for 3 min and 10 pc was loaded onto 12.5% SDS-PAGE gels.

For analysis of the location of imported proteins, chloroplasts recovered from

assays were lysed in 50 p1 10 mM Hepes/KOH pH 8.0, and incubated for 5 min.

Stroma was separated from membranes by centrifugation for 30 min at 37,500 x g.








82
The membranes were washed with 1 ml of import buffer, centrifuged for 30 min at

37,500 x g. and finally resuspended in 40 tl of 10mM Hepes/KOH pH 8.0.



Results

The tomato fruit cDNAs pTOM 111 and 41 were selected by Slater et al.

(1985) because transcripts homologous to these clones increased during ripening.

Data presented in the previous chapter have shown that in vitro transcripts from

these clones can be translated in vitro and the translation products can be imported

by pea chloroplasts. Further studies were performed in order to ensure that all the

criteria for successful import were met, such as the energy and time dependence of

the reaction along with sensitivity of the mature form of the protein to protease.



Import Studies of pTOM 111 Protein

The pTOM 111 in vitro transcript was translated into a 30 kD protein and

processed by the isolated plastids into a 24 kD form in an energy (Fig.4-1A) and

time (Fig.4-1C) dependent reaction. The production of this processed form was

virtually prevented by the addition of apyrase, an ATPase, to the translation mix in

the no ATP assay (Fig. 4-1A lanes 1 and 2 versus lanes 3 and 4). Only the 30 kD

precursor, which remains outside of the plastid, was removed by the protease

treatment (Fig. 4-1A lanes 1 and 4). The mature protein was sensitive to the protease

only after the plastids had been lysed prior to addition of this enzyme (Fig. 4-1B).





















+ +
- +
3 4


24 N in


Protease pg
Lysis
Lane #


jw 4


Min 0 1.5 2 4 6 8 10
Lane # 1 2 3 4 5 6 7

ii*."


The 30 kD pTOM 111 translation product can be imported into pea
chloroplasts and processed into a 24kD form in an energy and time
dependent process. In vitro transcribed RNA was translated with a
rabbit reticulocyte lysate system in the presence of [3S]-Met. Import
assays were conducted as described in the Materials and Methods. A)
Production of the mature form of the protein requires ATP and is
protected from protease digestion. A standard import assay was
conducted with (lanes 3 and 4) or without (lanes 1 and 2) 10mM Mg-
ATP. Plastids were pelleted and resuspended with (lanes 2 and 4) or
without (lanes 1 and 3) protease. B) The plastid membrane protects
the mature form of pTOM 111 from the added protease. Plastids were
(lanes 4, 5 and 6) and were not lysed (lanes 1, 2, and 3) after import
with 0 (lanes 1 and 4) 12 pg (lanes 2 and 5) or 24 pg (lanes 3 and 6)
of protease. C) The production of the mature form of pTOM 111 is
time dependent. Assays were of 100 pl of plastids (1mg/ml chlorophyll)
for each time point. The reactions were terminated with 3.3 mM HgCI2
to quickly stop the reaction.


ATP
Protease
Lane#


12 24
+ +
5 6


Figure 4-1








84
When HgClz was used to terminate the assays, a small amount of protease-resistant

precursor was observed in the early time points (Fig. 4-1B). This may reflect a stage

in the reaction prior to cleavage of the transit peptide but after import into the

organelle.

As will be discussed in the following section, sequence analysis identified

pTOM 111 as encoding a plastid-localized low MW heat shock protein (hsp).

Consequently, we asked whether our hsp would localize in vitro in the same manner

as a plastid localized low MW hsp that was isolated from heat stressed pea leaves.

Chloroplasts recovered from import assays were separated into membrane and

soluble fractions in Fig. 4-2. The proteins from both pTOM 111 and the pea cDNA

were recovered in the soluble fraction. Even though the tomato cDNA was isolated

from ripening fruit, the protein behaves in a manner similar to one isolated from

heat stressed leaves.



Import Studies of pTOM 41 Protein

A similar analysis of the import of an in vitro translation product from pTOM

41 was performed (Fig 4-3). In this case, a 47 kD protein was produced upon in vitro

translation of the pTOM 41 transcript (Fig. 4-3B). When the translation product was

incubated with isolated pea chloroplasts, a 41 kD protein was generated (Fig. 3B).

This predicts that a 6 kD transit peptide is cleaved upon import into the plastid.

Little of the 47 kD precursor was associated with chloroplasts recovered from assays

without added ATP, even without protease post-treatment (Fig. 4-3A, lane 1). Since

















pTOM 111
Tr St Mem
1 2 3


Figure 4-2.


pHSP 21
Tr St Mem
4 5 6


The pTOM 111 and an hsp 21 from heat stressed peas localize
predominantly to the stroma. In vitro transcript was translated with a
rabbit reticulocyte lysate system in the presence of [5S]-Met and
import assays were conducted as described in the Materials and
Methods. Translation products (Tr), stroma (St) and membrane
(Mem) fractions were analyzed by SDS-PAGE gels and fluorography.














Lysis
ATP
Protease
Lane #


- + -
- +
1 2 3


T St Mem

+ + + +
+ + + +
4 5 6 7


- ~ ~


Min
Lane #


01 2
1 23 4


8 10
7 8


47
41 .m.




Figure 4-3. The 47 kD pTOM 41 translation product can be imported into pea
chloroplasts and processed into a 41 kD form in an energy and time
dependent process. A) Production of the mature form of the protein
requires ATP and is protected from protease digestion. The mature
form of PTOM 41 can be digested by the protease if the plastids are
lysed. Import assays contained 10mM Mg-ATP (lanes 2, 4, 5, 6, 7, 8,
and 9). Thermolysin was added after import (lanes 3, 4, 5, 6, 7, and 9).
Plastids were lysed after import in 10mM Hepes/ KOH pH 8.0 and
treated with thermolysin (lane 9). Stromal (St) and membrane (mem)
fractions were separated from total (T) plastids following an import
reaction as described in the Materials and Methods. B) The
production of the mature form of pTOM 41 is time dependent. Assays
contained 50 gl of plastids (1mg/ml chlorophyll) for each time point.
[H]-leucine replaced [35]-methionine in the in vitro translation
reactions.









87
the plastids were repurified before the assays were analyzed, this suggests that little

of the pTOM 41 translation product attaches to the outside of the plastid.

Subfractionation of recovered chloroplasts showed the pTOM 41 imported protein

to be in the soluble fraction (Fig. 4-3A, lane 7). If plastids were lysed after import

and treated with protease, the pTOM 41 mature protein was digested (Fig.4-3A, lane

9 versus lane 8), which demonstrates that it is not inherently resistant to this

treatment A small amount of protease-resistant precursor was also found in the

early time points after import of the pTOM 41 protein (Fig. 4-3B).

These data demonstrate that the in vitro translation products of both pTOM

41 and 111 are imported into the plastid, are protected from protease by the

organellar envelope and are cleaved of their transit peptides. In order to further

characterize these chromoplast-associated proteins, the cDNAs were sequenced and

the corresponding amino acid sequences deduced (Materials and Methods).



The pTOM 111 cDNA Codes for a Low MW HSP

The pTOM 111 sequence (Fig. 4-4) shows significant homology to those of

low MW plastid-localized heat shock proteins isolated from heat stressed leaves.

Proteins from soybean, pea (Vierling et al., 1988) Arabidopsis, petunia (Chen and

Vierling, 1991) and maize (Mieto-Sotelo et al., 1990) have been cloned and

sequenced. The pTOM 111 sequence is 81% homologous to a cDNA isolated from

heat stressed petunia leaves. Low MW hsps from plants have been categorized on












88



-30 -10 10 30
GGGGCTATTTCTGTGATTAAAAATCTAAATAACTATAATGGCTTACACAAGTCTTACATCTTCTCCATTAGTGTCAAATG
----------........--------......-.......---------..-------------------.....................................
N A Y T S L T S S P L V S N V 15

50 70 90 110
TTAGCGTTGGTGGAACCTCTAAGATTAATAACAACAAGGTATCTGCACCTTGCTCTGTTTTCGTTCCGTCGATGAGAAGG
----------- ----------------......... --------------------------....... ................ .... ---------
15 S V G G T S K I N N N K V S A P C S V F V P S N R R 41

130 150 170 190
CCGACGACTAGCTGGTAGCTGCTGACcGGCGATAACAAAGATACGTCAGTCGACGTACACCACAGCAGTGCTCAAGG
---------..........--.-------4-----------------..-----------------------------------------
42 P T T R L V A R A T 6 D N K 0 T S V D V H H S S A 0 G 68

210 230 250 270
AGGAAACAACCAAGGCACTGCGTCGAACCGTTAAGATCGCTTGATGTTTCCCCTTTTGGAGTGTTAGATC
---------- ------.....................................---.......-------.........---------
69 G N N Q G T A V E R R P T R N A L 0 V S P F G V L o P 95

290 310 330 350
CAATGTCACCAATGAGAACAATGCGGCAGATGATAGACACTATGGACAGATTATTCGAAGATACAATGATTCCAGGAAGA
........ -----------..------------------------+.........-----------.......... -----------......... ........---------
95 S P M R T M R Q M I D T M D R L F E D T M I P G R 121

370 390 410 430
AACAGAGCATCAGGAACAGGGGAAATACGCACTCCATGGGACATTCATGATGATGAAAACGAAATCAAGATGCGTTTCGA
+----------.... --------... -------.. ------... ---------.. ................--........
122 N R A S G T G E I R T P U D I H D D E N E I K N R F D 148

450 470 490 510
CATGCCGGGACTGTCCAAGGAAGATGTAAAAGTATCAGTAGAAAATGACATGCTTGTCATTAAAGGGGAACACAAAAAGG
4.---------...-... ........ 4 .-- ..................-----------...---- ......------------------
149 N P G L S K E D V K V S V E N D N L V I K G E H K K E 175

530 550 570 590
AAGAAGACGGAAGAGATAAACACTCATGGGGTAGGAATTATAGCTCTTACGACACTCGTTTAGTCTCCAGATAATGTT
.-------- .-------.-----------.-----------4.-------.----------------------.-----------
175 E D G R D K H S W G R N Y S S Y D T R L S L P D N V 201

610 630 650 670
GTGAAGGATAAAATCAAAGCGGAACTGAAGAATGGAGTTCTTTTCATCTCGATTCCAAAGACTGAAGTGGAGAAAAAGGT
---......-----------..................................-----------...........------------------
202 V K D K I K A E L K N G V L F I S I P K T E V E K K V 228

690 710 730 750
GATTGATGTCCAAATTAACTAACATTCAGAATCGCCATGCTTTTGTTGTTCTGGTTCTTTGTAACCTTGTGTAAAATG
.---. ---.------ ------.. .---..-----------..... -----------....--........---------
229 I D V Q I N 255

770 790
TATGTTCGCATATGAATATAAAACAAGTAGTGCATCTTTGACA
---------... --------------------..... 4------- ......... -




Figure 4-4. Nucleotide and deduced amino acid sequence of pTOM 111. The
sequence was analyzed using the University of Wisconsin Genetics
Computer Group programs. The position of the specific nucleotides
appears above the sequence while the position of the specific amino
acid appears to the side.








89
the basis of deduced amino acid sequence similarity (Vierling, 1991). There are three

classes of cytoplasmically-localized low MW hsps, one class associated with the

chloroplast, and another affiliated with the endoplasmic reticulum. A comparison of

the deduced amino acid sequence of pTOM 111 to these proteins revealed the

greatest similarity with the petunia plastid hsp (77% identity) (Fig. 4-5). This is not

surprising since these species are evolutionarily most alike. Comparison to the only

other characterized low MW hsp from tomato fruit, pTOM 66 (Fray et al., 1990), a

cytoplasmically-localized hsp, revealed only 32% identity to pTOM 111 (Fig. 4-5).

The pTOM 66 protein shares more sequence similarity to cytosolically localized hsps

from other species (data not shown). Overall these hsp classes share more sequence

identity among members from different species than they do with hsps from other

families within the same species. The homology of pTOM 111 to a cDNA for a

known plastid-localized protein supports the use of import assays to identify cDNAs

for chromoplast-associated proteins.

Chen and Vierling (1991) have identified 3 conserved regions in 5 plastid

localized low MW hsps they have examined. While the first two regions are found

in all low MW hsps, the third region is unique to the plastid-localized proteins. It is

in these three regions that pTOM 111 has the highest homology to other low MW

hsps. A 90% amino acid identity to the petunia protein is found in these regions in

contrast with the 77% overall homology (Fig. 4-5).















111/hsp21
pTOM111
Petcphsp21
pTOM66



111/66
111/hsp21
pTOM111
Petcphsp21
pTOM66


++ ++ + +++++ + ++ ++ ++ ++ + + +
MAYTSLT.S. SPLVSNVSVG GTSKINNNK. VSAPCSVFVP ....SNRRPT
macktttcsa sptvsngws atsrtnnkkt ttapfsvcfp yskcsvrkpa
.......... .......... .......... .......... ..........

Putative Transit Peptide
51 100
+ + .+ + + +
+4-4+ +4"+4 +4+-H- + + +++++++++++ + ++ +4+++
TRLVARATGD NKDTSVDVHH S.SAQGGNNQ GTAVERRPTR MALDVSPFGV
srlvaqatgd nkdtsvdvhv snnnqggnnq gsaverrprr maldvspfgL
NSLIPRIFGD RRSSSN.... .....................FDPFSIDV


111/66
111/hsp21
pTOM111
Petcphsp21
pTOM66



111/66
111/hsp21
pTOM111
Petcphsp21
pTOM66



111/66
111/hsp21
pTOM111
Petcphsp21
pTOM66


+ + +


44+ 4-+H4a +4. 44++.+
LDPMSPMRTM RQNIDTMDRL
Ldpmspnirtm rqmmdtmdrl
FDP....... ..........


+ +


4++++ + -+++- 44+4- 4+4+
FEDTMIPGRN RASGTGEIRT PWDIHDDENE
fedtmtfpgs rnrgtgeira pwdikddene
FRELGFPSTN SGESSAFANT RIDUKETPEP


sensus Region III


+ +++ + + ++++ +

IKMRFDNPGL SKEDVKVSVE
ikmrfdmpgt skeevkvsve
HVFKVDLPGL KKEEVKVEVE


+ ++ ++44 + +
+ ++++ ++ +++ + + ++++++
ND.MLVIKGE HK..KEEDGR DKHSUGRNYS
dd.vtvikge hk..keesgk d.dswgrys
EDRVLQISGE RNVEKEDKND KWHRMERSSG


Consensus Region II


201
+ +44 + + +
++4+++4++444 4+4 +4 +4- +
SYDTRLSLPD NWKDKIKAE
sydtrlstpd nvdkdkvkae
KFMRRFRLPE NAKMDQVKAS


247
4*4+** *+ + +
+++++ +++ 4+4 +4+4 ++ ++ +
LKNGVLFISI PKTEVEK.KV IDVQIN.
Lkngvllisi pktkvek.kv tdveik.
MENGVLTVTV PKEEVKKPEV KSIEISG


Consensus Region I


Figure 4-5.


The pTOM 111 shares 77% amino acid sequence identity to an hsp 21
from petunia and 32% identity to pTOM 66 from tomato fruit. The
deduced amino acid sequences were compared with the University of
Wisconsin GCG program, "pileup", as described in the Materials and
Methods. The + symbol represents exact matches between proteins,
while dots represent spaces built into the protein by the pileup
alignment The putative transit peptide (Vierling, 1991) is delineated
by a solid underline. The three consensus regions described by Chen
and Vierling (1991) are also marked with a solid line.








91
The putative transit peptide of pTOM 111 protein is identified in Fig. 4-5.

(Vierling, 1991). Although this part of the protein is the least similar to the petunia

hsp shown in Fig. 4-5 (63% amino acid identity), the region still contains a number

of features characteristic of transit peptides (Keegstra et al., 1989). For example, it

is rich in serine, valine and alanine and deficient in the acidic amino acids glutamic

and aspartic acid. In addition, some basic amino acids (arginine and lysine) are also

present. Where the sequence varies from the petunia hsp within this region the

substitutions are generally those residues characteristic of transit peptides. The

observation that this portion of the protein is least similar to other plastid localized

low MW hsps is not surprising, since an examination of transit peptides for the 5

plastid-localized low MW hsps analyzed by Chen and Vierling (1991) has also shown

that they are the most diverged portion of the protein.

Even though the structure and function of the chromoplast differs drastically

from the chloroplast, a similar hsp is produced. Our data agree with the conclusion

of Vierling (1991) that localization to the plastid appears to predict more sequence

conservation than does the tissue or organism in which the protein is expressed.

Although the increases in low MW hsps pTOM 66 and pTOM 111 may be a general

result of ripening, sequence conservation suggests that their role may be more

pertinent to the compartment in which they function.



Sequence Analysis of pTOM 41

The sequence obtained for pTOM 41 is shown in Fig 4-6. The cDNA is 1423





















10 30 50 70
CTTCTTGAAATGGGTTGGGA TT TCATTTTTTCATTTTTTGTATTATTTCACTTGOAAACA TAMCTA
.-..........-- ..+----.............. -....... .......-+...........-
L 1
90 110 130 150
TOGCTTCCTGTATATTCTTTGTAGTGTCAAACCCCAATTGATTCTTTTAAAAAGATTGTTTTTTTTCAGTCATGGTC
---------.........+---------.........+----------........--..................4...........................
2 W L P V I F F V V 8 N P K L I L L K R V V F F 0 8 W 8 28

170 190 210 230
AAATAGCCACATTTCATCCTATTT CAAGAATATCCTTTTTTTAAAGAACATTTTGTTATTGTGAAGGCTTCAG
.........----------------...........................4........ ......... .........4-.........4
29 N R P H G 8 Y F N K N I 0 F R R N 8 F V I V K A 8 G 55

250 270 290 310
GTTCAAGAACTTAAAAAACAASTAGAGATAACATATATCCTGAAGAAAGATM TAAATTAGCTGATGMAGTGAT
.--.. ---...... ...-....- -.............. --...-....-4.......-...- ---.----.........
56 R T K K Q V E I T Y N P E E K F N K L A E V 82

330 350 370 390
AGAGAAGCTGGCTTTCAAGACTCACTCTTTTTCTCCTTCAAGATAAATGTTTTCTTGAGAATAACAAQCAAGAGGA
-------... .+......-..-.......+.........4.........-........4-.........--.........
82 R E A G L 8 R L T L F S P C K I N V F L R I T 8 K R 0 108

410 430 450 470
TGACGGATATCA TCGTCTCTCTTTCATTGATTAGTCTAGGAGATAAAATAAAGTTCTCCTGTCACCATCGA
.........-.........-.........4.........4........................... 4. ........4
109 D G Y H D L A 8 L F H V I 8 L 0 D K I K F 8 L 8 P 8 K 135
490 510 530 550
AGTCAAAGGATCGtTTATCTACTAATGTTGACGGAGTTCCATCGATGAGAGAAATCTGATTATAAAG CAATCTT
.........----------------................... ------.................----.......-... .........4
136 SKD R L T N V A V P L D E R N LII K A L N L 162

570 590 610 630
TATAG AAAGACTGGAACAGACMTTACTTTTGATTCATCTTGATAAAAAGTGCCTACTGGAGCTGGTCTTGGTG
----......-.........------.... .....-...........4.................------.........
162 Y R K K T 0 T 0 N Y F W I H L 0 K K V P T G A G L G G 188

650 670 690 710
TOGGAGCATAATGCTOCTCAAC CTCTGTGGCAGCAAATCAATTCAGTGGTTGTGTTGCCACTGAAAGCATCCAAG
-------------------------*--------- +--------- ---------------
................................................4. ...4..................4
189 G 8 8 N A A T T L W A A N 0 F S G C V A T E K E L Q E 215

730 750 770 790
AGTGGTCTOGTGAGATTGGTTCTGATATTCCTTTCTTTTCTCTCATGGACACCTACTTACGGTAGGGGTGAGGTT
..........................................................................4
216 W 8 E I G S D I P F F F 8 H G A A Y C T G R G E V 242
810 830 850 870
GTTCAGATATCCOTCACCCATACCATTTGACATTCCAATGTCCTCATAAAGCCTCAACACATOCTCCACTOCTGA
.........+.........4.........4.........4..........4..........4....................+
----------------------------------* ---------* -----.----*------
242 V Q D I P 8 P I P F 0 I P M V L I K P 0Q A C 8 T A E 268

890 910 930 950
AGTTTACAAGCGTTTCAGTTOGATCTOTCTAGTAAGGTTGATCCCTTGAGOTTACTOGAG ATCTCACTAGTOGAA
................ ....4.........4.........4.........4....................4
---------+---------+----------------------------------------
269 V Y K R F Q L D L 8 8 K V D P L 8 L L E K I 8 T 8 G I 295

970 990 1010 1030
TATCTCAAGATGTaTOTGTCAATGATTTAGAACCTCCTCCTTTGAAGTTCTTCCATCTCTTAAAAG TTAAACAACQA
---------*---------+--------------------- --------------------
.........-.........4.........4.-......4.4. + 4. 4..........
296 8 Q 0 V C V N 0 L E P P A F E V L P 8 L K R L K Q R 322

1050 1070 1090 1110
GTMTTGCTGCTGGCCTAGGACMATGATGCAGTCTTCATTCTGGAAGTGGAACACAATATAGGGGTTGGCTCTCC
......---.--.......---..-------.........-........4......... --..................
322 V I A A G R G Q Y D A V F M 8 G 8 G 8 T I V 6 V 6 8 P 348
1130 1150 1170 1190
AGATCCACCACAATTTGTCTATGATGATAAGAATACAAGGATGTCTTCTTGTCA aCAAGTTTCATCACTCGACCAG
.........---------+ ---.........----------......... -----.........4.........4.........-------4..........
349 D P P O F V Y 0 0 E E Y K D V F L 8 E A 8 F I T R P A 375

1210 1230 1250 1270
CCMACATGGTATGTTGAACCTGTTTCGGTACACTATTOGTGATCAATGAGTTTCTACATCTTTTGACATGTCT
---....-----.......-..-- ......+.-...... ........4-.........4.-........4-.........
376 N E W Y V E P V G T I D P E F 8 T 8 F D 402

1290 1310 1330 1350
TAAAACTCAG AGAGCTTAAAATTGMCAATAGGAGAAGTTTTTGTTGTAAATGTTATATCCTATTTTCTGTA
-...-------........- .........-- ........4.-................- -.......4-........-4

1370 1390 1410
TATCCT ACTTGATCCATTC AAAATAAAATTAGTCACAATCTGATCGAAA
---------... .. ..... ............---- -...........------- .. ..----

Figure 4-6. Nucleotide sequence and deduced amino acid sequence of pTOM 41.

Numbers appearing above the sequence refer to the nucleotide

sequence. Numbers appearing to the side of the sequence refer to the

deduced amino acid sequence.










9B 0




_-_ .- o








S o


-oo




-o "
ci
-- I 1












-D
_- _- M ,,.
- -o MSs













0 to1 1" 0
oe









-- ,^



4) 4) 0

n- =
-- o


-
= 1 g1




Ia
I1S








94
bases long. Only 1 long open reading frame (ORF) is present, but it starts with a

leucine, not a methionine (Fig. 4-6 and 4-7). This is hard to understand, given that

the pTOM 41 transcript directed production of a 47 kD translation product. A

methionine does occur within 12 nucleotides of the beginning of the long reading

frame; however, two stop codons are encountered within the next 57 bases. Another

small ORF of 28 residues is found two nucleotides 3' of the large ORF. This begins

with a methionine in a favorable initiation context (Kozak, 1991). Two possible

explanations for the observed anomaly are: 1) a sequencing error, or 2) a +1

frameshift during translation. The first possibility was addressed by additional

sequence analysis of the areas in the vicinity of the 5' and 3' ends of the long ORF.

The cDNA sequence in Fig. 4-6 shows that the long ORF starts at 78 bases

and ends at 1280. It is clear that an ORF of approximately this length is required to

produce the protein observed on SDS-PAGE, all other potential reading frames are

littered with stop codons, therefore, if a simple sequence error was generated, it

would be found within the first few hundred bases. The first 290 bases were

reexamined using a number of different modifications to the sequencing reactions.

Figure 4-8 is a compilation of these individual sequencing results. For example, both

strands of the sequence (R1 and Fl) were examined by comparing incorporation of

dITP instead of dGTP to the standard Sequenase double stranded sequencing

reactions. This region of the sequence was also analyzed by the ICBR DNA

sequencing core. They used either Klenow (R4) or TAQ polymerases (F2 and R2)

in generating these sequences. Since the template that was used for most of the




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PAGE 1

IDENTIFICATION AND CHARACTERIZATION OF cDNA CLONES FOR CHROMOPLAST-ASSOCIA TED PROTEINS IN TOMATO FRUIT By SUSAN D. LAWRENCE 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 1993

PAGE 2

ACKNOWLEDGEMENTS I wish to thank Drs. Donald Grierson and Wolfgang Schuch for providing us with clones from their ripening tomato fruit cDNA library. I would also like to take this opportunity to thank the members of my committee Drs. Gloria Moore, Ken Cline, Eduardo Vallejos, Don Huber and Bill Gurley for their advice and support. I am especially indebted to Eduardo Vallejos for extensive years of committee service. This dissertation is dedicated to Mike McCaffery for his tireless computing assistance and to Ralph Henry for his photographic skills. Finally, I would like to express my appreciation to all my Fifield Hall lab pals, past and present, for making this an experience I will fondly recollect. ii

PAGE 3

TABLE OF CONTENTS ACKNOWLEDGMENTS ...................................... 11 ABSTRACT ................................................ V CHAPTERS 1 INTRODUCTION ................................. 1 2 LITERATURE REVIEW ............................ 3 Morphological Changes .............................. 5 Biochemistry and Molecular Biology ..................... 6 Changes in Plastid Structure . . . . . . . . . . . . . 15 Regulation of Photosynthetic Proteins . . . . . . . . . 16 The Role of Ethylene in Tomato Fruit Ripening . . . . . 18 Transcriptional Regulation of Fruit Ripening . . . . . . . 19 Processes Associated with Chromoplast Development . . . . . . . . . . . . . . . . . . 20 Heat Shock Proteins and Oxidative Stress .......................................... 40 Conclusions ...................................... 51 3 CHROMOPLAST-TARGETED PROTEINS IN TOMATO FRUIT ........ ... .......... . .... 54 Introduction . . . . . . . . . . . . . . . . . . 54 Materials and Methods . . . . . . . . . . . . . . . 55 Results and Discussion . . . . . . . . . . . . . . . 58 4 CHARACTERIZATION OF TWO cDNAS FOR CHROMOPLAST-TARGETED PROTEINS ............. 74 Introduction . . . . . . . . . . . . . . . . . . 74 Materials and Methods . . . . . . . . . . . . . . . 75 Results ......................................... 82 Discussion . . . . . . . . . . . . . . . . . . . 105 5 CONCLUSIONS 118 iii

PAGE 4

APPENDIX CONSTRUCTION AND EXPRESSION OF ANTISENSE pTOM 41 AND Ptom 111 pLASMIDS IN TOBACCO ..... 120 REFERENCES .. .. ...... .. ... ........ .... .. .. ... .. .. 136 BIOGRAPHICAL SKETCH ... .. ... ....... .. . . .... .. .. . 153 iv

PAGE 5

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 IDENTIFICATION AND CHARACTERIZATION OF cDNA CLONES FOR CHROMOPLAST-ASSOCIATED PROTEINS IN TOMATO FRUIT By Susan D. Lawrence December 1993 Chairman: Dr. Gloria A. Moore Major Program: Plant Molecular and Cellular Biology The chloroplast to chromoplast transition during tomato fruit ripening is characterized by a dramatic change in plastid structure and function. The thylakoid membranes of photosynthetically competent chloroplasts are broken down and the carotenoid pigment lycopene is deposited within the developing chromoplast. A goal of this investigation was to determine whether this process is mediated by an increase in the steady-state level of RNA for plastid-targeted proteins. Assays for import of radiolabeled translation products into isolated pea chloroplasts were used to monitor levels of chromoplast-targeted proteins at four stages of tomato fruit development. During development, striking increases in levels of translatable RNA for two such proteins were found. Additionally, the import of in vitro translation products was examined for seven individual cDNA clones known to encode RNA that increases V

PAGE 6

during fruit ripening. Three of these clones produced in vitro translation products that imported into pea chloroplasts. One clone, pTOM5, has recently been shown by others to encode the enzyme responsible for the first committed step in the synthesis of lycopene. Subsequent sequence analysis of another cDNA, pTOM 111, revealed that it encodes a plastid localized low MW heat shock protein Not only does this transcript increase during development at normal temperatures, but it is increasingly inducible by elevated temperatures as ripening progresses. This is the first report of the developmental induction of a plastid-localized low MW heat shock protein. Identification of the induction of a heat stress protein during this transition suggests that either the plastid i s subjected to an increasingly stressful environment or, as is consistent for the developmental induction of other heat stress proteins, a massive structural reprogramming is underway. These studies imply that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast, and that the plastid conversion is an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus Furthermore, these results demonstrate the utility of this method for identification of structural genes involved in plastid morphogenesis vi

PAGE 7

CHAPTER 1 INTRODUCTION Ripening of tomato fruit involves a wide variety of developmental changes that include but are not limited to changes in cell wall structure, in metabolism, in ultrastructure, and an especially dramatic change in the form and function of the plastid. Biochemical and molecular studies suggest that most or all of these changes are mediated by the enhanced expression of ripening specific genes. I have chosen this system as a means to investigate plastid development and interconversion. We are interested in determining how the transition from chloroplast to chromoplast is regulated, e.g., does a subset of plastid proteins increase during this process? In an initial examination of this question, we have asked whether an increase in the accumulation of RNA for a subset of plastid proteins correlates with the chloroplast to chromoplast transition. Since transcription and/or translation of the plastid genome seems to diminish during chromoplast formation (Kobayashi, 1991 ), chromoplast-specific proteins are probably encoded in the nucleus. Nuclear encoded plastid proteins are synthesized in the cytosol and posttranslationally imported into plastids. The import of proteins into plastids is a highly specific process (Keegstra, 1989). The proteins are made as precursors with transient amino terminal "transit peptides," which are both necessary and sufficient for the correct 1

PAGE 8

2 targeting into this organelle. It is this latter observation that makes the import into plastids a potentially diagnostic assay for the identification of chromoplast associated proteins. The approach described within this dissertation has been to monitor chloroplast import of in vitro translation products from RNA of tomato fruit isolated at different ripening stages. We have also studied translation products programmed with transcripts from cDNAs (Slater et al., 1985) whose steady state RNA level increases during ripening. Since individual cDNAs were analyzed, subsequent sequence analysis allowed further characterization of the proteins that mediate this transition. Eventually the role of chromoplast-associated proteins in chromoplast development can be assessed directly via antisense technology. The final outcome of such a study should be an understanding of the role new protein synthesis plays in this process and the isolation, identification and characterization of genes that mediate this transition.

PAGE 9

CHAPTER 2 REVIEW OF LITERATURE The manner in which the transition from chloroplast to chromoplast is regulated is largely unknown. Electron micrographic analysis has shown that a massive reordering of plastid structure occurs during the conversion (Thelander et al., 1986). In tomato fruit, this is characterized by the overall breakdown of chloroplast specific components, e.g. the thylakoid membrane system and starch, with the concomitant accumulation of membrane-associated deposits of the carotenoid lycopene (Thelander et al., 1986). It has not been determined whether the breakdown of chloroplast structures is caused simply by the decrease in synthesis of photosynthetic proteins or if increases in degradative enzymes actually enhance this process. An examination of nuclear-encoded, plastid-targeted, photosynthetic proteins has shown that their transcription stops 5 to 10 days before the fruit reaches full size (Piechulla et al., 1986). Analysis of plastid-encoded transcripts has demonstrated that some are greatly reduced, whereas others persist, and one appears to increase during chromoplast formation (Kobayashi, 1991; Richards et al., 1991). Similarly, little has been ascertained about the function or expression of genes involved in the synthesis of chromoplast structures as few clones specific to this developmental stage have been available for analysis (for example, Kuntz et al., 1992). 3

PAGE 10

4 In considering what aspects of fruit ripening directly affect the proteins that are targeted to the plastid, it quickly became apparent that two types of functions could cause increases in proteins directed to the developing chromoplast. First, proteins involved in the degradation of the photosynthetic apparatus or the build up in carotenoids are excellent candidates for chromoplast-targeting and enhanced synthesis during fruit ripening. A broad description of plastid senescence will be considered, since that transition also involves the degradation of chloroplast structures and because an examination of RNA from senescing leaves has shown that many of the same transcripts are also present. Secondly, other aspects of maturation may have indirect effects on the environment in which the developing plastid resides. For example, the autocatalytic production of ethylene, the respiratory climacteric or an increase in oxidative stress during ripening could secondarily create increases in plastid-targeted proteins. Since the plastid is responsible for a number of basic biosynthetic functions, such as the synthesis of aromatic amino acids, fatty acids and 8-aminolevulinic acid these changes outside of the plastid could create a need for substrates synthesized within this organelle. A consequence of the work described in this dissertation was the characterization of a cDNA that encodes a plastid-localized low molecular weight (MW) heat shock protein (hsp ). Because transcript for this hsp increases during ripening at normal temperatures, it was necessary to assess how this unexpected finding fit into chromoplast development or fruit ripening. Since oxidative stress has been associated with senescence and hsps have been induced by oxidative stress in

PAGE 11

5 animal tissues, a consideration of heat stress proteins and oxidative stress during fruit ripening will also be examined Morphological Changes The overall changes that occur during the ripening of tomato fruit are as familiar to ones mother as they are to any post-harvest physiologist. Full sized green fruit harvested from the garden and brought into the kitchen will ripen normally. The color changes observed during the ripening of tomato fruit are due to the breakdown of chlorophyll and the deposition of Iycopene within the fruit plastids The breaker stage is identified by the appearance of external color at the blossom end. The exact fruit color that then develops is dependent on variety and consequently, the fruit may turn yellow pink, or orange-red. As the fruit becomes uniformly red, the cell walls begin to soften The characteristic flavor of ripe tomatoes is created by a combination of glucose fructose, citric and glutamic acids, which accumulate in the vacuoles, along with the synthesis of hundreds of volatile compounds (Davies and Hobson 1981 ; Grierson, 1986). The young fruit is green because it contains chlorophyll in photosynthetically functional chloroplasts It is considered immature green (IMG) until it reaches its full size. Growth is complete at mature green (MG) A green tomato may appear morphologically quiescent ; however, it is undergoing important ripening changes. Its Jocular tissue transforms from a solid to a gelatinous substance, it can respond rapidly to exogenous ethylene and the endogenous ethylene level also increases at

PAGE 12

6 this time By late MG, carotenoids begin to appear in the locules. Thus the color change begins in the interior of the fruit (Kader and Morris, 1976). After the fruit bas passed through mature green the transformation to a red ripe soft tomato proceeds rapidly Biochemistry and Molecular Biology Although the c h ange in fruit color is a convenient method of classifying tomato fruit ripening, a number of other biochemical changes also occur. For example a dramatic rise in respiration and ethylene production takes place. This phenomenon is known as the climacteric Climacteric ethylene levels reach about one half of their maximum by the breaker stage (Picton and Grierson 1988). The respiratory peak occurs soon after the rise in ethylene (Grierson, 1986) Alterations in Basic Metabolism During Fruit Ripening Changes take place in the type of carbohydrate that accumulates as tomato fruit ripens. Starch granules transiently appear between 14-28 days postanthesis in the immature green chloroplasts (Davies and Cocking, 1965). Although these plastids can fix carbon at up to 35 % of the rate attained by leaf plastids, the starch granules are mostly the product of sucrose imported from the leaves (Thelander et al., 1986). Since the import of carbohydrate from the leaves stops by MG (Thelander et al., 1986), fruit at this stage will develop normally if harvested from the plant. About 28-35 days after an thesis degradation of starch occurs as an increase in soluble

PAGE 13

7 sugars takes place. Consequently, little starch remains in the mature green fruit (Yelle et al., 1988). The rapid degradation of starch precedes the climacteric rise in respiration (Davies and Cocking, 1965) and this carbohydrate is stored as glucose and fructose in the vacuole (Grierson, 1986). The degradation of starch seems to occur via phosphorylase (Robinson et al., 1988), and the further steps of hexose to triose occur via the pentose phosphate pathway and glycolysis (Thelander et al., 1986) Therefore, as the tomato fruit matures, a shift of stored carbohydrate occurs from starch in the plastid to hexose sugars in the vacuole. In fact, the red ripe tomato contains no measurable starch and 47% hexose by dry weight (Davies and Hobson, 1981) The changes in stored carbohydrate precede the breakdown of the thylakoid membranes and consequently, any plastid associated enzymes responsible for starch degradation are not expected to increase during chromoplast development. It is unclear what causes the increase in CO 2 during ripening and, consequently, the molecular regulation of this increase is also unknown (Solomos, 1988). Fluctuations in enzymes involved in the tricarboxylic acid (TCA) cycle and glycolysis occur but it has not been determined whether these changes are due to a variation in the level of enzymatic effectors or are caused by alterations in the amount of de novo synthesis of these enzymes during ripening. Solomos (1988) reviewed the literature on respiration in ripening fruit and concluded that the respiratory burst measured is probably a result of an increase in substrate level phosphorylation caused by enhanced glycolysis An increase in the activities of cytosolic NADP linked malic enzyme (Goodenough et al., 1986) and the glycolytic

PAGE 14

8 enzyme phosphofructokinase (PFK) have been observed (Grierson, 1986). PFK is a control point in glycolysis and an increase in this enzyme activity probably results in an increase in carbon flow through this pathway (Solomos, 1988). Indeed, an increase in the accumulation of ATP is generally noted during the respiratory rise (Solomos, 1988). Investigation of mitochondrial electron transport in ripening fruit suggested that it remains unchanged (Solomos, 1988). However, the TCA cycle enzymes citrate synthase, malate dehydrogenase, NAO-linked malic enzyme, and isocitrate dehydrogenase decrease in ripening tomato fruit (Grierson, 1986; Jeffery et al., 1986). Overall, these data do not explain why an increase in respiration is measured during ripening. Since these changes are underway during the plastid conversion while photosynthesis is declining, it is unclear how this may effect the synthesis of proteins targeted to the plastid. Other Changes Associated with Fruit Ripening Apparently, the level of the anoxia-responsive enzyme, alcohol dehydrogenase (ADH), increases during ripening in tomato fruit (Longhurst et al., 1990; Van Der Straeten et al., 1991). Longhurst et al. (1990) concluded that the increase in ADH during ripening may be caused by the cytoplasmic increase in pH. It has been hypothesized that low oxygen levels could result within such a bulky organ, but by exposing ripening fruit to different levels of 0 2 and CO 2 these authors surmised that the change in pH leads to an induction of this enzyme.

PAGE 15

9 As fruit matures, the cell walls soften and an increase in soluble pectin occurs (Grierson, 1986). Characterization of the regulation of the enzymes involved in cell wall degradation has begun These data suggest that the pattern of expression for enzymes involved in cell wall degradation is complex and can even involve more than one isozyme (DellaPenna et al. 1986; Grierson et al., 1986 ; Fisher and Bennett, 1991; Harriman et al., 1991). For example, antisense mutagenesis of the cell wall softening enzyme, polygalacturonase (PG), has shown that although this enzyme is dramatically induced during fruit ripening, loss of this enzymatic function has little overall effect on maturation (Sheehy et al., 1988 ; Smith et al., 1988 and 1990). The synthesis of hundreds of volatile compounds occurs as tomato fruit ripens (Davies and Hobson, 1981). However, little is known about how these compounds are synthesized during maturation Oxidative Stress and Fruit Ripening Since a chromoplast-associated low MW heat shock protein was identified in this work it is important to understand the role this stress protein may play in fruit development. Oxidative stress occurs when the cell's ability to respond is exceeded by the level of reactive oxygen species present (Barja de Quiroga, 1992). The induction of hsps by compounds that cause oxidative stress has been shown in animal tissues (Drummond and Steinhardt, 1987; Cajone and Bernemmi-Zazzera, 1988 ; Courgeon et al. 1988). In addition, oxidative stress has often been associated with ripening and senescence. One theory postulates that a buildup in the deleterious

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10 effects of free radicals results in the physiological manifestations of aging (Harman, 1992) Free radical production during normal cellular metabolism Reactive oxygen compounds can cause damage to proteins and especially membrane components of the cell A number of normal cellular functions result in the production of these species, which can be a side effect of reactions that reduce molecular oxygen. For example, at least one percent of the oxygen utilized during respiratory electron transport is incompletely reduced (Fleming et al., 1992). Furthermore an increase in basal metabolism can result in an increase in the amount of reactive oxygen generated even without invoking a breakdown in oxidative phosphorylation (Barja de Quiroga, 1992). Photosynthesis is another normal cellular function during which a large amount of free radicals can form (Scandalios, 1993). In addition, some enzymatic reactions directly produce reactive molecules. Examples of this latter group include xanthine oxidase (an ATP degrading enzyme) and beta oxidation of fatty acids (Scandalios, 1993). Finally, certain environmental stresses can enhance the level of reactive molecules. For example, radiation, herbicides, pathogens, hyperoxia and temperature fluctuations can induce free radical production (Scandalios 1993) Perhaps a buildup of these deleterious compounds occurs during the ripening of tomato fruit. Since any cellular function that involves the reduction of oxygen causes production of reactive oxygen, either autocatalytic ethylene synthesis or the respiratory climacteric would be excellent sources of free radicals

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11 One source of reactive oxygen is lipoxygenase activity, which increases during ripening (Thompson et al., 1987). This enzyme incorporates oxygen into polyunsaturated fatty acids (Hatanaka et al., 1992) and leads to a build up of the reactive oxygen compounds hydroperoxide or superoxide. Recently, Bowsher et al. (1992) have purified a membrane associated Iipoxygenase from breaker stage tomato fruit. They find that the activity fractionates with the thylakoid membrane. Consequently, this could result in increases in the amount of reactive oxygen species in the developing chromoplast. The change in membrane integrity during fruit ripening A decrease in membrane fluidity, which may be created by oxidative stress, occurs during ripening and senescence along with a progressive loss of specialized membrane function (Thompson et al., 1987). Many aspects of membrane structure can affect the level of membrane viscosity. For example, the length or degree of unsaturation of the hydrocarbons and the level of sterols all can play a part. Although a decrease in membrane fluidity occurs in the plasma membrane of tomato fruit (Legge et al., 1986) and pepper microsomal membranes (Whitaker, 1991 ), no change in plastid membrane fluidity occurs in either tomato or pepper chromplasts. Neither fluorescent membrane probes, which directly assess the structure within specific environments of the membrane used by Legge et al. (1986), nor measurements of the free sterol or total sterol to phospholipid ratios determined by Whitaker (1991) could detect a change in the developing chromoplast membrane. Considering the increase in carotenoid biosynthesis during ripening, which is

PAGE 18

12 associated with plastid envelope membranes, it is appropriate that the integrity of this membrane persists. Free radical scavengers and oxidative stress Initially, an increase in free radicals results in an increase in enzymes or antioxidants that neutralize these reactive species. For example, a family of superoxide dismutases (SOD) acts to scavenge superoxide and create oxygen and hydrogen peroxide. Five isozymes of this enzyme have been described in tomatoes. Two are cytoplasmic, one is mitochondrial and two occur in chloroplasts (Perl-Treves and Galun, 1991). Also, ascorbic acid and a-tocopherol can act as antioxidants (Leshem, 1988). A number of enzymes are also involved in reducing the levels of hydrogen peroxide. Catalase, peroxidase and glutathione peroxidase all act in this manner (Leshem, 1988) Additionally, glutathione reductase is required to regenerate glutathione lost by interaction with hydrogen peroxide (Leshem, 1988). Consequently,the increase in these enzymes is correlated with an increase in reactive oxygen molecules. Any decrease in these enzymes with senescence, therefore, could contribute to degeneration of membranes and disturbance of cellular function. Increases in basal metabolism may induce SODs in plant tissue. Bowler et al. (1989) concluded that the mitochondrial SOD may respond to changes in the activity of respiratory electron transport, because the induction of this enzyme in tobacco correlated with increased activity for cytochrome oxidase. In addition, oxidative stress in one compartment can lead to the induction of free radical scavengers in another part of the cell. This was shown when paraquat,

PAGE 19

13 which is thought to act mainly on chloroplasts, was applied to tobacco and caused a strong induction of SOD transcript in the mitochondria, cytoplasm and chloroplast (Tsang et al., 1991) The level to which SOD is induced within a cellular compartment may depend on the amount of SOD that is currently present. This was the explanation of Perl Treves and Galun (1991) when they found that the addition of paraquat resulted in a greater induction of the cytoplasmic SOD in comparison to the plastid message in tomato leaves. The level of plastidial enzyme activity in mature leaves is already high in comparison with transcript level. They suggested that induction of plastidial SOD transcript may therefore be unnecessary. Ethylene has been shown to induce cytoplasmic and plastidial SOD transcripts and enzyme activity in tomato leaves (Perl-Treves and Galun, 1991) and mitochondrial SOD message in tobacco leaves (Bowler et al., 1989). In response to ethylene, therefore, an increase in reactive oxygen species has been noted and enzymes within all three cellular compartments can be affected. Free radical scavengers in ripening fruit The role of free radical scavengers during fruit ripening, however, is somewhat confusing. Peroxidase has been shown to decrease during ripening (Rabinowitch et al., 1982) or peak during the pink stage in tomato fruit (Rothan and Nicolas, 1989). Perhaps the techniques used by Rabinowith et al. (1982) were not sensitive enough to detect this peak of activity. The transient increase in basic peroxidase activity paralleled the increase in ethylene (Rothan and Nicolas, 1989). Since the conversion

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14 of 1-aminocyclopropane-1-carboxylate (ACC) to ethylene requires oxygen, this may lead to a transient increase in reactive oxygen molecules and an increase in this free radical scavenger. Changes in SOD have been reported during tomato fruit ripening but results were contradictory. SOD activity has been found to peak at the pink or orange phase (Rabinowitch et al., 1982; Livine and Gepstein, 1988). However, no change in the two plastidial SOD isozymes was reported by Perl-Treves and Galun (1991), nor was a peak of the cytoplasmic or plastidial SOD message noted during ripening. In fact, they found that the plastid-associated message actually decreased with maturation. They explain these contradictory results by suggesting that the enzyme could be quite stable and turnover very slowly. It is hard to explain all of the varied data reported by these three groups. More work needs to be done before a conclusion can be made about free radical scavengers and ripening in tomato fruit. Since the messages for the additional plastidial and cytoplasmic SODs have not been examined, perhaps these additional data will help reconcile these dissimilar observations. Pepper chromoplasts do appear to respond to an oxidative stress during fruit maturation (Romer et al., 1992). The antioxidant producing enzyme cysteine synthase was purified and a cDNA was isolated. They found not only did this enzyme increase during ripening but the activity of glutathione reductase, glutathione synthetase and glutathione content within chromoplasts also increased during their development. Since all these compounds protect organisms from oxidative damage and an increase in a-tocopherol also occurs during ripening, they concluded that these increases in

PAGE 21

15 antioxidants and peroxide scavenging compounds may allow protection of the overaccumulated carotenoids and other membrane components from oxidative stress. Since the results from tomato fruit on the induction of free radical scavengers is contradictory, information from other systems may help predict what occurs in tomato fruit chromoplasts. Leaf tissue responds to an increases in basal metabolism and the addition of ethylene by inducing SOD (Bowler et al., 1989; Perl-Treves and Galun, 1991) In addition, a compound expected to affect predominantly one compartment can induce SOD in other portions of the cell (Tsang et al., 1991). Finally, the level of induction may be dependent on current levels of enzyme activity (Perl-Treves and Galun, 1991) Perhaps the results from pepper chromoplasts where increases in a number of these free radical scavengers has been measured, explains how the chromoplast, unlike the plasma membrane, avoids oxidative damage and maintains membrane integrity (Whitaker, 1991; Romer et al., 1992). One might predict that the plastid can respond to an environment that contains an increasing amount of reactive oxygen and consequently it is able to maintain chromoplast membrane integrity during ripening. These data also suggest that the production of ethylene and/or the respiratory climacteric may induce the production of chromoplast-associated proteins involved in free radical scavenging. Changes in Plastid Structure Massive changes take place in plastid ultrastructure during the chloroplast to chromoplast transition. The thylakoid membranes are dismantled and chlorophyll

PAGE 22

16 degrades (Thelander et al., 1986). Concomitantly, lycopene begins to accumulate in the stroma. The green flesh mutant, which is inhibited in chlorophyll degradation during ripening, is helpful in the determination of how pigment accumulates in the developing chromoplast. Electron micrographs show that the granal stacks persist in this mutant even as pigment is deposited (Goodwin, 1980), and suggests that lycopene builds up around the disintegrating thylakoids. By the time a normal tomato is fully ripe, the chromoplasts contain little else but large stores of lycopene, which accumulate as lipophilic globules and crystals of pigment (Harris and Spurr, 1969). The type of chromoplast that develops may be dependent on the type of carotenoids that accumulate. The r' mutant, for example, with its low level of both lycopene and B-carotene, contains only plastoglobules (Goodwin, 1980). Regulation of Photosynthetic Proteins Transcripts for the nuclear-encoded components of photosynthesis decrease long before the breakdown of the thylakoid membranes. The mRNA for rbcS and cab (Rubisco small subunit and chlorophyll a/b binding protein respectively) are not detected after 30 days post an thesis (Piechulla et al., 1985, 1986). This loss of nuclear transcripts occurs 5 to 10 days before MG and precedes the end of chlorophyll synthesis and the disassembly of the thylakoid membranes by 2-3 weeks The plastid encoded messages rbcL and psbA (Rubisco large subunit and the 32kD photosystem II protein), howe v er, persist throughout development (Piechulla et al., 1986). Most of the other plastid transcribed proteins, such as the photosystem

PAGE 23

17 I and II proteins (psaA psbB psbC or psbD), all diminish in message levels throughout development. Once ripening has progressed to an intermediate stage of development beyond mature green, levels of photosynthetic activity have greatly diminished and this is reflected in decreased amounts of photosynthetic proteins. For example, by the yellow stage of development, the amount of LHCP protein has declined to 10% of the level present in IMG fruit (Livine and Gepstein, 1988). At the yellow-orange stage, the level of Rubisco activity has diminished to 2% of the level present at MG (Piechulla et al, 1987) while the amount of measurable protein for the large subunit has also decreased. The level of photosynthetic electron transport has decreased to 13% of what was observed at MG (Piechulla et al., 1987). Only plastocyanin and the 32kD protein were detected by this stage of development. These data underscore the dramatic change in the protein components of the plastid that occurs between mature green and this intermediate stage of ripening. It is unclear from this type of analysis whether the loss of photosynthetic proteins is simply due to a decrease in synthesis or is also caused by an enhanced production of degradative enzymes Examination of plastid gene expression during chromoplast development has shown a reduction in the level of plastid transcript during this process It is uncertain whether this is the result of posttranscriptional regulation (Gruissem et al., 1989; Marano and Carillo 1991) or whether the rate of transcription is decreased due to methylat i on of the plastid genome (Ngernprasirtsiri et al., 1988; Kobayashi et al.,

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18 1990) The level of translation is also dramatically reduced in Capsicum plastids (Kuntz et al., 1989) where ultrastructural analysis has shown that red fruit contain no ribosomes or rRNA (Carde et al., 1988). Richards et al. (1991) have identified a plastid transcript that is more abundant in chromoplast RNA than chloroplast RNA of tomato fruit. However, given the lack of functional translation in pepper chromoplasts and the decreased amount of transcripts present in tomato chromoplasts, it is expected that most proteins that are increasingly present in chromoplasts will be nuclear-encoded. The Role of Ethylene in Tomato Fruit Ripening The rise in ethylene during the climacteric is mediated by de novo synthesis of at least the final two enzymes in the biosynthetic pathway (Holdsworth et al. 1987; Olson et al., 1991; Rottmann et al., 1991) To date, only the genes for the enzymes that catalyze these final two steps in ethylene biosynthesis have been isolated Ethylene is synthesized from methionine via S-adenosyl-L-methionine and 1aminocyclopropane-1-carboxylic acid (ACC). The enzyme that synthesizes ACC (ACC synthase) is thought to be a pyridoxal 5'-phosphate utilizing enzyme (Yip et al., 1990). The final step in the synthesis of ethylene is mediated by ethylene oxidase (EFE), requires oxygen, and can be inhibited by the addition of free radical quenchers (Matoo and Aharoni, 1988). The synthesis of ACC is considered the rate limiting step in ethylene biosynthesis. However, EFE is also increasingly synthesized

PAGE 25

19 during ripening in tomato and avocado fruit, (Holdsworth et al., 1987, McGarvey et al., 1992) and during the senescence of carnation petals (Wang and Woodson, 1991). Production of transgenic tomato plants that contain antisense transcripts for the enzyme ACC synthase has shown that ripening involves both ethylene dependent and independent pathways (Theologis, 1992). A number of laboratories have produced transgenic tomato plants with reduced levels of ethylene (Hamilton et al., 1990, Klee et al., 1991, Oeller et al., 1991). The most dramatic decrease occurred in a transgenic plant carrying ten antisense copies of part of a gene for ACC synthase. The authors reported that the plant has a 99.5% reduction in the level of ethylene (Oeller et al., 1991) and produces fruit that remain firm and green until exogenous ethylene is applied. If no ethylene is added, the mutant fruit still express RNA for the ripening induced cell wall softening enzyme, polygalacturonase (PG) (Oeller et al., 1991). This result was unexpected because ethylene is required for the expression of PG. This suggests that the regulation of ripening by ethylene is more complex than just simple transcriptional control. While the transcription of PG is ethylene independent, the expression of the enzyme is dependent on the continued presence of this hormone. Transcriptional Regulation of Fruit Ripening A molecular analysis of fruit ripening in tomato has led to the conclusion that the de novo synthesis of ripening specific proteins occurs and is responsible for many of the changes associated with maturation. Several researchers have characterized

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20 changes in the profile of in vitro translation products that occur during development of tomato fruit (Rattanapanone et al., 1978; Speirs et al., 1984; Biggs et al., 1986; Lincoln et al., 1987). A number of groups have constructed cDNA libraries enriched in ripening specific sequences (Mannsson et al., 1985; and Slater et al., 1985; Lincoln et al., 1987). Generally, the onset of ripening in tomato fruit is mediated by an increase in a specific subset of transcripts, including those for PG, EFE and ACC synthase (DellaPenna et al., 1986; Grierson et al., 1986; Holdsworth et al., 1987; Olson et al., 1991; Rottman et al., 1991) Examination of the clones isolated by Grierson's group has shown that, for most of their cDNAs, RNA fails to accumulate when the fruit is held at high temperature (Picton and Grierson, 1988). High temperature (35C) has an inhibitory effect on fruit ripening, depressing the synthesis of lycopene, PG, and ethylene (Picton and Grierson, 1988). Perhaps this effect of high temperature represents a heat shock response (Picton and Grierson, 1988). This was not an overall effect for all clones, however, since RNA increased initially, for some clones, in response to an increase in temperature. Unfortunately, many of these clones have yet to be assigned a function. Processes Associated with Chromoplast Development Upon review of the work characterizing the molecular changes that occur during the chloroplast to chromoplast transition, it is notable that mainly photosynthetic proteins have been examined during this phase (Piechulla et al., 1985,

PAGE 27

21 1986, 1987; Livine and Gepstein, 1988). To undertake the investigation of proteins that increase during chromoplast development, a method must be found to identify proteins specific to this process. Isolation of chromoplasts and the subsequent comparison of proteins on SDS-PAGE from different developmental stages has been attempted and proteins that increase in amount have been identified (lwatsuki et al., 1984, Bathgate et al., 1985, Wrench et al., 1987, Hadjeb et al., 1988). Using this method, specific chromoplast-associated proteins have been isolated from only pepper chromoplasts (Hadjeb et al., 1988). Antibody has been purified for two proteins that increase during Capsicum chromoplast development (Newman et al., 1989). One of these proteins binds carotenoids and is a major constituent of Capsicum chromoplast membranes (Cervantes-Cervantes et al., 1990). However, this protein probably binds capsanthin since the antibody does not react to any protein present in chloroplasts or tomato fruit chromoplasts (Cervantes-Cervantes et al., 1990). While this type of approach has resulted in the characterization of a few proteins involved in chromoplast function, only abundant proteins can be identified in this manner. The plastid is involved in the synthesis of many compounds necessary for cell function. For example, the synthesis of fatty acids, aromatic amino acids and other substances such as <5-aminolevulinic acid have all been localized to plastids. Many of these compounds are precursors to a wide array of secondary metabolites, so it is possible that an increased synthesis of these plastid-synthesized compounds is required during chromoplast development. A recent review by Hrazdina and Jensen

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22 (1992) however documented that in many instances isozymes for the synthesis of amino acids involved in secondary metabolism may be present in the cytoplasm rather than in the plastids. More information on the compartmentalization of these pathways is needed before conclusions about the localization of enzymes for secondary metabolism can be drawn. Carotenoids Enzymes responsible for the synthesis of carotenoids are obviously the most likely candidates for proteins that may be increasingly produced during chromoplast development. Therefore it is necessary to consider what enzymes and cofactors may be involved in this pathway and how it may be regulated Carotenoid function In the chloroplast, carotenoids are an integral part of the photosynthetic apparatus, where they act as accessory pigments funneling light energy into the reaction centers. If light intensity becomes too great, the photosynthetic carotenoids can also trap excessive light energy. Without these peripheral pigments, the plants may photobleach (Mayfield et al., 1986). The necessity of carotenoids to forestall this deleterious event is apparent when synthesis of carotenoids is blocked by inhibitors or mutation Another function of carotenoids, however, is the pigmentation of many flowers and fruits. Most of the orange, yellow or red pigments found in these tissues can be attributed to carotenoids, which accumulate in the chromoplasts.

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23 Carotenoid structure Carotenoids are long chain hydrocarbons. The conjugated double bonds give them their color. The synthetic pathway produces carotenes, xanthophylls and abscissic acid. The desaturations that occur in the biosynthesis of carotenoids can create either a cis or trans configuration. Cyclization at one or both ends can occur (Goodwin, 1980). A beta, epsilon or gamma ring forms based on the position of the double bond (Goodwin, 1980). The end groups can be acyclic in the case of lycopene. If the pigment is a long chain hydrocarbon then it is a carotene. Xanthophylls on the other hand, are carotenoids that contain oxygen. A number of different modifications are possible to this basic long hydrocarbon chain; 500 different structures have been described. The complement of carotenoids found in green leaves are relatively uniform throughout the higher plant kingdom (Goodwin and Mercer, 1983). This may be expected considering the importance these compounds play in photosynthesis and the conservation of structure required of the proteins and pigments of the thylakoid membranes. However, wide structural variety occurs in carotenoids found in petals and fruit. The type of molecule that accumulates is species dependent. Carotenoid synthesis The basic scheme for the synthesis of higher plant carotenoids is shown in Figure 2-1. The production of phytoene represents the first committed step in the synthesis of carotenoids. Usually, step-wise desaturation of phytoene leads to all trans-lycopene. The normal product of phytoene synthase is 15-cis-phytoene. Where

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24 does the cis-trans isomerization take place? Beyond zeta-carotene, trans intermediates are involved. Four stepwise removals of 2 hydrogen molecules occur from phytoene to lycopene, as oxygen is reduced in this reaction. Proton attack of the Cl double bond of lycopene is involved in the formation of the ring structures. Different enzymes are responsible for the different rings that are formed. Little interconversion of these rings occurs. Xanthophyll formation results from the addition of hydroxyl groups. Location of carotenoid biosynthesis Where are the enzymes of carotenoid synthesis located? All activities after the synthesis of isopentyl pyrophosphate (IPP) have been shown to occur in the plastids (Gray, 1987). Compartmentalization of the enzyme activity has been best analyzed in chloroplasts as opposed to chromoplasts, since they are most amenable to fractionation. Mayfield (1986) examined the chloroplasts of spinach, and concluded that phytoene desaturase is located on the inner envelope membrane. Ultrastructural examination of developing chromoplasts suggests that pigments associate with membrane as they are produced in the inner envelope, which is followed by vesiculation of this membrane pigment complex (Camara and Brangeon, 1981). In addition, examination of the lipid component of these membranes confirms that they are a product of the inner envelope (Sitte et al., 1980). Genetic studies have mapped all the known tomato mutants to the nuclear genome (Tomato Genetic Cooperative Report, 1987), which suggests that the genes involved in the synthesis of carotenoids are nuclear-encoded. Furthermore, inhibition

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Cis-t-Coro1ene 1 Proneurosporene 1 Pro)ycopene lsopenlenyl pyropho 5pliu1E: I Geronylqeronyl pyropt.osphole T rons ( Corolene I I Neurosporene I Fig. 2-1 Pathway for the Biosynthesis of Carotenoids in Higher Plants 25

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26 of plastid translation bas demonstrated that this process is not necessary for carotenoid accumulation (Camara, 1984). Finally, the enhanced synthesis of phytoene caused by the addition of CPTA is blocked by the cytoplasmic translation inhibitor cycloheximide. Therefore, the evidence indicates that the enzymes required for carotenoid synthesis are imported into the plastid. Enzymology of carotenoid biosynthesis in tomato fruit The purification of the carotenoid biosynthetic enzymes is difficult because all the enzymes in this pathway beyond the synthesis of phytoene are associated with plastid membranes. Consequently, studies that characterize the number and type of enzymatic steps responsible for the synthesis of these compounds in higher plants have relied on adding radioactive precursors to solubilized crude enzyme preparations The distribution of radioactivity between the different carotenoids is then measured. These experiments have shown that when, for example, red tomato extracts are fed labeled lycopene, cyclic carotenoids are produced (Quershi et al., 1974). This suggests that the enzymes for the production of these compounds are present, but do not normally accept the lycopene that is produced in vivo. Perhaps an inhibitor is present in vivo that has been diluted out in vitro. Alternatively, a spatial separation between these enzymes may exist in vivo that is disrupted in vitro (Papastephanou et al., 1973). These in vitro studies involved the addition of quantities of substrate and cofactors that may drastically differ from the situation in vivo. These unexpected results demonstrate that the pathway for the synthesis of

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27 carotenoids is complicated and a more thorough understanding of the production of carotenoids requires analysis in less disrupted systems. Examination of mutants in the synthesis of tomato fruit carotenoids has predicted that the photosynthetic pigments are produced by different isozymes. A number of tomato mutants have been studied (for review see Goodwin and Goad, 1970). Many have been mapped, their complement of carotenoids has been measured and the position of the genetic lesion in the biosynthetic pathway has been predicted (Goodwin and Goad, 1970; and Khudari, 1972; Tomato Genetic Cooperative Report, 1987). Many of these mutants affect only fruit color, while the photosynthetic pigments found in the leaves are completely normal (Thelander et al., 1986). Genetic evidence suggests that fruit carotenoids are produced by two separate pathways. One pathway produces the abundant amount of lycopene generally associated with red ripe tomatoes, while the other pathway appears to make a small amount of 8-carotene. Evidence that two pathways exist includes the tomato mutant apricot, which is deficient in the accumulation of lycopene without affecting the level of 8-carotene in the fruit (Goodwin, 1980). Additional evidence includes the fact that the production of lycopene is sensitive to high temperatures in normal red tomatoes (Thelander et al., 1986), while the high f3 mutant has reduced lycopene and enhanced temperature sensitive synthesis of 8-carotene. In other words, the dominant high f3 tomato seems to have used the same temperature sensitive pathway to augment the levels of 8-carotene. Both the existence of the apricot mutant and the residual non

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28 temperature sensitive amount of B-carotene found in high J3 fruit imply that a second pathway exists. The residual, but ever present, level of total carotenoids found in some mutants, the presence of temperature sensitive B-carotene, and enzymes that function in vitro but fail to accumulate their product in vivo suggest that a major ripening enhanced inducible pathway for carotenoid biosynthesis may exist along with a minor, possibly constitutive, path. If the minor pathway is responsible for the production of the cyclic carotenoids, this predicts that enzymes for the production of cyclic carotenoids may not necessarily be enhanced during ripening in normal red tomatoes. Characterization of carotenoid biosynthesis in bacteria: a model for higher plant analysis The cloning and subsequent complementation of genes for carotenoid biosynthesis in bacteria demonstrates how powerful such a systematic analysis can be in the understanding of how the pathway functions. These genes are organized into clusters in the bacteria Erwinia herbicola and Erwinia uredovora. In addition, the carotenoids in these species are similar to the pigments that accumulate in higher plants. A number of investigators have used these genes to transform E. coli, Agrobacterium and Zymomonas (Misawa et al., 1990; Hundle et al., 1991; Misawa et al., 1991; Naagawa and Misawa, 1991). Deletion analysis of the cluster transformed into E. coli allowed an unambiguous ordering of the pathway by analysis of the carotenoids that accumulated in the resulting mutants (Misawa et al., 1990).

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29 The power of the characterization of carotenoid biosynthesis in E. coli is demonstrated by the analysis of individual genes from different species by Linden et al. (1991 ). A plasmid containing the genes responsible for the synthesis of phytoene in Erwinia uredovora can be transformed into E. coli and sequentially cotransformed with a plasmid containing a phytoene desaturase (PDS) gene. The type of pigment that is produced by the specific PDS enzymes can then be monitored. The PDS cDNAs cloned from Synechococcus, Rhodobacter and Erwinia uredovora were tested. Analysis of the carotenoids produced in these transgenic E. coli showed that the different enzymes produced a different complement of products. For example, the Synechococcus enzyme was able to introduce two double bonds in phytoene while the Rhodobacter enzyme introduces three double bonds. This analysis also shed some light on the level of cis-trans isomerizations that accumulate in the various complemented bacteria. The authors concluded that a gradual shift from cis to trans carotenoids occurs with an increase in the number of conjugated double bonds produced by the different enzymes. This suggests that a specific cis-trans isomerase may not be required for this interconversion. Finally, since the Rhodobacter PDS adds 3 double bonds, the specificity of the Erwinia lycopene cyclase and beta carotene hydroxylase on the asymmetrical molecule formed could be assessed. This experiment was performed by the introduction of a plasmid containing the entire Erwinia gene cluster except for a mutant PDS gene. Cotransformation of this plasmid with theRhodobacter PDS created unique carotenoids. This experiment demonstrated that the cyclase or the hydroxylase recognizes half of the carotenoid molecule. This

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30 type of analysis can allow a careful identification of the number and types of enzymes required to produce a specific complement of carotenoids. The sequential cloning and analysis of the different carotenoid biosynthetic genes in transgenic bacteria demonstrate how powerful such an approach could be for the dissection of this pathway in higher plants. If the higher plant carotenoid genes were cloned, a similar series of experiments could be undertaken. Molecular biology of carotenoid biosynthesis in higher plants The gene for phytoene synthesis has been isolated from tomato fruit. Originally, the clone for the phytoene synthase cDNA (pTOMS) was isolated by Slater et al. (1985) simply because it recognized increasing amounts of RNA as ripening progressed. Significant homology to the deduced amino acid sequence of the tomato cDNA, pTOM5, to a clone for phytoene synthase in Rhodobacter was identified (Armstrong et al., 1990a). Bartley et al. (1992) used this information to clone a similar cDNA from tomato fruit and provided evidence, by complimenting a mutant Rhodobacter, that it indeed coded for phytoene synthase. Recently, a normal red variety of tomato has been transformed with a constituitively expressed antisense version of pTOM5 (Bird et al., 1991 ). This resulted in yellow fruit and pale flowers. Chlorophyll a and b levels were normal in the leaves of the transformed plants, which suggested that the leaf carotenoids were unaffected by the constitutively expressed antisense message. These results confirm that pTOM5 codes for a phytoene synthase that is specific for ripening fruit. A more complete analysis of the transgenic plants by Bramley et al. (1992) confirmed that antisense pTOM5 alters

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31 carotenoid accumulation in the flower and fruit but not the leaves of transgenic plants. The gene for geranylgeranyl pyrophosphate (GGPP) synthase bas been cloned from pepper fruit (Kuntz et al., 1992). The enzyme was purified, antibody was produced, and a cDNA was isolated from a ripe pepper fruit expression library. Both the mRNA and the protein increased during fruit ripening. Immunological examination has shown that this stromal enzyme is mainly found concentrated around chromoplast plastoglobuli (Cheniclet et al., 1992). The deduced amino acid sequence from the cyanobacterium Synechococcus PDS allowed the cloning of a cDNA from soybean cotyledon by Scolnik's lab (Bartley et al., 1991). Similar work by Pecker et al. (1992) produced a PDS from ripening tomato fruit. An interesting observation resulting from this work is that, while the cyanobacterium and higher plant PDSs share more than 65% deduced amino acid identity, no homology beyond a short amino terminal region that contains a conserved dinucleotide-binding motif exists with the fungal, Rhodobacter or Erwinia PDS. This suggests that separate PDSs may have evolved for higher plants and green algae in contrast with other photosynthetic organisms. Following a different approach, Camara's lab purified a phytoene desaturase from Capsicum and used antibody to isolate a cDNA clone (Hugueney et al., 1992). The deduced amino acid sequence of that clone has striking homology to the tomato fruit sequence. Only slight increases in the steady-state level of RNA that hybridizes to this clone occur at MG. This is in contrast with the analysis of the protein and the enzyme activity,

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32 which increases significantly with ripening. It appears from the evidence provided by both laboratories (Hugueney et al., 1992; Pecker et al., 1992) that the quantity of this mRNA is low during ripening. Perhaps during chromoplast development large increases in mRNA occur only for the early enzymes involved in carotenoid biosynthesis. Mayer et al. (1992) have purified an oxidoreductase that is specific for chromoplasts and crucial for desaturation of carotenoids. The peripheral nature of the involvement of this enzyme in carotenoid synthesis is supported by the fact that antibody to this protein does not inhibit the synthesis of carotenoids. However oxidized quinones are required for carotenoid desaturation, so it is assumed that this enzyme acts to affect the redox state of the chromoplast membranes where a large amount of oxidized quinones and tocopherols found. Perhaps the o:xidoreductase and the increased amount of antioxidants in pepper chromoplasts point to an environment that contains an escalating amount of reactive oxygen (Romer et al., 1992). The desaturation of phytoene requires the transfer of electrons to oxygen. This might cause an increase in reactive oxygen and lead to the induction of enzymes that generate antioxidants. Two pathways are predicted for the synthesis of carotenoids in ripening tomato fruit. None of the genes for the enzymes responsible for steps in the pathway beyond the synthesis of zeta-carotene have been cloned. It is interesting that only minor increases in the steady-state level of RNA for PDS occur during fruit ripening. Since large increases in steady-state levels of RNA accumulate for both phytoene

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33 synthase in tomato and GGPP synthase in pepper fruit (Slater et al., 1985; Kuntz et al., 1992), perhaps only the very early steps of this pathway are transcriptionally induced during ripening, while transcript for the later steps will be constitutively expressed at a low level. Conceivably the dominant mutants de/+ and b+, which produce increased amounts of Sand B carotenoids respectively, will contain increased levels of RNA for the the enzymes responsible for these later steps in the biosynthetic pathway. Chloroplast Senescence Chloroplast senescence involves the degradation of chlorophyll and the dismantling of photosynthetic membranes Considering that the analysis of chloroplast breakdown during chromoplast formation has relied on ultrastructural analysis and characterization of mostly the decrease in steady-state levels of photosynthetic proteins, perhaps an understanding of how this process occurs during leaf sensecence will allow predictions for the plastid transition in ripening fruit. Indeed, 7 out of 12 fruit ripening enhanced clones share homology to transcripts that increase in senescing leaves (Davies and Grierson, 1989). During ripening and senescence starch, chlorophyll and photosynthetic proteins degrade (Davies and Grierson, 1989). A burst in ethylene synthesis has been correlated with senescence in leaf tissue (Davies and Grierson, 1989) and respiration also increases

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34 Photosynthetic protein synthesis during leaf senescence A decrease in the synthesis of photosynthetic proteins occurs during chloroplast senescence (Roberts et al., 1987). This loss of proteins follows a similar pattern as described for chromoplast development by Piechulla. The synthesis of protein in different ages of Phaseolus leaves was measured, and the amounts of cytochrome f, cytochrome b 6 a and b subunits of the A TPase, 68kD PSI protein, and LHCP decline in leaves. However, the 32 kD protein persists throughout senescence. Ultrastructural analysis of chloroplast senescence Plastid senescence is a gradual ordered process, which occurs within the plastid. It was initially assumed that chloroplasts were engulfed and degraded within vacuoles (reviewed by Gepstein, 1988). These early studies concluded that senescing leaves actually contained fewer plastids. However, reappraisal of this work suggested that the isolation of fewer plastids in senescing leaves was due to the increased difficulty in isolating these fragile organelles (reviewed by Gepstein, 1988). Additional ultrastructural analysis showed that plastids in senescing leaves undergo a transition in which thylakoids become unstacked and plastoglobuli accumulate (Gepstein, 1988). This suggests that the degradation of plastid components during senescence occurs within the plastid itself (Thayer et al., 1987), and is gradual and ordered. The plastid envelope maintains its integrity until very late in senescence, long after the degradation of thylakoid membranes is complete (Thomson and Platt Aloia, 1987). Furthermore, this means that if enhanced degradation of plastid

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35 components occurs during sensecence then it is possible that new proteins would be imported into the plastid for this purpose. The degradation of chlorophyll during chloroplast senescence The degradation of chlorophyll has been suggested to be the trigger for the proteolysis of the protein components of the thylakoid membranes. Unfortunately, the pathway for the catabolism of chlorophyll is not well understood. Work with the non-yellowing mutant of the temperate grassfestuca suggested that an initial step in thylakoid degradation involves dephytlation of the chlorophyll molecule while it remains in the pigment-protein complex (Thomas et al., 1989). A polar green chlorophyll degradation product can be isolated from purified light harvesting complexes (LHCs) of these mutant plastids. This degradation product is also produced in normal plastids exposed to anoxic conditions. The buildup of this intermediate occurs in the mutant plant because a downstream step in the chlorophyll catabolism pathway is missing. Since the product could be isolated from purified LHCs the authors concluded that the first step in the degradation of chlorophyll occurs within the protein-pigment complexes of the tbylakoid membrane. Chlorophyllase bas been isolated from chloroplasts and could perform this function (Matile et al., 1989). However, the protein does not seem to increase during senescence. Alternatively, the enzyme could remain latent in the plastid and be activated when necessary. Perhaps the activation of the cbloropbyllase requires a newly synthesized protein?

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36 The next step in the catabolism of chlorophyll is probably an oxidation. However, it is not clear whether this occurs in the plastid or vacuole (Matile et al., 1989). One theory suggested that a thylakoid associated chlorophyll oxidase is induced by an increase in linolenic acid during senescence (Luthy et al., 1984), which triggers the catabolism of chlorophyll and the resulting cascade of degradative reactions. This idea may require reappraisal in light of the fact that oxidized pyyroles have been found in vacuoles and not isolated from senescing plastids (Matile et al., 1989). Degradation of the thylakoid membranes during chloroplast senescence The analysis of stay-green mutants in soybean and the previously described mutant in festuca demonstrates that the degradation of the thylakoid membrane is mediated by at least two separate pathways and is the result of both nuclear and plastid-encoded proteins. For example, the cytoplasmic mutant cytG of soybean specifically retains the (LHCs) (Guiamet et al., 1991). Clearly the breakdown of the core proteins involves a separate path from the degradation of the LHCs Since chlorophyll a and b are still catabolized in this plant, these data suggest that a plastid-encoded protease is lacking and is involved in LHC breakdown. When two homeologous recessive nuclear-encoded genes ( d 1 d 2 ) of soybean are present in this tetraploid plant, most of the chlorophyll is retained and all of the thylakoid proteins appear to be present, in contrast with nonmutant leaves (Guiamet

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37 et al., 1991). These data imply that the degradation of the thylakoid membrane in soybean is also mediated by nuclear-encoded genes. Evidence from the mutant festuca underscores the complexity of the degradation of thylakoids. The phenotype of this recessive mutant is a selective maintenance of components of the photosynthetic membranes. For example, the level of LHCP is maintained in the mutant plant long after the amount of this protein has begun to diminish in normal plants (Thomas and Hilditch, 1987). Additionally, the degradation of the plastid DI protein, which normally turns over in either the light or the dark, is inhibited in the dark, but not the light (Thomas and Hilditch, 1987). The 33Kd protein, however, degrades normally in this mutant (Thomas and Hilditch, 1987). These mutant plants are also able to photobleach if they are subjected to high light intensities (Thomas and Hilditch, 1987). These data suggest that two separate protease systems are responsible for the degradation of thylakoid membranes. One system is light activated and unaffected by this mutation and another functions in the dark and is inhibited by this genetic lesion. Degradation of Rubisco An examination of the degradation of Rubisco suggests how a stromal protein may be broken down during senescence. The breakdown of Rubisco can be induced by oxidative stress, in a process that appears to mimic natural senescence (Mehta et al., 1992). A specific and highly conserved cysteine is oxidized by the addition of CuS0 4 and leads to dimerization of the protein. This is followed by a transient accumulation of this stromal protein on the membrane, and, finally, degradation. In

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38 vitro studies have also demonstrated that the oxidized form of Rubisco is inactive and more susceptible to proteolytic digestion (Penarrubia and Moreno, 1990). This suggests that the turnover of stromal protein is selective and may involve membrane associated proteases. It is uncertain whether this response to oxidative damage is analogous to senescence, however, it does demonstrate that an ordered system of stromal protein degradation exists in the plastid. It remains to be seen if natural senescence involves a build up of oxidized protein that enters into this degradation pathway, or if it is developmentally manifested in some other manner. Changes in other components of the chloroplast during senescence A decrease in the level of galactolipids has been noted in senescing chloroplasts (Woolhouse, 1984). However, Woolhouse (1984) pointed out that it is uncertain whether this change in lipid content is caused by decreased biosynthesis or increased degradation The isolation of proteases involved in plastid protein degradation has been fairly unsuccessful (Dalling, 1987) The majority of proteases are found in the vacuole, and their presence masks an examination of changes in plastid proteases during senescence. Control of chloroplast senescence Overall, plastid senescence requires continued protein synthesis, since the process can be inhibited by cycloheximide (reviewed by Gepstein, 1988). Although plastid senescence can be triggered by a number of environmental stimuli such as darkness, oxidative compounds, or nitrogen deprivation, it is still unclear whether this

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39 process is mediated by an increase in de novo synthesis of specific proteins. In the case of Rubisco, turnover is slow until a nuclear encoded event (assumed since it is inhibited by cycloheximide or kinetin) triggers the rapid degradation of this abundant photosynthetic protein (Thayer et al. 1987) Perhaps the breakdown of chlorophyll acts as a signal for the degradation of photosynthetic membrane proteins (Nock et al., 1992). This has been suggested by the results from studies of the mutant festuca where a mutation in chlorophyll catabolism inhibits the breakdown of the protein components of the thylakoid membranes. The production of the dephytlated chlorophyll degradation product requires protein synthesis (Thomas et al., 1989), which implies that the synthesis of a cytoplasmic protein is required for induction of chloroplast senescence. Conclusions about chloroplast senescence To date ultrastructural analysis has suggested that the senescence of chloroplasts is a gradual event within the organelle. The degradation of plastid proteins is a complex process involving multiple proteases with different properties Since the breakdown of chloroplasts is inhibited by cycloheximide, this suggests that senescence requires continued protein synthesis (Gepstein, 1988). Additionally, the rapid degradation of Rubisco during senescence implies that this upsurge in protein loss may require a de novo increase in proteins involved in this degradative process rather than simply continued constitutive expression It is difficult to draw generalizations about what may trigger senescence from the analysis of the few mutants available. The mutant phenotype could simply result

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40 from the accumulation of particular plastid components due to loss of a single function and may not represent a protein that is developmentally induced during senescence. It seems safe to assume that the non-yellowingfestuca demonstrates that although the thylakoid membranes can degrade in response to photooxidative stress, the more gradual and perhaps dark associated breakdown requires proteases that are not active until the pigment component has been modified. It would be extremely helpful if proteins or the genes responsible for the senescence of chloroplasts could be isolated. An understanding of their regulation could suggest how senescence is controlled. It is possible that the conversion from chloroplast to chromoplast may represent an ideal system for the isolation and identification of cDNA clones for proteins involved in this degradative process since the chromoplast transition does not take place in a sensecing tissue Heat Shock Proteins and Oxidative Stress The identification of a chromoplast associated low MW heat shock proteins (hsp) during this study provoked an investigation into the literature on why this hsp may have been induced during the chloroplast to chromoplast transition. One possibility is that the low MW hsp is induced during certain developmental stages. Historically, the induction of low MW hsps occurs during times when rapid restructuring of the tissue is underway (Arrigo 1987) It is also feasible that the hsp is induced because the tissue is enduring an oxidative stress. Hsp induction by compounds that create oxidative damage has been described in animal tissues

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41 (Drummond and Steinhardt, 1987; Cajone and Bernelli-Zazzera, 1988; Courgeon et al., 1988). Both the induction of hsps during development and oxidative stress will be considered. Introduction to Heat Shock Proteins Heat shock proteins were first identified by their massive induction upon increases of about 10C above ambient temperature (recently reviewed for plants by Vierling, 1991; and generally by Jaattela and Wissing, 1992). Hsps have been found in a diverse range of organisms from bacteria to mammals. These proteins are broadly categorized by their molecular weight in SOS-PAGE. An individual organism may contain a number of spatially and functionally distinct proteins from each size class. For example, a number of different hsp 70 proteins have been described, some of which are present constitutively (hsc) The function of these proteins has been best characterized for the constitutive members of the hsp60 and 70 classes. The 60 kD protein plays a role in the assembly of the multisubunit Rubisco enzyme complex (Roy, 1989). Involvement in the assembly of multimeric proteins in the mitochondria bas also been shown (Cheng et al., 1989). The 70 kD proteins have been implicated in moving newly synthesized proteins from ribosomes to the cellular compartment in which they function (Weber, 1992). The role of the hsps during heat shock has been inferred from the function of their constitutive forms. It is thought that the hsps bind to proteins denatured by superoptimal temperatures and may facilitate refolding (Weber, 1992). A role for

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42 hsps in providing thermotolerance has been suggested by experiments that create protection from severe heat shock by the induction of these proteins with a brief or moderate increase in temperature (reviewed by Lindquist and Craig, 1988). The induction of hsp messages is rapid and transient (reviewed by Neumann et al., 1989) Examination of the steady-state level of RNA for a number of different hsps in soybean revealed that levels decay more quickly at normal temperatures than during heat stress and the absolute amount of hsps induced is lower in plants that have been previously exposed to a small heat stress (Kimpel et al., 1990). This is reminiscent of the thermotolerance response in that the reaction to heat shock is reduced by heat pretreatment. The transient nature of this response is apparent in the fact that the hsp message had already begun to decay while the temperature was still high, and the message was undetectable within 17 hours after the peak temperature (Kimpel et al., 1990). The induction of heat shock proteins in field grown plants has also been demonstrated (Burke et al., 1985; Kimpel and Key, 1985). Kimpel and Key (1985) found that a transient increase in the message for both low and high MW hsps occurs under fairly normal Georgia summer field conditions with soybean. This induction is dramatically enhanced in non-irrigated plants. Burke et al. (1985) also showed similar responses in material from irrigated versus non irrigated fields. However, they compared the canopy temperatures and found that the non-irrigated field was 10C above the temperature in the irrigated field. This is an important point since the quantity of hsp message produced increases with increasing temperature (Chen et al., 1990; Kimpel et al., 1990).

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43 Low MW Heat Shock Proteins In plants the low MW hsps are generally not present at detectable levels in leaves under nonstress conditions (Vierling, 1991 ). The low MW hsps represent a small multigene family that has been subdivided on the basis of amino acid sequence similarity (Vierling, 1991). Classes of this family have been found in the cytosol, plastid, and endoplasmic reticulum. In fact, one class of low MW hsp is induced during meiosis (Bouchard, 1990). Upon induction, the low MW hsps can accumulate to massive amounts and form large crystalline deposits within the cytoplasm. Low MW Heat Shock Proteins and Development The low MW hsps have been found to increase transiently at different times during development. The best characterized developmental induction of these proteins is found during Drosophila embryonic development. Arrigo (1987) suggested that the period of development in which these proteins are most highly induced represents a time of massive developmental change when the cell may need carefully controlled mechanisms for overall transformation. Four low MW hsps (ranging in size from 22 to 27 kD) follow a complex pattern of expression that appears to be mediated by the molting hormone ecdysterone. Addition of this hormone to embryonic cell lines induces these proteins (reviewed in Bond and Schlesinger, 1987). Deletion analysis of their promoters indicated that regions of the DNA responsive to hormone and heat induction reside in separate portions of the gene. The same hsp is, therefore, induced developmentally and by heat stress. Since heat causes

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44 destabilization and unfolding of proteins and embryo development suggests rapid protein biogenesis, the induction of the same group of proteins during both events implies that they are involved in either stabilizing or degrading incorrectly folded proteins. Evidence for a developmental change in low MW hsps has begun to accumulate in higher plants. The 14 and 40 kD hsps are present in dry embryos of wheat and their mRNA has also been isolated from quiescent wheat embryos not subjected to heat stress (Helm and Abernethy, 1990). These authors propose that these hsps may act to protect the seed early in development from the effects of excess heat. Since the germinating wheat embryo has fewer mechanisms to control ambient temperature in comparison to an older seedling, maybe the production of hsps allows the organism to tolerate this transiently harsh environment (Helms and Abernethy, 1990). The wheat embryo hsps also demonstrate how the genes for hsps may respond differently to heat stress at different stages of development. These proteins are heat inducible at early times during imbibition. However, they are not induced after as little as 12 hours of imbibition (Helm et al., 1989). The wheat seed is initially quite tolerant to heat stress, such that pretreatment with a low but hsp inducing temperature does not improve this thermotolerance (Abernethy et al., 1989). By 9 hours of imbibition, however, a pretreatment can improve the survival of the seed to heat stress (Abernethy et al., 1989). Another interpretation of these data from wheat, however, could be that rather than protecting the young embryo from heat

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45 stress perhaps the hsps are present in preparation for development and consequently, additional induction by heat may be unnecessary. Messages for low MW hsps have been found in dry embryos in other plant species. The mRNA to a 17.6 kD hsp is present in dry seed and accumulates during embryo development of sunflower (Almoguera and Jordano, 1992). This cDNA also hybridizes to mRNA from 3-day-old seedlings subjected to ABA, mannitol or heat stress. The mRNA for an alfalfa hsp 18.1 has been found to be present in microcallus suspension and increases transiently during embryo formation at normal temperatures (Gyorgyey et al., 1991). This cDNA also hybridizes to RNA from callus subjected to CdC1 2 sucrose and heat stress. These data demonstrate that transcripts for low MW hsps have been found in plant embryos and appear to be developmentally produced at nonheat stress temperatures. However, it is unclear from these data whether the transcript induced by stress is encoded by the same developmentally induced gene or represents another member of a multigene family. Plastid-Localized Low MW Heat Shock Proteins A structural analysis of plastid-localized low MW heat shock proteins may suggest a possible function. Chen and Vierling (1991) have compared the deduced amino acid sequences of 5 of these plastid proteins, and have reported three conserved regions. Two of these regions are also present in the low MW hsps found in the cytoplasm. A third carboxyl terminal consensus region, which is abundant in methionine, is unique to the plastid-localized low MW proteins. Computer analysis

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46 of the tertiary structure of the protein in this methionine rich region suggested that an alpha helix with a hydrophobic and hydrophilic face could form (Chen and Vierling, 1991). This same type of three dimensional structure is predicted for three such methionine rich regions of the SRP 54 (signal recognition particle), which is involved in transporting newly synthesized proteins in the endoplasmic reticulum (Lutke et al., 1992). Careful structure/function analysis of this region in SRP 54 and its interaction with signal peptides using crosslinking reagents, antibodies produced against specific fragments of the SRP 54 and deletion analysis of this protein revealed that binding of signal sequences of nascent proteins occurs here (Lutcke et al., 1992). Binding experiments with the 7S RNA showed that this same region is involved in interaction with the RNA. High and Dobberstein (1991) suggested that the SRP 54 interacts with signal peptide on the methionine rich hydrophobic face and with RNA on the hydrophilic face. It is tempting to speculate that the low MW plastid-localized hsp is involved in a similar protein-protein interaction. Perhaps the need for these hsps is increased during times when excessive incorrectly folded proteins are present. The sublocalization of the plastid-localized low MW hsp has been controversial. Work by Kloppstech and colleagues suggested that this hsp becomes associated with thylakoid membranes upon heat stress (Kloppstech et al., 1985). However, this occurs only with plants grown at low light intensities (Glaczinski and Kloppstech, 1988). Binding of hsp to the membrane occurs most abundantly at a light intensity below the level at which net carbon is fixed. Considering the transient

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47 nature of these proteins, and that their transcription usually requires heat stress, this combination of circumstances could occur at dusk in summer or on the floor of a tropical rain forest. Chen et al. (1990) have shown that under heat stress and higher light levels this hsp remains predominantly in the stroma. They reported that the only plastid-localized low MW hsp from pea associates into 200 kD stromal complexes. Clarke and Critchley (1992) showed that one of the two low MW plastid-localized hsps, the 32 kD form, from heat stressed barley leaves associates into 250 to 265 kD complexes within the stroma and contains no RNA, DNA or the a-subunit of the Rubisco binding protein. This protein is reminiscent of an hsp 28 found in soybean plastids, which also contains two low MW hsps of 22 and 28 kD (Clark and Critchley, 1992). The idea that the methionine rich region could interact with denatured proteins during heat stress suggests that these complexes could sequester unfolded proteins. The hsp 60 also associates into an 800-900 kD oligomer of 14 subunits (Roy, 1989). Perhaps the role of the plastid-localized low MW hsp will be easier to resolve if, like the hsp 60 and 70s, a constitutive or developmentally associated form can be identified. Maybe the chromoplast-associated low MW hsp described within this work is induced by the degradation of proteins for the photosynthetic apparatus that occurs between mature green and intermediate stages of fruit ripening. Induction of Heat Shock Proteins By Other Stresses Hsps can also be induced by heavy metals, ethanol, amino acid analogues and glucose starvation (Vierling, 1991). Arsenite and cadmium produced the greatest

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48 induction of hsps (Czarnecka et al., 1984; Edelman et al., 1988). At lower concentrations of these compounds, the response, like the heat stress response, was also transient. By 12 hours of exposure, the messages had begun to diminish (Edelman et al., 1988). Oxidative stress and the induction of hsps The initial correlation between heat and oxidative stress was made in an examination of Drosophila salivary glands. Puffs, representing high levels of transcription, may occur at the same loci along the chromosome when either a heat or oxidative stress is applied (reviewed by Loven, 1988). A combination of heat treatment and application of an oxidizing agent can act synergistically to reduce the deleterious effect of a subsequent heat stress (Issels et al., 1987) This thermotolerance developed from oxidative stress inducers can be blocked by the addition of catalase during the induction period (Issels et al., 1987). Studies in Drosophila cells (Drummond and Steinhardt, 1987; Courgeon et al., 1988) and rat hepatocytes (Cajone and Bernelli-Zazzera, 1988) show a mild induction of some hsps by oxidative stress. In addition, different hsps were induced by different chemicals (Cajone and Bernelli-Zazzera, 1988). Hsps are also induced by oxidative stress in Drosophila cells (Courgeon et al., 1988) where hsp 70 and hsp 23 were induced by hydrogen peroxide. These experiments point to the conclusion that oxidative stress and heat shock, which induce many of the same proteins, do not provoke an identical response

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49 Heat stress and the induction of free radical scavenging enzymes It still is not entirely clear how heat and oxidative stress are related. Heat shock uncouples oxidative phosphorylation and this creates an increased level of reactive oxygen species (Loven, 1988). Additionally, a link has been made between the application of a heat stress and an induction of free radical scavenging enzymes. A hyperthermia induced increase in SOD, CAT and glutathione levels occurs with exercise training of skeletal muscle (reviewed in Barja de Quiroga, 1992). The implication is that these antioxidants are induced by mild heat stress produced during exercise. These additional antioxidants reduce the long term deleterious effects of free radicals produced in this tissue from the uncoupling of oxidative phosphorylation. The heat induced induction of plant SOD was also noted by Tsang et al. (1991) in tobacco The cytosolic form of SOD was induced most dramatically compared with the mitochondrial or plastid form in tobacco. This infers that plants also may respond to heat stress by inducing enzymes involved in oxidative stress. Conclusions about the relationship of heat and oxidative stress Since both heat and oxidative stresses produce a similar response, a number of hypotheses have been developed to explain how they are related Some authors have suggested that the deleterious effects of heat stress are due solely to oxidative damage (Loven, 1988). This idea may be a bit extreme in view of the fact that hsps can be induced under anoxia in Drosophila cells (Drummond and Steinhardt, 1987). Rather than the induction of antioxidants, therefore, this type of assertion requires

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50 that direct oxidative damage be measured, which can be done by examining the amount of lipid peroxidation. However, few studies have been preformed in which hyperthermia is directly correlated with oxidative stress (Barja de Quiroga, 1992). A corollary to this idea was developed in Ame's lab (Lee et al., 1983), in which the synthesis of bisnucleoside polyphosphates, also termed alarmones, acts as the signal for oxidative stress induction of hsps. Apparently an increase in these compounds is observed during both of these stresses. However, it was soon demonstrated that the synthesis of alarmones is inhibited by heat stress rather than induced. A buildup in these compounds occurs because their degradation is actually inhibited by beat. Addition of alarmones to cells was also shown to have no effect on the induction of hsps and hsps could actually be induced without an increase in the amount of these compounds (Guedon et al., 1986). The hypothesis that is currently favored for the induction of hsps by oxidative stress is that both heat and free radicals create aberrant proteins that lead to the production of hsps (Neumann et al., 1989). This probably explains why the two stress responses do not completely overlap. Since hyperthermia creates unstable proteins and uncouples oxidative phosphorylation, the increase in free radicals and the resulting response may occur secondarily. Oxidative stress may mainly induce free radical scavengers, which can forestall the production of large numbers of aberrant proteins.

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51 Conclusions The molecular characterization of fruit ripening has shown that the events that characterize the maturation of tomato fruit involve the de novo synthesis of a subset of ripening associated transcripts. At this point, however, only a few genes involved in this process have been identified and their mode of regulation assessed. Even though the conversion of chloroplasts into chromoplasts involves the degradation of chlorophyll and the breakdown of thylakoid membranes along with a rapid synthesis of carotenoids, only a small number of genes have been identified that encode proteins that may be involved in this transformation. To date, two genes in tomato fruit and one in pepper that code for proteins involved in carotenoid biosynthesis have been isolated. In addition, only a single gene for synthesis of cysteine synthase bas been identified in pepper chromoplasts. Therefore, it is unclear how many proteins may be involved in this process and whether their synthesis increases during development. Historically the characterization of carotenoid biosyntbesis in higher plants involved following the incorportaion of radiolabeled precursors into crude enzyme preparations or the identification of the compliment of carotenoids produced by mutants in the biosynthetic pathway. This type of examination was complicated by the presence of multiple pathways and by the complexity inherent in assays of disrupted tissue. The analysis of carotenoid biosynthesis, as demonstrated in bacteria, in which individual genes can be cloned and the activity can be assessed by complementation in E. coli may now be possible in higher plants. In this way, an

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52 unambiguous assessment of the role an individual enzyme plays on the synthesis of carotenoids can finally be evaluated. The determination of how many enzymes are involved in the biosynthesis of fruit carotenoids can be determined. In addition, the cloning and characterization of the genes involved in this pathway will allow an understanding of how the synthesis of carotenoids is controlled during chromoplast formation. The senescence of chloroplasts in leaves involves the breakdown of photosynthetic structures, in a gradual process within the plastid. It is clear from evidence accumulated from the analysis of stay-green mutants that the degradation of thylakoid membranes involves at least two different systems of protein degradation, portions of which are encoded in both the nucleus and the cytoplasm. However, it is uncertain whether de novo synthesis of new proteins occurs during chloroplast senescence in the leaves. During the transition of chloroplast to chromoplast in pepper chromoplasts, an increase of compounds that scavenge free radicals occurs. Since the induction of hsps has been observed during oxidative stress, it is possible that the induction of an hsp during fruit ripening is caused by such an increasingly harsh environment. Clearly, the ripening-associated hormone ethylene can induce SOD in tomato and tobacco leaves. However, the evidence for an increase in enzymes involved in oxidative stress in tomatoes remains contradictory. More information is needed before conclusions can be drawn about what role oxidative stress plays during tomato fruit ripening.

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53 Clearly the transition within the developing chromoplast involves a rapid degradation of proteins. The period between mature green and yellow fruit appears to be one in which a particularly rapid degradation of plastid proteins occurs Since an increase in hsps has been associated with developmental stages where a great deal of structural change occurs, the chloroplast to chromoplast transition may be an ideal environment for the induction of these proteins

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CHAPTER 3 CHROMOPAST-TARGETED PROTEINS IN TOMATO FRUIT Introduction The transformation of chloroplasts into chromoplasts results in a dramatic alteration of organellar structure The thylakoid membranes are dismantled in a ordered process while carotenoids build up within the plastid (Thelander et al., 1986) The structural changes that occur during this transition have been documented by electron microscopy (Harris and Spurr, 1969). However, it is unclear bow this event is regulated. One of the objectives of this project was to determine whether the development of chromoplasts during tomato fruit ripening is mediated by an increase in nuclear-encoded transcripts of chromoplast-associated proteins. Since plastid transcription and translation is greatly reduced during chromoplast development (Kobayashi, 1991) we have focused on proteins that are nuclear-encoded, translated in the cytoplasm and imported into the plastid. The import of proteins into plastids is highly specific and requires an amino-terminal transit peptide, which is necessary and sufficient for targeting into the organelle (Keegstra, 1989). We have monitored chloroplast import of in vitro translation products from RNA of tomato fruit isolated at different ripening stages. We have also examined translation products from 54

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55 transcripts encoded from cONAs (slater et al, 1985) whose steady-state RNA level increases during ripening. Materials and Methods Plant Material Tomato plants (Lycopersicon esculentum Mill. cv. Rutgers) were grown in a greenhouse in Gainesville, Florida. Fruit were harvested at four ripening stages. MG 1 was characterized by full-size green fruit with firm Jocular tissue; MG3 fruit had completely developed locular gel; the 20-50% ripe fruit were red over less than half of the outer pericarp; and >80% ripe fruit were red over 80% of the outer pericarp. The pericarp was frozen in liquid nitrogen immediately after harvesting. Preparation and Translation of RNA Total RNA was extracted by combining the protocols of McCarty (1986) and Grierson et al.(1985). Briefly, this involved grinding the frozen tissue with a coffee grinder and homogenizing in extraction buffer with a polytron. The extraction buffer consisted of 100 mM Tris-HCI pH 9.0, 200 mM NaCl, 5 mM OTT, 1 % SOS and 20 mM EOTA. The insoluble material was pelleted and reextracted with 1/2 volume of extraction buffer The supernatant was extracted 2 times with phenoVchloroform (1:1) and one time with chloroform The final supernatant was brought to O.lM KCI and precipitated overnight with 2 volumes of ethanol at 4C. The precipitate was

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56 washed several times as described in Grierson et al. (1985) and the soluble material was applied to a cellulose (Sigmacell type 50) column to remove substances that interfere with extraction of poly (At RNA (Grierson et al., 1985). Poly (At RNA was isolated from total RNA by one passage over oligo(dT)-cellulose (BRL). Individual pTOM clones pTOM 5 25 36 41 92, 111, and 114 (Slater et al., 1985) were subcloned into the i n vitro transcription vector pSport (BRL) at the Pstl site Plasmid DNA was isolated by the alkaline lysis method of Lee and Rasheed (1990). The plasmid DNA was linearized and transcribed with either SP6 polymerase or T7 polymerase as described in Cline (1988). The poly (At RNA and the in vitro transcribed RNA were translated (see Figure legends for specific experiments) with either a wheat germ system (Cline 1988) or a rabbit reticulocyte lysate system from Promega (Madison Wi.) in the presence of [ 35 S]-methionine. Chloroplast Isolation and Import Assay Chloroplasts were isolated from pea (Pisum sativum L. cv Laxton's Progress 9) shoots by a combination of differential centrifugation and Percoll gradient centrifugation (Cline et al., 1989). Import assays were conducted as described by Cline (1988) except that translation products were diluted 1:3 with unlabelled methionine in import buffer ; 50 l of the diluted translation products were added to 100 l of chloroplasts (1 mg chlorophyll/ml) Import assays were conducted for 20 min at 25 with white light and lOmM Mg-ATP. After incubation, intact chloroplasts were reisolated by centrifugation through 35 % Percoll with or without a treatment

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57 of the protease thermolysin to remove surface bound proteins (Cline, 1988). Repurified plastids were lysed in 30 l of lOmM Hepes/KOH pH 8.0. Experiments testing the requirement of ATP were conducted in foil wrapped tubes to maintain darkness. Subfractionation and Treatment of Plastids after Import In experiments examining import of translation products from total poly (At RNA, the repurified plastids were lysed in lOmM Hepes/KOH pH 8.0 and subsequently subfractionated into stromal (soluble) and membrane (pellet) fractions by centrifugation for 10 min at 10,000 x g In experiments with translation products from individual cDNA clones, the soluble fraction was isolated from the membranes by lysing the plastids in lOmM Hepes/KOH pH 8.0 on ice for 5 min and centrifuging 30 min at 37,500 x g. The membranes were washed one time with 1 ml of lOmM Hepes/KOH pH 8.0 and resuspended in the same solution Alternatively, the membrane fraction was washed with 0.1 N NaOH (Cline, 1986). Envelopes were separated from thylakoid membranes as described in Cline (1986), however, 1.3 ml of plastids (1 mg chlorophylVml) were used for each import reaction. Sensitivity of the imported proteins to thermolysin was tested as follows: A standard import assay was carried out and divided into two equal fractions. These fractions were either resuspended in import buffer or lysed with lOmM Hepes/KOH pH 8 0. Aliquots of 25 I (1mg chlorophyll/ml) were incubated with O or 20 g of thermolysin for 45 min at 4 C. EDTA was added to 5mM and the samples were

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58 immediately frozen An equal volume of 2X-SDS PAGE buffer (Cline, 1986) was added to freshly thawed samples and the tubes were heated to 98 C for 3 min. Analysis of Import Products Import products from total poly (At RNA were examined by SDS-PAGE and fluorography on 7 5%-20% gradient gels (Cline, 1986). Gel lanes received equivalent amounts of protein as determined by chlorophyll estimation of the recovered plastids. Import products from in vitro transcribed RNA were examined on 12.5% SDS PAGE gels. Results and Discussion Profiles of in vitro translation products of poly (At RNA differed markedly between ripe fruit and mature green tissue (Fig.3-lA) The profiles of the two later ripening stages closely resembled one another, as did the translation products from the two samples of mature green tissue. Speirs et al. (1984) reported a similar result from a more detailed analysis of the polypeptide profiles of total mRNA from ripening tomato fruit. Pea chloroplasts were chosen to analyze import into plastids because of their ease of isolation The isolation of tomato chromoplasts has been reported by others (lwatsuki et al. 1984 ; Bathgate et al., 1985 ; Hunt et al., 1986 ; Wrench et al., 1987), but we were unable to isolate pure and intact fruit chromoplasts that withstand the handling required to demonstrate successful import. However, in vitro import of an

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59 amyloplast specific protein into chloroplasts (Klosgen et al., 1989) and the in vivo import of a chloroplast protein into non-green plastids using transgenic plants ( deBoer et al., 1988) implied that chromoplast proteins should also be imported into pea chloroplasts Changes in the profile of imported proteins during chromoplast development were detected (Fig.3-lB). A number of proteins decreased during this period (Fig. 3-lB) For example, a 30kD and a 14kD soluble fraction protein diminished as ripening progressed Two imported translation products dramatically increased in the later ripening stages. A 26 kD protein fractionated with the soluble plastid proteins, whereas a 17 kD protein was associated with the membrane fraction. These results imply that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast and that the plastid conversion may be an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus. We next asked whether an import assay could be used to identify individual clones coding for chromoplast-targeted proteins. A specific cDNA clone, pTOM5, was selected to evaluate this system. Slater et al. (1985) originally isolated this cDNA because its RNA becomes more abundant as ripening proceeds. Armstrong et al. (1990) recognized that the deduced protein sequence shares approximately 25% identity with the predicted polypeptide of a bacterial prephytoene synthase. Bird et al. (1991) confirmed the involvement of pTOM5 in carotenoid synthesis by creating

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Figure 3-1. Profiles of in vitro translation products and import products are altered during tomato fruit ripening A, In vitro translation of total poly (At RNA with four different ripening stages from tomato fruit pericarp. 1 ug of total poly (At RNA per 50 ul reaction was translated in vitro in a wheat germ system in the presence of [ 35 S]-methionine Translation products were examined by SDS-PAGE and fluorography on 7.5%-20% gradient gels. (MGl) mature green 1, (MG3) mature green 3, (20-50%) 20-50% red over the outer pericarp, (>80%) greater than 80% red over the outer pericarp B, Import into pea chloroplasts with in vitro translation products of total poly (At RNA extracted from tomato fruit pericarp at four different ripening stages. Import assays were conducted and fractionated as described in Materials and Methods The (soluble) fraction was isolated from the (membranes) of the re-purified lysed plastids by centrifugation for 10 min at 10 000 x g Equivalent amounts of plastid protein (as determined by chlorophyll estimation) from the +/protease treated plastids were separated on 7.5 % -20% SDS-PAGE gradient gels and fluorographed. [ 14 C]-labelled 10 to 70 kD molecular weight standards (Sigma) appear as arrows on the left side of the figure. The size of the two import products were derived from a standard curve and are designated by arrows on the right side of the figure.

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\0 I 80% .l!! .Q 20-50% ee Q. Q) MG3 -i" E MG1 ;~ >80 % I I >80% l~ 20 50 % l ' Q. 0 +"' MG3 MG1 20-50% MG3 MG1 ct: Q) > 80 % "' 80 %
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62 an antisense pTOM5 tomato transformant that produces yellow rather than normal red fruit. The transgenic fruit are blocked in the synthesis of phytoene from its immediate precursor, implying that pTOM5 encodes phytoene synthase (Bramley et al., 1992). We selected this clone because enzymological data suggest that synthesis of this nonpigmented carotenoid occurs in plastids (Lutke-Brinkhaus et al., 1982; Mayfield et al., 1986; Dogbo et al. 1987;). The major translation product of the pTOM5 transcript was 46 kD (Fig. 3-2, lane 1); this is in agreement with the size of the hybrid-selected translation product for pTOM5 identified by Slater et al. (1985). Minor lower molecular weight translation products were also produced from this transcript and may represent commencement of translation from internal methionines because these products are not imported into plastids Incubation with pea chloroplasts resulted in the appearance of a 41 kD polypeptide that fractionated with intact plastids and was resistant to protease treatment of the chloroplasts (Fig 3-2, lane 4) Protease resistance was not due to an inherent property of the 41 kD protein because it was degraded by thermolysin if the plastids were lysed prior to treatment ( data not shown). This result is expected of a protein produced in the cytosol, imported into the plastid, protected from protease by the organellar envelope, and cleaved of its transit peptide. Additional criteria must also be demonstrated for the confirmation of import into plastids Import is an energy dependent process (Keegstra, 1989); white light or

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63 + + Protease + + ATP 1 2 3 4 5 Figure 3-2. The 41 kD form of pTOM5 is protected from protease digestion by the plastid envelope and its appearance requires the addition of ATP. Lane 1) In vitro transcribed RNA was translated with a rabbit reticulocyte lysate system in the presence of [3 5 S]-methionine. Import assays and post assay treatments were conducted as described in Materials and Methods. Lanes 2-5) Import of pTOM5 in vitro translation products was conducted with (lanes 2,4) or without (lanes 3,5) lOmM Mg-ATP in darkness. Intact chloroplasts were re-isolated with (lanes 4,5) or without (lanes 2,3) protease post-treatment. Proteins from lysed plastids were separated on 12.5% SDS-PAGE gels and flurographed.

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64 exogenous ATP must be provided for the successful import of proteins. Fig. 3-2, lanes 2 and 3 show that processing of the 46 kD protein into the 41 kD peptide required exogenous ATP. In the absence of ATP, the 46 kD putative precursor bound to the chloroplasts but was destroyed by the protease (Fig. 3-2, lanes 3 and 5). The import into plastids should also be time dependent; this was found to be the case for the production of the 41 kD protein (Fig. 3-3). Thus, the successful import of the pTOM5 translation product verified the utility of import assays for the identification of genes encoding chromoplast-targeted proteins. Pea chloroplasts were fractionated into soluble and membrane fractions to allow analysis of the sublocalization of the 41 kD protein. The protein distributed between these two compartments (Fig 3-3). Parallel import assays with the thylakoid localized light-harvesting chlorophyll a/b protein (LHCP) and the stromal small subunit of Rubisco (SS) were used to assess the quality of our subfractionation. LHCP was exclusively found in the membrane fraction whereas virtually all of the SS was recovered in the soluble fraction ( data not shown). During preparation of this manuscript, Bartley et al. (1992) have reported the isolation of a cDNA from a tomato fruit library by hybridization with a PCR fragment produced with oligonucleotides derived from the sequence of pTOM5. They found that their clone differs from pTOM5 in 19 amino acids that reside 5 amino acids away from the deduced carboxyl tenninal end They also demonstrated that their in vitro translation product imports via an ATP requiring process and that their import product also appears in both stromal and membrane fractions of pea chloroplasts.

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65 Stroma Membrane 0 1 2 4 6 8 10 0 1 2 4 6 8 10 Min ... Figure 3-3. The production of the 41 kD form of pTOM5 was time dependent. This protein associated with both stromal and membrane fractions. pTOM5 import assays were conducted as described in Materials and Methods. One half of a standard import assay was immediately repurified on a 35% Percoll cushion after 0, 1, 2, 4 6, 8, or 10 minutes of incubation Chloroplasts were protease treated, repurified and subfractionated as described in the Materials and Methods Samples were analyzed by SOS-PAGE and fluorography

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66 The membranes of pea chloroplasts were further subfractionated into envelopes and thylakoid membranes (Cline, 1986); upon fractionation of the membranes after import of pTOM5, virtually all of the mature-sized protein was found to be associated with the thylakoid fraction (data not shown). A mild NaOH wash removed the pTOM5 protein from the membrane (data not shown) This suggested that the protein was only peripherally associated with this fraction (Cline, 1986). Previous attempts by others to localize the site of phytoene synthesis within the plastid via enzyme assays have resulted in conflicting conclusions. Lutke Brinkhaus et al. (1982) concluded that the production of phytoene is associated with the plastid envelope However, Mayfield et al. (1986), and independently, Dogbo et al. (1987) deduced that phytoene is synthesized in the stroma. The unexpected association of the protein with thylakoids in import assays and its peripheral association with the membranes demonstrated by the NaOH extraction may have provided additional evidence that this protein is functionally associated with the stroma Alternatively, perhaps the 41 Kd protein has not folded or localized correctly in pea chloroplasts Bartley et al. (1991) reported the import of phytoene desaturase from soybean leaf into pea chloroplasts. Although this protein imported effectively, Bartley et al. found that it does not also fractionate into the plastid compartment expected from biochemical analysis. Immunocytolocalization may clarify these contradicting results. Therefore, it appears that import assays can be useful for the identification of chromoplast-targeted proteins ; however, it is currently unclear what

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67 conclusions can be made from the in vitro organellar sublocalization when using heterologous systems. Six additional ripening enhanced pTOM cDNA clones (pTOM 25, 36, 41, 92, 111, 114) of unknown function (Slater et al., 1985) were tested to determine whether they encode plastid targeted proteins. These particular clones were selected because a comparison of the published sizes of the cDNA inserts with the size of their respective mRNA suggested that they might be full length (Slater et al., 1985; Maunders et al., 1987). In vitro transcription of the cDNAs was carried out in both directions because the orientation of the clones within the vector was unknown. In vitro translation products of these transcript pairs failed to import in four of the six clones. Fig. 3-4, lanes 1 to 7 shows examples of the translation products and lack of import products produced from these clones However, since the transit peptide, which is required for successful import of the protein, is located on the amino terminal end, and since in vitro translation can begin from any internally situated methionine, any cDNAs that produced an in vitro translation product but not an imported product may simply not include the entire coding sequence. Translation products from pTOM41 and pTOMlll were imported successfully into pea chloroplasts (Fig. 3-4, lanes 9 and 11 ). The imported product from both proteins was smaller than the translation product, which is consistent with processing of the plastid transit peptide. These proteins were sensitive to protease treatment (see chapter 4), but resistant after import into plastids (Fig. 3-4). As with pTOM5, import of pTOMlll and pTOM41 translation products were ATP and time dependent (see

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68 1 2 3 4 5 6 7 8 9 10 11 Figure 3-4. Translat j on products from transcripts of additional pTOM clones were tested for their ability to import into isolated pea chloroplasts. In vitro translation products from pTOM41 and pTOMl 11 imported into isolated pea chloroplasts. In vitro transcribed RNA was translated with a rabbit reticulocyte lysate system in the presence of [ 35 S]-methionine. Import assays were conducted as described in Materials and Methods. Samples were analyzed by SOS-PAGE and fluorography In vitro translation with no added RNA (lane 1 ). pTOM92 transcribed in vitro with SP6 polymerase, translated in vitro, (lane 2) and imported (lane 3). pTOM92 transcribed in vitro with TI polymerase, translated in vitro, (lane 4) and imported (lane 5). pTOM36 transcribed in vitro with TI polymerase, translated in vitro, (lane 6) and imported (lane 7). pTOM41 transcribed in vitro with TI polymerase, translated in vitro, (lane 8) and imported (lane 9). pTOMl 11 transcribed in vitro with TI polymerase, translated in vitro, (lane 10) and imported (lane 11).

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69 chapter 4). These data support the idea that the cDNAs do indeed code for plastidtargeted genes We have begun sequence analysis (see chapter 4) and antisense transformation experiments of these cDNAs (see Appendix) to investigate their role in chromoplast function. We wondered whether our initial examination of import from total poly (At translation products (Fig. 3-1B) might underestimate the number of chromoplast targeted proteins. Therefore, we asked whether the pTOM import products could be recognized among the total poly (At RNA import products. The pTOM5 protein comigrated with a band shown to increase in the import products from the membrane fraction of late stage fruit (Fig. 3-5, lanes 5 and 6). It is interesting that the 26kD soluble import product (Fig. 3-1B) migrates similarly to the pTOMlll import product (Fig. 3-5). However, analysis of these two proteins on several gels suggested that the pTOMlll product was slightly larger. Consequently, it is unclear if pTOMlll represents a different isozyme or a completely dissimilar protein. Since the import of the in vitro translation product of pTOM41 associates with the stromal fraction, this was compared with the soluble fraction of total poly A+ import products. No protein appears to increase in the later stages of ripening fruit which comigrates with this band (Fig. 3-5, lanes 2 and 3). We concluded, therefore, that not all of the chromoplast-targeted proteins could be detected in our analysis of total poly (A/ RNA import products

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Figure 3-5. Import products corresponding i n size to those for pTOMlll and pTOM5 were enhanced in late stage tomato fruit. However, no polypeptide was seen that coincided to the pTOM41 import product. pTOM41, pTOM5 and pTOMlll were transcribed in vitro with 17 polymerase. RNA was translated with rabbit reticulocyte lysate in the presence of ( 35 S]-methionine In vitro translation, import assays and subfractionation were conducted as described for analysis of individual translation products in Materials and Methods. Lane 1) Soluble fraction of the pTOMl 11 import product. Lane 2) Soluble fraction of the pTOM41 import product. Lane 3) Soluble fraction of imported in vitro translation products of total poly (At RNA from >80% ripe fruit. Lane 4) Soluble fraction from imported, in vitro translation products of immature green total poly (At RNA. Lane 5) Membrane fraction of the pTOM5 import product. Lane 6) Membrane fraction from imported, in vitro translation products of total poly (At RNA from >80% red ripe fruit. Lane 7) Membrane fraction from imported, in vitro translation products of immature green total poly (At RNA.

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71 1 2 3 4 5 6 7

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72 It is presently unclear how many plastid targeted proteins increase during the chloroplast to chromoplast transition. The data presented in Fig. 3-4 and 5 clearly demonstrate that our original examination of chromoplast targeted proteins shown in Fig. 3-1 underrepresents the number of proteins that increase during this process. In fact, this is not surprising when considering the limited sensitivity of import of total poly (A+) translation products. Therefore, we believe that individual testing of ripening enhanced cDNA clones, with the inherent increase in the quantity of the individual protein product, is required to clearly identify all the chromoplast targeted proteins whose RNA increases during this developmental process. Little detailed analysis of the regulation of ripening enhanced genes has been undertaken. Lincoln and Fisher (1988) and DellaPenna et al. (1989) concluded that increases in transcription rate were mainly responsible for the increases in steady state RNA levels for the ripening enhanced genes they have analyzed. However, they also invoked post-transcriptional mechanisms to explain differences between the rate of transcription and accumulation. Although we cannot exclude post-transcriptional mechanisms for the increase in steady-state levels of the RNAs we have analyzed, the simplest interpretation for our data that is consistent with other ripening enhanced genes is that the processes involved in the development of the chromoplast are directed at a transcriptional level in the nucleus. Utilizing an in vitro assay for the import of proteins into plastids we have identified two proteins from in vitro translation products of total poly (At RNA that increase during the ripening of tomato fruit. Additionally, we have shown that the

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73 in vitro translation products from three individual ripening enhanced cDNA clones import into plastids. Therefore, the transition from chloroplast to chromoplast coincides with an increase in steady-state levels of RNA for proteins destined to this organelle. This suggests that this process is not a simple decline in the synthesis of photosynthetic proteins that causes a breakdown of the thylakoid membranes and a shunting of substrates into the carotenoid pathway, but an active developmental program.

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CHAPTER 4 CHARACTERIZATION OF TWO cDNAS FOR CHROMOPLAST TARGETED PROTEINS Introduction As tomato fruit ripens, cell walls soften, hexoses are deposited into vacuoles, and carotenoids accumulate in developing chromoplasts The transformation of chloroplasts into chromoplasts is distinguished by the breakdown of the photosynthetic apparatus and a massive synthesis and deposition of carotenoids. Little is known about how the catabolism of thylakoid membranes or the synthesis of carotenoids is regulated. Only two tomato genes for early steps in the carotenoid biosynthetic pathway have been cloned (Bird et al., 1991; Bartley et al., 1992; Pecker et al., 1992). Besides proteins involved in the synthesis of carotenoids, only the plastidial superoxide dismutase, characterized by Livine and Gepstein (1988), has been shown to increase during tomato fruit ripening. However, Romer et al. (1992) have also isolated and cloned a cysteine synthase from pepper chromoplasts. These latter findings suggest, therefore, that the chromoplast may be responding to an increase in oxidative stress during development. In order to understand how the transformation from chloroplast to chromoplast is mediated one objective of the present study was to identify and clone 74

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75 genes for proteins involved in chromoplast development. We have identified plastid proteins that may be increasingly synthesized during chromoplast development by examining cDNAs isolated from a ripening enhanced tomato fruit library, translating their transcripts in vitro and testing whether the resulting proteins can be imported into isolated pea chloroplasts. This chapter describes the characterization of two cDNAs that code for transcripts whose steady-state level increases during chromoplast development and whose proteins can be imported into plastids. The characterization of these two cDNAs represents a novel use of import assays to examine chromoplast development. Since this type of analysis is free of functional bias, the identification of unexpected components of chromoplast development is possible. Materials and Methods Plant Material Tomato plants (Lycopersicon esculentum Mill cv. Sunbeam) were grown in the field at the Horticultural Unit of the University of Florida in Gainesville, Florida. Fruit were harvested at 9:00 am on June 19, 1992 from five different ripening stages. Mature green 2 (MG2) was characterized by full size green fruit with fully developed gel in at least one but not all of the locules; mature green 3 (MG3) fruit had completely developed Jocular gel; breaker stage fruit were characterized by the appearance of carotenoids at the blossom end; 20-50% ripe fruit were red over less

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76 than half but more than 20% of the outer pericarp; and >80% ripe fruit were red over 80% of the outer pericarp Pericarp from fruit of each stage was frozen in liquid nitrogen immediately after harvesting at -70C for analysis at a later date. For the examination of ripening under controlled temperature, MG fruit were placed in jars with a flow through system of humidified air and allowed to ripen at 22C in the dark This prevented water stress or a buildup of respiratory CO 2 Fruit from three different ripening stages (MG3, breaker, and 20-50% ripe) were selected and exposed for 6 hours to 36C in this humidified environment. Following the incubation period, pericarp was frozen in liquid nitrogen. Preparation of RNA Total RNA was extracted by combining the methods of Chomczyinski and Sacchi (1987) and Cathala et al. (1983). Briefly, this involved grinding 30 g of frozen tissue in a coffee grinder and homogenizing in 60 ml of extraction buffer with three 30 sec bursts of a Brinckman polytron The extraction buffer contained 4M guanidine isothiocyanate (Bethesda Research Laboratories), 0.5% sarkosyl, 25 mM sodium citrate pH 7.0, and O lM 2-mercaptoethanol Sodium acetate pH 4.0 was added to 0 18M, followed by one volume of phenol and 1/10 volume chloroform The homogenate was shaken for 10 sec after each addition and then cooled on ice for 15 min. The phases were separated by centrifugation at 10 000 x g for 20 min, the aqueous phase filtered through 2 layers of Miracloth (Calbiochem ), and nucleic acids precipitated with an equal volume of isopropanol for one hour at -20C. The

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77 precipitate was collected by centrifugation for 45 min at 10,000 x g. The pellet was dissolved in extraction buffer and RNA was precipitated with 5 volumes of 4M LiCI for 15 hours at 4C Following centrifugation for 90 min at 10,000 x g, the pellet was washed with 3M LiCl and recovered by centrifugation for 1 hour. The RNA was dissolved in 0.1 % SDS, extracted with 1 volume of phenoV chloroform (1:1), then reextracted with 1 volume chloroform, and precipitated with 0.3M sodium acetate and 2.5 volumes of ethanol. Precipitated RNA was dissolved in sterile water and stored at -70C Template Preparation. In Vitro Transcription and In Vitro Translation A cDNA for the hsp 21 from heat stressed pea leaves, pHSP 21, was provided by E Vierling (Vierling et al., 1988) The pTOM 111 and pTOM 41 cDNA were provided by D Grierson (Slater et al., 1985) The pTOM cDNAs were digested with Pstl and ligated into pSport 1 (Bethesda Research Laboratories) at thePstl site, and transformed into E coli strain TBl. Plasmid DNA was isolated by the alkaline lysis method of Lee and Rasheed (1990). The pHSP 21 plasmid was linearized with Bam HI and in v itro transcribed with SP6 polymerase as described in Cline (1988). The pTOM 41 and pTOM 111 plasmid DNAs were linearized with Eco RI and in v itro transcribed with T7 polymerase (Bethesda Research Laboratories). The in vitro transcribed RNA transcripts were translated with a rabbit reticulocyte lysate system from Promega (Madison Wi.) in the presence of either [ 35 S]-Met or [ 3 H]-Leu according to procedures described in the Promega manual.

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78 Northern Hybridization Total RNA, quantified by spectrophotometric absorbance at A261J, was loaded (15 g/lane) onto a formaldehyde denaturing gel (Sambrook et al., 1989). After electrophoresis the gel was washed in sterile water and stained in ethidium bromide (lg/ml) for 30 min and destained in 20X SSC, which contained 0.3 M sodiun citrate and 3 M sodium chloride, for 45 min. The amount of ribosomal RNA was visually assessed to ensure that approximately equivalent amounts of RNA were loaded. The RNA was capillary blotted onto nylon membrane (Amersham Corp.) in 20X SSC overnight. The blot was washed with lOX SSC and exposed to 1.5 J/cm 2 UV light with a transilluminator (Bios). For hybridization probes, the entire cDNA insert was excised with Pstl, isolated by gel electrophoresis, and electro-eluted. 100 ng of DNA was radiolabeled with 75 Ci of a 3 2 P-dCTP by random primer labelling with a kit (Boehringer Mannheim) Prehybridization and hybridization conditions were as described in Church and Gilbert (1984). The prehybridization buffer contained 0.5M sodium phosphate pH 7.2, 7% SDS and 1 % bovine serum albumin (Sigma, fraction V) at 65C. Blots were prehybridized in 40 ml of buffer for at least 1 hour at 65C. The labeled probe was denatured at 100C for 5 min and added to the blot with 5 ml of prehybridization solution. This was incubated overnight at 65C. The blot was washed one time in lX SSC and 0.1% SDS at room temperature and then 3 times in 0.2X SSC and 0.1% SDS for 30 min X-ray film (Kodak, X-OMAT) was exposed to the blot overnight

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79 with two intensifying screens (DuPont) to locate bands Bands were excised from the membrane and quantified by liquid scintillation counting. DNA Sequencing The Pst/ excised pTOM 111 cDNA insert was subcloned into pBluescript (SK) plasmid (Stratagene) at the Pstl site. This was restricted with either Xbal and Sstl or Kpnl and Eco RV for production of Exolll/Mungbean nested deletion plasmids following the protocol described in the manual (Stratagene ). The resulting plasmids were precipitated, resuspended in TE, ligated directly, and transformed into E. coli strain TBl. Plasmids containing nested deletions from either direction were selected and sequenced using the T3 or M13-40 primer of the pBluescript vector. Double stranded plasmid DNA was used as a template and prepared as described by Lee and Rasheed (1990) DNA sequencing procedures were as described in the Sequenase sequencing kit manual (US Biochemical) The pTOM 41 cDNA was blunt end ligated (Sambrook et al., 1989) into pGEM-4Z (Promega) at the Smal site. Both the pGem constructs described above and the pSport constructs described in the section on in vitro transcription were used as template for double stranded DNA sequencing as described above. Initially the TI or M13-40 sites of the pSport or pGem vectors were used as primer sites Oligomers (15mers) specific to pTOM 41 were synthesized by the UF-ICBR DNA Synthesis Core and used in subsequent sequencing reactions Some portions of the sequence were generated by the ICBR DNA Sequencing Core Sequences were

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80 analyzed with the University of Wisconsin Genetics Computer Group computer programs (Devereux et al., 1984). Protein sequence comparisons were performed at the National Center for Biotechnology Information using the GENINFO(R) program of the Experimental Blast Network Service (Altschul et al., 1990) made available by the ICBR Biological Computing Facility. The petunia hsp 21 sequence (Chen and Vierling, 1991) is available as EMBL accession #X54103. The pTOM66 sequence (Fray et al.,1990) is available as EMBL accession #X56138. The sequence for the hypothetical 30.9 Kd protein from E. coli (Post et al., 1992) is available in GenBank accession # M77236 Chloroplast Isolation and Import Assays Chloroplasts were isolated from pea (Pisum sativum L. cv Laxton's Progress 9) shoots by a combination of differential centrifugation and Percoll gradient centrifugation (Cline et al., 1989). Import assays were conducted as described by Cline (1988) except that translation products were diluted 1:3 with unlabelled methionine or leucine in import buffer; and that 50 l of diluted translation products were added to 100 l of chloroplasts (1 mg chlorophylVml) for a 300 l total assay volume. Import assays were conducted for 15 minutes at 25C with white light and lOmM Mg-ATP unless indicated otherwise. Experiments testing the requirement of ATP for import of pTOM 111 protein involved incubation of 200 .l of the translation mixture with or without 4 units of apyrase (Sigma) for 5 min at 25C. The import assays were conducted in foil wrapped tubes to maintain darkness at 25C.

PAGE 87

81 After incubation, intact chloroplasts were reisolated by centrifugation through 35% Percell with or without a treatment of the protease thermolysin (Sigma) to remove surface bound proteins (Cline, 1988). Plastids were treated with 50 g thermolysin for 40 min at 4C unless indicated otherwise. Import products were diluted 1:1 with 2X-SDS PAGE buffer, incubated 8 min at 60C and analyzed by SDS-PAGE and fluorography (Cline, 1986) on 12.5% gels. In order to demonstrate that the imported protein was sensitive to thermolysin, an import assay was performed and the chloroplasts were lysed before the addition of the protease. One fraction of a standard assay was resuspended in 100 l import buffer, the other fraction was lysed with the same amount of lOmM Hepes/KOH pH 8.0. Thirty I aliquots (0.5mg chlorophylVml) were incubated with 0, 12, and 24 g of thermolysin for 45 min at 4C. For the pTOM 41 protein, [ 3 H] leucine was used in the in vitro translation and 1/2 of a standard import reaction (50 I of plastids at 1mg chlorophylVml) was resuspended in 25 I lOmM Hepes/KOH pH 8.0. This sample was divided in half and 5 g of thermolysin was added to an aliquot. After 40 min incubation at 4C, EDTA was added to a final concentration of 5mM and the samples were immediately frozen. An equal volume of 2X-SDS PAGE buffer (Cline, 1986) was added to freshly thawed samples, the tubes were heated at 98C for 3 min and 10 l was loaded onto 12.5% SDS-PAGE gels. For analysis of the location of imported proteins, chloroplasts recovered from assays were lysed in 50 l 10 mM Hepes/KOH pH 8.0, and incubated for 5 min. Stroma was separated from membranes by centrifugation for 30 min at 37,500 x g.

PAGE 88

82 The membranes were washed with 1 ml of import buffer, centrifuged for 30 min at 37,500 x g. and finally resuspended in 40 l of lOmM Hepes/KOH pH 8.0. Results The tomato fruit cDNAs pTOM 111 and 41 were selected by Slater et al. (1985) because transcripts homologous to these clones increased during ripening. Data presented in the previous chapter have shown that in vitro transcripts from these clones can be translated in vitro and the translation products can be imported by pea chloroplasts. Further studies were performed in order to ensure that all the criteria for successful import were met, such as the energy and time dependence of the reaction along with sensitivity of the mature form of the protein to protease Import Studies of pTOM 111 Protein The pTOM 111 in vitro transcript was translated into a 30 kD protein and processed by the isolated plastids into a 24 kD form in an energy (Fig.4-lA) and time (Fig.4-lC) dependent reaction. The production of this processed form was virtually prevented by the addition of apyrase, an A TPase, to the translation mix in the no ATP assay (Fig. 4-lA lanes 1 and 2 versus lanes 3 and 4). Only the 30 kD precursor, which remains outside of the plastid, was removed by the protease treatment (Fig. 4-lA lanes 1 and 4). The mature protein was sensitive to the protease only after the plastids had been lysed prior to addition of this enzyme (Fig. 4-lB).

PAGE 89

A A T P P r o teas e La ne # B + + + + Protease g 1 2 3 4 Lysis kD Lane# 3C.,.. 24 ... C M i n O 1 .5 2 Lane# 1 2 3 4 4 6 5 0 12 24 0 + 2 3 4 ----8 6 10 7 83 12 24 + + 5 6 Figure 4-1 The 30 kD pTOM 111 translation product can be imported into pea chloroplasts and processed into a 24kD form in an energy and time dependent process. In vitro transcribed RNA was translated with a rabbit reticulocyte lysate system in the presence of ( 35 S]-Met. Import assays were conducted as described in the Materials and Methods A) Production of the mature form of the protein requires ATP and is protected from protease digestion. A standard import assay was conducted with (lanes 3 and 4) or without (lanes 1 and 2) lOm~ Mg A TP. Plastids were pelleted and resuspended with (lanes 2 and 4) or without (lanes 1 and 3) protease B) The plastid membrane protects the mature form of pTOM 111 from the added protease. Plastids were (lanes 4 5 and 6) and were not lysed (lanes 1, 2, and 3) after import with O (lanes 1 and 4) 12 g (lanes 2 and 5) or 24 g (lanes 3 and 6) of protease C) The production of the mature form of pTOM 111 is time dependent. Assays were of 100 l of plastids (lmg/ml chlorophyll) for each time point. The reactions were terminated with 3.3 mM HgCl 2 to quickly stop the reaction.

PAGE 90

84 When HgC1 2 was used to terminate the assays, a small amount of protease-resistant precursor was obseived in the early time points (Fig. 4-lB). This may reflect a stage in the reaction prior to cleavage of the transit peptide but after import into the organelle. As will be discussed in the following section, sequence analysis identified pTOM 111 as encoding a plastid-localized low MW heat shock protein (hsp). Consequently, we asked whether our hsp would localize in vitro in the same manner as a plastid localized low MW hsp that was isolated from heat stressed pea leaves. Chloroplasts recovered from import assays were separated into membrane and soluble fractions in Fig. 4-2. The proteins from both pTOM 111 and the pea cDNA were recovered in the soluble fraction. Even though the tomato cDNA was isolated from ripening fruit, the protein behaves in a manner similar to one isolated from heat stressed leaves Import Studies of pTOM 41 Protein A similar analysis of the import of an in vitro translation product from pTOM 41 was performed (Fig 4-3). In this case, a 47 kD protein was produced upon in vitro translation of the pTOM 41 transcript (Fig. 4-3B). When the translation product was incubated with isolated pea chloroplasts, a 41 kD protein was generated (Fig. 3B). This predicts that a 6 kD transit peptide is cleaved upon import into the plastid. Little of the 47 kD precursor was associated with chloroplasts recovered from assays without added ATP, even without protease post-treatment (Fig. 4-3A, lane 1). Since

PAGE 91

pTOM Tr St 1 2 111 Mem 3 pHSP Tr St 4 5 21 Mem 6 85 Figure 4-2. The pTOM 111 and an hsp 21 from heat stressed peas localize predominantly to the stroma. In vitro transcript was translated with a rabbit reticulocyte lysate system in the presence of ( 35 S]-Met and import assays were conducted as described in the Materials and Methods. Translation products (Tr), stroma (St) and membrane (Mem) fractions were analyzed by SOS-PAGE gels and fluorography.

PAGE 92

86 A T St Mem Lysis + + ATP + + + + + + + Protease + + + + + + Lane# 1 2 3 4 5 6 7 8 9 -~-8 kD Min 0 1 2 4 6 8 10 Lane# 1 2 3 4 5 6 7 8 47 41 Figure 4-3 The 47 kD pTOM 41 translation product can be imported into pea chloroplasts and processed into a 41 kD form in an energy and time dependent process A) Production of the mature form of the protein requires ATP and is protected from protease digestion The mature form of PTOM 41 can be digested by the protease if the plastids are lysed. Import assays contained lOmM Mg-ATP (lanes 2, 4, 5 6, 7 8, and 9). Thermolysin was added after import (lanes 3, 4, 5, 6, 7, and 9) Plastids were lysed after import in lOmM Hepes/ KOH pH 8.0 and treated with thermolysin (lane 9). Stromal (St) and membrane (mem) fr actions were separated from total (T) plastids following an import reaction as described in the Materials and Methods B) The production of the mature form of pTOM 41 is time dependent. Assays contained 50 l of plastids (lmg/ml chlorophyll) for each time point. [ 3 H]-leucine replaced [ 35 S]-methionine in the in vitro translation reactions.

PAGE 93

87 the plastids were repurified before the assays were analyzed, this suggests that little of the pTOM 41 translation product attaches to the outside of the plastid Subfractionation of recovered chloroplasts showed the pTOM 41 imported protein to be in the soluble fraction (Fig. 4-3A, lane 7) If plastids were lysed after import and treated with protease, the pTOM 41 mature protein was digested (Fig.4-3A, lane 9 versus lane 8), which demonstrates that it is not inherently resistant to this treatment. A small amount of protease-resistant precursor was also found in the early time points after import of the pTOM 41 protein (Fig. 4-3B). These data demonstrate that the in vitro translation products of both pTOM 41 and 111 are imported into the plastid, are protected from protease by the organellar envelope and are cleaved of their transit peptides. In order to further characterize these chromoplast-associated proteins, the cDNAs were sequenced and the corresponding amino acid sequences deduced (Materials and Methods). The pTOM 111 cDNA Codes for a Low MW HSP The pTOM 111 sequence (Fig 4-4) shows significant homology to those of low MW plastid-localized heat shock proteins isolated from heat stressed leaves Proteins from soybean, pea (Vierling et al., 1988) Arabidopsis, petunia (Chen and Vierling, 1991) and maize (Mieto-Sotelo et al., 1990) have been cloned and sequenced. The pTOM 111 sequence is 81% homologous to a cDNA isolated from heat stressed petunia leaves Low MW hsps from plants have been categorized on

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88 -30 -10 10 30 GGGGCTATTTCTGTGATTAAAAATCTAAATAACTATAATGGCTTACACAAGTCTTACATCTTCTCCATTAGTGTCAAATG ----------+---------+---------+---------+---------+---------+---------+--------" A Y T S L T S S P L V S N V 15 50 70 90 110 TTAGCGTTGGTGGAACCTCTAAGATTAATAACAACAAGGTATCTGCACCTTGCTCTGTTTTCGTTCCGTCGATGAGAAGG +---------+---------+---------+---------+---------+---------+---------+--------15 s V G G T S K I N N N K V S A P C S V F V P S M R R 41 130 150 170 190 CCGACGACTAGGCTGGTAGCTCGGGCTACcGGCGATAACAAAGATACGTCAGTCGACGTACACCACAGCAGTGCTCAAGG +---------+---------+---------+---------+---------+---------+---------+--------42 P T T R L V A R A T G D N K D T S V D V H H S S A Q G 68 210 230 250 270 AGGAAACAACCAAGGCACTGCcGTCGAACGCCGTCCTACGAGGATGGCGCTTGATGTTTCCCCTTTTGGAGTGTTAGATC +---------+---------+---------+---------+---------+---------+---------+--------69 G N N Q G T A V E R R P T R M A L D V S P F G V L D P 95 290 310 330 350 CAATGTCACCAATGAGAACAATGCGGCAGATGATAGACACTATGGACAGATTATTCGAAGATACAATGATTCCAGGAAGA +---------+---------+---------+---------+---------+---------+---------+--------95 S P M R T M R Q M I D T M D R L F E D T M I P G R 121 370 390 410 430 AACAGAGCATCAGGAACAGGGGAAATACGCACTCCATGGGACATTCATGATGATGAAAACGAAATCAAGATGCGTTTCGA +---------+---------+---------+---------+---------+---------+---------+--------122 N R A S G T G E I R T P W D I H D D E N E I K M R F D 148 450 470 490 510 CATGCCGGGACTGTCCAAGGAAGATGTAAAAGTATCAGTAGAAAATGACATGCTTGTCATTAAAGGGGAACACAAAAAGG +---------+---------+---------+---------+---------+---------+---------+--------149 M P G L S K E D V K V S V E N D M L V I K G E H K K E 175 530 550 570 590 AAGAAGACGGAAGAGATAAACACTCATGGGGTAGGAATTATAGCTCTTACGACACTCGTTTAAGTCTCCCAGATAATGTT +---------+---------+---------+---------+---------+---------+---------+--------175 E D G R D K H S W G R N Y S S Y D T R L S L P D N V 201 610 630 650 670 GTGAAGGATAAAATCAAAGCGGAACTGAAGAATGGAGTTCTTTTCATCTCGATTCCAAAGACTGAAGTGGAGAAAAAGGT +---------+-------+---------+---------+---------+---------+---------+--------202 V K D K I K A E L K N G V L F I S I P K T E V E K K V 228 690 710 730 750 GATTGATGTCCAAATTAACTAACATTCAGAATCGCCATGCTTTTGTTGTTCTAGGTTCATTTGTAACCTTGTGTAAAATG +---------+---------+---------+---------+---------+---------+---------+--------229 I D V Q I N 255 770 790 TATGTTCGCATATGAATATAAAACAAGTAGTGCATCTTTGACA +---------+---------+---------+---------+-Figure 4-4. Nucleotide and deduced amino acid sequence of pTOM 111. The sequence was analyzed using the University of Wisconsin Genetics Computer Group programs. The position of the specific nucleotides appears above the sequence while the position of the specific amino acid appears to the side.

PAGE 95

89 the basis of deduced amino acid sequence similarity (Vierling, 1991). There are three classes of cytoplasmically-localized low MW hsps, one class associated with the chloroplast, and another affiliated with the endoplasmic reticulum. A comparison of the deduced amino acid sequence of pTOM 111 to these proteins revealed the greatest similarity with the petunia plastid hsp (77% identity) (Fig. 4-5). This is not surprising since these species are evolutionarily most alike. Comparison to the only other characterized low MW hsp from tomato fruit, pTOM 66 (Fray et al., 1990), a cytoplasmically-localized hsp, revealed only 32% identity to pTOM 111 (Fig. 4-5). The pTOM 66 protein shares more sequence similarity to cytosolically localized hsps from other species ( data not shown). Overall these hsp classes share more sequence identity among members from different species than they do with hsps from other families within the same species. The homology of pTOM 111 to a cDNA for a known plastid-localized protein supports the use of import assays to identify cDNAs for chromoplast-associated proteins. Chen and Vierling (1991) have identified 3 conserved regions in 5 plastid localized low MW hsps they have examined. While the first two regions are found in all low MW hsps, the third region is unique to the plastid-localized proteins. It is in these three regions that pTOM 111 has the highest homology to other low MW hsps. A 90% amino acid identity to the petunia protein is found in these regions in contrast with the 77% overall homology (Fig. 4-5).

PAGE 96

111/hsp21 pTOM111 Petcphsp21 pTOM66 111/66 111/hsp21 pTOM111 Petcphsp21 pTOM66 111/66 111/hsp21 pTOM111 Petcphsp21 pTOM66 111/66 111/hsp21 pTOM111 Petcphsp21 pTOM66 111/66 111/hsp21 pTOM111 Petcphsp21 pTOM66 so ++ ++ + ++++++ + ++ ++ + ++ ++ + + + + MAYTSLT.S. SPLVSNVSVG GTSKINNNK. VSAPCSVFVP SMRRPT macktltcsa splvsngvvs atsrtmkkt ttapfsvcfp yskcsvrkpa Putative Transit Peptide 51 100 + + ++ + + + ++++ ++++ +++++++++ + +++++ + +++++ + +++++++++ TRLVARATGO NICDTSVOVHH S.SAQGGNNQ GTAVERRPTR MALDVSPFGV srlvaqatgd nkdtsvdvhv Sl'VVlqggnnq gsaverrprr maldvspfgl MSLIPRIFGO RRSSSM FDPFSIDV Con101 150 + + + + + + ++++++++++ +++ ++++++ +++++ + ++++++ ++++ +++++ LDPMSPMRTM RQMIDTMDRL FEDTMIPGRN RASGTGEIRT PWDIHDDENE ldpmspmrtm rCJlllldtmdrl fedtmtfpgs rnrgtgeira pwdikddene FDP FRELGFPSTN SGESSAFANT RIDIJKETPEP sensus Region Ill 151 + +++ ++ +++ ++ + + ++ ++ 200 + + ++++++++++ +++ ++++++ + +++ ++ ++ +++ + + +++++++ IKMRFDMPGL SKEDVKVSVE ND.MLVIKGE HK KEEDGR DKHSWGRNYS ikmrfdmpgl skeevkvsve dd.vlvikge hk keesgk d.dswgrnys HVFKVOLPGL KKEEVKVEVE EDRVLQISGE RNVEKEDKND ICWHRMERSSG Consensus Region 11 201 + ++ + + ++ ++++ 247 ++ ++ + + ++++++++++ ++ +++ +++ ++++++ +++ +++ +++ ++ ++ + SYDTRLSLPD NWKDKIKAE LKNGVLFISI PKTEVEK.KV IDVQIN. sydtrlslpd nvdkdkvkae lkngvllisi plctkvek.kv tdveik. KFMRRFRLPE NAKMDQVKAS MENGVLTVTV PKEEVKKPEV KSIEISG Consensus Region I 90 Figure 4-5. The pTOM 111 shares 77% amino acid sequence identity to an hsp 21 from petunia and 32% identity to pTOM 66 from tomato fruit. The deduced amino acid sequences were compared with the University of Wisconsin GCG program, "pileup", as described in the Materials and Methods. The + symbol represents exact matches between proteins, while dots represent spaces built into the protein by the pileup alignment. The putative transit peptide (Vierling, 1991) is delineated by a solid underline. The three consensus regions described by Chen and Vierling (1991) are also marked with a solid line.

PAGE 97

91 The putative transit peptide of pTOM 111 protein is identified in Fig. 4-5. (Vierling, 1991). Although this part of the protein is the least similar to the petunia hsp shown in Fig 4-5 (63% amino acid identity), the region still contains a number of features characteristic of transit peptides (Keegstra et al., 1989). For example, it is rich in serine, valine and alanine and deficient in the acidic amino acids glutamic and aspartic acid In addition, some basic amino acids ( arginine and lysine) are also present. Where the sequence varies from the petunia hsp within this region the substitutions are generally those residues characteristic of transit peptides. The observation that this portion of the protein is least similar to other plastid localized low MW hsps is not surprising, since an examination of transit peptides for the 5 plastid-localized low MW hsps analyzed by Chen and Vierling (1991) has also shown that they are the most diverged portion of the protein Even though the structure and function of the chromoplast differs drastically from the chloroplast, a similar hsp is produced Our data agree with the conclusion of Vierling (1991) that localization to the plastid appears to predict more sequence conservation than does the tissue or organism in which the protein is expressed. Although the increases in low MW hsps pTOM 66 and pTOM 111 may be a general result of ripening, sequence conservation suggests that their role may be more pertinent to the compartment in which they function. Sequence Analysis of pTOM 41 The sequence obtained for pTOM 41 is shown in Fig 4-6. The cDNA is 1423

PAGE 98

92 10 30 50 70 CTTCTTGMAMTGGGGTTGAGGM TTGMTCA TTTTGTTCMATTTTGCTTTTT ATTTCACTTGGAMCACAGT AGCTA -+ -+ --+ --+--+---+ ----+ L 90 110 130 150 TGGCTTCCTGTMTATTCTTTGTAGTGTCOACCCCOATTGATTCTTTTMMAGAGTTGTTTTTTTTCAGTCATGG T C + -+ .... .. -+ -+ -+.. .... + .... .. +.. -+ 2 W L P V I F F V V S N P K L I L L K R V V F F Q S W S U 170 190 210 230 MAT AGGCCACATGGTTCATCCTATTTT MCANJN.TATCCMTTTAGAAG,AAACAGTTTTGTTATTGTGAAGGCTTCAG .... ...... + .. .. + .. .. -+ .. -+-+ .. .... .. +.. + -+ 29 N R P H G S S Y F N K N I Q F R R N S F V I V K A S G 250 270 290 310 GTTCMGAACT AGT MAAMCAAGT AGAGATAACATAT MTCCTGAAGAGAAGTTTMT MATT AGCTGATGAAGTGGAT ... ++ + + + .. .. .. .... + 56 SRTSKKQVEITYNPEEKFNKLADEVD 82 330 350 370 390 AGAGAAGCTGGGCTTTCMGACTCACTCTTTTTTCTCCTTGCAAGAT MATGTTTTCTTGAGAAT AACAAGCAAGAGGGA .. .. + + + + .. + .. .. .. -+ .... .. .. ++ R EA G L SR L T L F SP C K I NVF L R I T SK R D 1~ 4 1 0 430 450 470 TGACGGATATCATGATTTGGCGTCTCTCTTTCATGTMTTAGTCTNJIJNJl,,.TMMTMAGTTCTCGCTGTCACCATCGA .. ..... .. + .. + + .. .. + + .. .. ........ +-++ 109 D G Y H D L A S L F H V I S L G D K I K F S L S P S K 135 490 510 530 550 AGTCAMGGATCGtTT ATCT ACT MTGTTGCTGGAGTTCCACTCGA TGAGAGAMTCTGATT AT MAGGCCCTCMTCTT -+ -+ --+ -. -+---+ ----+ -. --+ 1~ S K D R L ST NV AG VP L DE RN L I I KA L NL 1e2 570 590 e1 o e30 TAT OG,' o GACTOG,'ACAGACOTT ACTTTTGGATTCATCTTGAT AAGMAGTGCCT ACTGGAGCTGGTCTTGGTGG + -+ .. -+.. .. -+ + .. .. .. + 1 ~ YR K KT GT D NY F WI H L DK KVP T GAG LG G 1~ e50 e10 ego 110 TGGGAGCAGT MTGCTGCAACAACTCTGTGGGCAGCMATCMTTCAGTGGTTGTGTTGCCACTGA GGAGCTCCAAG .. -+.. -+.. -+ -+ .. -+ + .. + .. ...... + 189 G S S N A A T T L W A A N Q F S G C V A T E K E L Q E 215 730 750 770 790 AGTGGTCTGGTGAGATTGGTTCTGA TATTCCTTTCTTCTTCTCTCATOG,'GCAGCCT ACT GT ACGGGT AGGGGTGAGGTT -. ... -.. .. -+-+ ----+. --...... -+ 21 e W S G E I G S D I P F F F S H G A A Y C T G R G E V 242 810 830 850 870 GTTCAGGATATCCCGTCACCCAT ACCATTTGACATTCCMTGGTCCTCAT MAGCCTCAACNJGC>.TGCTCCACTGCTGA + -+ + -+ ... +.. + .... .. .. + ~2 V Q D I PS P I P F D I PM V L I KP Q QA CST A E ~8 890 910 930 950 AGTTTACAAGCGTTTTCAGTTGGATCTGTCT AGT AAGGTTGATCCCTTGAGCTTACTGGAGMMTCTCAACT AGTGGAA .. .... .. + .. +.. + .. .. ++ + .. .. .. + ~9 VYKRFQLDLSSKVDPLSLLEKISTSGI 295 970 990 1010 1030 TATCTCAAGATGTGTGTGTCMTGATTTAGAACCTCCTGCCTTTGAAGTTCTTCCATCTCTTMMGGTTc.v,cGA ... .. + ... + ... .. .. + 2911 S Q D V C V N D L E P P A F E V L P S L K R L K Q R 322 1050 1070 1090 1110 GT MTTGCTGCTGGCCGAGGACAATATGATGCAGTCTTCATGTCTGGAAGTGGAAGCACAAT AGTAGGGGTTGGCTCTCC +. -+ -. + -+-++ + -+ ~2 V I A AG R G Q YD AV F MSG SGS T I VG VG S P 348 1130 1150 1170 1190 AGATCCACCACMTTTGTCTATGA TGA TGMGMT ACMGGA TGTCTTCTTGTCAGAAGCAAGTTTCATCACTCGACCAG + -. + . ++ -+-+ + + 349 D P P Q F V Y D D E E Y K D V F L S E A S F I T R P A 375 1210 1230 1250 1270 CCMCGAGTGGTATGTTGAACCTGTTTCAGGT AGCACTATTGGTGATCAACCTGAGTTCTCT ACATCTTTTGACATGTCT + + -+ -+++ -+ 3n NEW Y VE P VS GS T I GD Q P E F ST S F D MS 4~ 1290 1310 1330 1350 TMMGGCTCAGMGAGCTGTMMTTGMGCMTAGGAGAAGTTTTTGTTGTMATGTTATATCCTATMTTTCTGTAG -+-+-+ -+ + -++ + 1370 1390 1410 TATCATCCTTTTACTTTGGATGCACTATTCAAGMAATMMTTAGTCACMTCTGATCGAM -+ -+ Figure 4-6. Nucleotide sequence and deduced amino acid sequence of pTOM 41. Numbers appearing above the sequence refer to the nucleotide sequence. Numbers appearing to the side of the sequence refer to the deduced amino acid sequence.

PAGE 99

1111 I I II I I s I I I 11 II II Ill 3 n Li_ I 11177 I I 11 111 II I 111 [II 111 11 II II 11 I I I I I n Li r7l nn I 500 TTl I 11 I I II I II I II I I I II I I 11 I IIJ fTT I II u TT7 rn I 1 000 111 I I I 11 I 1111 I II II I 3 LI II T7l n n 5' m Figure 4-7. Graphic representation of potential reading frames of pTOM 41. Methionines are represented by lines appearing above the reading frame. Stop codons are represented by lines appearing below the reading frame. The top three frames were produced by reading the sequence from an arbitrary 5' end. The bottom three frames refer to the reading frames produced from the opposite strand. (0 (u

PAGE 100

94 bases long. Only 1 long open reading frame (ORF) is present, but it starts with a leucine, not a methionine (Fig. 4-6 and 4-7). This is hard to understand, given that the pTOM 41 transcript directed production of a 47 kD translation product. A methionine does occur within 12 nucleotides of the beginning of the long reading frame; however, two stop codons are encountered within the next 57 bases. Another small ORF of 28 residues is found two nucleotides 3' of the large ORF. This begins with a methionine in a favorable initiation context (Kozak, 1991). Two possible explanations for the observed anomaly are: 1) a sequencing error, or 2) a + 1 frameshift during translation. The first possibility was addressed by additional sequence analysis of the areas in the vicinity of the 5' and 3' ends of the long ORF. The cDNA sequence in Fig. 4-6 shows that the long ORF starts at 78 bases and ends at 1280. It is clear that an ORF of approximately this length is required to produce the protein observed on SOS-PAGE, all other potential reading frames are littered with stop codons, therefore, if a simple sequence error was generated, it would be found within the first few hundred bases. The first 290 bases were reexamined using a number of different modifications to the sequencing reactions. Figure 4-8 is a compilation of these individual sequencing results. For example, both strands of the sequence (Rl and Fl) were examined by comparing incorporation of dlTP instead of dGTP to the standard Sequenase double stranded sequencing reactions. This region of the sequence was also analyzed by the ICBR DNA sequencing core. They used either Kienow (R4) or TAQ polymerases (F2 and R2) in generating these sequences. Since the template that was used for most of the

PAGE 101

Figure 4-8 Individual sequencing results of the first 290 bases of pTOM 41. Numbers appearing above the line refer to the position of the nucleotide within the sequence. The (consensus) sequence was deduced from the individual runs. Inconsistencies to the consensus sequence are underlined. (Fl) was obtained by comparing sequence reactions run with dGTP or dITP in the forward direction or (Rl) from the opposite strand. (F2) and (R2) were obtained in sequence reactions with primers specific for either strand using TAQ polymerase at the ICBR DNA sequencing core. (F3) and (R3) were obtained with primers specific for either strand using a standard Sequenase sequencing reaction. (F3a) was obtained with a pSport based template while (F3b) was derived from a pGEM-4Z based template. (R4) was obtained by the ICBR DNA sequencing core using Klenow polymerase.

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Consensus F1 R1 F2 R2 F3a F3b R3 R4 CONSENSUS F1 R1 F2 R2 F3a F3b R3 R4 CONSENSUS F1 R1 F2 R2 F3a F3b R3 R4 CONSENSUS F1 R1 F2 R2 F3a F3b R3 R4 CONSENSUS F1 R1 F2 R2 F3a F3b R3 R4 CONSENSUS F1 R1 F2 R2 F3a F3b R3 R4 50 CTTCTTGAAA MTGGGGTTG AGGAATTGAA TCATTTTGTT CAMTTTTGC cttcttgaaa aatggggttg aggaattgaa tcattttgtt caaattttgc cttgaga aatggggttg aggaattgaa tcattttgtt caaattttgc CTTGGTNAAA MTGGGGTTG AGGAATTNM TCATTTTNTN CAAATTTTGC CTTCTTGAM MTGGGGTTG AGGAATTGM TCATTTTGTT CAAATTTTGC CTTCTTGAAA MTGGGGTTG AGGAATTGAA TCATTTTGTT CAMTTTTGC .ttcttgaaa aatggggttg aggaattgaa tcattttgtt caaattttgc gctcttgaaa aatggggttg aggaattgaa .cattttgtt caaattttgc cttcttgaaa aatggggttg aggaattgaa tcattttgtn caaattttgc 51 100 TTTTTATTTC ACTTGGAAAC ACAGTAGCTA TGGCTTCCTG TMTATTCTT tttttatttc acttggaaac acagtagcta tggcttcctg taatattctT tttttatttc acttggaaac acagtagcta tggcttcctg taatattctT TTTTHATTTC ACTHGGAAAC ACAGTAGCTA TGGCTTCCTG TMTATHCTT TTTTTATTTC ACTTGGAAAC ACAGTAGCTA TGGCTTCCTG TMTATTCTT TTTTTATTTC ACTTGGAAAC ACAGTAGCTA TGGCTTCCTG TAATATTCTT tttttatttc acttggaaac acagtagcta tggcttcctg taatattctt tttttatttc acttggaaac acagtagcta tggcttcctg taatattctT tttttatttc acttggaaac acagtagcta tggcttcctg taatattctT 101 150 TGTAGTGTCA AACCCCAAAT TGATTCTTTT AAAAAGAGTT GTTTTTTTTC tgtagtgtca aaccccaaat tgattctttt aaaaagagtt gttttttttc tgtagtgtca aaccccaaat tgattctttt aaaaagagtt gttttttttc TGTAGTGTHA AACCCCAAAT TGATTCTTTT AAAAAGAGTT GTTTTTTTTC TGTAGTGTCA MCCCCAAAT TGATTCTTTT AAAAAGAGTT GTTTTTTTTC TGTAGTGTCA AACCCCAAAT TGATTCTTTT AAAAAGAGTT GTTTTTTTTC tgtagtgtca aaccccaaat tgattctttt aaaaagagtt gttttttttc tgtagtgtca aaccccaaat tgattctttt aaaaagagtt gttttttttc tgtagtgtca aaccccaaat tgattctttt aaaaagagtt gttttttttc 151 200 AGTCATGGTC AAATAGGCCA CATGGTTCAT CCTATTTTAA CAAGAATAT! agtcatggtc aaataggcca catggttcat cctattttaa caagaatatc agtcatggtc aaataggcca catggttcat cctattttaa caagaatatc AGTCATGGTC AAATAGGCCA CATGGTTCAT CCTATTTGAA CAAGANTATC AGTCATGGTC AAATAGHCCA CATGGTTCAT CCTATTTTM CAAGAATATC AGTCATGGTC AAATAGGCCA CATGGTTCAT CCTATTTTM CMGAATATC agtcatggtc aaataggcca catggttcat cctattttaa caagaatatc agtcatggtc aaataggcca catggttcat cctattttaa caagaatatc agtcatggcc aaatagncca catggttcat cctattttaa caagaatatc 201 250 CAATTTAGAA GAAACAGTTT TGTTATTGTG AAGGCTTCAG GTTCMGAAC caatttagaa gaaacagttt tgttattgtg aaggcttcag gttcaagaac caatttagaa gaaacagttt tgttattgtg aaggcttcag gttcaagaac CMTTTAGAA GAAACAGTTT TGTTATHGTG MGHCTTCAG GTTCAGGAAC CAATTTAGAA GAAACAGTTT TGTTATTGTG AAGGCTTCAG GTTCMGAAC CAATTTAGAQ GAAACAGTTT TGTTATTGTG MGGCTTCAG GTTCMGAAC caatttagaa gaaacagttt tgttattgtg aaggcttcag gttcaagaac caatttagaa gaaacagttt tgttattgtg aaggcttcag gttcaagaac caatttagaa gaaacagttt tgttattgtg aaggcttcag gttcaagaac 251 290 TAGTAAAAAA CAAGTAGAGA TAACATATAA TCCTGAAGAG tagtaaaaaa caagtagaga taacatataa tcctgaagag tagtaaaaaa caagtagaga taacatataa tcctgaaga. TAGTAAAAAN CAAGTAGGGA TAACATATM TCCTGA TAGTAAAAAA CAAGTAGAGA TAACATATAA TCCTGAAGAG TAGTAAAAAC CAGGTGGAGA TAACATHTAA TCCGAQGQG tagtaaaaaa caagtagaga taacatataa tc.tgaagag tagtaaaaaa caagtagaga t tagtaaaaaa caagtagaga taacatataa tcctgaagag 96

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97 sequencing was cloned into pGEM, and therefore represents a second generation away from the pSport cDNA that was used for in vitro transcription, the pSport cDNA (F3a) was also sequenced in this region. Small single base differences were obtained with the different sequence reactions. However, no difference in the sequence was observed between the reaction that used the pSport template and the consensus sequence derived from other individual sequencing reactions (Fig. 4-8). These results verify that no simple sequence error was made within the first 290 bases. To establish that an error at the end of the sequence had not produced a prematurely smaller ORF, the 100 bases around the stop codon of the long ORF were also reexamined (Fig. 4-9). The sequences shown in Fig. 4-9 have all been generated by standard Sequenase sequencing. Introduction of any changes to the consensus, (found in individual sequencing reactions) singly or in combination does not alter the length of the ORF. In addition, since adjustments to the first 300 bases are required to place a methionine in the long ORF, any changes to the end of the sequence cannot significantly alter the conclusions. These data suggest that this particular cDNA generates a transcript that is either altered during in vitro transcription or is translated in an unusual fashion. Attempts to directly sequence the in vitro transcript in this region of pTOM 41 were not successful (data not shown). This area of the cDNA is extremely A:T rich (67.6%). For example, an 8 base run of thymines between 142 and 149 nucleotides completely stops further RNA sequence characterization downstream.

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98 1263 1312 Consensus ACATCTTTTG ACATGTCTTA AAAGGCTCAG MGAGC.TGT MMTTGMG Gam43 acatcttttg acatgtctta aaaggctcag aagagc.tgt aaaattgaag Gam41b acatcttti9 acatgtctta aaiggctcag aagagc.tgt aaaattgaag Gam31b acatcttttg acatgtctta aaaggctcag aagagcftgt aaaattgaag Gam31 acatcttttg acatgtctta aaagictcag aagagcctgt aaaattgaag 1313 1362 CONSENSUS CMTAGGAGA AGTTTTTGTT GTAAATGTTA TATCCTATM TTTCTGTAGT Gam43 caataggaga agtttttgtt ctaaatgtta tatcctataa tttctgtagt Gam41b caataggaga i9tttiigtt gtaiatgita tatictatai tttctgtag. Gam31b caataggaga agtttttgtt gtaaatgtta tatcctataa tttctgtagt Gam31 caataggaga agtttttgti gt ....... ............. Figure 4-9. Individual sequence results obtained by sequencing both strands of the pTOM 41 cDNA between nucleotide 1263 and 1362 using standard Sequenase sequencing reactions. The (consensus) sequence was obtained by comparison to the individual sequencing results. Garn 43, 41 and 31 represent specific primers for the pTOM 41 sequence.

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99 This result may explain how an error by the reverse transcriptase could have been made when the pTOM 41 cDNA was originally produced. The pTOM 41 ORF Shares Homolog_y to an E. Coli ORF Comparison of the long ORF of pTOM 41 to other deduced amino acid sequences revealed 51% sequence similarity to an ORF from E.coli (Fig. 4-10). A glycine rich region between peptide 183 and 196 is particularly striking. Post et al. (1992) identified this region in the E. coli ORF as a nucleotide binding domain based on similarity to other proteins. The sequence similarity between these two proteins starts at the 88th amino acid of pTOM 41. However, it runs throughout the entire bacterial sequence. If the first 87 amino acids of the pTOM 41 ORF are removed, a 10 kD transit peptide and a 34.5 kD mature protein would be predicted. The molecular weight on SOS-PAGE suggested that a 6 kD transit peptide would be produced. Bartley et al. (1992) showed a similar result when they analyzed a tomato phytoene synthase they isolated. A 50 amino acid transit peptide would be cleaved upon import into plastids while homology to the bacterial phytoene synthase predicted a transit peptide of 120 amino acids. These authors suggested that perhaps the amino terminal end of the plant protein is simply longer than the bacterial peptide. Steady State RNA Analysis of the Chromoplast-Associated Hsp It was important to determine whether the accumulation of pTOM 111

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Length: 294 Gaps: 7 Percent Similarity: 51.119 Percent Identity: 31.343 . . 2 RTQWPSPAKLNLFLYITGQRADGYHTLQTLFQFLDYGDTISIEL.RDDGB0 I : 11.1:1:ILll:,1.1111.1 .. 11:.: .. 11.1.:.1 ... :. 88 RLTLFSPCKINVF RITSKRDDGYHDLASLFHVISLGDKIKFSLSPSKSK37 . . 51 IRLLTPVEGVE.HEDNLIVRAARLLMKTAADSGRLPTGSGANISIDKRL99 II I 1-11-1 111::1 .1 .. 1 .. : ... : I :11::1 138 DRLSTNVAGVPLDERNLIIKALNLYRKKTGTDNYF ..... WIHLDKKV181 . . 100 MGGGLGGGSSNAATVLVALNHLWQCGLSMDELAEMGLTLGADVPVFVRG~49 1:11111111111,11 1:: .. 1 ... 11.1: .:1.1:1.1 .. 182 TGAGLGGGSSN tTLWAANQFSGCVATEKELQEWSGEIGSDIPFFFSH031 . . 150 AAFAEGVGEILTPVDPPEKW ... YLVAHPGVSIPTPVIFKDPELPRNTPK96 232 il ~ci-!Rb~~~ao=I 0 PSIP i ~FD I p~~L i kPaOAcb~ A:E~~Kfi~OLDLSSK1Z81 . . 197 RSIETLLKCEFSNDCEVIARKRFR .... EVDAVLSWLL ........ EYA33 I I I 11 I 1 J . . . . . . . . 282 DPLSLLEKISTSGISQDVCVNDLEPPAFEVLPSLKRLKQRVIAAGR QYB31 . . 234 PSRLTGTGACVFAEFDTESEARQVLEOAPEWLNGFVAKGANLS~77 332 lvF~sbsbsi-iv~ .. VGS~ciPJa~v~oo!~v~o~~L.S~AiFiHB73 100 Figure 4-10. Comparison of the deduced amino acid sequences of the putative 30.9 kD protein from E. Coli to pTOM 41 using Bestfit from the University of Wisconsin Genetics Computer Group programs. The E. coli sequence appears at the top, while the pTOM 41 sequence is underneath. Identical matches are noted by a solid line. Conservative substitutions are denoted by two dots, while amino acids broadly sharing similar properties are identified by a single dot.

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101 transcripts in ripe fruit was the result of ripening or of heat stress. This was accomplished by measuring the steady state level of transcript in fruit harvested from the field and comparing that to transcript from fruit that had been picked and held at 22C during ripening or alternatively elevated to 36C. The field grown fruit represented tissue that presumably differs only in ripening stage. Fruit was isolated from field grown plants at 9:00am in order to avoid prior heat induction. The pTOM 111 transcript was increasingly abundant in field-ripened fruit as development proceeded (Fig. 4-11, lanes 1-5). For example, breaker fruit contained 8 times as much transcript as MG. Fruit was also harvested and stored in jars with a flow through system of humidified air at 22C. No change in internal temperature was observed during ripening. Fruit maintained at 22C took 2 days to reach 50% ripe from the breaker stage. Examination of the steady-state RNA that hybridizes to pTOM 111 revealed that an increase in transcript occurred as development progressed (Fig. 4-11, lane 68). About 2.5 to 3 times more pTOM 111 specific RNA was present by the 50% ripe stage in fruit held at nonstress temperatures (Fig. 4-11, lanes 6-8). Approximately 4 times more RNA hybridized to pTOM 111 from field grown breaker stage tissue in comparison to fruit held at 22C (Fig. 4-11, lane 3 versus lane 7) More pTOM 111 hybridizing RNA was found in all later stage field-grown tissue in comparison to fruit held at 22C. This difference may be caused by an increase in some stress inducing factor in the field-grown fruit. Clearly, conditions in the field

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22c MG2 MG3 Br 50 >80 MG3 Br 50 1 2 3 4 5 6 7 8 36C MG3 Br 50 pTOM 111 9 10 11 12 13 102 Figure 4-11. The steady state level of RNA for pTOM 111 increases during development in tomato fruit isolated from field grown fruit (lanes 1-5) and fruit harvested at MG and held at 22C (lanes 6-8) The amount of RNA that is induced during heat stress also increases as fruit ripens (lane 9-11). RNA from mature green 2 (MG2) mature green 3 (MG3), breaker (Br), 20-50% red ripe (50), and more than 80% red ripe (>80) pericarp tissue was separated by electrophoresis on denaturing agarose gels (15 g total RNA per lane) and blotted onto nylon membrane. Lanes 12 and 13 contain pTOM 111 in vitro transcript and a 1:5 dilution respectively for comparison between different blots. The membranes were probed with[ 32 P]-labeled pTOM 111. Films were exposed for 4 hours with 2 intensifying screens.

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103 led to a greater accumulation of this transcript during ripening than if fruit was hatvested and ripened in the dark at a uniformly nonheat stress temperature. Fruit at 3 different developmental stages was subjected to 6 hours of 36C. The internal temperature of fruit transferred from 22C to 36C took about 4 hours to rise above 33C. Tomatoes held continuously at this temperature were visibly inhibited in carotenoid accumulation. Exposure to heat stress also resulted in increased accumulation of pTOM 111 transcript in all ripening stages (Fig. 4-11, lanes 6 versus 9, 7 versus 10 or 8 versus 11). The MG fruit accumulated about half of the RNA upon heat stress than did more mature fruit. These data show that this gene codes for a message that is associated with ripening. It also shows that the same gene or a highly homologous gene is induced by heat stress. Characterization of pTOM 41 Transcripts in Vivo RNA from the same tissue was also probed with pTOM 41. The amount of transcript present in tomato fruit that hybridizes to this cDNA increased as ripening progressed (Fig. 4-12, lane 1-5) A comparison of RNA from tissue held at high and low temperature revealed that a second smaller hybridizing transcript ( about 400 bases less) was produced in material that had been exposed to heat stress (Fig. 4-12, lanes 9-11). The RNA isolated from field-grown fruit contained proportionally less of this smaller transcript than fruit held at 36C. Because of the production of this smaller transcript, heat stressed material contained more total message that hybridized to pTOM 41. Since the discovery of a smaller sized transcript and an

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104 22 36 MG2 MG3 Br 50 >80 MG3 Br 50 MG3 Br 50 pT0M41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Figure 4-12. The steady state level of RNA for pTOM 41 increases during ripening in tomato fruit isolated from the field (lanes 1-5) and fruit harvested at MG and held at 22C (lanes 6-8). A second smaller transcript appears, which also increases with developmental stage, when fruit is subjected to heat stress at 36C for 6 hours (lanes 9-11). RNA from mature green 2 (MG2), mature green 3 (MG3) breaker (Br), 20-50% red ripe (50), and more than 80% red ripe (>80) pericarp tissue was separated by electrophoresis on denaturing agarose gels (15 g total RNA per lane) and blotted onto nylon membrane. Lanes 12, 13 and 14 contain pTOM 41 in vitro transcript and 1:5 dilutions respectively for comparison between different blots The membranes were probed with [3 2 P]-labeled pTOM 41. Films were exposed overnight with 2 intensifying screens.

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105 increase in pTOM 41 transcript during heat stress was unexpected, the same samples were hybridized with a sequence of known function. The pTOM 6 cDNA, which codes for the ripening associated enzyme polygalacturonase (PG), was chosen because the level of its message was not expected to change after such a short heat stress. As expected, the transcript for pTOM 6 increased during ripening in all three treatment groups. However, no new sized transcript or dramatic change in the amount of transcript that hybridized to pTOM 6 due to heat stress was observed ( data not shown). Discussion To begin an examination of the transition of chloroplasts into chromoplasts, we were interested in identifying plastid proteins that are increasingly synthesized during this period. Previous studies have either focused on the decrease in synthesis of photosynthetic proteins (Piechulla et al., 1985, 1986 and 1987; Livine and Gepstein, 1988) or have relied on the identification of specific chromoplast associated proteins from total plastid extracts (Newman et al., 1989). Because the latter method can only be applied to abundant proteins, we chose to use import assays to identify cDNAs for any transcripts that are ripening enhanced and encode chromoplast-targeted proteins. The identification of a plastid localized low MW hsp was both gratifying and puzzling. Since the fruit used to clone pTOM 111 was not known to be specifically

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106 heat stressed, the finding of such a protein during ripening was unexpected. Other hsp cDNAs have been isolated from heat stressed material and later the proteins have been localized to the plastid. Consequently, the results from sequence analysis of this cDNA supported the plastid localization identified by the import assay. The lack of a standard ORF in pTOM 41 could be the result of a cDNA synthesis artifact, or could represent a novel method of regulatory control. Since the first sequenced cDNA from this library for pTOM 13 was also altered in a similar manner (Hamilton et al., 1991 ), the first explanation cannot be ignored. The fact that pTOM 41 can produce an in vitro translation product capable of being imported suggests that the cDNA is, at worst, only slightly changed from its functional sequence. Translation of the in vitro transcript can be explained as frameshifting caused by the presence of numerous consecutive runs of individual bases within the first 180 nucleotides of the pTOM 41 sequence (Fig. 4-6). Such single base repeats have been called "slippery runs" (Atkins et al., 1990) because they can facilitate +/frameshifts during translation. The best characterized examples of this phenomenon have been documented in the (RF)2 gene of E. coli and in retroviruses. These sequences are considered to be a mechanism for translational control because they can alter the efficiency of translation for specific genes. In the retrotransposon Tyl of yeast a simple sequence CUU AGG C has been shown to cause a + 1 frameshift up to 50% of the time (Weiss, 1991). The tRNA1eu recognizes the CUU codon; however, it can also utilize the + 1 frameshift codon UUA. An additional requirement for efficient

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107 translational frameshift in this sequence is the presence of the rare arginine codon AGG (present 8.1/1000 codons in yeast). Perhaps when this rare AGG codon is encountered, the ribosomes pause while waiting for the minor tRNA, and this allows the resident leucine tRNA to slip. The ribosomes can actually stack up along the transcript because translation effeciency is so compromised. Overexpression of the arginine tRNA, by transformation of its gene on a high copy plasmid, reduces the level of frameshifting. The conservation of this type of translational mechanism is demonstrated by the ability of rabbit reticulocytes to produce the frameshifted (RF)2 protein of E. coli (reviewed by Weiss, 1991). A hypothesis of how a frameshifted pTOM 41 protein could be produced is presented in Fig. 4-13. The first 28 codons of the 2nd and 3rd reading frames are shown. A + 1 frameshift could occur at codon 18. Not only is the same codon used in either frame, so that the same tRNA could be utilized, but if the codon preference is examined, which has been tabulated for numerous species (Wada et al.,1992), it is apparent that the following 20th and 21st codons of frame 2 are fairly rare codons. A comparison between codons used in tomato versus rabbit and wheat (Fig. 4-13B) revealed that the 20th and 21st codons of the second reading frame are even less common in these organisms from which the in vitro translation systems have been isolated. Since the successful frameshift in Tyl of yeast occurred because AGG represented every 8.1/1000 codons (Wada et al., 1992), it seems plausible that the rabbit and wheat systems may be particularly underrepresented for the tRNAs needed at codon 20 and 21 (found 7.3 and 6.4/1000 in rabbit). This suggests that, like

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108 A COOON# 2 3 4 5 6 7 8 9 10 11 12 13 14 GCT TCC TGT MT ATT CTT TGT AGT GTC AM CCC CM ATT FRAME# 2 A s C N I L C s V I( p Q I TcATO 23.4 8.7 8.5 27.8 31.7 25 8.5 12. 1 10.6 30.3 7.4 21.4 31.7 CTT CCT GTA ATA TTC TTT GTA GTG TCA MC CCC AM TTG FRAME# 3 L p V I F F V V s N p I( L TcATO 25 17.3 9.9 10.6 19.5 23.7 9.9 17.8 16.7 20.8 7.4 30.3 24.6 COOON# 15 16 17 18 19 20 21 22 23 24 25 26 27 28 GAT TCT TTT AM MG AGT TGT TTT TTT TCA GTC ATG GTC AM FRAME# 2 D s F I( I( s C F F s V M V I( TcATO 33.5 17.4 23.7 30.3 34 4 12. 1 8.5 23.7 23.7 16.7 10.6 26.4 10.6 30.3 ATT CTT TTA AM AGA GTT GTT TTT TTT CAG TCA TGG TCA MT FRAME# 3 I L L I( R V V F F Q s w s TcATO 31. 7 25 10.3 30.3 14.6 31.7 31.7 23.7 23.7 11. 1 16.7 13.2 16.7 27.8 B C COOON# 17 18 19 20 21 COOON# 18 19 20 21 TTT AM MG AGT TGT FRAME# 2 AM MG AGT TGT FRAME# 2 F I( I( s C I 11 11 11 1-11-11-11TcATO 23.7 30.3 34.4 12.1 8.5 FRAME# 3 AM AGA GTT GTT RABBIT 16.4 18.4 36.4 7.3 6.4 WHEAT 13.4 5.9 25.1 3.9 4. 1 Figure 4-13. Model explaining a possible + 1 frameshift in the pTOM 41 transcript. A) Analysis of the codon usage for the second and third forward reading frame of pTOM 41. The 3rd frame is offset from the 2nd frame by a single base. Reading frame 2 appears above reading frame 3. Codon usage was tabulated by Wada et al. (1992) as the number of times the codon appears per 1000 codons. B) Comparison of codon usage in tomato, rabbit, and wheat for the codons appearing in position 17 to 21 in the 2nd frame of the pTOM 41 sequence. C) Model for possible + 1 frameshift at codon 18.

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109 the scenario in yeast, a large pTOM 41 protein could occur by a + 1 frameshift caused by inefficient translation in vitro Since the import assay involves the translation and import of a single transcript, it is possible that even an inefficiently translated protein could be detected. Perhaps the only other partially import competent protein produced in this reaction is the 28 amino acid peptide found in the second reading frame. A protein of this size is observed in the translation mixture (data not shown). This small protein could be imported and quickly degraded inside of the plastids, since the cleaved transit peptides are also not found inside the organelle. Alternatively, the smaller protein, which does not contain the entire transit peptide, may be outcompeted by the larger pTOM 41 translation product. Until it can be determined that the pTOM 41 cDNA is a functional representative of the gene in vivo, attempts to clarify a precise frameshift mechanism seem premature. In order to determine how pTOM 41 is translated in vivo a genomic clone of pTOM 41 should be isolated and sequenced in this region. Hamilton et al. (1991) resolved the error generated in the pTOM 13 cDNA by direct RNA sequencing However, given the A:T richness of this region in pTOM 41 and the relatively low abundance of this message, direct sequencing is probably not practical. If additional cloning and sequencing demonstrate that this unusual sequence is normally present in vivo, then it would be important to make an antibody to the long ORF produced in E. coli in order to detemine that this protein is actually present in ripening tomato fruit. At this point we have only shown that the transcript

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110 is increasingly present during fruit maturation Additionally, the antisense mutagenesis of tomato with pTOM 41 (see Appendix) is presently underway This may produce a demonstrable mutant phenotype proving that the transcript is important in vivo Finally, it is possible that the frameshift outlined above is a mechanism used in vivo to control translation of the pTOM 41 transcript. It is possible to sequence radiolabeled translation products, so that a determination can be made about what reading frame has been used to produce the pTOM 41 translation product (Bassham et al., 1991) This technique determines where the radiolabeled amino acid is incorporated in the sequence. For example if [ 3 H]-leucine were used in the sequencing reaction the long unmodified ORF in frame 3 would be labeled at the 1st, 3rd, 15th, 17th and 18th position In the first 28 amino acids of frame 2 the same reaction would label only the 7th amino acid (Fig 4-13). Alternatively lysine could be used to confirm the initial protein sequencing results. In this way, the precise position of the frameshift could be demonstrated. More information is needed about the function of the E coli ORF before conclusions can be made about what the homology to pTOM 41 means to the understanding of chromoplast development. This ORF, which is referred to as ORF2, is transcribed as a tricistronic message with phosphoribosylpyrophosphate (PRPP) synthetase PRPP is an intermediate in the synthesis of pyridine nucleotide coenzymes, histidine, tryptophan, and the pyrimidine and purine nucleotides. The tricistronic message only represents about 5% of the total amount of PRPP

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111 synthetase message produced (Post et al., 1992). The rest is synthesized from a promoter within the ORF2 on a monocistronic transcript that is inducible by pyrimidine starvation In addition, the ORFl has been shown to complement mutants in 8-aminolevulinic acid (ALA) synthesis (Ikemi et al., 1992). The ALA mutants are deficient in cytochromes, and consequently, result in respiratory deficiencies. These authors have suggested that the ORFl may synthesize one of the subunits for a tRNA glutamyl NADPH dehydrogenase, which is a step in the 5 carbon pathway of ALA synthesis. It is unclear why the synthesis of PRPP and ALA should be coordinated. The only obvious involvement of PRPP in the ALA pathway may be in the production of the NADPH. In addition, these data do not suggest a function for the ORF2 or pTOM 41. Until a mutant phenotype for the ORF2 or pTOM 41 is produced, or a connection is made between the synthesis of ALA and PRPP, the function of these two ORFs will remain enigmatic. The second transcript that hybridizes to pTOM 41 may be produced by a second gene or may result from processing of pTOM 41 itself. Normally, heat stress can inhibit the transcription, processing, stability of messages and the translation of constitutively expressed proteins (Gurley and Key, 1991). However, the opposite seems to occur with pTOM 41. In this case, not only is more total pTOM 41 hybridizing message produced during heat stress, but a smaller transcript is formed. Southern analysis of tomato DNA with pTOM 41 suggests that only 1 or 2 genes are present (data not shown). Since more steady-state RNA is present in the heat stressed material, it is unlikely that a stress related transcript degradation is the

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112 predominant factor for the increase in total amount of RNA that hybridizes to pTOM 41 in heat stressed material. The simplest interpretation of these data is that heat stress actually induces the production of the second transcript. Perhaps a second pTOM 41 protein is produced that is not targeted to plastids If the pTOM 41 transcript did not contain a transit peptide, comparison to the bacterial ORF suggests that the transcript would be approximately 340 bases shorter. This second transcript could be produced by a second gene or result from altered processing of pTOM 41. Recently, Chiu et al.(1992) found that the same gene in yeast produced two transcripts for a histidyl-tRNA synthetase. One of these proteins was targeted to the mitochondria while a shorter transcript coded for a cytoplasmic form of the enzyme. Site directed mutagenesis of the gene and transformation back into yeast confirmed what sequence analysis had suggested Antisense RNA transformation of pTOM 41 is presently underway (see Appendix). Hopefully the production of an antisense mutant in tomato will help to clarify the role of pTOM41 during chromoplast development. The results reported in Fig. 4-11 confirm that the pTOM 111 transcript is ripening associated at normal temperatures and also that more transcript is produced in response to heat stress as ripening proceeds. These results are strikingly reminiscent of data shown by Olson et al. (1991) for the accumulation of ACC synthase 1 in tomato fruit during ripening, with and without wounding The ACC synthase 1 transcript codes for a member of a small multigene family that is involved in the synthesis of ethylene The induction of this hormone is known to occur during

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113 both ripening and wounding. Fruit wounded at different stages of maturity showed an increasing amount of wound induced ACC synthase 1 transcript as ripening progressed. In other words, the combination of ripening and wounding acts synergistically to affect the steady state level of this transcript. This analogy proposes that the increase in hsp transcript is due to chromoplast development and is enhanced by heat stress. The role for the low MW heat shock proteins remains obscure (Vierling, 1991 ). It is generally accepted that these proteins are induced by the presence of unfolded or aberrant proteins (Neumann et al., 1989). Unlike the hsp 60s and 70s, the low MW hsps in plants are usually only observed during times of stress. However, a few exceptions to this have been noted. For example, low MW hsp transcript is present in dry and germinating embryos of wheat (Helm and Abernethy, 1990) and sunflower (Almoguera and Jordano, 1992). It has also been observed during somatic embryo formation in alfalfa (Gyorgyey et al., 1991). However, there have been no previous reports of a developmentally induced plastid-localized low MW hsp. Perhaps this type of evidence will suggest a function for these enigmatic proteins. The low MW hsps have also been associated with cadmium, arsenite, and osmotic stress in plants in some instances (Czarnecka et al., 1984; Edelman et al.,1988; Gyorgyey et al., 1991; Almoguera and Jordano, 1992). Low MW hsps have also been associated with oxidative stress in Drosophila and mammalian cells (Drummond and Steinhardt, 1987; Cajone and Bernelli-Zazzera, 1988; Courgeon et

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114 al., 1988) Both heat and oxidative stress are believed to create aberrant and unfolded proteins (Neumann et al., 1989). Although the response to these stresses is usually lower than the response to heat stress, the fact that the low MW hsps are induced by other stresses does suggest that changes in the environment of ripening fruit should be considered. The question that immediately arises from these observations is, what role does a plastid localized low MW hsp perform during ripening? Is the production of this transcript due to some massive reprogramming of protein structure during the formation of chromoplasts or does some other aspect of the environment within the ripening fruit induce expression of this hsp? A recent paper by Romer et al. (1992) found that several free radical scavenging enzymes and compounds increase during the development of chromoplasts in pepper fruit. However, the evidence for oxidative stress during chromoplast formation in tomato fruit is contradictory. Perl-Treves and Galun (1991) found no increase in either of the plastidial free radical scavenging superoxide dismutase (SOD) isozymes during tomato fruit development. Additionally, they found that the single plastidial transcript they followed is low at MG and actually undetectable later in development. Livine and Gepstein (1988) however, found that cross-reacting material to a plastidial SOD peaks at the orange stage. Consequently, it is unclear whether tomato chromoplasts are oxidatively stressed during ripening. One result of oxidative stress can be a loss in membrane integrity. Examination of the plastid membrane during chromoplast development in pepper

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115 and tomato fruit has shown that no loss in integrity occurs (Legge et al., 1986; Whitaker, 1991). In other words, although there is evidence for an increase in oxidative stress in pepper chromoplasts, there is currently no evidence for damage of plastid membranes due to oxidative stress during chromoplast development. Consequently, even if there is some increase in free radical scavengers during ripening it is uncertain if this changing environment leads to an increase in damaged plastid proteins. One possible explanation for the induction of pTOM 111 during ripening is that the stress response is a general one caused by changes in the cytoplasm during development. Since pTOM 66 also increases during ripening in the absence of heat stress (Picton and Grierson, 1989), perhaps the induction is dependent on the tissue rather than the compartment in which the protein resides. However, this idea requires that the induction of some hsps during ripening may be superfluous. The ultimate location within the plastid may help predict the function of the plastid-localized low MW hsps, however, these data have been somewhat conflicting. Kloppstech et al. (1985) have proposed that the plastid hsp protects the photosynthetic membranes from heat stress because they found it associated with the thylakoid membranes. However, later studies revealed that only plastids exposed to heat stress under low light intensities have an hsp attached to this membrane (Glaczinski and Kloppstech, 1988). Work by Chen et al. (1990) and Clark and Crithley (1992), show that this protein accumulates predominantly in the stroma under normal light conditions even in heat stressed chloroplasts. Since Chen et al.

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116 (1990) have found this hsp in heat stressed pea root amyloplasts and our data showed an increasing abundance of the transcript as the thylakoid membranes degrade, a broader role for this protein than simply protecting photosynthetic membranes must be postulated An examination of the conserved domains of plastid-localized low MW hsps has shown that the third domain, which is unique to these hsps, is similar to the methionine rich region in SRP54 a protein found in the endoplasmic reticulum and known to be responsible for binding signal sequences and transporting newly translated proteins (Chen and Vierling, 1991). It is tempting to speculate that the hsp accumulates in the stroma and is involved in sequestering unfolded proteins This role could be important during heat or oxidative stress and during developmental changes as well, since all three of these conditions may cause an accumulation of unfolded proteins. The breakdown of the photosynthetic membranes during chomoplast development may be an important factor in the induction of pTOM 111. Piechulla et al. (1987) and Livine and Gepstein (1988) have examined the loss of photosynthetic proteins during chromoplast development in tomato fruit. They find that a dramatic loss of LHCP between mature green and the yellow stage Considering the abundance of this protein in the plastid, this could represent a significant increase in unfolded proteins. Our data show an increase in transcript for the hsp only after MG H the induction of this protein is correlated with chloroplast breakdown, then increase in this hsp transcript may be expected at this time.

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117 Characterization of the accumulation of this hsp in the tomato mutant green flesh might be useful since the thylakoid membranes persist longer in mutant fruit than in normal fruit. If the plastid hsp is also deterred in this mutant then perhaps an involvement of this hsp in plastid protein breakdown would be suggested In addition, the antisense mutagenesis of tomato with an antisense pTOM 111 construct is currently underway (see Appendix) Perhaps if this protein is responsible for the ordered dismantling of thylakoid membranes during chromoplast formation then the antisense mutant will be inhibited in breakdown of the photosynthetic membranes, or may result in diminished carotenoid synthesis because the plastid cannot adequately respond to this developmental stress. An increase in the amount of transcript for two low MW hsps occurs during tomato fruit ripening at nonheat stress temperatures. This suggests that either both cytoplasm and plastid are undergoing massive structural reprogramming or a general increase in stress occurs in this tissue during ripening. A correlation between the breakdown of plastid proteins and the increase in the steady-state level of this transcript may exist. However, it probably is not such a simple correlation, since the transcript remains high after plastid conversion and a cytoplasmic low MW hsp is also induced. Examination of the breakdown of the photosynthetic membranes in the antisense pTOM 111 compared to normal fruit may help to clarify the involvement of this protein in the chloroplast to chromoplast transition.

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CHAPTER 5 CONCLUSIONS Chromoplast development is correlated with an increase in the steady-state level of a subset of transcripts encoding proteins directed to this organelle. We have identified 2 proteins from experiments examining the import of in vitro translation products from total poly A+ RNA, and 3 proteins from the analysis of proteins encoded by cDNAs that are increasingly synthesized during this transition Since pTOMS ( chapter 3) and PDS (Pecker et al., 1992) are increasingly synthesized as tomato fruit ripens, these data suggest that the deposition of carotenoids is not simply due to a repartitioning of the breakdown products of photosynthesis. In addition, these findings imply that the import apparatus remains functional during this period. The characterization of a chromoplast-associated low MW hsp brings up more questions then it answers Does this imply that the chromoplast is under stress during its development? Is ripening tomato fruit generally undergoing some type of stress? Does the transformation of the chloroplast into a chromoplast result in the induction of this hsp? The correlation between the degradation of thylakoid membrane proteins and the induction of the plastid-localized low MW hsp implies that induction of this protein may be caused by an increase in the amount of unfolded 118

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119 proteins in the chromoplast. However, since induction of the hsp transcript appears to persist after the breakdown of the photosynthetic apparatus, perhaps an increase in oxidative stress during ripening also contributes to the induction of this protein. The identification of the low MW hsp during chromoplast development was unexpected It demonstrates the power such an unbiased assay can have on the characterization of a developmental process. Proteins not generally associated with chromoplast development or proteins of low abundance can be identified In this way, a broader understanding of this transition is possible.

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APPENDIX CONSTRUCTION AND EXPRESSION OF ANTISENSE pTOM 41 AND pTOM 111 PLASMIDS IN TOBACCO Introduction A complete examination of the proteins involved in chromoplast development includes a functional analysis of the protein's role in this developmental process. An advantage of the experimental approach described within this dissertation is that once the chromoplast-associated protein has been identified, it has also been cloned. This permits production of plasmids that encode sense and ailtisense transcripts that can be transformed into tomato plants and hopefully produce a mutant phenotype. Not only can the transition from chloroplast to chromoplast be described, but the loss of function for particular proteins involved in this process can be monitored. Antisense transformation has been utilized successfully for a number of ripening-associated genes. The expression of the cell wall softening enzymes polygalacturonase (PG) and pectin methylesterase, the ethylene biosynthetic enzymes ACC oxydase and ACC synthetase, and the carotenoid biosynthetic enzyme phytoene synthase have all been successfully inhibited by introduction of plasmids encoding antisense transcripts (Sheehy et al., 1988; Smith et al., 1988; 1990; Hamilton et al., 1990; Bird et al., 1991; Oeller et al., 1991; Tieman et al.,1992). 120

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121 Antisense mutagenesis has produced dramatic consequences with ACC synthetase, where mutant fruit fail to soften, undergo the respiratory climacteric or produce carotenoids (Oeller et al., 1991). The application of ethylene is required before this fruit will proceed through the processes associated with ripening. PG, for example, is transcribed but it is not expressed. In this manner, the requirement of ethylene for the induction of ripening-associated proteins can be examined. The results of antisense mutagenesis for PG, however, have been less striking (Sheehy et al., 1988; Smith et al., 1988; 1990). It was initially assumed that, since the transcript was enhanced during fruit ripening, and because this protein was produced in such vast amounts that it could be identified on a Commassie stained gel, inhibition of PG expression during ripening would lead to obvious reductions in cell wall softening. Although the production of the protein can be inhibited by antisense mutagenesis, cell wall softening is almost unaffected. This type of functional analysis was required to clarify the significance of PG during fruit ripening. In the following section, the construction of a vector for production of antisense transcripts will be described. The ability of this vector to express transcripts was tested in tobacco. The transformation of sense and antisense pTOM 41 and pTOM 111 plasmids into tomato plants is currently underway. Given our current knowledge about these genes, the potential phenotype for successful antisense mutagenesis will be discussed.

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122 Materials and Methods Construction of Vectors for Production of Senses and Antisense Transcripts and Transformation into Agrobacterium The vector system constructed for production of sense and antisense transcripts depends on three separate plasmids. First, both orientations of the pTOM cDNAs were subcloned into pGEM-4Z (Promega) in order to benefit from the galactosidase selection built into this plasmid (Fig. lA and B) The cDNAs can then be directionally excised from pGEM-4Z and ligated into a cauliflower mosaic virus (CaMV) 35S promoter/ nopaline synthase (nos) terminator cloning cassette cloned into Peth 3c (Fig 2A) (McCarty et al., 1991). Finally, the cloning cassettes containing the sense and antisense pTOM cDNAs are ligated into an Agrobacterium vector pGA472 (An et al., 1985), which has also been modified to contain the gene for glucuronidase (GUS). Details of the vector construction and directional cloning of the pTOM cDNAs are given below. Initially, the pTOM 111 and pTOM 41 cDNAs were blunt-end ligated into pGEM-4Z (Fig. 1). All molecular methods described in this section were taken from Sambrook et al.(1989) unless indicated otherwise. The pTOM 111 cDNA was isolated from pBluescript (KS) (Stratagene) ( see chapter 4) by digestion with Eco RV and Smal and ligated directly into pGEM-4Z at the Smal site. The pTOM 41 cDNA was isolated from pSport 1 (Bethesda Research Laboratories) by digestion with Pstl and made blunt ended with T4 DNA polymerase (Bethesda Research Laboratories) before it was ligated into the pGEM-4Z Smal site. Plasmids were transformed into the E. coli strain TBl and DNA was isolated by an alkaline lysis miniprep method

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123 of Lee and Rasheed (1990). Sense and antisense orientations of the cDNAs in pGEM-4Z were confirmed by the presence of asymmetrical Eco RV sites in pTOM 41 (Fig. lA) and a Ndel site in pTOM 111 (Fig lB) The pGEM-4Z vector contains Kpnl and BamHI sites on either side of the Smal restriction site used for the initial cloning (Fig. 1A and B) The plasmids containing the sense and antisense orientations of the pTOM cDNAs were ligated into the vector outlined in Fig 2A This vector was constructed in the following manner The plasmid pBI221 (Clonetech) contains the coding region of GUS between the CaMV 35S promoter and the nos terminator. A polylinker shown in Fig. 2A was created at the ICBR DNA Synthesis Core and ligated into the Xbal and Sstl sites of pBI221. This excised the GUS coding region and placed the polylinker between the CaMV 35S promoter and the nos terminator The entire cloning cassette, which contained the CaMV 35S promoter, polylinker and nos terminator was then excised by restriction with Hindlll and Eco RI and ligated into Peth 3c. Sense and antisense orientations of the pTOM cDNAs were excised from the pGEM plasmids by restriction with Kpnl and BamHI and ligated into the polylinker of the cloning cassette at the Kpnl and BamHI sites. The pTOM cDNAs were now cloned in both orientations between the CaMV 35S promoter and the nos terminator In this manner, both sense and antisense transcripts would be produced. The vector for transformation into Agrobacterium and ultimately into plant material was prepared as follows. The vector pGA472 was restricted with Seal The entire gene for the production of GUS with CaMV promoter and nos terminator,

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124 was excised from pBI221 by restriction with Hindlll and EcoRJ. The GUS gene was made blunt ended with T4 DNA polymerase and ligated into the unique Seal site of pGA472 (Fig. 2B). The cloning cassettes, which contained the sense and antisense pTOM cDNAs were excised from Peth 3c with Hindlll and Bglll and ligated into pGA472 GUS, which had been restricted with the same enzymes. TheAgrobacterium strain AGLl (Lazo et al., 1991) was transformed (An, 1987) with the plasmid DNA. Successful transformation into AGLl was confirmed by PCR amplification of bacterial DNA with primers specific for the NPTII and the GUS genes of the modified pGA472. The template for the PCR reaction was prepared by growing the transformed AGLl at 28C overnight. A 5 l aliquot of this culture was diluted in 95 l of water and boiled for 1 min. The PCR reaction contained 10 l of this bacterial culture, 10 l lOX TAQ polymerase buffer (Bethesda Research Laboratories), 200 Meach dNTP, 100 ng each primer, 1.25 of TAQ polymerase in 100 l final volume. Plant Material Tobacco plants (Nicotiana tabacum L. cv. Kentucky 14) were grown in culture cups in sterile soil. Transformation of Tobacco Tobacco transformation were performed according to the procedure outlined by Horsch et al.(1985). I did not personally perform this particular procedure, so details of the protocol will not be outlined here.

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125 Preparation of RNA Tobacco leaf RNA was isolated as described by Dunsmuir et al. (1988). Briefly, this involved grinding 2 g of leaf tissue in liquid N 2 with a mortar and pestle A 4.5 ml aliquot of NTS buffer, which contained 0.lM NaCl, 0 01M Tris-HCl pH 7.5, 1 mM EDTA and 1 % SDS was added to the finely ground tissue. A 3 ml volume of phenoVchloroform/isoamyl alcohol (25:24:1) (PCI) was added and the tissue was ground until the mixture thawed. This was poured into a 50 ml capped tube and the mortar and pestle was rinsed with an additional 4.5 ml of NTS buffer and 3 ml of PCI. The mixture was vortexed for 5 min and the phases separated by centrifugation at 1000 x g for 10 min. The aqueous layer was saved and the phenol layer was washed with 2 ml of NTS and separated again by centrifugation. The combined aqueous layers were extracted with phenoVchloroform (1:1), reextracted with chloroform and precipitated by incubation overnight at -20C with 0.1 volumes of 3M sodium acetate and 2 volumes of ethanol. The nucleic acids were collected by centrifugation at 10,000 x g for 10 min. The pellet was washed with 70% ethanol and repelleted. The nucleic acids were resuspended in 2 ml of st~rile water and 2 ml of 4 M lithium acetate was added. The RNA was precipitated by incubation for 3 hours at 4C and pelleted by centrifugation for 10 min at 10,000 x g The pellet was resuspended in 0.9 ml sterile water, reprecipitated with 0.1 ml 3 M sodium acetate and 2 ml ethanol for 2 hours at -20C and collected by centrifugation for 10 min at

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126 10,000 x g The pellet was rinsed with 70% ethanol, dissolved in 400 l water and stored at -10C Northern Hybridization Total leaf RNA (10 g/lane) was loaded onto formaldehyde denaturing gels, blotted, prehybridized, hybridized and washed as described in chapter 4. Probes for pTOM 111 and pTOM 41 were prepared as described in the previous chapter. A portion of the GUS gene from the Xbal to the EcoRV site of pBI221 was used as a probe and prepared for hybridization as described in the previous chapter Results Agrobacterium transformation plasmids were constructed, as described in the Materials and Methods, that produce sense or antisense transcripts of pTOM 111 and pTOM 41 (Fig.A-1 and A-2). Tobacco leaf pieces were transformed with these plasmids, selected with kanamycin and tested for GUS activity (Jefferson, 1987). I did not perform the tobacco transformation or GUS staining so these technical details will not be discussed at this time Total RNA was extracted from leaf tissue of greenhouse grown transgenic tobacco plants, run on 1 % agarose formaldehyde denaturing gels, blotted and hybridized as described in the previous chapter Each blot was probed with either pTOM 111 or pTOM 41 cDNA, stripped and reprobed with a portion of the gus

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A pTOM 41 in pGEM-42 Sense _.., pTOM41 EcoR1 Kpn1 EcoRV EsoRV pGEM-42 8 p T OM 111 in pGEM-42 Sense _.. pTOM 111 pGEM-42 127 Figure A-1. Cloning of pTOM 41 and pTOM 111 into pGEM-4Z in the sense orientation. A) pTOM 41 contains two EcoRV sites One of these restriction sites is within the first 400 bases of the 5 end of the clone. The pTOM 41 was cloned into pGEM-4Z at the Sma/ site which is bounded by BamHI and KpnL B) pTOM 111 contains a Nde/ site 30 bases from the 3' end of the clone The pGEM-4Z contains an Nde/ site ~ 290 bases from the Kpnl site

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128 B Hind~III pGMnGUS .~ J/ Figure A-2. The vectors used for cloning pTOM 41 and pTOM 111 cDNAs in the sense and antisense orientation. A) The cloning cassette contains a CaMV 35S promoter, polylinker and nos terminator The polylinker contains an Xbal, BamHI, Smal, Kpnl, Ncol, Xhol, and Sstl restriction sites. The pTOM cDNAs were cloned into this cassette at the Kpnl and BamHI sites of the polylinker. B) The Agrobacterium vector pGA472 contains a GUS gene at the Seal site. The cloning cassette is cloned into this vector at the Hindlll and Bglll sites.

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129 gene as described in the Materials and Methods. A representative blot is presented for pTOM 111 (Fig. A-3) and for pTOM 41 (Fig. A-4). A total of 30 separate transgenic plants were tested for the ability to express transcript. Tobacco plants do not normally appear to contain any RNA that hybridizes to pTOM 111 or pTOM 41 cDNA. This was shown by examining plants that have been transformed with a plasmid containing the GUS gene alone (Fig. A-3, lanes 11 and 12; and Fig. A-4, lanes 2 and 8). Most plants transformed with these constructs expressed the GUS gene, although, slight differences in the level of expression were seen in the individual plants. This demonstrates that these newly constructed vectors were capable of being stably transformed and expressed in tobacco (Fig. A-3 and A-4) However, 7 of the 30 putatively transformed plants did not contain RNA that hybridized to the GUS probe. Either the gene or expression of the GUS gene was selectively lost in 6 of these plants, because 6 GUS negative transformants were positive for expression of pTOM transcript. A striking example of this is shown in Fig. A-3, lane 9. This plant is producing copious amounts of pTOM 111 sense transcript, but apparently no GUS message is produced. Partial loss of T-DNA during transformation is not uncommon, nor is inhibition of expression of individual genes following transformation (Jones et al., 1987; Deroles and Gardner, 1988). The level of expression of the sense and antisense transcripts of the pTOM cDNAs varied dramatically between individual transformants (Fig. A-3 and A-4). In addition, different orientations expressed very different levels of transcript. For

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130 Lane# 1 2 3 4 5 6 7 8 9 10 11 12 13 gus p T O M1 11 Figure A-3. Individual transgenic tobacco plants were transformed with constructs of the pTOM 111 cDNA that produced either sense or antisense transcripts. RNA was extracted from leaf tissue Total RNA (lOg/lane) was loaded onto 1 % agarose formaldehyde denaturing gels, blotted and probed as described in the Materials and Methods The bottom panel represents the results obtained when the blot was probed with the entire pTOM 111 cDNA The blot was stripped and reprobed with an Xbal to EcoRV fragment of the GUS gene. Lanes 2, 7, 8, 9 and 10 were individual transgenic plants that produced sense transcripts of the pTOM 111 cDNA. Lanes 1 3, 4, 5, and 6 represent RNA from individual transgenic plants containing constructs that. produce antisense transcripts of pTOM 111. Lanes 10 and 11 contain RNA from plants transformed with pGA472 GUS alone Lane 13 contains 5 g total RNA from >80% red ripe fruit.

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131 Lane# 1 2 3 4 5 6 7 8 9 gus pTOM 41 Figure A-4 Individual transgenic tobacco plants transformed with constructs of the pTOM 41 cDNA that produced either sense of antisense transcripts. RNA was extracted from leaf tissue. Total RNA (10 g,'lane) was loaded onto 1 % agarose formaldehyde denaturing gels, blotted and probed as described in the Materials and Methods. The bottom panel represents the results obtained when the blot was probed with the entire pTOM 41 cDNA The blot was stripped and reprobed with an Xbal to EcoRV fragment of the GUS gene. This is shown in the top panel. Lanes 3, 4, and 7 contain RNA isolated from individual transgenic plants transformed with a plasmid that produces antisense pTOM 41. Lanes 1, 5 and 6 contain RNA from individual transgenic plants transformed with a plasmid that produces sense transcripts of pTOM 41. Lanes 2 and 8 contain RNA from individual transgenic plants that have been transformed with pGA472 GUS alone Lane 9 contains RNA from 50% red ripe tomato fruit.

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132 example the sense orientation of pTOM 111 produced large amounts of transcript whereas the antisense orientation did not (Fig. A-3) Plants transformed with the antisense orientation of pTOM 111 often contained hybridizing RNA that produced a low level smear when hybridized with pTOM 111 as a probe. However this was not a general property of the RNA since hybridization of the same blot with the GUS probe produced a discrete band (Fig. A-3). This appears to be an inherent result of the antisense pTOM 111 construct because this was observed in at least 9 out of 11 individual transformants In contrast, 4 out of 5 plants transformed with the sense orientation expressed RNA at levels similar to the amount expressed during chromoplast development in tomato fruit. This is shown by comparison of the RNA produced by transformed plants to the RNA from >80% red ripe tomato fruit (Fig A-3, lanes 2 8, 9 and 10 versus lane 13). These results confirm that the cloning cassettes containing either the sense or antisense pTOM 111 constructs are capable of expressing transcript. A similar analysis was performed with tobacco plants transformed with constructs capable of producing sense and antisense transcripts of pTOM 41. Fig. A4 shows that in this case 3 individual transgenic plants of 3 tested, which contain antisense pTOM 41 produced large amounts of antisense transcript. Comparison of the amount of RNA produced by these plants to the amount normally found in 50% red ripe tomato fruit shows that a large excess of antisense RNA is produced by the transgenic plants (Fig A-4, lane 9 versus lanes 3, 4 and 7). Unfortunately, no RNA was produced with the sense constructs of pTOM 41 in the 9 individual transgenic

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133 plants tested. It is unclear why the expression of the sense transcript of pTOM 41 is inhibited. These plants produce measurable amounts of GUS transcript; therefore, they have been successfully transformed. Either a portion of the plasmid containing the pTOM 41 cloning cassette bas been selectively lost or the expression of this transcript bas been selectively inhibited. No obvious phenotype resulted from the transformation of tobacco with these constructs. However, the plants have not set seed yet so it is too early to tell if embryo development will be affected by the new genes. Discussion The purpose of this study was to test whether the constructed plasmids could effectively produce transcripts so that ultimately they could be used in tomato transformation The constructs were first tested in tobacco, because, although there are reports in the literature on tomato transformation, it is not an entirely straight forward procedure. We have shown that the GUS gene placed at the Seal site of pGA472 is stably expressed in mature tobacco leaves. The cloning cassette can also be effective in stable expression of large amounts of RNA. These plasmids will have utility beyond this study, since they can be used in other situations and with other species. The 11 plants transformed with the antisense construct of pTOM 111, however, do not appear to maintain high levels of this transcript. This result may occur for a number of reasons. First, the transformed tobacco plants may selectively

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134 lose expression of the gene during transformation and growth of the transgenic plant. Secondly, the antisense RNA may interact with a tobacco leaf plastid-localized low MW hsp transcript. A loss in antisense RNA was observed when endogenous message to pTOM 13 was induced by wounding antisense pTOM 13 plants (Hamilton et al., 1990). The production of the endogenous transcript caused degradation of both messages. It is unclear how homologous the tobacco hsp gene is to pTOM 111. However, considering the fact that a petunia hsp gene is 81 % homologous to the tomato hsp and that successful reduction in tobacco chalcone synthase message occurred with transformation of a petunia antisense construct that was only 86% homologous to the endogenous transcript, the idea that an interaction could occur between the pTOM 111 antisense transcript and the endogenous message seems feasible (van der Krol et al., 1988). Perhaps the plants should be maintained in conditions that do not lead to selection against expression of the antisense transcript. ff an interaction is occurring between the antisense construct and the tobacco plant, then high level expression of a stable transformant in tomato tissue will be difficult The tomato transformation experiment could be performed with a fruit specific promoter rather than the nonspecific CaMV 35S promoter. This would not be difficult since I have also constructed a cloning vector with the fruit specific promoter E8 (Fischer, personal communication). In this way, even if the endogenous hsp is produced in the leaves it would not be inhibited by the antisense construct. Therefore, the effect of inhibiting hsp induction would be studied only during chromoplast development

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135 What type of phenotype may be expected by the transformation of antisense transcripts of pTOM 41 into tomato plants? This question is extremely difficult to answer. It is unclear from sequence comparison to the ORF2 from E. coli what function pTOM 41 performs during chromoplast development. Since the synthesis of the ORF2 is coordinated with the synthesis of PRPP synthetase and a gene involved in c5-ALA synthesis, perhaps it is involved with the synthesis of NADPH. The stepwise dehydrogenation of phytoene requires pyridine nucleotides to function. Perhaps pTOM 41 is involved in the synthesis of these cofactors. In that case, the inhibition of pTOM 41 could result in loss of carotenoid synthesis. The loss of pTOM 111 during chromoplast development via antisense mutagenesis may result in a failure in the ordered breakdown of the thylakoid membranes during the chloroplast to chromoplast conversion. This has been suggested by the correlation between the induction of the pTOM 111 transcript with the loss of LHCP during chromoplast development. If the induction of pTOM 111 occurs because of an increase in unfolded proteins during the degradation of photosynthetic proteins, then it is possible that the degradative process will continue. Perhaps the loss of LHCP will be slowed in the mutant plants and this may be demonstrated by comparisons of developmental profiles of proteins from tomato fruit extracts. It is likely that the continued synthesis and deposition of carotenoids could be inhibited by the plastids inability to cope with a buildup in partially degraded proteins. If a function of this protein is uncovered during chromoplast development, perhaps it will suggest a function for these hsps during heat stress.

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REFERENCES Abernethy, R.H D S. Thiel N.S Petersen, and K. Helm. 1989. Thermotolerance is developmentally dependent in germinating wheat seed Plant Physiol. 89:596-576 Almoguera, C and J. Jordana. 1992 Developmental and environmental concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAs Plant Mol. Biol. 19:781-792 Altschul S.F., W Gish, W. Miller, E W Myers, and D.J Lipman 1990. A basic local alignment search tool. J. Mol. Biol. 215 : 403-410 An, G 1987 Binary Ti vectors for plant transformation and promoter analysis Methods in Enzymol. 153:292-305 An., G., B.D. Watson, S Stachel, M.P. Gordon, E.W. Nester 1985. New cloning vehicles for transformation of higher plants. Embo Journal 4 : 277-284 Armstrong, G A M. Alberti, J.E. Hearst. 1990a Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes. Proc. Natl. Acad. Sci. USA. 87:9975-9979 Armstrong, G.A., A Schmidt, G. Sandmann, and J.E. Hearst. 1990b. Genetic and biochemical characterization of carotenoid biosynthesis mutants of Rhodobacter capsulatus. The J Biol. Chem 265(14):8329-8338 Arrigo, A.P. 1987. Cellular localization of hsp23 during Drosophila development and following subsequent heat shock Develop. Biol. 122:39-48 Atkins, J F R B Weiss, and R F Gesteland 1990 Ribosome gymnastics-Degree of difficulty 9 5, style 10 0. Cell 62 : 413-423 Barja de Quiroga, G. 1992. Brown fat thermogenesis and exercise: two examples of physiological oxidative stress. Free Radical Biology and Medicine. 13:325-340 136

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150 Roy, H. 1989. Rubisco assembly: A model system for studying the mechanism of chaperonin action. The Plant Cell. 1:1035-1042 Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning a Laboratory Manual. 2nd edition Cold Spring Harbor Laboratory Press. NY Scandalios, J.G. 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101:712 Sheehy, R.E., M. Kramer, and W.R. Hiatt. 1988. Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc. Natl. Acad. Sci. USA. 85:8805-8809 Sitte, P., H Falk and B Liedvogel. 1980. Chromoplasts. In: Czygan, F.C. (ed.) Pigments in Plants. Fischer Stuttgart pp.117-148 Slater, A., M.J. Maunders, K. Edwards, W. Schuch and D. Grierson. 1985. Isolation and characterization of cDNA clones for tomato polygalacturonase and other ripening-related proteins. Plant Molecular Biology. 5:137-147 Smith, C.J.S., C.F. Watson, J.Ray, C.R.Bird, P.C. Morris, W. Schuch, and D. Grierson. 1988. Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature. 334(25):724726 Smith, C.J.S., C.F. Watson, P.C. Morris, C.R. Bird, G.B. Seymour, J.E. Gray, C. Arnold, G.A. Tucker, W. Schuch, S. Harding, and D. Grierson. 1990. Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol. Biol. 14:369-379 Solomos, T. 1988. Respiration in senescing plant organs: Its nature, regulation, and physiological significance. In: L.D. Nooden, and A C. Leopold (eds) Senescence and aging in plants. Academic Press Inc. New York. pp.111-145 Speirs, J., C.J. Brady, D. Grierson and E. Lee. 1984. Changes in ribosome organization and messenger RNA abundance in ripening tomato fruits. Aust. J. Plant Physiol. 11:225-33 Thayer, S.S., H.T. Choe, A. Tang, and R.C. Huffaker. 1987. Protein turnover during senescence. In: W W Thomson, E.A. Nothnagel, and R.C. Huffaker (eds.) Plant senescence: Its biochemistry and physiology, American Society of Plant Physiologists. Rockville, Maryland. pp 71-80 Thelander, M., J.O. Narita, and W. Gruissem. 1986. Plastid differentiation and pigment biosynthesis during tomato fruit ripening. Current Topics in Plant Biochemistry and Physiology 5:128-141

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152 Vierling, E., R.T. Nagao, A.E. DeRocher, and L.M. Harris. 1988. A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins. The Embo J. 7(3): 575-581 Vierling, E. 1991. The role of heat shock proteins in plants. Annu. Rev. Plant Physiology Plant Mol. Biol. 42:579-620 Wada, K.-N., Y. Wada, F. Ishiashi, T. Gojobori, and T. Ikemura. 1992. Codon usage tabulated from the GenBank genetic sequence data Nucl. Acid. Res. 20, Suppl.:21112118 Wang, H., and W.R. Woodson. 1991. A flower senescence-related mRNA from carnation shares sequence similarity with fruit ripening-related mRNAs involved in ethylene biosynthesis. Plant physiol. 96:1000-1001 Weber, L.A. 1992. Relationship of heat shock proteins and induced thermal resistance. Cell Prolif. 25:101-113 Weiss, R.B. Ribosomal frameshifting, jumping and readthrough. 1991. Curr. Opin. in Cell Biol. 3:1051-1055 Whitaker, B.D. 1991. Growth conditions and ripening influence plastid and microsomal membrane lipid composition in bell pepper fruit. J. Amer. Soc. Hort. Sci. 116(3):528-533 Woolhouse, H.W. 1984. The biochemistry and regulation of senescence in chloroplasts. Can. J. Bot. 62:2934-2942 Wrench, P.M., M. Olive, R.G. Hiller, C.J. Brady, and J. Speirs. 1987. Changes in plastid proteins during ripening of tomato fruits. J. Plant Physiol. 129 : 89-122 Yelle, S., J.D. Hewitt, N.L. Robinson, S. Damon, and A.B. Bennett. 1988. Sink metabolism in tomato fruit III. Analysis of carbohydrate assimilation in a wild species. Plant Physiol. 87:737-740 Yip, W-K., J-G. Dong, J.W. Kenney, G.A. Thompson, And S.F. Yang. 1990 Characterization and sequencing of the active site of 1-aminocyclopropane-1carboxylate synthase. Proc. Natl. Acad. Sci. USA. 87:7930-7934

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BIOGRAPHICAL SKETCH I grew up in Minnesota thinking 3 months of tough sledding was a normal way of life. My family moved to the Smoky Mountains of Asheville, North Carolina, where I spent the last two years of high school. I graduated from a very small experimental school, in a graduating class of 16 students. The experience gave me a fairly utopian perspective of education, which was augmented by my undergraduate education I pursued a Bachelor of Sciences degree from Beloit College, a small liberal arts college in southern Wisconsin Upon graduation from college, I took a research position in the Department of Veterinary Sciences at the University of Wisconsin for a year and at McCardle Labs for Cancer Research the following three years. I decided to go back to school in plant molecular biology This was not really a change in direction, since my undergraduate education specialized in botany. 153

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ~~Q_ ~Q _,, Gloria A. Moore, Chair Professor of Plant Molecular and Cellular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree o~ of Philosop~y 1 L(2 Kenneth Cline Associate Professor of Plant Molecular and Cellular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosoph C. Eduardo Vallejos Associate Professor of Plant Molecular and Cellular Biology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor Philosophy r ",,~ .' ' ~ \ :::0 \''-\ Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. William Gurley ~7 Associate Professor of Plant Molecular and Cellular Biology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1993 Dean, Dean, Graduate School