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PRESTORAGE HEAT TREATMENT TO INHIBIT CHILLING INJURY AND
SYNCHRONIZE RIPENING 1N TOMATO (LYCOPERSICON ESCULENTUM
MILL.) FRUIT

By

Konstantinos E. Vlachonasios

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1999

ABSTRACT

PRESTORAGE HEAT TREATMENT TO INHIBIT CHILLING INJURY AND
SYNCHRONIZE RIPENING IN TOMATO (LYCOPERSICON ESCULENTUM
MILL.) FRUIT

By

Konstantinos E. Vlaehonasios

The effect of heat treatment on attenuating development of chilling injury of
tomato ﬁuit was investigated. Mature green tomatoes were held at different high
temperature regimes prior to storage at 2°C for 2 or 3 weeks. Chilling injury (CI) was
circumvented by heat treatment at 42°C for 36 or 48 h while non-heated fruits stored at
2°C developed typical CI symptoms and failed to ripen when returned to permissive
ripening temperatures. Heat treatment-induced protection from C1 was abolished if the
tomatoes were transferred from high temperatures to 20°C for 1 or 3 days before the low
temperature storage. The heat-treated fruits ripened normally although more slowly that
non-treated tomatoes. We hypothesized that heat shock proteins (HSPs) may be
responsible for increased tolerance to CI. Using mRN A differential display, we cloned
and characterized a full-length cDNA that is related to heat-induced chilling tolerance
(HCT); it encodes a 17.6-kD cytosolic class H smHSP (named LeHSP17.6). In a screen
of a cDNA library from heat-treated tomato fruit, a 17.4-kD cytosolic class II smHSP
(LeHSP17.4) was isolated. In addition, three cDNAs encoding a cytosolic class I smHSP
and partial cDNA clones for a chloroplastic smHSP, a mitochondrial smHSP and a heat-

inducible HSP9O member were isolated. Northern analyses indicated that the expression

of the smHSPs genes induced by heat continued at low temperature only if the fruits had
been exposed to heat shock. Fruit that received only low temperatures were not
stimulated to express smHSPs (cytosolic class I and II as well as chloroplastic smHSP).
The re-induction of the transcripts in the cold was more favorable after continued
exposure to high temperatures. LeHSP90 and its constitutively expressed homologue
LeHSC80 were induced by low temperatures. However, the heat-treated fruits had a
higher LeHSP90 transcript level than the non-heated fruits at low temperatures. In
addition to mRNA expression pattern of the smHSPs and HSP9OS, the protein level
followed a similar pattern. More importantly, these proteins were still detectable when
the fruits were transferred to 20°C for 3 days. The cold-inducible expression of HSP
genes in the heat-treated fruits raised the possibility that both high- and low-molecular-
weight HSPs may play critical roles in resistance to chilling stress. The heat-shock-
induced protection against CI was lost when the fruits were transferred to 20°C after heat
shock and before cold storage. Surprisingly, the re-induction of the HSPs at low
temperatures was not prevented. These data suggest that heat shock proteins are not the

only factor(s) that contribute to the chilling—tolerant phenotype of tomatoes.

To my parents, Epaminondas and Anna, and my brother Ioannis

ACKNOWLEDGMENTS

I would like to express my thanks to all my committee members; to my major
adviser Dr. David R. Dilley for his guidance, support, and encouragement throughout the
completion of my thesis; Drs. Randolph Beaudry, Rebecca Grumet, Hans Kende and
Michael Thomashow for their help and support.

The completion of this thesis would not have been possible without the ﬁnancial
support provided by the State Scholarships Foundation of the Republic of Greece. I
would like to thank also the Michigan State University Graduate School for the
dissertation completion fellowship and the Department of Horticulture of Michigan State
University for the educational support.

I owe special thanks to Dr. Dina Kadyrjanova for her help, guidance and
assistance throughout the completion of this study. I would also like to express my
appreciation to Dr. Phillippos Ververidis for his help and advice in the beginning of this
study.

Special thanks to my parents and my brother for their support and encouragement.
Finally, I am indebted to my wife Ekaterini Papadopoulou for her unlimited love,

encouragement, inspiration and understanding.

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................... x
LIST OF FIGURES ........................................................................................................ xi
INTRODUCTION ........................................................................................................... 1
REFERENCES ................................................................................................................ 3
LITERATURE REVIEW ................................................................................................ 5
1. Prestorage Heat Treatments ...................................................................................... 5
1.1 Heat Treatment for Control of Postharvest Diseases and Insect Pest of Fruits ..... 5
1.2 The Effect of High Temperature on Fruit Ripening ............................................... 7
The effect of heat treatment on ethylene biosynthesis and action. ......................... 8
The effect of heat treatment on respiration ........................................................... 9
The effect of heat treatment on color development ............................................... 9
The effect of heat treatment on ﬁrmness ............................................................. 10
The effect of heat treatment on sugar/ acid ratio .................................................. 11
The effect of high temperature on gene expression during fruit ripening ............ 11
1.3 Heat Treatment Induces Resistance to Chilling Injury ......................................... l3
2. Heat Shock Response, Heat Shock Proteins and Cellular Metabolism ................. 19
3. Small Heat Shock Proteins ..................................................................................... 21
4. Function of the Heat Shock Proteins ...................................................................... 24
REFERENCES .............................................................................................................. 29
CHAPTER I .................................................................................................................. 45
PRESTORAGE HEAT TREATMENT REDUCES CHILLING INJURY IN TOMATO
FRUIT ............................................................................................................... 45
ABSTRACT .................................................................................................................. 45
INTRODUCTION ......................................................................................................... 47

vi

MATERIAL AND METHODS ..................................................................................... 48

Experiments Conducted in 1994 ................................................................................. 48
Experiments Conducted in 1995 ................................................................................. 49
Experiments Conducted in 1997 ................................................................................. 49
RESULTS ..................................................................................................................... 50
Experiments Conducted in 1994 ................................................................................. 50
Experiments Conducted in 1995 ................................................................................. 54
Experiments Conducted in 1997 ................................................................................. 54
DISCUSSION ......................................................................................................................... 62
REFERENCES .............................................................................................................. 65
CHAPTER II ................................................................................................................. 68

MOLECULAR CLONING OF A NOVEL HEAT-INDUCED CHILLING-
TOLERANCE RELATED cDNA IN TOMATO FRUIT BY USE OF mRN A

DIFFERENTIAL DISPLAY .............................................................................. 68
ABSTRACT .................................................................................................................. 68
INTRODUCTION ......................................................................................................... 7O
MATERIAL AND METHODS ..................................................................................... 72

Plant Material and Temperature Treatments ....................................................... 72

Nucleic Acid Isolation ....................................................................................... 72

Differential Display ........................................................................................... 73

Isolation of cDNA Bands ................................................................................... 74

RNA Gel Blot Analysis ...................................................................................... 74

The Cloning of cDNA Bands ............................................................................. 75

Library Construction and Screening ................................................................... 76

DNA Sequencing and Analysis .......................................................................... 76

Southern Blot Analysis ...................................................................................... 77

vii

RESULTS ..................................................................................................................... 77

Differential Display ........................................................................................... 77
Expression Pattern Of the Speciﬁc Fragments Corresponding mRN As during Heat
Shock and Subsequent Chilling Temperatures .................................................... 80
Cloning and Sequence Analysis of the Speciﬁc cDNA Bands ............................ 82
Construction, Screening of the cDNA Library and Sequence Analysis of a HC T 1
cDNA ................................................................................................................ 84
Southern Blot Analysis ...................................................................................... 87
DISCUSSION ......................................................................................................................... 87
REFERENCES .............................................................................................................. 93
CHAPTER III ............................................................................................................... 99

HEAT TREATMENT ATTENUATES CHILLING INJURY: INVESTIGATION OF
THE INVOLVEMENT OF HEAT SHOCK GENES AND HEAT SHOCK
PROTEINS IN THE RESISTANCE OF TOMATO FRUIT TO LOW

TEMPERATURES ............................................................................................ 99
ABSTRACT .................................................................................................................. 99
INTRODUCTION ....................................................................................................... 101
MATERIAL AND METHODS ................................................................................... 102

Nucleic Acid Isolation ..................................................................................... 102

Differential Display ......................................................................................... 103

cDNA Library Screening ................................................................................. 104

DNA Sequencing ............................................................................................ 104

RNA Gel Blot Analysis .................................................................................... 104

Protein Isolation and Western Analysis ............................................................ 105
RESULTS ................................................................................................................... 106

Cloning and Characterization of Cytosolic Class II smHSP LeHSP17.4 ........... 106

Differential Display, Cloning and Sequence of Heat-Induced Chilling Tolerance—
Related HSPs ................................................................................................... 112

viii

Expression Patterns of the smHSPs, HSP90s and Ripening Related Genes ....... 137

Immunodetection of the smHSP and HSP90 in Heat-Treated Cold Stored Tomato

Fruit ................................................................................................................. 140
Expression Patterns of the Tomato smHSPs and HSP905 Genes under Different
Heat treatment Regimes .............................................................................. 142
DISCUSSION ....................................................................................................................... 151
REFERENCES ...................................................................................................................... 158
CHAPTER IV ............................................................................................................. 171
IDENTIFICATION OF THE GENOMIC SEQUENCE OF LeHSP17. 6 AND ITS CIS-
ACTING ELEMENTS ..................................................................................... 171
ABSTRACT ................................................................................................................ 171
INTRODUCTION ....................................................................................................... 172
MATERIAL AND METHODS ................................................................................... 173
Genomic Library Screening ............................................................................. 173
DNA Sequencing ............................................................................................ 173
Promoter Analysis ............................................................................................ 174
RESULTS ................................................................................................................... 174
DISCUSSION ....................................................................................................................... 178
HSE (Heat Shock Element) .............................................................................. 178
AT-rich Promoter Element ............................................................................... 181
CCAAT (Y-box) and REa Elements ................................................................ 182
STRE (Stress Response Element) ..................................................................... 183
NIT2 or GATA Core Elements ........................................................................ 187
REFERENCES ...................................................................................................................... 189
CONCLUSIONS AND FUTURE RESEARCH .................................................................. 198

LIST OF TABLES

CHAPTER II ................................................................................................................. 68
Table 1. Analysis Of differential display cDNA bands corresponding to H, HC, C and

MG tomato fruit RNA ........................................................................................ 81
CHAPTER III ............................................................................................................... 99

Table I. Amino acid sequence identity of LeHSPl7.4 to those of the cytosolic class II
smHSPs from various species .......................................................................... 109

Table II. Characteristics of the cDNA clones corresponding to heat-inducible chilling

tolerance genes ................................................................................................ 114
Table III. Cytosolic class I smHSP similarity to LeHSPl7.7 ....................................... 120
Table IV. HSP90 Accession Numbers ........................................................................ 131
CHAPTER IV ............................................................................................................. 171

Table 1. Potential binding sites for transcription factors and cis-acting elements in the
LeHSPI 7. 6 gene promoter ............................................................................... 177

LIST OF FIGURES

CHAPTER I .................................................................................................................. 45

Figure 1. The effect of heat treatment on ﬂesh ﬁrmness of tomato fruit stored at 2°C for
7 days and then ripened at 20°C (year 1994) ...................................................... 51

Figure 2. The effect of heat treatment on color development of tomato ﬁuit aﬁer storage at
2°C for 7 or 14 days and during ripening at 20°C (year 1994) .................................. 52

Figure 3. The effect of heat treatment on chilling injury Of tomato fruit after storage at 2°C
for 7 or 14 days and during ripening at 20°C (year 1994) ......................................... 53

Figure 4. The effect of heat treatment on color development of tomato fruit after storage at
2°C for 14 or 21 days and during ripening at 20°C (year 1995) ................................ 55

Figure 5. The effect of heat treatment on chilling injury of tomato fruits aﬁer storage at 2°C
for 14 or 21 days and during ripening at 20°C (year 1995) ....................................... 56

Figure 6. The effect of heat treatment and post-treatment at 20°C on color development
of tomato ﬁ'uits subsequemly stored at 2°C for 14 days and during ripening at
20°C (year 1997) ............................................................................................... 58

Figure 7. The effect of heat treatment and post-treatment at 20°C on chilling injury of
tomato ﬁuits subsequently stored at 2°C for 14 days and during ripening at 20°C
(year 1997) ........................................................................................................ 59

Figure 8. The effect of heat treatment (42°C for 36 h) and post-treatment at 20°C for 1
day on chilling injury of tomato fruits subsequently stored at 2°C for 14 days and
ripened at 20°C .................................................................................................. 60

Figure 9. The effect of heat treatment (42°C for 48 h) and post-treatment at 20°C for 1
day on chilling injury of tomato fruits subsequently stored at 2°C for 14 days and

ripened at 20°C .................................................................................................. 61
CHAPTER II ................................................................................................................. 68
Figure 1. Differential display of total RNA from four different treatments of tomato ﬁuit

.......................................................................................................................... 78
Figure 2. RNA blot hybridization analysis of total RNA from tomato fruit ................... 83

Figure 3. Nucleotide and deduced amino acid sequences of the LeHSPI 7.6 cDNA ....... 85
Figure 4. Genomic Southern blot analysis of the tomato LeHSP17. 6 ............................. 86

Figure 5. LeHSPl7.6 is a cytosolic class II small HSP .................................................. 89

xi

CHAPTER III ............................................................................................................... 99

Figure 1. Nucleotide and deduced amino acid sequences of the LeHSPI 7.4 (LeHCTZ)
cDNA .............................................................................................................. 108

Figure 2. Amino acid sequence comparison among the cytosolic class H smHSPs ...... 110

Figure 3. Nucleotide and deduced amino acid sequences of the cytosolic class I
LeHSPI 7. 7 (LeHCT 3) cDNA ........................................................................... 115

Figure 4. Nucleotide and deduced amino acid sequences of the cytosolic class I
LeHSPI 7.8 (LeHC T 8) cDNA ........................................................................... 1 16

Figure 5. Nucleotide and deduced amino acid sequences of the cytosolic class I
LeHSPI 7.6 (LeHCT 4) cDNA ........................................................................... 117

Figure 6. Amino acid sequence comparison among the cytosolic class I smHSPs ....... 122

Figure 7. Nucleotide and deduced amino acid sequences of the LeHSP21.0 cDNA
(LeHCT 5) ........................................................................................................ 126

Figure 8. Nucleotide and deduced amino acid sequences of the putative LeHSP22. 0
cDNA (LeHCT 6) ............................................................................................. 126

Figure 9. Nucleotide and deduced amino acid sequences of the LeHSP90 cDNA ........ 128
Figure 10. Amino acid sequence comparison among the HSP9OS ................................ 132

Figure 11. The expression patterns of the tomato smHSPs, HSP90s and E8 are affected
by heat treatment and low temperature storage of the fruits .............................. 138

Figure 12. Immunodetection of cytosolic small HSPs and HSP90 protein levels in heat-
treated/cold stored tomato fruits ....................................................................... 141

Figure 13. The effect of heat shock on the level of transcripts for the smHSPs and
HSP903 in tomato fruits ................................................................................... 144

Figure 14. The effect of heat treatment on smHSPs, HSP90 and E8 mRNA levels in
tomato fruits subsequently stored at 2°C for 14 days ........................................ 145

Figure 15. The effect of heat treatment and post-treatment at 20°C on smHSPs, HSP90
and E8 mRNA levels in tomato fruits subsequently stored at 2°C for 14 days .. 149

CHAPTER IV ............................................................................................................. 171

Figure 1. Nucleotide sequence of tomato cytosolic class II smHSP LeHSPI 7.6 gene .. 176

xii

INTRODUCTION

Tomatoes as well as other subtropical fruits develop chilling injury if held below
12°C during low temperature storage and/or after being returned to permissive ripening
temperatures. The degree of injury is related to the magnitude and duration at chilling
temperatures. Typical symptoms appear after transfer from storage and include failure to
ripen, irregular ripening, surface pitting, and increased susceptibility to decay. Chilling
injury can be prevented by storage at temperatures slightly above the critical chilling range
(Hatton, 1990), or holding the tissue above 35°C prior to chilling (Lurie and Klein, 1991).

Postharvest heat treatment of temperate, subtropical and tropical ﬁuit has been
examined by numerous research groups with the objective of developing non-chemical,
alternative strategies for insect and disease control of fruits exported to or imported from
various countries (Couey, 1989; Lay-Yee and Rose, 1994; Shellie and Mangan, 1994;
Ben-Yehoshua et al., 1995). These tests and others (Dentener et al., 1996) have
employed hot water or heated air at various temperatures ranging from 38 to 50°C for
brief periods (minutes) at the higher temperatures or extended periods (days) at the lower
temperatures. Interestingly in these studies, heat treatment of several fruits diminished
the incidence of some important physiological disorders related to low temperature
storage such as chilling injury and superﬁcial scald (Klein and Lurie, 1992). When
apples were heat-treated at 38°C for 3 days and subsequently stored for 6 months at low

temperatures (0-3 °C), they ripened and softened slower than non-heated fruits.

Moreover, the incidence of superﬁcial scald was dramatically reduced (Lurie et a1. ,
1990).

Mature green tomato fruits normally must be stored at temperatures above 10°C
to avoid chilling injury and, therefore, have a temperature-limited postharvest life of only
a few days to a week before ripening is initiated. Heat treatment of tomato fruits has very
signiﬁcant potential for commercial application for several reasons: (1) Wholesomeness
of the fruit may be improved, avoiding the use of applied chemicals to control some
important physiological and pathological disorders; 2) ripening can be delayed; and 3) the
storage duration can be doubled without evoking chilling injury disorders and the
accompanying decay.

More importantly, heat treatment endows the mature green tomatoes with the
ability to be stored in air at 2°C for several weeks and subsequently ripen normally
without symptoms of chilling injury (Lurie and Klein, 1991). Heat treatment was
recemly found to induce resistance to chilling injury in avocado (Sanxter et al., 1994;
Woolf et al., 1995; Florissen et al., 1996), citrus fruits (Rodov et al., 1995) cucumber
(McCollum et al., 1995), mango (McCollum et al., 1993), mung bean hypocotyls (Collins
et al., 1993; 1995), sweet pepper (Mencarelli et al., 1993), persimmons (Burmeister et al.,
1997; Lay-Yee et al., 1997), and zucchini squash (Wang, 1994). It has been
hypothesized that the induction of heat-shock proteins during heat treatment is
responsible for acquisition of chilling injury tolerance.

The purpose of this study was i) to develop, optimize and characterize a

commercially applicable method of postharvest heat treatment for storage to chilling

injury and ii) to identify and investigate the involvement of heat-shock proteins in the

resistance to chilling injury.

REFERENCES

Ben-Yehoslma S, Rodov V, Fang DQ, Kim JJ (1995) Performed antiﬁrngal
compounds of citrus fruit: effect of postharvest treatments with heat and growth
regulators. J Agric Food Chem 43: 1062-1066

Burmeister DM, Ball S, Green S, Woolf AB (1997) Interaction of hot water treatments
and controlled atmosphere storage on quality of 'Fuyu' persimmons. Postharvest
Biol Technol 12: 71-81

Collins GG, Nie X, Saltveit ME (1993) Heat shock increases chilling tolerance of mung
bean hypocotyl tissue. Physiol Plant 89: 117-124

Collins GG, Nie X, Saltveit ME (1995) Heat shock proteins and chilling sensitivity of
mung bean hypocotyls. J Exp Bot 46: 795-802

Coney HM (1989) Heat treatment for control of postharvest diseases and insect pests of
fruits. HortScience 24: 198-202

Dentener PR, Alexander SM, Lester PJ, Petry RJ, Maindonald JH, McDonald RM
(1996) Hot air treatment for disinfestation of lightbrown apple moth and
longtailed mealy bug on persimmons. Postharvest Biol Technol 8: 143-152

Florissen P, Ekman JS, Blurnenthal C, McGlasson WB, Conroy J, Holford P (1996)
The effects of short heat treatments on the induction of chilling injury in avocado
fruit (Persea americana Mill). Postharvest Biol Technol 8: 129-141

Hatton '11 (1990) Reduction of chilling injury with temperature manipulation. In CY
Wang, ed, Chilling Injury of Horticultural Crops. CRC Press, Boca Raton,
Florida, pp 269-280

Klein JD, Lurie S (1992) Heat treatments for improved postharvest quality of
horticultural crops. HortTechnology 2: 316-320

Lay-Yea M, Bali S, Forbes SK, Woolf AB (1997) Hot-water treatment for insect
disinfestation and reduction of chilling injury of 'Fuyu' persimmon. Postharvest
Biol Technol 10: 81-87

Lay-Yee M, Rose KJ (1994) Quality of "Fantasia" nectarines following forced-air heat
treatments for insect disinfestation. HortScience 29: 663-666

Lurie S, Klein JD (1991) Acquisition of low temperature tolerance in tomatoes by
exposure to high temperatures. J Amer Soc Hort Sci 116: 1007-1012

Lorie S, Klein JD, Ben Arie R (1990) Postharvest heat treatment as possible means of
reducing superﬁcial scald of apples. J Hort Sci 65: 503-509

McCollnm TG, D'Aquino S, McDonald RE (1993) Heat treatment inhibits mango
chilling injury. HortScience 28: 197-198

McCollum TG, Doostdar H, Mayer RT, McDonald RE (1995) Immersion of
cucumber fruit in heated water alters chilling-induced physiological changes.
PostharvestBiol Technol 6: 55-64

Mencarelli F, Ceccantoni B, Bolini A, Anelli G (1993) Inﬂuence of heat treatment on
the physiological response of sweet pepper kept at chilling temperature. Acta
Hort 343: 23 8-243

Rodov V, Ben-Yehoshua S, Albagli R, Fang DO (1995) Reducing chilling injury and
decay of stored citrus fruit by hot water dips. Postharvest Biol Technol 5: 119-
127

Sanxter SS, Nishijima KA, Chan HT Jr (1994) Heat-treating "Sharwil" avocado for
cold tolerance in quarantine cold treatments. HortScience 29: 1166-1168

Shellie KC, Mangan RL (1994) Postharvest quality of "Valencia" orange after exposure
to hot, moist, forced air for ﬁ'uit ﬂy disinfestation. HortScience 29: 1542-1527

Wang CY (1994) Combined treatment of heat shock and low temperature conditioning
reduces chilling injury in zucchini squash. Postharvest Biol Technol 4: 65-73

Woolf AB, Watkins CB, Bowen JH, Lay-Yee M, Maindonald JH, Ferguson [B
(1995) Reducing external chilling injury in stored “Hess” avocados with dry heat
treatments. J Amer Soc Hort Sci 120: 1050-1056

LITERATURE REVIEW

1. Prestorage Heat treatments

Heat treatments have been used to control diseases and insect infestation of ﬁuits for
many years (Baker, 1952). However, with the development of effective fungicides and
insecticides, which could be applied easie' and cheaper, interest in heat treatments waned.
Recently, EPA (Environmental Protection Agency) has withdrawn the registration of many
agricultural chemicals due to safety issues (Couey, 1989). The cost of developing new
fumigants increased due to regulatory restrictions, and therefore interest in heat treatments
or alternative methods has been revived (Klein and Lurie, 1992). Heat may be applied in
several ways: by exposure to hot water, to vapor heat (water-saturated hot air), to hot dry air,
to infrared and microwave radiation (Couey, 1989). Commercially, only vapor heat or hot
water treatments have been used. Heat treatments have the advantage of effective
insecticidal and ﬁrngicidal action, easy of application, and absence of chemical residues. In
contrast, the potential of fruit damage and the relatively high cost of application are the

major disadvantages.

1.1 Heat treatment for Control of Postharvest Diseases and Insect Pest of Fruits
Prestorage heating of fruits is promising as a nonchemical method for managing

postharvest pathological disorders (Paull, 1990; Klein and Lurie, 1991). In the early 1920’s,

citrus packers in California started using hot water to improve the effectiveness of a

detergent wash Evidently by accident, this treatment reduced decay of oranges (F awcett,

1922). Fungal germination and growth are inhibited by heat shock (Barkai-Golan and
Phillips, 1991). Hot water has been used more widely to control postharvest diseases than
for insect control (Couey 1989). Water dips at 38°C to 60°C for 2 to 60 min has been
reported to control in vivo and in vitro spore germination and decay development of
postharvest ﬁrngi in apples (Edney and Burchill, 1967), mangoes (Coates and Johnson,
1993; Johnson et al., 1997), melons (Teitel, et al., 1991), papayas (Akamine and Arisumi,
1953), strawberries (Couey and Follstad, 1966) and tomatoes (Barkai-Golan, 1973).

In contrast to the beneﬁcial effects of short hot-water dips used to control fungal
pathogens, these treatments were insufficient to disinfest fruit and vegetables. Therefore,
vapor heat treatment regimes between 42 and 48°C have been used (Couey, 1989). Heat-
treatments for disinfesting fruit were ﬁrst developed by Baker and co-workers before 1929
(Baker, 1952) in order to protect the citrus region from the Mediterranean ﬁ'uit ﬂy (Ceratitis
apitata). Citrus were treated using an 8h approach time and treatment at 43°C for another
8h. Vapor heat has been used as disinfestation treatment providing an assurance to the
authorities of an importing country that the commodity is free of pests (Johnson and
Heather, 1995). For instance, treatment of 46°C from 30 min to 6b is used to disinfect
mangoes from fruit ﬂies (Johnson et al., 1997). Moreover, the USDA-APHIS-PPQ (The
United States Department of Agriculture, Animal and Plant Health Inspection Service, Plant
Protection and Quarantine) approved high-temperature forced-air as a quarantine treatment
for mangoes imported into USA from Taiwan. In 1993, APHIS approved a similar heat-
treatment to disinfest grapefruit of the Mexican ﬁuit ﬂy (US. Department of Agriculture,
1993). Vapor heat or hot water disinfestation treatments has been used on mango (Johnson

et al., 1997), papaya (Akamine and Arisumi, 1953), persimmons (Derrtener et al., 1996).

1.2 The Effect of High Temperature on Fruit Ripening

Ripening, the phase of fruit development preceding senescence, includes
developmental changes to enhance the probability of seed dispersal (Giovanonni, 1993).
The ripening process involves biochemical and physiological changes occurring by various
cellular and subcellular compartments and organelles, which contribute to the desired
organolyptic attributes of texture, ﬂavor and aroma observed during ripening (Brady, 1987).
These changes include; ﬂesh softening, increase in sugar/acid ratio, enhanced color
development, and increase in aromatic compounds (volatiles related to ﬂavor development).
These physio-chemical changes are preceded by a dramatic increase in ethylene production
which sparks an increase in metabolic activities as seen by ascent in respiratory activity, .
inaease in activity of alternative oxidase and elevated rates of mRN A and protein synthesis
(Frankel et al., 1968). Molecular and genetic studies showed that ripening in tomato is
regulated at the level of gene expression (Gray et al., 1992; Fray and Grierson, 1993;
Giovanonni, 1993). Regulation by posttranscriptional events is also implicated (Blume and
Grierson, 1997). Ethylene biosynthesis and action plays an important role in initiating and
sustaining the metabolism in ripening ﬁ'uit (Zarembinski and Theologis, 1994; Lelievre et
al., 1997).

More than 50 years ago Hansen (1942) reported that ripening of pear fruit was
reversibly inhibited by holding them at 40°C for extended periods. Heat treatment evokes a
reversible inhibition of ripening in several other fruits including apple (Ponitt and
Lidster, 1978; Klein, 1989; Lurieand Klein, 1990), avocado (Eaks, 1978), papaya (Chan,
1986), pears (Maxie et al., 1974), and tomato (Biggs et al., 1988; Lurie and Klein, 1991;

Lurie et al., 1996).

The eﬁ‘ect of heat treatment on ethylene biosynthesis and action.

A common observation derived from all of the heat treatment studies with
climacteric fruits is reversible inhibition of ethylene synthesis and action (Burg and
Thimann, 1959; Maxie et al., 1974; Eaks, 1978; Biggs et al., 1988; Lurie et al., 1996).
The effect of high temperature on ethylene production in excised tissues has been
investigated (Saltviet and Dilley, 1978; Field, 1981). They demonstrated that basal and
wound-induced ethylene increased up to 37°C, thereafter declined rapidly and there was no
detectable ethylene production above 42°C.

Tomato ﬁ'uits incubated at 37°C showed reduction of ripening- associated ethylene,
regardless of the ripening-stage (Biggs et al., 1988). Both ACC synthase and ACC oxidase
activity were reduced by high temperatures (Atta-Aly, 1992; Biggs et al., 1988). Moreover,
a rapid loss of ACC oxidase activity occurs in papaya and othe’ fruits exposed for short
periods to temperatures greater than 40°C (Chan, 1986; Klein and Lurie, 1990). Upon
transferring the fruits to 25°C, there is a rapid increase in ACC synthase activity and a
slower recovery for ACC oxidase activity. Full recovery of ACC oxidase occurs within 3
days for apples (Klein and Lurie, 1990). The recovery of ethylene production required de
novo synthesis of protein since cycloheximide inhibits that process (Biggs et al., 1988).
Picton and Grierson (1988) reported that high temperatures not only inhibit ethylene
biosynthesis but also its action, since ethylene application during heat shock, did not

overcome the high temperature inhibition of fruit ripening.

The eject of heat treatment on respiration.

Carbon dioxide production and 02 uptake (respiratory activity) rise as the fruits
ripen. In the case of heated tomatoes and avocados, an increase in CO; production occurs
during exposure to high temperatures (Kerbel et al., 1987; Lurie and Klein, 1991). When
the fruits are subsequently transferred to 20°C, the respiration rate plunges to a lower level
than the control fruit (Lurie and Klein, 1991). In persimmons, however, C02 production
increases after heat treatment and then decreases (within 24 h), but remains at a higher

level than the non-heated control (Burmeister et al., 1997).

The eﬁ'ect of heat treatment on color development.

In tomato ﬁuit, heat stress delays color development during postharvest ripening
(Buescher, 1979; Cheng et al., 1988). The retardation in color development is attributed to
an induced attenuation of lycopene formation (Vogele, 1937; Sayre et al., 1953; Ogura et al.,
1975; Yang et al., 1990) and chlorophyll degradation (Tomes, 1963; Lurie and Klein, 1991).
The color development of heated fruit resumes after the temperature stress is removed
(Cheng et al., 1988; Lurie and Klein, 1992). Furthermore, hot water treatment can delay
yellowing of green tissues (Kazami et al., 1991; Tian et al., 1996, 1997; Wang, 1998). For
example, immersion of broccoli in 50°C water for 2 minutes is the most effective
treatment for reducing yellowing and decay while not inducing off-odors or accelerating

weight loss.

The eﬂect of heat treatment on ﬁrmness

Tissue softening is another parameter affected by high temperatures. Prestorage
heating of tomatoes leads to enhanced retention of fruit ﬁrmness during ripening (Hall,
1964; Ogura et al., 1975; Yoshida, et al. 1984; Cheng, et a1. 1988; Sozzi et al., 1996). The
rate of softening increased when the heat-treated ﬁuit were returned to 20°C, but it was still
less than the control fruit. Mitcham and McDonald (1992) reported that the rate of cell wall
degradation is reduced while the synthesis of soluble polyuronides continues after heat
treatment, resulting in ﬁrmer ﬁ'uits. BenShalom et al. (1996) showed that heat treatment
partially inhibited the degradation of uronic acids in the apple ﬁ'uit cell wall. In heat-
treated apple fruit, the content of galactose and arabinose in the cell wall decreased
during storage, suggesting that the inhibition of solubilization of the carbonate soluble
pectin fraction is one of the main factors contributing to the ﬁrmness retention caused by
heat- treatment. In contrast, softening-related phenomena, such as loss of neutral sugars,
pectin methylesterase activity and pectin deesteriﬁcation, were not changed by heating
(BenShalom et al., 1993; Klein et al., 1995). The retardation of softening by heat
treatment is accounted for by the absence of polygalactorunase (PG) activity (Y oshida et
al., 1984). For many year PG was thought to be the primary enzyme for tomato fruit
softening. However, recent work with transgenic plants proved that the breakdown of cell
wall polyuronides by PG was not sufficient to induce softening (Smith et al., 1988;
Giovanonni et al., 1989; DellaPenna et al., 1990). Other enzymes, including. a- and B-
galactosidases and an endo-B-mannanase, that appear earlier than PG in ﬁuit ripening
could play alternative role in fruit softening (Fischer and Bennett, 1991). Heat treatment

at 40°C for 2 days had a profound incidence on the levels of a- and B—galactosidase

10

activity in tomatoes (Sozzi et al., 1996). The recovery in B-galactosidase activity upon
transfer of fruits to 21°C was complete after 10 days. On the contrary, the recovery of
the a-galactosidase activity was very slow. Moreover, endo-B-mannanase activity, an
enzyme involved in the breakdown of mannose—containing polymer, was undetectable
after heat treatment at 40°C for 2 days, and the recovery of the activity at the permissive
temperature was incomplete (Sozzi et al., 1996). The level of cellulase activity, which is
associated with softening in avocados, was lower in heat-treated ﬁ'uit than in nonheated

controls (Klein and Lurie, 1991).

The eﬂ'ect of heat treatment on sugar/acid ratio.

The taste of tomato fruit is mainly determined by sweetness induced by the
reducing sugars and the soumess or acidity caused by the organic acid content. Heat-
treatment increases the sugar/acid ratio by 10 to 30%, which make the fruit more
attractive to the consumers (Lurie and Klein, 1992). Higher ratio of sugar versus acid has
been reported also for heat-treated apples (Klein and Lurie, 1990; 1992). Similar results
have been reported in broccoli ﬂorets (Tian et al., 1997). Sucrose content in broccoli
stems and ﬂorets increased dramatically within a short period of hot water treatment

(47°C for 7.5 min) and then declined.

The eﬂ'ect of high temperature on gene expression during fruit ripening.
Fruit ripening, involves a co-ordinated and complex set of biochemical changes and
requires the expression of speciﬁc genes (Giovanomri, 1993). As fruit ripens protein

synthesis (Frenkel et al., 1968) occurs. Molecular and genetic studies showed that ripening

ll

in tomato is regulated at the level of gene expression (Gray et al., 1992; Fray and Grierson,
1993; Giovanonni, 1993).

Picton and Grierson (1988) demonstrated that inhibition of ripening by high
temperature treatments occrured at the level of gene expression. Incubation of tomato ﬁuit
at 35°C dramatically altered the level of ripening-related mRNAs. For example, there is a
reduction in the expression of proteinase inhibitor, phytoene synthase, ACC oxidase, N-
hydroxycinnamoyl/benzoyl transferase, and membrane channel protein following heat shock
stress (Picton and Grierson, 1988). At elevated temperatures (>35°C) a decline in the
activities of polygalactrnonase (PG) (Chan et al., 1981; Yoshida, et al., 1984; Picton and
Grierson, 1988), pectinmethylesterase (PME) (Kagan-Zur, et al., 1995), ACC synthase
(Biggs, et al., 1988), ACC oxidase (Biggs, et al., 1988; Atta-Aly, 1992; Chan, et al., 1996;
Lurie, et al., 1996) and phytoene synthase (Lurie, et al., 1996) was observed. However, full
recovery of the ACC synthase, ACC oxidase, phytoene synthase (Biggs, et al. 1988; Lurie,
et al., 1996) and PME (Kagan-Zur, et al., 1995) activities were reported when the heat-
treated fruit were returned to room temperatures. The level of the PG mRN A was partially
restored at room temperature, but the rate of PG mRNA accumulation declined
exponentially with the duration of heat stress suggesting that PG gene expression was
gradually and irreversible shut-off during heat stress (Picton and Grierson, 1988; Kagan-
Zur, et al., 1995). The recovery of the PME gene expression was unaffected by the duration
of heat stress (Kagan-Zur et al., 1996). Although the accumulation of PMEs mRNA was
reduced by heat shock, PME protein and activity were unaffected, suggesting a greater

stability of the PME to elevated temperatures (Kagan-Zur et al., 1996).

12

Heat treatment appears to evoke a fundamental mechanism in gene regulation that
operates to temper metabolism while the tissue is under heat stress and restore normal
metabolism when the temperature is subsequently lowered. This might be similar to the
heat shock response common to prokaryotes and eukaryotes (V ierling, 1991). The heat
shock response involves up-regulation of synthesis and action of heat shock proteins, while
at the same time causes down-regulation of many proteins and metabolic enzymes at high
temperatures and this process is reversed when the temperature is subsequently lowered.
Kato et al., (1993) reported that heat treatment (37°C for 24 hours) induces substantial
amounts of nuclear and cytosolic small HSPs in tomato fruit. Further, cytosolic class I
smHSPs (Lurie et al., 1996; Sabehat et al., 1998a; This study), cytosolic class II smHSP
(Kadyrzhanova et al., 1998; This study) and chloroplastic smHSP (Sabehat et al., 1998a;
This study) were induced after exposure of tomato fruit at 38°C or 42°C. Moreover,

cytosolic class I smHSP has been isolated during tomato ﬁuit ripening (Fray et al., 1990).

1.3 Heat treatment Induces Resistance to Chilling Injury

While therrnotolerance can be induced in plants and plant organs by heat
treatment, heat stress is also known to induce tolerance to some other environmental
stresses (Orzeck and Burke, 1988; Kuznetsov et al., 1997). Prestorage heat treatment of
apples was found to inhibit superﬁcial scald (a chilling injury phenomenon) during
storage at 0°C (Lurie et al., 1990) while heat treatment inhibited chilling injury of
avocados (Woolf et al., 1995; Florrisen et al., 1996). Mature green tomatoes are very

susceptible to chilling injury by storage for only a few days at temperatures below 10°C;

13

symptoms normally become evident subsequent to returning them to 20°C. However, if
tomatoes are heated at 38 to 40°C for 3 days prior to storage at 2°C for 3 weeks they were
found to ripen normally and without development of chilling injury symptoms (Lurie and
Klein 1991; 1992; This study). This protection disappears if the tomatoes are transferred
from 38 to 20° C for 4 days before 2° C storage (Lurie and Sabehat, 1997). Nonheated
tomatoes remained green after removal from 2°C storage and developed typical surface
pitting and extensive microbial spoilage. Protection of tomatoes from chilling injury
afforded by prestorage heat treatment has been correlated with the induction of
transcription of smHSPs mRN A during the heat treatment and translation of the smHSPs
which persisted during subsequent storage of the ﬁuit at 2°C (Sabehat et al., 1996; 1998;
Kadyrzhanova et al., 1998). An interim of holding the tomatoes at 20°C after heat and
before storage at low temperatures, however without decreasing the expression in HSPs led
to loss of resistance to low temperatures (Sabehat et al., 1998; This study). This suggests
that the synthesis and action of HSPs afforded by heat treatment may be involved in
protecting the fruit during heating and during subsequent exposure to chilling
temperature. However, it appears that the HSPs are not the only factor contributing to
chilling tolerance phenotype (This study).

Neven et al. (1992) identiﬁed that HSP70 is involved with a metabolic adjustment of
spinach seedlings to low non-ﬁeezing temperature during cold acclimation Low
temperature stimulated the expression of most spinach HSP70 members, with cytosolic
HSC70-12 to be the most inducible and the HSC70-2 (an ER luminal member) the least
inducible (Guy and Li, 1998). This ﬁndings is consistent with the identiﬁcation of the cold

shock DnaK homologue in Ecoli (Levilent and Kawula, 1995) and the cold sensitive

I4

phenotypes derived by mutations in yeast stress 70 members (Schilke et al., 1996).
Moreover, Krishna et al. (1995) reported a role for HSP90 in adaptation to cold temperature
in B. ncpus tissues. Interestingly, an endoplasmic reticulum smHSP from potato has been
isolated during cold storage (Van Berke] et al., 1994). The cold-induced expression of HSP
genes raises the possibility that heat shock proteins may play a critical role in the cold
acclimation or the chilling resistance process in plants.

Chilling injury is thought to be a membrane-related disorder (Lyons, 1973). Murata
(1983) found that a higher degree of fatty acid unsaturation in phosphatidylglycerol was
related to chilling tolerance. Although many attempts to ﬁnd a causal relationship between
unsaturation of fatty acids and chilling have been made in higher plants, the results are
controversial (Raison and Orr, 1990). Recent experiments, (Murata et al., 1992; Welter et
al., 1992) indicate that an increase in saturation of acyl chains attached to chloroplastic
phosphatidylglycerol can increase cold-induced photoinhibition. In contrast, Wu and
Browse (1995) reported that the fabl Arabidopsis mutant, which contain 43% of high
melting point phosphatidylglycerol, was unaffected by chilling temperatures. Moreover,
recent evidence indicates that at low temperature, chs 1 Arabidops'is mutants are deﬁcient in
the accumulation of chloroplast proteins, probably due to the lack in a chaperonin that is
required to fold chloroplast proteins at low temperature (Schneider et al., 1995). On the
other hand, the function of the unsaturated fatty acids in chilling tolerance has been
demonstrated using the fad mutants of Arabidopsis thaliana, which are defective in
desaturation of membrane lipids (Somerville and Browse, 1991). The fad mutants (fora,
fad5 and faab) have reduced amounts of polyunsaturated fatty acids and are more sensitive

to chilling (Hugly and Somerville, 1992; Miquel et al., 1993). The facﬂ mutants is deﬁcient

15

.LM

“'3”.
g b.-

-' a
~a

5 II

V' A ‘
ht'l.

b».

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r":
"a"...

in chloroplast (D-3 fatty acid desaturase activity, which is responsible for the formation of
trienoic fatty acids (16:3 and 18:3) in leaf tissue (The, et al., 1993). Kodama et al. (1994)
overexpressed the FAD7 cDNA in tobacco plants and chilling injury was reduced,
suggesting the importance of the polyunsaturated fatty acids in chilling tolerance. Recent
data indicated that the increased fatty acid desaturation during chilling acclimation at 15°C
is one of the factors involved in normal leaf development at low, .nonﬁ'eezing temperatures
(Kodama et al., 1995). Similarly, genes for mitochondrial catalase 3 isozyme (Prasad et al.,
1994) and HSP70 related proteins (Cabane et al., 1993) are induced during chilling
acclimation at 10-20°C and may act in concert to increase chilling tolerance.

Organisms adapt to low temperature by increasing the proportion of cis-acting
unsaturated fatty-acyl groups in their membrane lipids and thus increase membrane ﬂuidity
(Hazel, 1995). Fatty acid desaturase activity is induced during low temperature (Nrshida
and Murata, 1996). Recently, Murata and Wade (1995) produced cyanobacteria that display
chilling tolerance by transforming them with A12-desaturase. A decrease in membrane
ﬂuidity was reported to induce desaturase genes (Vigh et al., 1993).

It has been suggested that the heat treatment institutes a response to high temperature
stress in the tissue that leads to strengthened membranes (Lurie et al., 1997). These
treatments led to an increase in phospholipid content, a lower sterol to phospholipid ratio
and more unsaturated fatty acids, relative to the unheated tissues (Lurie et al., 1997).

Interestingly, Carratu et al. (1996), using yeast as a model, proposed that the ratio of
saturated fatty acid versus unsaturated fatty acid and disturbance of membrane lipoprotein
complexes are involved in the perception of rapid temperatures changes, and that under heat

shock conditions perturbation of the preexisting physical state of the membranes causes

16

transduction of a signal that induces transcription of heat shock genes. The induced HSPs
may bind to membrane lipoproteins (Moczko et al., 1995) leading to stabilization of
membrane topology. Indeed interaction between heat shock proteins and membranes has
been reported in many organisms (V igh et al., 1998). In Synechocystis cells, following heat
shoclg both GroEL and Hsp17 are associated with thylakoids (Kovacs et al., 1994; Horvath
et al., 1988). Membrane bound HSPs might act by preventing denaturation of membrane
localized enzymes (T orok et al., 1997). Therefore, the membrane acts as a sensor and the
altered saturation remotely activates a mechanism, which enhances HSP transcription
(Carratu et al., 1996). Similar speculation has been proposed for the desaturase genes (V igh
et al., 1993). In contrast, no correlation was found between changes in fatty acid
composition and the thermotolerance in plants (Rikin et al., 1993). Moreover, changes in
heat tolerance are caused by protein factors (Nishiyama et al., 1993).

Interestingly, induction of gene expression by low temperature has been
demonstrated in different plant tissues (Guy et al., 1985; Mohapatra et al., 1989;
Thomashow et al., 1993). Schatfer and Fischer (1988) have shown in tomato ﬁuit that the
expression of three mRN As including one that homologues to a cDNA clone encoding a
thiol protease, increased at 4°C suggesting that proteolysis might occur during chilling. The
level of these transcripts was different in cold sensitive and cold tolerant varieties,
suggesting that genetically determined cold tolerance inﬂuences cold inducible gene
expression. Lately, Yu et al. (1996) compared the changes in gene expression between
two different chilling tolerant tomato fruits, the hybrid “NY” (L. esculentum x L.
pimpinellifolium) known to be chilling tolerant, and the chilling sensitive “EC”. Two low

molecular weight basic translation products (18kDa, pI 8.0 and 14kDa, pI 8.2,

17

respectively) were noticed. The ﬁrst polypeptide increased during chilling while the
latter was sharply reduced during chilling and reaccumulated after transfer to 20°C. The
down-regulation of many translation products might be related to the delay in ripening at
low temperature. Conversely, chilling-induced translation products could play an
important role in metabolic adjustments at lew temperature and in the protection of
existing cellular structures. The chilling sensitivity of the “EC” might involve inhibition
of phosphatidylcholine transfer from the endoplasmic reticulum to the chloroplast at low
temperature.

Heat shock proteins have been reported to inﬂuence protein folding (Weish et al.,
1992) and probably plant HSPs assist in refolding of proteins denatured by cold temperature
stress. Neven et al., (1992), proposed that accumulation of HSPs at low temperature may
not only be indicative of a stress condition but also may have adaptive values. During
heating of tomato ﬁuit at 38°C ﬁve proteins of molecular weights between 15-22 kD, a HSP
at 70 kD, and two above 80 kD were newly and abundantly synthesized (Lurie and Klein,
1991). Similarly sized HSPs have been described in tomato shoot and root apices (Koning
et a1, 1992), in tean cell cultures (Never et al., 1989), and tomato leaves (Fender and
O'Cenell, 1990). Additionally, normal proteins synthesized at 20°C are not detectable by
SDS-PAGE dming the heat treatment. Furthermore, mRNA for the two heat shock proteins
HSP17 and HSP 70, remained abundant in heat-treated tomatoes stored at 2°C for three
weeks while it disappeared rapidly from tomatoes placed at 20°C after heat treatment (Lurie

et al., 1996). Recently, two other members of heat shock proteins with molecular weights

23 and 18 kD were found in heat-treated tomato fruits stored at 2°C for 21 days (Sabehat et

18

al., 1996). Therefore, the heat shock proteins in tomato fruits may act as chaperones and

their continued presence at low temperature suggests their importance in chilling tolerance.

2. Heat Shock Response, Heat Shock Proteins and Cellular Metabolism

Thermal stress evokes rapid induction of speciﬁc proteins termed heat shock
proteins (HSPs) (Key et al., 1981; Lindquist and Craig, 1988; Vierling, 1991; Parsell and
Lindquist, 1994). In plants, Key et al. (1981) have demonstrated the rapid induction of
speciﬁc proteins under thermal stress. The induction of genes encoding heat shock proteins
is triggered by an abrupt increase in temperature of 5-10°C above the normal growing
temperature (Kimpel and Key, 1985). Proteins induced by heat stress can be assigned to
11 families conserved among all organisms. They vary in size from 15 to 110 kD and
within size classes share considerable homology at the amino acid level (Vierling, 1991;
Never and Scharf 1997). Isoferms within a HSP family have similar or identical
biochemical ﬁrnction but differ in their intracellular localization and their regulation.
Partiwlarly, the low molecular weight HSPs in higher plants are encoded by six nuclear
gene families (Waters et al., 1996). Classes I and II encode cytosolic proteins while
classes III and IV encode chloroplast and endoplasmic reticulum localized proteins
(Vierling, 1991; Helm et. al, 1993). A ﬁfth class encodes the mitochondrial proteins
(Lenne and Deuce, 1994; Dong and Dunstan, 1996). For class VI, which is represented
by a single 22.3 kD HSP from soybean, the intracellular location is proposed to be the

endoplasmic reticulum (Lafayette et. al., 1996). In many cells it has been observed that

19

HSPs or homologues are expressed constitutively, under developmental or cell cycle control
(Lindquist and Craig, 1988).

According to Vierling (1991), the induction of HSPs is an autonomous cellular
phenomenon Heat stress induces rapid transcriptional regulation of the HSPs by binding
of the heat shock transcription factor (HSP) to a common promoter sequence called the
heat shock element (HSE) (Never et al., 1990; Gurley and Key, 1991; Sorger, 1991;
Merimete et al., 1996). HSFs in plants are encoded by at least 3 members and some of
them are heat-inducible (Never et al., 1996).

Heat shock disrupts splicing of numerous mRNA precursors (Y est and Lindquist,
1988), thus determining the nature of mature mRNAs to be transcribed. Heat stress also
aﬁ'ects gene expression at the translational level by blocking translation of preexisting
mRNAs while pre-existing pelysemes decline (Brostrom and Brostronr, 1998). HSP
mRN As appear rapidly in response to heat, become associated with pelysemes and
translation of HSPs accounts for them becoming the major products of protein synthesis
(Lindquist, 1980). Heat shock in plants was recently found to increase mRN A stability of
a reporter mRNA (Gallie et al., 1995). Mature mRNAs existing prior to heat stress
remain eligible for translation in cell-free systems but in vivo HSP mRNAs are
preferentially translated (Lindquist, 1981). This major alteration in protein synthesis
while the cells are under thermal stress is fully reversible upon lowering the temperature.
A normal pattern of protein synthesis is restored, depending upon the severity and
duration of the heat stress and synthesis of HSPs diminishes (DiDomenico et al., 1982a).
In addition, cells transferred gradually to high temperatures recover much more rapidly than

cells raised suddenly to the same temperature (Lindquist and DiDomenico, 1985). The

20

repression of general translation likely serves to restrict the production of missense,
incomplete, or misfolded proteins that might subsequently have a deleterious effect on cell
growth (Duncan, 1996). The magnitude of preferential translation of some and repression of
other normal proteins differs between cell types, species and heat shock conditions. For
example, in Drosophila cells, inhibition of general translation becomes detectable at around
32°C and reaches maximum inhibition at 37°C (10 degrees above the normal temperature),
while in mammalian cells, protein synthesis is inhibited at 41°C (Duncan, 1996).
Following the heat stress, cells recover full translational activity. Contrary to the repression,
the recovery process requires several hours (DiDomenice et al., 1982b). When more severe
heat shocks are administrated, the recovery is delayed (DiDomenice et al., 1982b; Lindquist,

1993 ), although the non-heat shock mRN As appear to recover synchronously.

3. Small Heat Shock Proteins

Small heat shock proteins dominate the protein synthesis proﬁle of many plants
during heat stress and particularly the cytosolic classes can accumulate to over 1.0% of
the total leaf or root cell protein under certain heat shock conditions (I-Isieh et al., 1992;
DeRocher et al., 1991). Vierling (1991) has grouped the smHSPs irrte four nuclear gene
families based on amino acid sequence, immunological cross-reactivity and intracellular
localization. Two classes are cytosolic proteins and the other two are organelle-localized,
targeting to endoplasmic reticulum (ER) (Helm et al., 1993) and chloroplasts,

respectively. Recently, LaFayette et a1. (1996) proposed to expand the classes to six

21

including a mitochondrial class (Lenne, 1995) and an additional endemembrane class of
smHSPs. This diversiﬁcation of the small heat shock proteins is unique to plants.

The small heat shock proteins showed less sequence similarity than the highly
conserved HSP70 proteins, including both comparisons between divergent species and
between diﬁ'erent classes of plant smHSPs (Waters et al., 1996). One of the major
characteristics of the lOw molecular weight HSPs is the conserved carbexyl-terminal
domain (heat-shock domain) of about 100 amino acids, which is also found in the or-
crystallin proteins of the vertebrate eye lens (DeJong et al., 1993). This domain is further
subdivided into two subdomains, consensus I and II, separated by a variable hydrophilic
region. The consensus I subdomain includes the motif P-meGVL which is
characteristic for all the smHSPs. Interestingly, a similar motif appears in the consensus
H subdomain region P-X(14)-X-V/L/I-V/L/I (Waters et al., 1996). Moreover, these
regions have the same hydropathy proﬁles and secondary structure prediction (Caspers et
al., 1995).

In contrast to the carboxyl terminus, the amino-terrninal region is more variable
with little homology among the different classes of low molecular weight HSPs
(V ierling, 1991). It has been proposed that the amino-terminal region interacts with
different substrates er relates to distinct ﬁrnctions (V ierling, 1991). Furthermore, the N-
tenninal region also includes sequences that determine localization of organelle-
associated HSPs (Waters et al., 1996). However, within the amino-terminal regions of
the mature proteins, consensus domains Unique to each class of smHSP have been
identiﬁed (Waters, 1995). The presence of these highly conserved domains suggests that

they serve important roles in protein ﬁrnction (Waters et al., 1996).

22

With a few exceptions, the smHSPs are not expressed by vegetative tissues in the
absence of heat stress. The small heat shock proteins accumulate rapidly during high
temperature stress and in proportion to the temperature and duration of the stress
(Vierling, 1991). Maximum synthesis and accumulation of smHSPs is observed at
temperatures just below lethal temperatures (Howarth, 1991). Quantitative analysis
indicates that class 1 proteins accumulate to ever 1% of total leaf protein during heat
stress (DeRocher et al., 1991; Hsieh et al., 1992) and they are considered as the most
abundant class of smHSPs. The chloroplast heat shock proteins have been estimated to
be 0.02% of the total protein content (Chen et al., 1990). The organelle-localized HSPs
are less abundant than the cytoplasmic class I and II HSPs, which may reﬂect a lower
protein content of the endemembrane system relative to the cytosol. The half-life of the
small HSPs following the heat stress is 30-50 h (Chen et al., 1990; DeRocher et al.,
1991), indicating their function may be critical for the recovery period.

The plant smHSPs, like the other HSPs, are regulated at the transcriptional level
in response to heat stress (Gurley and Key, 1991). The presence of multiple genes
encoding heat shock factors that bind to the heat shock element are differentially
regulated by heat shock, suggesting that these transcription factors may regulate genes in
response to signals other than heat stress (Waters et al., 1996).

The small heat shock proteins are also regulated by a variety of other
environmental and developmental factors (Arrige and Landry, 1994). Contrary to the
high molecular weight heat shock proteins, the smHSPs are not constitutively expressed
in the normal cells, indicating that the smHSP ﬁrnction is restricted to specialized cellular

conditions shared by developmental and stressed stages (Waters et al., 1996). The best

23

characterized cases of developmental regulation are expression during pollen
development (Beuchard, 1990; Dietrich et al., 1991; Atkinson et al., 1993; Kobayashi et
al., 1994) and during seed maturation (Vierling 1991; DeRocher and Vierling, 1994).
Small HSPs have been identiﬁed also during fruit ripening (Fray et al., 1990; Lawrence
1997). In addition, heat shock proteins are induced by other stress such as cold, drought,
salinity, ethanol, heavy metals, ozone and oxidative stress (Andersen et al., 1994;
Celmenero-Flores et al., 1997; Ruis and Schuller, 1995; Eckey-Kaltenbach et al., 1997;
Banzet et al., 1998). These stress conditions could damage or denature cell proteins. In
yeast, inhibition of protein breakdown with the preteasome inhibitors caused a coordinate
induction of many heat shock proteins (Bush et al., 1997; Lee and Goldberg, 1998).
Further, treatments with amino acids analogues as well as introduction of unfolded
proteins triggers the induction of HSPs (Ananthan et al., 1986; Lee et al., 1996). The
induction of the heat shock response can protect cells against a variety of other toxic
insults, such as ethanol and hydrogen peroxide (Ruis and Schuller, 1995; Storz and Pella,

1996).

4. Function of the Heat Shock Proteins

Speciﬁc functions of HSPs such as HSP60, 70 and 90 have been ascribed to
protein/protein interactions in protein folding and aggregation (Boston et al., 1996;
Buchner, 1996; Hth 1996; Bukau and Horwich, 1998). The HSPs could refold stress-
damaged proteins by functioning as molecular chaperenes. These proteins prevent

denaturatien and aggregation as well as promote the refolding of denatured proteins.

24

Speciﬁcally, the HSP90, 70, 60 classes are thought to aid in the proper folding of the newly
synthesized nascent peptides, transport across membranes, assembly of oligomeric
complexes and maintenance of steroid receptor conformation The small HSP proteins
which are unique to plant kingdom, have been shown recently to maintain proper folding
of reporter proteins and exhibit in vitro chaperone activity (Ehrnsperger et al., 1997; Lee
et al., 1997; Lee et al, 1995a). The recombinant HSP 18.1 and HSP 17.7, representing
class I and class H cytosolic smHSPs from pea, were able to enhance the refolding of
chemically denatured model substrates citrate synthase and lactate dehydrogenase and
prevented their aggregation and irreversible inactivation (Lee et al, 1997). In soybean,
HSPs-enriched ﬁactions were able to therrnostabilize mainly . membrane-associated
proteins (Jinn et al., 1993). Based on these data, it is suggested that smHSPs act in vivo
as a type of molecular chaperone to bind partially denatured proteins preventing
irreversible protein inactivation and aggregation, and that smHSP chaperone activity
contributes to the development of therrnetolerance (Waters et al., 1996). In contrast to
high molecular weight HSPs, the activities of the small heat shock proteins are not
stimulated by nucleotides (Boston et al., 1996).

Under non-denaturing conditions in different organisms, the small heat shock
proteins have been found to form high molecular weight complexes, between 200-800
kD. The class I smHSPs complexes are approximately 200-300 kD in size (Helm et al.,
1993; Jinn et al., 1995). Similar sizes have been observed in class H (Helm et al, 1997),
chloroplast (Clarke and Critchley, 1994; Chen et al., 1994; Osteryeung and Vierling,
1994) and mitochondrial (Lenne and Deuce, 1994) smHSPs. In the presence of high salt

concentration and nonionic detergent, the complexes keep their integrity (Jinn et al.,

25

1995) suggesting that strong ionic interactions and hydrophobic forces stabilize the high
molecular weight complexes (Chen et al., 1994). Jinn and colleagues (1995) observed
that the isolated 280 kD smHSP complex from soybean was able to protect up to 75% of
the total soluble proteins of the cell from heat denaturation in vitro. These smHSP
complexes can associate into insoluble larger cytoplasmic aggregates termed as “heat
shock granules” (Never et al., 1990). It has been suggested that these HS granules are
transient sites for non-heat shock mRN A, preventing its degradation during heat stress
(Never 1991). These large structures may be common to all smHSPs (Osteryeung and
Vierling, 1994) and their formation may be reversible and occur mainly at highly
stressing temperatures (Waters et al., 1996).

The HSP18.1 (cytosolic class I from pea) has been shown to facilitate the
refolding of chemically or heat-denatured model substrates (Lee et al., 1997).
Furthermore, HSP 18.1 prevents aggregation of substrate proteins heated at 45°C. Using
size exclusion chromatography, Lee et al., 1997 found that the binding of the
mitochondrial malate dehydrogenase to smHSPs is hydrophobic in nature and that high
temperatures increase the available binding surface. Moreover, based on ﬂuorescent
probe bis-ANS [1,1’-bi(4-anilino) naphthalene-5,5’-disulfonic acid] incorporation and
substrate protection experiments, the consensus H region is critical for substrate binding
(Lee et al., 1997). Similarly, Jinn et al. (1995) reported that immunodetectable smHSPs
from heated plant extracts shifts to a higher molecular weight on native gels, while the
puriﬁed smHSPs when heated alone did not show size shitting. Based on these data,
Boston et al. (1996) suggested a model in which the smHSPs capture unfolding

polypeptides by hydrophobic interactions and keep them in state capable to refeld.

26

However, separation of the smHSP/ substrate complex was insufﬁcient without
disasseciation of the smHSPs eligemers themselves. This observation suggests that
substrate release in vivo is triggered by synergistic interactions with other chaperones
(Boston et al., 1996). In a different way, the substrate/smHSP interactions may facilitate
proteolytic degradation of the substrate (Boston at al., 1996). Interestingly, Forreiter et
al. (1997) using a stable transformed Arabidopsis cell suspension culture overexpressing
luciferase as a reporter gene, demonstrated that the cytosolic class I smHSP showed
chaperone activity in vivo, while the HSP90 protects the Arabidopsis cells only during
recovery from the heat stress. More importantly, although the HSP70 was unable to
protect the luciferase during stress, the HSP70 and the smHSPs could act synergistically
during the refolding process. This is in agreement with the notion that the
substrate/smHSPs complexes association is stimulated by interaction with the high
molecular weight HSP.

Another important function of certain HSPs is to promote the rapid degradation of
abnormal proteins (Hershke, 1988; Vierstra, 1993; Sherman and Goldberg, 1996). In
eukaryotes, ubiquitin and certain ubiquitin-conjugating enzymes are involved in the rapid
breakdown of denatured proteins (Parsell and Lindquist, 1994). In addition, certain
molecular chaperones have been shown to serve as cofactors in the selective degradation
of abnOrmal polypeptides (Kandror et al., 1994; Lee et al., 1996). Some of these proteins
are proteases whereas others are additional components involved in substrate recognition.

Although in vitro experiments have demonstrated that the smHSPs potentially act
as molecular chaperones, evidence for the role of smHSP in stress conditions varies

dramatically among organisms. Until recently there has only been correlative evidence

27

that HSPs protect cells from deleterious effects of heat stress or other environmental
stresses (Never, 1991; Vierling, 1991). Lee et al. (1995b) have shown by genetically
engineering Arabidopsis to derepress the activity of heat shock factor (AtHSFI) that
constitutive synthesis of HSPs is enhanced and this increased thermotolerance. Schoffl
and his colleagues (Prandl et al., 1998) had isolated another two HSFs from Arabidopsis
(HSF 3 and HSF 4). Transgenic Arabidopsis that overexpress constitutively the HSF 3
derepressed the heat shock response and conferred thermotolerance. Conversely,
Arabidopsis 35S: :HSF 4 transgenic plants were unable to synthesize HSPs constitutively
and as a results were incapable of bestowing thermotolerance. The AtHSFl and AtHSF3
are expressed in the wild type plants (Hubel and Schoﬁl, 1994; Prandl et al., 1998) but
they require heat for activation of DNA binding and this involves aggregation ﬁ'om
monomer to trimer forms (Hubel et al., 1995). Moreover, Lee and Schoffl (1996)
indicated that the plant HSP70 is involved in early stages of HSF inactivation, possible
by dissociation of the HSF trimers. It is highly plausible that the mechanism of heat
activation of HSPs in Arabidopsis may serve as a general model for other plants.
Alternatively, in vivo studies of the HSFs from soybean, Arabidopsis and tomato,
conducted in a transient expression system using GUS as a reporter gene driven by HSE,
demonstrated that plants have two classes of HSFs; the HSF class A which activate
transcription (activators), and the HSF class B which are relatively abundant, albeit
repressors of the heat shock response (Czarnecka-Vemer et al., 1997). Thus, the active
HSFs may be replaced by competition from repressor. The mechanism by which heat

treatment evokes thermotolerance of tomato fruit to heat may ﬁt this model.

28

Despite the extensive research concerning the mechanisms that regulate activation of
HS gene expression, the nature of the “primary sensor” or how the signal is transferred to

the nucleus is not known (Morimote, 1993).

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43

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conservation. Plant Mol Biol 26: 1579-1597

CHAPTER I

PRESTORAGE HEAT TREATMENT REDUCES CHILLING INJURY IN

TOMATO FRUIT

ABSTRACT

The ripening of the tomato (Lycopersicum esculentwn Mill. cv. Mountain
Springs) fruit and the development of chilling injury in relation to heat treatments was
studied for three different years. Mature green tomatoes were held at 38°C, 42°C or 45°C
for 8 to 48 hours prior to storage at 2°C for 2 or 3 weeks. After cold storage they were
transferred to 20°C for 3 to 9 days. Fruits were also stored at 2°C without prior heat
treatment and others were ripened at 20°C as controls. Chilling injury (CI) was prevented
by heat treatment at 42°C for 36 or 48 hours prior to cold storage, whereas ﬁuit stored at
2°C without preheating developed typical CI symptoms and failed to ripen at 20°C.
Some protection was lost if the tomatoes were transferred ﬁom 42°C to 20°C for l to 3
days before low temperature storage; however, the CI was less than that of control fruits.
The heat-treated ﬁuits ripened normally although more slowly than non-treated tomatoes.
In contrast, the non-heated tomatoes stored at 2°C remained green and developed typical

CI symptoms upon return to a permissive ripening temperature. The heat-treated and

45

 

 

non-heated fruits were ﬁrmer after storage at low temperatures than the freshly harvested
tomatoes. In summary, prestorage heat treatment permits storage for longer periods at
low temperatures without CI development than is the case for non-treated fruits and with

no loss of their ability to ripen normally.

 

INTRODUCTION

Mature green tomatoes, and other tropical and subtropical fruits like bananas,
develop chilling injury (CI) if held below 10°C. Chilling injury is progressively more
severe as the storage temperature is lowered and storage duration at low temperatures is
extended (Wang, 1994a). Symptoms of CI (failure to ripen, non-uniform ripening,
surface pitting and increased incidence of decay) may not be evident while the fruits are
at low temperature but appear after the hits are returned to permissive ripening
temperatures of 15 to 25°C (Cheng and Shewfelt, 1988). Ripening of tomatoes is only
minimally retarded at temperatures that do not cause CI; therefore, the postharvest
storage life is limited to only a few days or a few weeks at best.

In recent years, there has been growing interest in heat treatments as a method to
reduce chilling injury in horticultural crops, thus permitting extended storage times
(Hatton, 1990; Klein and Lurie, 1991). Elevated temperatures alter tomato fruit ripening
characteristics such as lycopene formation (Ogura et al., 1975; Lurie and Klein, 1991),
chlorophyll degradation (Lurie and Klein, 1992), tissue softening (Y oshida, et al. 1984;
Cheng, et al. 1988; Mitcham and McDonald, 1992), lipid composition (Whitaker, 1994),
volatile production (McDonald et al., 1996), as well as both respiration rate (Lurie and
Klein, 1991; 1992) and ethylene production (Biggs et al., 1988; Lurie and Klein, 1991).
Prestorage high temperature treatments that reduce CI can be divided into two categories:
longterm(12hto4daysinair)at38to46°C and shortterm(upto60mininwater)at45 to
60°C (Klein and Lurie, 1991).

The objective of this study was to develop, optimize, and characterize a

commercially applicable postharvest heat treatment for cold storage of tomato ﬁuit. Herein,

47

 

 

we present data ﬁom three years of several long-term heat treatments on chilling tolerance
of tomatoes. Parameters evaluated included chilling injury symptoms, color development
and ﬂesh ﬁrmness. We conclude that heat treatment at 42°C for 36 or 48 hours was

appropriate not only to provide chilling tolerance but also to permit normal ripening after

storage.

MATERIAL AND METHODS

Experiments Conducted in 1994

Mature green tomato fruits (cv. Mountain Springs) were purchased from a
grower/shipper in Southwest Michigan. Two replicates of ten fruits each were used per
treatment at the various temperature regimens. The maturity/ripening indices monitored
were: i) ﬁrmness with a modiﬁed Chatillon NY gauge taking 4 penetrations into the
internal pericarp of the ﬂesh of the fruit; ii) color development using a Michigan State
University tomato chart scale, namely 1 (MG) to 5 (Dark red); and iii) chilling injury (CI)
development expressed as chilling incidence index (1; 0% CI, 2; 10% CI, 3; 30% CI, 4;
>50% CI and 5; >80% CI). The temperatures used for the heat treatment were 38, 42, and
45°C with 3 different durations for each treatment: 24, 48, and 72 h for the 38°C, 12, 24,
and 48 h for the 42°C treatment, and 8, l6, and 24 h for the 45°C heat treatment.
Immediately after the heat treatment, the hit were stored at 2°C for up to 2 weeks. At
weekly intervals, two replicates of each treatment were taken to 20°C, and

maturity/ripening indices were determined after 1- or 7-day shelf life at 20°C. Fruits

48

maintained continuously at 20°C and at 2°C for 2 weeks before ripening at 20°C,

respectively, served as controls.

Experiments Conducted in 1995

The 1994 experiment was repeated in 1995 with the same tomato cultivar. Three
replicates of ten ﬁuits per treatment were used. The temperatures used for the heat
treatment were 38, 42, and 45°C with different durations for each treatment: 48, 72 h at
38°C, 12, 24, and 48 h at 42°C, and 12 h at 45°C. Immediately after the heat treatment,
the fruits were stored at 2°C for up to 3 weeks. Color and chilling index were monitored

as above.

Experiments Conducted in 1997

Mature green tomato ﬁuits (cv. Mountain Springs) were purchased from a
grower/shipper in Southwest Michigan Three replicates of ten ﬁuits were used per
treatment at the various temperature regimens. The maturity/ripening indices monitored
were: i) color, development using the chromatomaer (Minolta, Japan) and ii) CI
development expressed as chilling index (1; 0% chilling injury (CI), 2; 10% CI, 3; 30% CI,
4; >50% CI and 5; >80% CI). Color development was measured as the Hunter “a” value.
Three measurements were taken per fruit in the middle of the pericarp. Based on the
previous year’s data, the temperature used for the heat treatment was 42°C with 4 different
durations for each treatment: 12, 24, 36 and 48 h. Immediately after the heat treatment, the
ﬁuits were stored at 2°C for up to 3 weeks. In addition, following heat treatment mm were

transferred to 20°C for 1 or 3 days and then stored at low temperatures. At weekly intervals,

49

three replicates of fruits ﬁom each treatment were taken to 20°C and maturity/ripening
indices were determined after 3, 6 or 9 days shelf life at 20°C. Fruits maintained
continuously at 20°C and at 2°C before ripening at 20°C, respectively, served as untreated

and chilling controls, respectively. Standard error (SE) is indicated by bars.

RESULTS

Experiments Conducted in 1994

Flesh ﬁrmness of heated and nonheated ﬁuits was not affected by low-
temperature storage at 2°C for 7 days. However, low-temperature storage delayed tissue
softening during ripening compared to the control fruits (Figure 1). After 7 days at 20°C,
heat-treated and non-heated ﬁuits were at the pink stage or color index 3 (Figure 2).
There was no signiﬁcant difference among the treatments. The heat-treated fruits required
about twice as long at 20°C to develop full red color after chilling compared to the non-
chilled control ﬁ'uits. The major signiﬁcant difference observed between heated and non-
heated ﬁuits was in the chilling injury incidence (Figure 3). Seven days at low
temperatures was not enough to induce CI in control fruits, but 14 days was enough to
cause typical symptoms. Only heat treatment at 42°C for 48 h attenuated the CI
symptoms, which developed severely for ﬁ'uits of the other heat treatments in relation to
the control at 2°C without previous heat treatment (Figure 3). The ﬁ'uits kept at 45°C
ripened slowly but suffered from internal breakdown as well as heat injury. In addition,
40% of the ﬁuits developed irregular color during 7 days at 20°C, which may be due to

both heat and cold injury.

50

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Experiments Conducted in 1995

Similar to the 1994 data, color development was delayed by low temperature
storage in heated and nonheated ﬁuits but the fruits ripened normally (Figure 4).
However, after 3 days at 20°C there were no signiﬁcant differences between the heat
treatments at a given heat regimen for fruits stored for 2 or 3 weeks at 2°C. After 9 days
at 20°C, the color index ranged from 2 (in the 42°C 48-h treatment) to 3.5 (in the 42°C
12-h treatment). The control ﬁ'uits developed red color aﬁer 9 days at room temperature.
Similar to the previous year’s results, chilling injury was prevented by heat-treating
tomato fruits at 42°C for 48-h (Figure 5). Control ﬁuits developed chilling injury
symptoms after storage for 3 weeks at low temperature. Surprisingly, two weeks of low
temperature storage was not enough to induce chilling injury. Fruits from the other heat
treatments developed chilling injury, but the incidence of the injury was lower or similar
to that of the control ﬁuits. The symptoms of the chilling injury become more evident as

ripening of the stored ﬁuits proceed.

Experiments Conducted in 1997

Color development was measured as the Hunter “a” value. Three measurements
were taken per hit in the middle of the pericarp. Negative values were obtained for
mature green ﬁ'uit, values near to 0 were given to turning fruit and the positive values for
pink and red ﬁ'uits (Figure 6). Nonheated fruits developed color normally at 20°C and
reached positive values in 6 days. In contrast, ﬁuits receiving low temperature for 2
weeks failed to develop red color and remain green even after 6 days at 20°C. The heat-

treated fruits ripened slowly, and came to the pink stage after 15 days at room

54

 

 

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Figure 4. The effect of heat treatment on color development of tomato fruits after storage at 2°C for 14 or 21 days and during ripening at

20°C (year 1995).

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temperature. Overall, the shelf life of the heat-treated/cold stored tomatoes was extended
to 32 days after harvest, while the control ﬁ'uits attained the same ripening stage in 6
days.

Chilling injury was circumvented by heat-treating mature green tomatoes at 42°C
for 36 or 48 hours prior to storing them at 2°C for two weeks, whereas ﬁuits stored at
2°C without preheating developed typical chilling injury symptoms and failed to ripen at
20°C (Figure 7). This protection was lost if the tomatoes were transferred from 42°C to
room temperature for l or 3 days before cold storage; nevertheless the chilling injury was
less than that of the control fruits (Figure 7). Likewise, heat shock of tomatoes at 42°C
for 12 or 24 hours was not sufficient to protect the ﬁuit from low temperature. These
treatments, however, were able to reduce decay signiﬁcantly compared to the control
ﬁuits (data not shown). Visual difference in the incidence of chilling injury and the
ability to ripen, between the heat-treated fruit at 42°C for 36 or 48 hours and the control
ﬁuits stored at 2°C for 14 days, are shown in Figures 8 and 9, respectively.

Collectively, heat treatment of 42°C for 36 and 48 h attenuates chilling injury of

tomato ﬁuit stored at 2°C up to 2 or 3 weeks; the response varied among years.

57

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Figure 8. The effect of heat treatment (42°C for 36-h) and post-treatment at 20°C for 1
day on chilling injury of tomato fruits subsequently stored at 2°C for 14 days and

ripened at 20°C.

    

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day on chilling injury of tomato ﬁ'uits subsequemly stored at 2°C for 14 days and
ripened at 20°C.

61

DISCUSSION

The effect of 42°C treatment for 36 h or 48 h on reducing C1 are in agreement with
previous studies showing that long-term (3 8°C for 48 h) heat treatment conferred chilling
tolerance to tomatoes (Lun'e and Klein, 1991; 1992, McDonald et al., 1996; Lurie and
Sabehat, 1997). Short-term heat treatment (42°C for 60 min) was also reported to beneﬁcial
for maintaining fruit quality after low temperature storage (McCollum and McDonald, 1993;
McDonald et al., 1996; 1998). In our studies, however, the 38°C for 48 h did not prevent CI
following storage.

Depending on the year in which the experiment was conducted, heat treatment was
effective in preventing chilling injury of fruits stored at 2°C for up to 3 weeks. It appears
that pre-harvest or the growing season environment factors may impact on the effectiveness
of heat- treatment on CI attenuation In addition, cultivar differences may determine
whether a heat- treatment will be effective. For example, the cultivar that Lune and Sabehat
(1997) used, “Daniella”, is a slow ripening variety that increases the shelf life and allows
storage up to 4 weeks at 2°C after heat treatment. The variety used in the present study,
“Mountain Springs” or the “Rutgers” used in other studies (Whitaker, 1994), do not appear
to respond favorable to the same heat treatment (38°C for 48 h).

Heat treatment was also found to induce resistance/tolerance to chilling injury in
avocado (Sanxter et al., 1994; Woolf et al., 1995; Florissen et al., 1996), citrus ﬁuits
(Rodov et al., 1995) cucumber (McCollum et al., 1995), mango (McCollum et al., 1993),

mung bean hypocotyls (Collins et al., 1993; 1995), sweet pepper (Mencarelli et al.,

62

1993), persimmons (Burmeister et al., 1997; Lay-Yee et al., 1997), and zucchini squash
(Wang, 1994b).

We wanted to test if the protection against CI conferred by heat treatment before
storage would be lost if the heated fruit were held at 20°C for 1 or 3 days prior to storage at
2°C for 14 days (Figure 7). Indeed, the protection against CI was lost especially in the
short-term heat treatment (42°C for 12 or 24 h). Similar results were reported by Lurie and
Sabelmt (1997).

The protective role of heat treatment against chilling injury in tomatoes has been
correlated with the accumulation of heat shock proteins (Sabehat et al., 1996; 1998;
Kadyrzhanova et al., 1998, chapter III of this study also). HSPs and especially the small
HSPsarenotpreseminﬁuitsstoredatlowtemperatureunlesstheyhadbeenheat-treated
(Sabehat et al., 1998; this study chapter Ill). HSPs accumulated in the cold-stored fruits that
were held at room temperature after heat shock and prior to low temperature storage
(Sabehat et al., 1998; this study chapter III). These results suggest that heat-shock proteins
are not the only factor responsible for the protection of the tomato ﬁuits from C1. Sabehat
et al. (1998) suggested that the expression of the small HSP is correlated with the protection
against some, but not all, symptoms of CI. Small HSPs are important components of the
heat shock response and possess molecular chaperone activity in vitro and in viva (Forreiter
et al., 1997; Lee et al., 1997). At this moment, however, there is no report suggesting
chaperone activity of the smHSP at low temperatures.

Another possible reason for the protection against chilling injury is related to the
production of ethylene. A number of studies (Kosiyachinda and Young, 1976; Chaplin et

al., 1983; Lee and Young, 1984; Florissen et al., 1996), some of them contradictory (Lipton

63

and Aharoni, 1979) have suggested that increased levels of ethylene are associated with
increased levels of chilling injury. Avocados were more chilling sensitive during the
climacteric rise and at the climacteric peak (Kosiyachinda and Young, 1976). Ethylene
application increased the severity and the threshold temperature of chilling injury (Chaplin
et al., 1983; Lee and Young, 1984; Florissen et al., 1996). In contrast, Lipton and Aharoni
(1979) reported that ethylene reduces CI in melons. The longer the period of the heat
treatment, the lower were the peak levels of ethylene production (Biggs et al., 1988; Lurie
and Klein, 1991). Tomato fruits that received prolonged heat shock had gained chilling
insensitivity (Lurie and Klein, 1991). Antisense ACC oxidase expression in transgenic
melon has been reported to confer chilling tolerance, while ethylene application restored
chilling sensitivity (J C Pech, personal communication).

In conclusion, the protection against CI afforded by heat treatment may be due to
the production of HSPs as well as to the reduction in ethylene production or sensitivity.
Therefore, heat treatment of tomato fruits has very signiﬁcant potential for commercial
application for several reasons: (1) Wholesomeness of the fruit may be improved,
avoiding the use of postharvest-applied chemicals to control some important
physiological and pathological disorders; 2) ripening can be delayed; and 3) the storage
duration can be more than doubled without evoking chilling injury disorders and the

accompanying decay.

REFERENCES

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inhibition of ethylene biosynthesis in ripening tomato fruit. Physiol Plant 72:
572-578

Burmeister DM, Ball S, Green S, Woolf AB (1997) Interaction of hot water treatments
and controlled atmosphere storage on quality of 'Fuyu‘ persimmons. Postharvest
Biol Technol 12: 71-81

Chaplin GR, Wills RBH, Graham D (1983) Induction of chilling injury in stored
avocados with exogenous ethylene. HortScience 18: 952-953

Cheng TS, Shewfelt RL (1988) Effects of chilling exposure of tomatoes during
subsequent ripening. J Food Sci 53: 1160-1162

Cheng, TS, Floros JD, Shewfelt RL, Chang CJ (1988) The effect of high temperature
stress on ripening of tomatoes (Lycopersicon esculentum). J Plant Physiol 132: 459-
464

Collins GG, Nie X, Saltviet ME (1993) Heat Shock increases chilling tolerance of mung
bean hypocotyl tissue. Physiol Plant 89: 117-124

Collins GG, Nie X, Saltviet ME (1995) Heat shock proteins and chilling sensitivity of
mung bean hypocotyls. J Exp Bot 46: 795-802

Florissen P, Ekman JS, Blumenthal C, McGlasson WB, Conroy J, Holford P (1996)
The effects of short heat treatments on the induction of chilling injury in avocado
fruit (Persea americana Mill). Postharvest Biol Technol 8: 129-141

Forreiter C, Kirschner M, Nover L (1997) Stable transformation of an Arabidopsis cell
suspension culture with ﬁreﬂy luciferase providing a cellular system for analysis
of chaperone activity in viva. Plant Cell 9: 2171-2181

Hatton '11 (1990) Reduction of chilling injury with temperature manipulation. In CY
Wang, ed, Chilling Injury of Horticultural Crops. CRC Press, Boca Raton,
Florida, pp 269-280

Kadynhanova DK, Vlaehonasios KE, Ververidis P, Dilley DR (1998) Molecular
cloning of a novel heat induced/chilling tolerance related cDNA in tomato fruit by
use of mRNA differential display. Plant Mol Biol 36: 885-895

Klein JD, Lurie S (1991) Postharvest heat treatment and fruit quality. Postharvest
News Info 2: 15-19

65

Kosiyachinda S, Young RE (1976) Chilling sensitivity of avocado fruit at different
stage of the respiratory climacteric. J Amer Hort Sci 101: 665-667

Lay-Yee M, Ball S, Forbes SK, Woolf AB (1997) Hot-water treatment for insect
disinfestation and reduction of chilling injury of 'Fuyu' persimmon. Postharvest
Biol Technol 10: 81-87

Lee GJ, Roseman AM, Saibil HR, Vierling E (1997) A small heat shock protein stable
binds heat-denatured model substrates and can maintain a substrate in a folding-
competent state. EMBO J 16: 659-671

Lee SK, Young RE (1984) Temperature sensitivity of avocado fruit in relation to
ethylene treatment. J Amer Hort Sci 109: 689-692

Lipton WJ, Aharoni Y (1979) Chilling injury and ripening of “Honey Dew”
muskmelons stored at 2.5 or 5C aﬁer ethylene treatment at 20C. J Amer Soc Hort
Sci 104: 327-330

Lurie S, Klein JD (1991) Acquisition of low temperature tolerance in tomatoes by
exposureto high temperatures. J Amer Soc Hort Sci 116: 1007-1012

Lurie S, Klein JD (1992) Ripening characteristics of tomatoes stored at 12°C and 2°C
following a prestorage heat treatment. Sci Hort 51: 55-64

Lnrie S, Sabehat A (1997) Prestorage temperature manipulations to reduce chilling
injury in tomatoes. Postharvest Biol Technol 1: 57-62

McCollum TG, D'Aquino S, McDonald RE (1993) Heat treatment inhibits mango
chilling injury. HortScience 28: 197-198

McCollum TG, Doostdar H, Mayer RT, McDonald RE (1995) Immersion of
cucumber fruit in heated water alters chilling-induced physiological changes.
Postharvest Biol Technol 6: 55-64

McDonald RE, McCollum TG, Baldwin EA (1998) Heat treatment of mature-green
tomatoes: Differential effects of ethylene and partial ripening. J Amer Soc Hort
Sci 123: 457-462

McDonald RE, McCollum TG, Baldwin EA (1996) Prestorage heat treatments
inﬂuence free sterols and ﬂavor volatiles of tomatoes stored at chilling
temperature. J Amer Soc Hort Sci 121: 531-536

Mencarelli F, Ceccantoni B, Bolini A, Anelli G (1993) Inﬂuence of heat treatment on
the physiological response of sweet pepper kept at chilling temperature. Acta
Hort 343: 23 8-243

Mitcham EJ, McDonald RE (1992) Effect of high temperature on cell wall modiﬁcations
associated with tomato fruit ripening. Postharvest Biol Technol 1: 257-264

Ogura N, Nakagawa H, Takehana H (1975) Effect of high temperature-short term
storage of mature green tomato fruits on changes in their chemical composition alter
ripening at room temperature. J Agr Chem Soc Japan 49: 189-196

Rodov V, Ben-Yehoshua S, Alhagli R, Fang DO (1995) Reducing chilling injury and
decay of stored citrus fruit by hot water dips. Postharvest Biol Technol 5: 119-
127

Sabehat A, Lurie S, Weiss D (1998) Expression of small heat-shock proteins at low
temperatures. A possible role in protecting against chilling injuries. Plant Physiol
117: 651-658

Sabehat A, Weiss D, Lurie S (1996) The correlation of heat-shock protein accumulation
and persistence and chilling tolerance in tomato fruit. Plant Physiol 110: 531-537

Sanxter SS, Nishijima KA, Chan HT Jr (1994) Heat-treating "Sharwil" avocado for
cold tolerance in quarantine cold treatments. HortScience 29: 1166-1168

Wang CY (1994a) Chilling injury of tropical horticultural commodities. HortScience
29: 986-988

Wang CY (1994b) Combined treatment of heat shock and low temperature conditioning
reduces chilling injury in zucchini squash. Postharvest Biol Technol 4: 65-73

Whitaker BD (1994) A reassessment of heat treatment as a means of reducing chilling
injury in tomato ﬁ'uit. Postharvest Biol Technol 4: 75-83

Woolf AB, Watkins CB, Bowen JH, Lay-Yee M, Maindonald JH, Ferguson ID
(1995) Reducing external chilling injury in stored “Hass” avocados with dry heat
treatments. J Amer Soc Hort Sci 120: 1050-1056

Yoshida O, Nakagawa H, Ogura N, Sato T (1984) Effect of heat treatment-on the
development of polygalacturonase activity in tomato ﬁuit during ripening. Plant
Cell Physiol 25: 505-509

67

CHAPTER II

MOLECULAR CLONING OF A NOVEL HEAT-INDUCED CHILLING-
TOLERANCE RELATED cDNA IN TOMATO FRUIT BY USE OF mRNA

DIFFERENTIAL DISPLAY

ABSTRACT

Chilling injury was circumvented by heat-treating mature green tomatoes
(Lycopersicon esculentum, Mill. cv. Mountain Springs) at 42°C for two days prior to
storing them at 2°C for one or two weeks, whereas fruits stored at 2°C without preheating
developed typical chilling injury symptoms and failed to ripen at 20°C. Using
diﬁ‘erential display of mRN A and screening of the cDNA libraries, we have cloned from
tomato hit a full-length HC T 1 cDNA (heat induced/chilling tolerance related). The
protein (17.6 kD) predicted from the coding region of HCT 1 cDNA has high identity with
cytosolic class 11 small HSPs. The gene corresponding to HCTI cDNA was termed
LeHSPI 7. 6. Southem-blot hybridization indicates that LeHSPI 7. 6 belongs to a two-
member gene family. Northern blot analysis indicates the heat-induced transcript of the
LeHSP17. 6 remains up-regulated during subsequent exposure of the fruit to chilling

temperatures for at least one week and upon transfer to ripening temperatures for one

68

day. Fruits that were only chilled show a low level of expression of the LeHSPl 7. 6
transcript. We hypothesize that LeHSP17. 6 may be involved in protecting the cell from
metabolic dysfunctions leading to ripening failure caused by chilling injury. This is the

ﬁrst report of a cytosolic class H smHSP-encoding gene in tomato.

69

INTRODUCTION

The useﬁrl postharvest life of many fruits of tropical or subtropical origin is
limited because they must be stored above 10-12°C to avoid chilling injury (Lyons,
1973). For example, if mature green tomatoes are stored for a few days at temperatures
below 10°C and subsequently returned to a normally permissive ripening temperature,
they fail to ripen and become susceptible to microbial spoilage (Hobson, 1987).
Prestorage heat treatment has been found to increase chilling tolerance of tomato and
other subtropical fruits (Hirose, 1985; Lurie and Klein, 1991; McCollum et al., 1993;
Sanxter a al., 1994). Increased tolerance to chilling injury by high temperature treatment
has been related to the accumulation of heat-shock proteins (HSPs) (Lafuente et al., 1991;
Lurie and Klein, 1991). Plant HSPs consist of a few high molecular weight classes 60
kD, 70 kD, 90 kD, 100 kD and a complex group of low molecular weight proteins with
molecular sizes ranging from 17 to 30 kD (Key et al., 1981; Nover et al., 1990; Vierling,
1991). The small HSPs are structurally related and are encoded by six discrete gene
families (Waters et al., 1996). Classes I and II encode cytosolic proteins while classes III
and IV encode chloroplast and endoplasmic reticulum localized proteins (V ierling, 1991;
Helm et. al, 1993). A ﬁﬁh class encodes the mitochondrial proteins (Lenne and Douce,
1994; Dong and Dunstan, 1996). For class VI, which is represented by a single 22.3 kD
HSP from soybean, the intracellular location is proposed to be the endoplasmic reticulum
(Lafayette et. al., 1996). Cytosolic small HSPs are particularly abundant in plants
subjected to stress conditions but their speciﬁc functions are not known. Recently, in

vitro studies demonstrated that small HSPs as well as high molecular weight HSPs

70

display elements suggesting molecular chaperone activities (Lee et al., 1995a). While
they ostensibly function in the development of thermotolerance (Lee et al., 1994; Lee et
al., 1995b; Yeh et al., 1995), some plant HSPs are expressed when the plant is subjected
to other stresses such as water stress (Almoguera et al., 1993), heavy-metal toxicity
(Neumann et al., 1994) and cold stress (Neven et al., 1992; Cabane et al., 1993;
VanBerkel et al., 1994; Krishna et al., 1995).

To gain insight about the molecular mechanisms by which heat treatment affords
subsequent protection against chilling injury in tomato fruits, we employed the
differential display of mRNA technique. The differential display of mRNA method
(Liang and Pardee, 1992) has been proving to be a good tool for detecting speciﬁcally
expressed genes in plant tissues (Goorrnachtig et al., 1995; Johnson et al., 1995; Oh et al.,
1995; Sharma and Davis, 1995; Wilkinson et al., 1995; Van der Knaap and Kende, 1995;
Tieman and Handa, 1996). We herein report the identiﬁcation and cloning of a full-
length HC T 1 cDNA (heat-induced/chilling tolerance related). HC T 1 cDNA encodes a
putative 17.6 kD protein which has high identity with cytosolic class 11 small HSPs of
other plants. The gene corresponding to HCTl cDNA was termed as LeHSP17. 6
(Kadyrzhanova et al., 1998). The transcript ofLeHSP17.6 is induced by heat treatment at
42°C for 2 days and remains up-regulated during a subsequent one week exposure to
chilling temperatures of 2°C. The heat-treated fruits ripened normally following the low
temperature storage while non-heated fruits showed typical chilling injury symptoms.
We hypothesize that the translation product of LeHSPI 7. 6 may be involved in protecting

the cell from the metabolic dysfunctions leading to ripening failure caused by chilling

injury.

71

MATERIALS AND METHODS

Plant Material and Temperature Treatments

Tomato (Lycopersicon esculentum Mill. cv. Mountain Springs) hits were
harvested at the mature green stage and subjected to heat treatment at 42°C for 2 days
(designated as H) followed by cold storage at 2°C for 7 days and 1 day at 20°C
(designated as HC). Fruits that received only cold storage at 2°C for 7 days plus 1 day at
20°C (designated as C); links at the mature green stage (MG) served as controls. Fruit

pericarp tissue from each treatment was frozen in liquid nitrogen and stored at -80°C.

Nucleic Acid Isolation.

Total RNA was extracted and puriﬁed according to (Grierson and Covey, 1976)
and Fray and Grierson (1993) with some modiﬁcations. Frozen fruit pericarp was ground
in liquid nitrogen with a mortar and pestle and extracted with buffer containing: 1%
triisopropylnaphthalene sulfonate, 6% p—aminosalicylate, 5% (v/v) Tris-saturated phenol
and 50 mM Tris-HCl, pH 8.0). The homogenate was extracted 4 times with an equal
volume of phenolzchloroform (1:1) then once with chloroform. RNA was precipitated at
-20°C by addition of 0.1 vol 3M sodium acetate, pH 5.6 and 2.5 vol of ethanol in two
steps: ﬁrst, 1 vol of ethanol was added and if a carbohydrate and DNA precipitate
formed, this was removed and the remaining 1.5 vol of ethanol was added. RNA was
precipitated with 4M lithium chloride and the pellet was dissolved in 250mM potassium

acetate pH 7.0. The RNA was precipitated with 3 vols of ethanol, the pellet washed with

72

80% (v/v) ethanol, dried and dissolved in diethylpyrocarbonate (DEPC)-treated water.
RNA was quantiﬁed and qualiﬁed by spectrophotometrically. Integrity of RNA was
evaluated by fractionation of an aliquot on 1.2% agarose/formaldehyde gel.

Poly (A)+ mRN A was isolated from total RNA using the PolyAT-tractR mRNA
isolation kit (Promega, Madison, WI). Concentration of poly(A)+ mRN A was measured
using the DNA DipStickm kit (Invitrogen, San Diego, CA).

Genomic DNA was isolated from tomato leaves as described by Doyle and Doyle
(1987) except that SDS was added to the extraction buffer instead of CTAB. DNA
concentration, purity and integrity were determined spectrophotometrically and by

running an aliquot on 0.8% agarose gel.

Differential Display.

Diﬂ‘erential mRN A display (Liang and Pardee, 1992) was performed using the
RNA mapTM kit (GenHunter, Brookline, MA) according to the manufacturer’s
recommendation. Total RNA samples were DNase I treated as described by Liang et al.,
(1993) using RNase-free DNase I and human placental ribonuclease inhibitor
(Boehringer Mannheim, Indianapolis, IN). DNase-free total RNA samples (0.2 pg) were
used for the cDNA synthesis. The reverse transcription (RT) reaction consists of TuMN
anchor primers (where M is degeneration of A, C and G, while N is one of the four
nucleotides). PCR ampliﬁcation of one-tenth of the RT products was done in the
presence of [”P] dATP (Bauer et.al., 1993). AmpliTaq polymerase (Perkin-Elmer Cetus
Norwalk, CT) was used. Five decamers (AP1 to AP;) were used in combination with the

respective TuMN. Control reactions were performed in the absence of reverse

73

transcriptase. The ampliﬁed cDNAs were separated by electrophoresis on 6% denaturing
polyacrylamide gel, containing 7 M urea at constant 60-W for 3 to 4 h, followed by
vacuum-drying at 80°C on Whatman 3MM without ﬁxation. X-ray ﬁlm was exposed to
the dried ﬁlm overnight. The 32read labeled (pX174/Hae m (GIBCO-BRL, Life

Technologies, Gaithersburg, MD) was used as the molecular weight markers.

Isolation of cDNA Bands.

The appropriate differential display cDNA bands were excised and eluted
according to the manufacturer’s instructions. Each gel was reexposed to X-ray ﬁlm to
verify that the band of interest had been correctly excised (Zimmerman and Schultz,
1994). cDNAs were then reampliﬁed using the same PCR conditions and primers as
above. The size of the PCR products was determined on a 1.2% agarose gel. cDNAs

were puriﬁed from agarose slices using the QIAEXIITM kit (Qiagen, Chatsworth, CA).

RNA Gel Blot Analysis.

Total RNA (15 pg) was separated on a 1.2% agarose gel containing 2.2 M
formaldehyde and transferred to Hybond-N nylon membranes (Amersham, Aylesbury,
UK) as described by Sambrook et al. (1989). The membranes were ﬁxed by using a
microwave oven for 2 min at ﬁrll setting (700 W) (Angeletti et al., 1995).
Prehybridization and hybridization was performed according to standard procedures
(Sambrook et al., 1989). The blots were prehybridized for 3 h in hybridization buffer
(50% formarnide, 5X SSC, 25mM potassium phosphate, pH 7 .4, 5X Denhardt’s solution,

50 pg mL'l denatured salmon sperm DNA) at 42°C. The eluted cDNAs were labeled

74

with [ct-”PJdCTP (DuPont/NEN Products, Boston, MA) using the random primer
labeling kit (GIBCO-BRL, Life Technologies, Gaithersburg, MD) in the presence of the
TuMN oligomer. After alkaline denaturation, 32P-labeled probe was added to
hybridization buffer containing dextran sulfate in 5% (w/v) ﬁnal concentration and
hybridized for 16 h at 42°C. Membranes were washed twice with 1X SSC and 0.1% SDS
at room temperature for 15 min and once with 0.2x SSC and 0.1% SDS at 60°C for 30
min before exposing X-ray ﬁlm to the membranes overnight or periods up to 7 days.
Transcript size was estimated by comparing the position of the hybridizing band to RNA

molecular-weight markers (GIBCO-BRL).

The Cloning of cDNA Bands.

Following RNA blot hybridization, cDNA bands were recovered using the
Northern-blot afﬁnity capturing method (Li et al., 1994). A piece of the nylon
membrane containing the captured probe was cut out, using the autoradiograrn for band
localization. The membrane pieces were stripped by boiling in water for 5 min, and the
DNA was precipitated with 0.3 M sodium acetate and ethanol using glycogen as
carrier. The reconstituted probe was reampliﬁed by PCR using the same conditions as
described above. An aliquot of the remaining reaction was ligated into pCRlI vecror of
the TA—Cloning kit (Invitrogen, San Diego, CA). Multiple plasmid preparations were
performed for each clone using the standard alkaline lysis method (Sambrook et al. ,
1989) and analyzed by restriction digestion with EcoRI. cDNA band inserts were
puriﬁed from agarose slices, random primed radiolabeled and used in RNA blot

analysis.

75

Library Construction and Screening.

Ampliﬁed cDNA libraries were constructed from heat-treated (H) and heat- and
cold-treated (HC) tomato fruit poly (A)+ mRNA in Agt 11 vector using the CapFinderm
PCR cDNA Library Construction kit (Clontech, Palo Alto, CA) according to
manufacturer's instructions. Screening of the (H) and (HC) cDNA libraries was done
by the Long-Distance (LD)-PCR based method (Bames, 1994; Cheng et al., 1994; Ali-
Osrnan and Akande, 1995). In a 50 pL reaction mixture, 106 phages from (H) or (HC)
cDNA libraries were screened by ampliﬁcation with 50 ng of the 3' gene-speciﬁc
primer (30-mer) derived from a sequence near the poly (A) tail of HCTI cDNA' band
and 50 ng of the 5'-PCR primer (30-mer) supplied in the CapFinder1M PCR cDNA
Library Construction kit (Clontech, Palo Alo, CA). The LD-PCR band was eluted

from the agarose gel and cloned into pCRlI vector by using TA—cloning.

DNA Sequencing and Analysis.

Double-stranded DNA sequencing was performed on an Applied Biosystems
373A (Foster City, CA) at the Michigan State University DNA Sequencing Facility,
East Lansing, MI. Sequences were compared to the National Center of Biotechnology
Information nonredundanr sequence database (www.ncbi.nlm.nih. gov) using the default
settings of BLASTN or BLASTX (Altschul et al., 1990). DNA sequence data were

assembled and analyzed using DNA STAR (DNA STAR, Madison, WI).

76

Southern Blot Analysis.

Genomic DNA (20 pg) was digested with appropriate restriction enzymes and
separated by electrophoresis on a 0.8% agarose gel. Blotting and hybridization
conditions were perfonned as described by Sambrook et al. (1989). DNA blots were
washed three times with 2X SSC and 0.1% SDS at room temperature for 15 min and
twice with 0.1x SSC and 0.5% SDS at 42°C for 15 min before X-ray ﬁlm was exposed to

the membrane overnight with double intensifying screens at -80°C.

RESULTS

Differential Display.

The effect of heat and/or cold treatment on tomato fruit gene expression at the
transcriptional level was studied using the differential display of mRNA (Liang and
Pardee, 1992). Twenty RT-PCR reactions for each total RNA samples were conducted
by combining ﬁve 5’ arbitrary decamers (APl-AP5) and four 3’ anchor primers (TnMG,
TnMC, TuMA, TnM'I'). An example of the diﬁ‘erential display pattern is presented in
Figure 1. About 40-80 ampliﬁed bands ranging ﬁ'om 100 to 600 bp in size were visible
for each primer set. The anchor primers T ”MA and TuMT showed lower speciﬁcity and
selectivity than the TnMG and TnMC primers. From a total of about 1200 cDNA

bands, 142 showed an increase as a result of heat shock (H), 41 showed high intensity

77

Figure 1. Differential display of total RNA from four different treatments of tomato
fruit. (1) RNA was isolated from control fruits at the mature green (MG) stage, (2) heat-
treated at 42°C for 2 days (H), (3) heat-treated at 42°C for 2 days followed by storage at
2°C for one week and then transferred to 20°C for 1 day (HC), and (4) ﬁuits that were
stored at 2°C for one week followed by one day at 20°C (C). Total RNA was reverse
transcribed with the anchored primer TuMC. The anchor primer and the arbitrary
primer (AP1, APz, AP3, AP4, and M5) were used for the ampliﬁcation step of differential
display. 32P-end labeled <pX174/Hae 111 (Gibco BRL) was used as molecular weight
marker. The arrows indicate putative candidate, bands that were induced by heat shock
(2) and maintained during low temperature storage (3).

78

TrzMC

APS

AP4

AP:

AP:

Al’r

 

  

 

 

 

“4.8 ‘8‘?! wﬁe.
. a .4. we 8...?
Wang“... . ﬂ. ......utu a... ....

 

 

 

 

Am). . w Q
a!

 

 

 

4

e8. e.

.. mirOmnﬁsﬁ. ﬁe? .. I.

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79

upon heat shock and subsequently cold temperatures (HC), while 18 were up-regulated
during cold treatment (C) (Table I). Twenty-nine cDNAs were speciﬁcally expressed in
both H and HC treatments and selected for further evaluation. After elution from the gel,
these bands were reampliﬁed with the same set of primers as used for the differential
display under the same PCR conditions. The size of the fragments was veriﬁed on 1.2%
agarose gels by electrophoresis. Other differential display bands derived from the same
sets of primer combinations in RT-PCR reactions were detected but have not been

completely characterized.

Expression Pattern of the Speciﬁc Fragments Corresponding mRNAs during Heat
Shock and Subsequent Chilling Temperatures.

To verify the authenticity of the differential display bands, the reampliﬁed
cDNAs were used as probes for RNA blot hybridization. Upon Northern analysis,
twenty-one cDNAs showed levels of induction that conﬁrmed the differential display
pattern (Figure 2). The cDNAs were absent in mature green tomato fruit, while they
were rapidly up-regulated at 42°C. Interestingly, the level of induction remained elevated
during the subsequent period of 7 days at 2°C followed by one day at room temperature.
However, transcripts homologous to the selected cDNAs were detected at a lower level of
induction in fruits, which had only been chilled. The length of the transcript was
approximately 0.8 kb. Northern blot analysis could detect but not conﬁrm the speciﬁc
pattern for some cDNA ﬁ'agments, while other ﬁagments did not reveal any detectable

hybridization signals.

80

Table 1. Analysis of differential display cDNA bands corresponding to H, HC, C and

 

 

 

 

 

 

 

MG tomato fruit RNA.
Number of cDNA bands
Primer combinations in Total
RT-PCR reactions H HC C (including MG)
AP; 8 2 4 58
AP; 9 1 1 53
thMG AP; 15 5 0 54
AP4 13 2 1 57
APs 5 l O 41
AP: 16 5 10 69
AP; 7 5 0 64
TnMC AP; 1 l 1 1 73
AR; 10 3 l 61
AP; 8 2 0 65
AP; 4 2 0 77
AP; 1 4 0 69
TnMA AP; 1 1 O 67
AP4 5 1 0 57
AP; 3 l O 41
AP; 7 1 0 54
AP; 4 1 0 59
TnMT AP; 7 1 0 64
AP4 2 l _ 0 39
AP; 6 l O 56
Total 142 41 l 8 l 196

 

81

Cloning and Sequence Analysis of the Specific cDNA Bands.

The cDNA bands conﬁrmed by RNA blot analysis were recovered using the
Northern blot afﬁnity capturing method (Li et al., 1994), and cloned into the pCRIl
plasmid vector by using TA Cloning. The cloned cDNA ﬁagments were released from
the pCRII vector by digestion with EcoRI and random prime labeled for use as a probe in
RNA blot hybridization. Northern blot analysis resulted in an autoradiogram (Figure
2)very similar to that obtained previously.

The partial cDNA clones were termed HC T (heat-induced/chilling tolerance
related). Herein, we present one representative named as HCTl. The HCTl nucleotide
sequence contained the decamer and the oligo (dT) primer used for the differential
display as well as part of the open reading frame (ORF) (underlined in Figure 3).
Database searches revealed the following similarities: At the nucleotide level, the 273-bp
HC T1 ﬁagment showed 79% similarity with the cytosolic class 11 small HSP from
soybean (Raschke et al., 1988), and pea (Lauzon et al., 1990). The carboxyl end (40
amino acids) of the ORF present in the HCTI encoding protein had 92 % identity to the
soybean cytosolic class 11 small HSP and contained the conserved GVL motif that is
common to all the small heat-shock proteins (Waters et al., 1996). The 3’ non-coding
region of the HCTl contained a putative polyadenylation signal AATAAA 104 bp

upstream of the poly (A) tail (Joshi, 1987).

82

 

4— 0.8“)

   

 

 

 

Figure 2. RNA blot hybridization analysis of total RNA from tomato fruit.
Approximately 15 pg of total RNA from control mature green tomato fruit (MG), heat-
treated fruit at 42°C for 2 days (H), heat-treated fruit as (H) followed by storage at 2°C
for one week and then placed at 20°C for one day (HC) and non heat-treated fruit stored
at 2°C for one week and transferred at 20°C for one day (C) was hybridized with 32P
random primed labeled reampliﬁed HCTI cDNA. The mRNA molecular weight is
indicated.

83

Construction, Screening of the cDNA Library and Sequence Analysis of a HCTI
cDNA.

To isolate a full-length HCTI cDNA, ampliﬁed cDNA libraries were constructed
from heat-treated and from heat- and cold-treated tomato fruit poly (A)+ mRNA,
respectively, in Agtll (Clontech) according to the manufacturer’s instructions. Screening
of the cDNA libraries was performed by LD-PCR based screening (Ali-Osman and
Akande, 1995). The screening of 106 independent phages using a speciﬁc 3’UTR primer
(5’-GGCATCAAAACAAACAATACCCATGGCTAT-3’) from HCTI and the speciﬁc
5’ cap primer (30-mer) supplied by the CapFinderTM PCR cDNA Library Construction kit
(Clontech) conﬁrmed the presence of about 0.8 kb PCR products. DNA sequencing of
the cloned LD-PCR product revealed a full-length HC T 1 cDNA 738 bp in size. The
entire sequence of one of them (designated as LeHSP 17. 6) is presented in Figure 3.
Translation of the LeHSPI 7. 6 cDNA sequence revealed an uninterrupted reading frame
of 477 nucleotides starting at the ﬁrst ATG (108) and ending with a TGA 154 bases
upstream of the poly (A) tail.

The deduced amino acid sequence of the LeHSPl7.6 (Figure 3) corresponds to a
polypeptide of 158 amino acids with an expected molecular mass of 17.6 kD and a pI of
6.63. A BLAST search of the nonredundant protein database showed that LeHSP17 .6
protein is highly similar (>70%) over its entire length to cytosolic class 11 small heat-
shock proteins (Vierling, 1991). The highest identity (75%) was found to the 17.7 kD
HSP from pea (Lauzon et al., 1990), 74% to the 17.9 kD HSP from soybean (Raschke et

al., 1988), 73% to the 17.9 kD HSP from parsley (Eckey-Kaltenbach, et al., 1997)

84

TACGGCTGCGAGAAGACGACAGAAGGGGACTGCAATTACAAATCAAACCAAAATT 55
GACAAATTTCACGCACAAAATCACAATATCCAAAAATTTCTCAATACTGAAAATG 110
M 1
GATTTGAGGTTGTTGGGTATCGATAACACACCACTCTTCCACACTCTCCACCATA 165
D L R L L G I D N r P L F n r L a H 19
TGATGGAAGCTGCCGGTGAAGATTCCGACAAGTCTGTCAATGCACCATCAAGGAA 220
hr at a A. A. G a D s D K S ‘v 'N .A. P s R. N 38
CTATGTTCGTGATGCTAAGGCCATGGCTGCTACACCAGCGGATGTGAAGGAGTAT 275
Y 'v R D .A K .A.:M .A .A r P A. D 'v K n Y 56
CCTAATTCGTATGTTTTTGTTGTGGATATGCCAGGGTTGAAATCTGGAGATATCA 330
P N s Y ‘v F ‘v 'v D :M P G L K s G D I 74
AAGTGCAGGTGGAAGAAGACAATGTGCTGTTGATTAGTGGTGAAAGGAAGAGGGA 385
K ‘v Q ‘v a a D N ‘v L L I s G a R K R a 93
AGAAGAGAAAGAAGGTGCAAAGTTTATTAGGATGGAGAGAAGGGTTGGGAAATTC 440
a a K E G .A K r I R. M: a R R ‘v G K r 111
ATGAGGAAGTTTAGTCTGCCAGAGAATGCGAATACTGATGCAATTTCTGCAGTTT 495
M R K F s L P a N .A. N T D .A. I s A.'V 129
GTCAAGATGGAGTTCTGACTGTTACTGTTCAGAAATTGCCTCCTCCTGAGCCAAA 550
c Q D G 'v L r 'v r V’ Q K L P P P a P K 148
GAAACCCAAAACAATTGAGGTGAAAGTTGCTTGAAGTTATGGACTCTGTTTTGAT 605
K P K r I a 'v K ‘v .A . 158
GGTTTGTGGTATGATGTAGTAGAEEEERRGTTGTAGGAGTAGTGAACTTTTCCTT 660
TCATCTTTCTGCTATGTTTTCACGTCTGTTTGAATGTTACAATAGCCATGGGTAT 715
TGTTTGTTTTGATGCCAAAAAAA 738

 

 

 

Figure 3. Nucleotide and deduced amino acid sequences of the LeHSPI 7.6 cDNA.
The putative polyadenylation signal is boxed. The differential display HCTI nucleotide
sequence is underlined. The GenBank Accession Number of the LeHSPI 7. 6 sequence is
U723 96.

85

BamHl HindIII EcoRI

 

‘1
at

 

 

 

Figure 4. Genomic Southern blot analysis of the tomato LeHSP17.6. Tomato
genomic DNA (20 pg) was digested with BamHI, Hindﬂl or EcoRI. The restriction
fragments were separated on an agarose gel, blotted on nylon membrane and probed with
LeHSP17.6 cDNA without its poly(A) tail under low stringency hybridization and
washing conditions.

and 70% with small HSPs from alfalfa (Keleman et al., unpublished) as well as with the

17.2 kD HSP from Phcn'bitis nil (Krishna et al., 1992).

Southern Blot Analysis.

To estimate the LeHSP17.6 gene copy number, Southern blot analysis of genomic
tomato DNA digested with three different restriction enzymes (EcoRI, BamI-Il, HindIII)
was conducted, using the entire LeHSPI 7. 6 cDNA without its poly(A) tail as the probe
under low-stringency hybridization and washing conditions. As shown in Figure 4, two
bands were detected in each restriction digestion, indicating that the LeHSPl 7. 6

transcript is encoded by a two-member gene family in the tomato genome.

DISCUSSION

Employing the differential display of mRN A techniques and screening the cDNA
libraries, we have identiﬁed and cloned a ﬁrll-length LeHSPI 7. 6 cDNA (heat-
induced/chilling tolerance related) whose expression pattern changes during heat
treatment and subsequent cold storage of tomato ﬁ'uits. Northern blots conﬁrmed the
speciﬁc expression of the LeHSPI 7. 6 cDNA. Only transcripts ﬁ'om heated- and heated-
and chilled-tomato tissues were preferentially expressed. These data Show that
LeHSP17. 6 cDNA originates ﬁ’om a gene(s) whose transcription is activated by heat
treatment and maintained up-regulated during subsequent cold storage. The protein (1 7 .6

kD) predicted from the coding region of LeHSP17. 6 cDNA has high identity with

87

cytosolic class H small HSPs ﬁ'om other plants. This is the ﬁrst report of a cytosolic class
H smHSP encoding gene in tomato.

The optimal multiple sequence alignment identiﬁed several conserved amino
acids among the LeHSP17.6 and the other cytosolic class II smHSPs (Figure 5). The
carboxyl end of LeHSP17 .6 contains the conserved “heat shock” domain. These
conserved domains consist of two subdomains I and H separated by a variable length
hydrophilic region (Waters et al., 1996). The subdomain I contains the motif P-th-GVL
(where X is any amino acid) and the subdomain H consists of a similar motif P-X14-N-
V/L/I-V/L/I (Waters et al., 1996). The signiﬁcance of these conserved carboxyl terminus
domains for the structure and ﬁlnction of smHSP has not been determined (Waters et al.,
1996). Contrary to the carboxyl end, the amino terminus of the LeHSP17 .6 differs from
that of the other smHSPs. This may confer some functional speciﬁcity to the protein.

Interestingly, LeHSP17.6 includes a putative protein kinase C phosphorylation
site S28DK in the N-terminal region where the serine residue is preferentially
phosphorylated by protein kinase C (Woodget et al., 1986) and a putative cAMP
dependent protein kinase phosphorylation site RKFS” (Zetterqvist, 1990) at the C-
terminus, suggesting that phosphorylation of the LeHSP17 .6 might occur. However, in
early studies, Nover and Scharf (1984) failed to detect phosphorylation of the smHSP in
tomato culture cells. Similarly, Suzuki et al. (1998) reported that the HSP21 and the
cytoplasmic class I and class H smHSP do not appear to be phosphorylated during heat
stress. Waters et al. (1996) suggested that the lack of the consensus RXXS

phosphorylation motif is a possible explanation of the insufﬁcient smHSP

88

 

1 60

Pi sum sa tivum MD ..... LDS . PLFNTL .HHIMDLTDDTT . . EIQILNAPTRTYVRDAIGMAATPADVKEHP
Glycine max MDFRVMGLES . PLFHTL . QHMMDMSEDGAGDNKTHNAPTWS YVRDAKAMAATPADVKEYP

P. crispum MDFRLMGFN . HPLLNTLSPHCDEDNQDSSNKNKSEQA. . RSYVRDAIO-XMATTPADVKEYP
Pharbi tis nil MDLRLMGFD.HPLF. . . .HHIMDYAGDD. .KSSNSSAPSRTFMLDAIQMAATPADVIGYP
Medi on go 55! t iva WFRIMGIJQQC . YTAL . HQMMDLS DENVEKS S SHNAPTRS YVRDAIQMAATPADVKENP
LeHSP17.6 MDLRLLGIDNTPLFHTL.HHMMEAAGEDg..QESVNAPSRNYVRDAKAMAATPADVKEYP
Consensus ID A Dunn 11PM P

61 I 120
Pisum sa tivum NSYVFMVDMPGVKSGDIKVQVEDENVLLISGERKRE. EEKEGVKYLKIE DIGKLMRKFV
Glycin e max NSYVFE I DMPGLKSGDI KVQVEDDNLLLI CGEl RKRD . EEIGGAKYLRIE GKLMRKFV
P . cri spum N S YVFWDMPGLKS GDI KVQVEEDNVLWSGE RKRE . EEKEGVKYVRME GIG'MRKFV
Pharbi ti s Hi 1 NSYVFI I DMPGLKSGDI KVQVDGDNVLSI SGE RIGREAEEKEGAKYVRMERF VGKLMRKFV
Madicago sativa NSYVFVIDMPGLKSGDIKVQVEDDNVLVI5GBRKREEEKEGGAKYLRMERFVGKFMRKFV
LeHSPl 7 . 6 NSYVFWDMPGLKSGDI KVQVEEDNVLLI SGE RKRE . EEKEGAIG‘IRMERF VGKFMRES

Consensus NSYVF MPG MIW N In W a m
s a

Consensus region II
121 II 163
Pisum sativum LPENANIEAISAISQDGVLTVTVNKEPPPEPKKPkTIQVKVA
Glycine max LPENANTDAI SAVCQDGVLSV‘I'VQKE PPPEPKKP' RTI QVKVA
P. crispum LPENADLENINAVCQDGVLSVTVQKI PPPEPKKP' KTI EVKIA
Pha rbi tis nil LPENANKEKITAVCQDGVLTVTVENV PPPEPKKP‘ RTIEVKIG
Mbdicago sativa LPENPNTDAVSPVCQDGVLTVTVQKEPPPRPKKPRTIEVQIA
LeHSPl 7. 6 LPENANTDAISAVCQDGVLTVTVQKIl PPPEPKKP IEVKVA

Consensus LPEN QDGVL VTV TI V
a: see

 

 

 

 

 

 

Consensus region I

Figure 5. LeHSP17.6 is a cytosolic class 11 small HSP. The amino acid sequence
alignment of the LeHSP17.6 tomato protein with cytosolic class H smHSPs ﬁ’om pea
(M33901), soybean (X07159), parsley (X95716), Pharbitr’s nil (M99429) and alfalfa
(X98617). Consensus sequence appears below the alignment typed in bold. A putative
nuclear localization signal is indicated in box 1. Box H indicates a potential polyproline
motif. The underlined sequences suggest a putative protein kinase C phosphorylation
sites. Asterisks deﬁne the important residues within the heat shock domain. Gaps within
the alignment were introduced to optimize the alignment.

89

phosphorylation.

In the ﬁrst box of the alignment (Figure 5) a conserved basic amino acid sequence
is present (RKR) and corresponds to a putative Xenopus type nuclear localization signal
(NLS) (Robbins et al., 1991). According to that motif, 2 basic amino acids are followed
by 10 residues and the next 5 residues contain at least 3 basic residues. The two basic
regions cooperate in binding, whereas the spacer may facilitate their cooperative
interaction (Raikhel, 1992). Alternatively, the LeHSP17.6 at the C-terminus (KmKKPK)
contains a putative SV40 large T-antigen nuclear targeting signal (Garcia-Bustos et al.,
1991). Recent studies by Wollgiehn et al. (1994) have suggested that the smHSPs move
between the nucleus and the cytoplasm in a stress-dependent fashion. Small proteins,
like the 13.8 kD heat-shock protein from yeast (Moreland ct al., 1987), have been shown
to have an NLS capable of redirecting a reporter protein to the nucleus. By histochemical
analysis, it has been shown that the SV40 sequence can function as an NLS in transgenic
tobacco (Van der Krol and Chua, 1991). However, not all the sequences similar to SV40
NLS are recognized by the plant nuclear import machinery (Silver, 1991). Perhaps, these
putative nuclear localization signals are responsible for the shuttling of the smHSPs ﬁom
the cytoplasm to the nucleus during stress.

Finally, the carboxyl end of the cytosolic class H smHSPs contains a polyproline
motif PPPEPKKP (Figure 5). This motif, particularly the diproline sequence PXXP, is
recognized by proteins with Src homology 3 (SH3) domains (Rickles et al. 1995; Ren et
al., 1993). In a number of cell types, the SH3 domains function to regulate cellular
events such as protein localization, enzyme activity and substrate requirement (Cohen et

al., 1995). The sequences of these proteins determine speciﬁc signal transduction

pathways (Pawson, 1995). It remains to be elucidated if the smHSPs bind to proteins
with 8H3 domains.

Genomic Southern-blot hybridization indicates that LeHSPI 7. 6 belongs to a two-
member gene family (Figure 5). Schoffl and Key (1983) reported that the soybean
cytosolic class H smHSP is encoded by single gene. In contrast, the Pharbitr's nil
cytosolic class H smHSPs are encoded by a multigene family, with at least four
representatives (Krishna et al., 1992).

The transcription of the LeHSPI 7. 6 gene is heat induced and is maintained up-
regulated during subsequent exposure to chilling temperature and this correlated with
tolerance to chilling injury. The mechanism remains to be determined and the function of
the putative LeHSP17 .6 is unknown. However, our data are in good agreement with
results of S. Lurie and colleagues (Sabehat et al., 1996). They found that protection of
tomatoes from chilling injury afforded by prestorage heat treatment is correlated with the
induction of transcription of HSP] 7 and HSP70 mRN As and with translation of HSP17
and HSP23 proteins which persist during subsequent storage of the ﬁuit at chilling
temperature. Other heat-induced transcripts may be involved as well. Recently, Collins
et al. (1995) reported that heat shock of mung bean hypocotyls induced synthesis of
several HSPs and only de novo synthesized HSP 79 and HSP 70 remained at signiﬁcantly
higher levels in tissue during a subsequent chilling period. These data, together with our
results, suggest that the synthesis and action of HSPs attained by heat treatment may be
involved in protecting the fruit and other parts of the plant from heat and chilling stress
damage. One of the possible models for the mechanism by which heat treatment

attenuate heat and chilling injury may be attributed to molecular chaperone activities of

91

HSPs (Vierling, 1991). Molecular chaperones are a group of intracellular proteins that
control correct folding, oligomeric assembly, transport across membranes or disposal by
degradation of other conformer unstable proteins by binding to them and release of them.
Additionally, molecular chaperones prevent incorrect interaction within and between
non-native polypeptides, which result in their irreversible aggregation (Hartl, 1996).
Recently, the in vitro evidence for molecular chaperone activity of plant smHSPs was
demonstrated by Vierling and colleagues (Lee et al., 1995a). They observed that
recombinant HSP 18.1 and HSP 17.7 representing cytosolic class I and class H smHSPs
ﬁ'om pea were able to enhance the refolding of chemically denatured model substrates
citrate synthase and lactate dehydrogenase and prevented their aggregation and
irreversible inactivation.

Plant smHSPs can assemble into multimeric units and form soluble high
molecular weight complexes between 200-400 kD (Lee et al., 1995a; Waters et al., 1996).
Lin and colleagues (Jinn et al., 1995) observed that the isolated 280 kD smHSP complex
from soybean was able to protect up to 7 5% of the total soluble proteins of the cell from
heat denaturation in vitro. These smHSP complexes can associate into insoluble larger
cytoplasmic aggregates termed as “heat shock granules” (Nover et al., 1989). It has been
suggested that these HS granules are transient sites for non-heat shock mRN A,
preventing its degradation during heat stress (N over, 1991). These large structures may
be common to all smHSPs (Osteryoung and Vierling, 1994) and their formation may be
reversible and occur mainly at highly stressing temperatures (Waters et al., 1996).
Another role of plant HSPs is the protection of some proteins from and the targeting of

other proteins to proteolysis (V ierstra, 1993). This putative function of HSPs may be

92

involved in the protection of plants from different stress damages such as heat shock and
chilling injury. Genetically engineering transgenic plants that constitutive overexpress
LeHSP17. 6 should prove useﬁrl to elucidate the function of the LeHSPl7 .6 in attenuation

of chilling injury in tomatoes.

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98

CHAPTER IH

HEAT TREATMENT ATTENUATES CHILLIN G INJURY: INVESTIGATION
OF THE INVOLVEMENT OF HEAT SHOCK GENES AND HEAT SHOCK
PROTEINS IN THE RESISTANCE OF TOMATO FRUIT TO LOW

TEMPERATURES

ABSTRACT

Chilling injury (CI) was prevented by heat treating mature green tomato
(Lycopersicum esculenturn cv. Mountain Springs) fruit at 42°C for 36 or 48 h prior to
storage at 2°C for two weeks, whereas fruit stored at 2°C without preheating developed
typical CI symptoms and failed to ripen at 20°C. The protection afforded by heat
treatment was lost if the tomatoes were transferred from 42°C to 20°C for 1 to 3 days
before low temperature storage, however the CI was less than that of control ﬁuits. We
hypothesized that heat-shock proteins (HSP) may be responsible for increased tolerance
to CI. Using differential display of mRN A we recently cloned and characterized a ﬁlll-
length cDNA that encodes a cytosolic class H smHSP (LeHSPl7 .6) termed HC T 1 (heat
induced/chilling tolerance). Screening a cDNA library from heat-treated tomato ﬁuits, a
17.4 kD cytosolic class H smHSP (HCI?) was isolated. It has about 90% similarity at the

amino acid level to LeHSP17.6. Northern analysis indicates that both transcripts were

99

induced during heat shock, and the level of transcription remained high during
subsequent storage at chilling temperatures. Using gene-speciﬁc differential display, six
heat shock genes were identiﬁed including, three members of cytosolic class I smHSP, a
chloroplastic smHSP, a mitochondrial smHSP and a cytosolic HSP90. The transcripts for
the cytosolic class I smHSPs and the chloroplastic smHSP were up-regulated by heat
treatment and are increased slightly during cold storage; fruits receiving only chilling
temperatures had a low level of expression. When the fruits were warmed to 20°C for 3
days, the mRN A for all the smHSP declined slowly. HSP90 was induced by heat, but
when the fruits were exposed to 2°C, the mRNA level increased 5-fold, suggesting that
the expression of HSP90 is cold regulated. In a similar manner to the smHSPs, the
HSP90 mRNA decreased slowly upon return to 20°C. HSP90 has 70% similarity at the
amino acid level with the developmentally-regulated LeHSC80 protein and is more
similar to stress-induced cytosolic HSP90 ﬁ'om other species. These data suggest that
the synthesis and action of several HSPs whose transcripts increase during heat treatment

may be involved in protecting the ﬁuit from chilling stress.

100

INTRODUCTION

When plants are exposed to temperatures that exceed their normal growth
temperature, they respond by inducing the synthesis of several polypeptides referred to as
heat-shock proteins (Vierling, 1991). Pre-exposing plants to a mild heat shock, will
induce resistance to higher lethal temperatures, a phenomenon called thermotolerance
(Nover, 1991; Vierling, 1991). While thermotolerance can be induced in plants by heat-
treatrnent, heat stress is also known to develop tolerance against other environmental
stresses (Bonham-Smith et a1, 1987; Orzeck and Burke, 1988; Kuznetsov et al., 1997;
Sabehat et al., 1998b). The cross-protection response observed in plants is similar to that
observed in yeast (Mager and Varela, 1993; Piper, 1993; Schuller et al.,‘1994); Exposure
to one stress induces tolerance to another stress unrelated to the ﬁrst one. Heat shock
proteins are induced by other stress such as cold, ethanol, drought, amino acid analogues,
salinity, ozone and oxidative stress (Anderson et al., 1994; Ruis and Schuller, 1995; Coca
et al., 1996; Lee et al., 1996; Colmenero-Flores et al., 1997; Eckey-Kaltenbach et al.,
1997; Banzet et al., 1998). The induction of the heat shock response can protect cells
against a variety of other toxic insults, such as ethanol and hydrogen peroxide (Ruis and
Schuller, 1995; Storz and Polla, 1996).

Mature green tomatoes are very susceptible to chilling injury by storage for only a
few days at temperatures below 10°C. Heat treatment has been found to protect tomatoes
against chilling injury (Klein and Lurie, 1991). We hypothesized that heat-shockproteins

(HSP) may be responsible for increased tolerance to chilling injury following heat

101

treatment. Using differential display of mRNA we cloned and characterized a full-length
cDNA (LeHSPI 7. 6) that encodes a cytosolic class H small heat-shock protein
(Kadyrzhanova et al., 1998). We conﬁrmed our hypothesis that heat-shockproteins are
correlated with chilling tolerance. This raised question whether other members of the

HSPs are induced during heat shock and are stable during cold temperatures.

MATERIALS AND METHODS

Nucleic Acid Isolation

Total RNA was extracted and puriﬁed according to (Grierson and Covey, 1976)
and Fray and Grierson (1993) with some modiﬁcations. Frozen fruit pericarp was ground
in liquid nitrogen with a mortal and pestle and extracted with buffer containing: 1%
triisopropylnaphthalene sulfonate, 6% p-aminosalicylate, 5% (v/v) Tris-saturated phenol
and 50 mM Tris-HCl, pH 8.0). The homogenate was extracted 4 times with an equal
volume ofphenolzchloroform (1:1) and once with chloroform. RNA was precipitated at -
20°C by addition of 0.1 vol 3M sodium acetate, pH 5.6 and 2.5 vols of ethanol in two
steps: ﬁrst, 1 vol of ethanol was added and if a carbohydrate and DNA precipitate
formed, this was removed and the remaining 1.5 vol of ethanol was added. RNA was
precipitated with 4M lithium chloride and the pellet was dissolved in 250mM potassium
acetate pH 7 .0. The RNA was precipitated with 3 vols of ethanol, the pellet washed with

80% (v/v) ethanol, dried and dissolved in diethylpyrocarbonate (DEPC)-treated water.

102

RNA was quantiﬁed and qualiﬁed by spectrophotometrically. Integrity of RNA was

evaluated by fractionation of an aliquot on 1.2% agarose/formaldehyde gel.

Differential Display

Differential display was performed as described by (Kadyrzhanova et al., 1998)
with a some modiﬁcations. Instead of the arbitrary decamer, primers designed ﬁ'orn a
conserved amino acid region of the speciﬁc HSP (mitochondrial, chloroplastic and
cytosolic class I smHSP as well as HSP90) were combined with the anchored primers
(TnMN). The primers used were: 5’-GATCAAAGGTCCCAGGGTGGTGAGCATTGC-
3 ’ for mitochondrial smHSP, 5’-GGGGGAACACAAAAAGGAAGAAGACGGAG-3 ’
for the chloroplastic smHSP, 5’-GGATCAAGTTAAGGCGTCTATGGAGAATGG-3’
for the cytosolic class I smHSP and 5-GAGACTGCCCTTCTCACCTCAGGTTTCAGG-
3’ for the HSP90. Total RNA isolated from ﬁuits, at the mature green (MG) stage, or
heat-treated at 42°C for 2 days (H), or heat-treated at 42°C for 2 days following by
storage at 2°C for one week and then transferred at 20°C for 1 day (HC). RNA from
fruits that were stored at 2°C for one week following by one day at 20°C (C) was also
obtained. Total RNA was DNAse I treated as described by Liang et al., (1993). DNAse-
free RNA was reversed transcribed with the anchored primer TnMN. PCR ampliﬁcation
of 1/10 of the ﬁrst strand synthesis cDNA product was done in the presence of [or-”P]
dATP. Ampliﬁed cDNAs were separated through a 6% denaturing polyacrylamide gel
containing 7M urea. Electrophoresis was done at constant 60W for 3-4hrs followed by
drying on Whatman paper. The PCR fragments were resolved on 1.2% agarose gel,

puriﬁed and then inserted into a EcoRI linearized plasmid pCRH vector using TA-cloning

103

system (Invitrogen). The cDNA clones released from the pCRH were random primer

labeled and used as probes in Northern blots.

cDNA Library Screening

Screening of the cDNA library was done by the Long-Distance (LD)-PCR based
method using gene-speciﬁc primers (Barnes, 1994; Cheng, 1994; Ali-Osman and
Akande, 1995). About 10‘5 phages from a heat- and cold-treated tomato ﬁuit cDNA
library (Kadyrzhanova et al., 1998) were screened by ampliﬁcation with 3’gene-speciﬁc
primer derived from sequence close to the poly (A) tail of the diﬂ’erential display
products and the 5’-PCR primer provided by the CapFinterTM PCR cDNA library
Construction kit (Clontech). The screening of the cDNA library for the isolation of the

LeHC T2 cDNA was performed according standard procedures (Sambrook et al., 1989).

DNA sequencing

The putative gene-speciﬁc clones were sequenced by double-stranded DNA
sequencing. DNA sequence determination was performed on Applied Biosystems 37 3A
(Foster City, CA) at the MSU Instrumentation Facility. DNA sequence data were

assembled and analyzed using DNA STAR (DNA STAR, Madison, WI).

RNA Gel Blot Analysis

Total RNA (25pg) was separated on a 1.2% agarose gel containing 2.2 M
formaldehyde and transferred to Hybond-N nylon membranes (Amersham, Aylesbury,
UK) as described by Sambrook et al. (1989). The membranes were ﬁxed by using a

microwave oven for 2 min at full setting (700W). The blots were prehybridized for 3 hrs

104

(50% formamide, SXSSC, 25mM potassium phosphate, pH 7.4, 5X Denhardt’s solution,
50pg/ml denatured salmon sperm DNA) at 42°C. The eluted cDNAs were [ct-”PMCTP
labeled using Random Primer Labeling kit (Gibco BRL, Gaithersburg, MD) in the
presence of the TuMN oligomer. After denaturation, the radiolabeled probe was added
to hybridization buffer containing 5% (w/v) dextran sulfate and hybridized for 16 hrs at
42°C. Membranes were washed twice for 15 min at room temperature with 1X SSC and
0.1% SDS, followed by 15 min at 60°C with 0.2 X SSC and 0.1% SDS before exposure

to X-ray ﬁlm overnight to 7 days.

Protein Isolation and Western Analysis

Total proteins were extracted by grinding tissue in SDS sample buffer containing
60mM Tris-HCl pH 8.0, 60mM DTT, 2% SDS, 15% sucrose, lmM PMSF, using 5 ntLg'l
fresh weight of tomato pericarp. After grinding, samples were boiled for 5 min, and the
insoluble debris was removed by centrifugation (10 min at 17,000g). Proteins were
precipitated with 4 volume of cold acetone overnight at -20°C. Protein concentration was
determined using Bradford assay (Bio-Rad). Twenty pg of each sample were separated
on a 10% or 13% (w/v) polyacrylamide gel using the method of Laemmli (1970). After
electrophoresis, gels were either stained with Coomassie brilliant blue R or transferred to
a nitrocellulose membrane (Bio-Rad) for Western blot analysis. Membranes were
blocked with TBST (20mM Tris-HCl, pH 7.5, SOOmM NaCl and 0.5% (v/v) Tween 20)
supplemented with 5% (w/v) non-fat dry milk for 1 h and then incubated for 1 or 2 h with
the same solution containing anti-HSP antiserum. Antibodies used were the Arabidopsis
thaliana cytosolic class I HSP 17 .6 (1:2000 dilution) and the wheat cytosolic class H HSP

18.0 (1:500 dilution), both were gifts from Dr. Vierling, (University of Arizona, Tuscon),

105

as well as the Pharbitis nil HSP83 (1:5000 dilution), kindly provided by Dr. Krishna
(University of Western Ontario, Canada). Bound antibodies were detected using goat
anti-rabbit horseradish peroxidase diluted 1:2000 (v/v), an enhanced chemiluminescent
system (Amersham), and exposed to x-ray ﬁlm (Amersham) for l to 5 min, depending on

signal strength.

RESULTS

Cloning and Characterization of Cytosolic Class H smHSP LeHSP 1 7.4.

Genomic Southern blot hybridization indicates that the cytosolic class 11 smHSP
LeHSP17. 6 belongs to a two-member gene family (Kadyrzhanova et al., 1998). To isolate
the other member of the family we screened 106 phages of the ampliﬁed cDNA library
from heat-and cold-treated tomato fruit with the LeHSPI 7. 6 cDNA The screening
resulted in isolation of a full-length cDNA LeHC T2 (heat induced/cold tolerance). Figure
1 shows the nucleotide sequence and the deduced amino acid sequence of the LeHCT2.
The cDNA consists of 759 bp upstream of the polyadenylation tail, which includes a 79-
bp 5’ leader sequence, followed by 466 bp of an open reading frame (ORF), and a 213-bp
3 ’-untranslated region. A polyadenylation signal 174bp before the poly(A) tail is
marked. The translation product of the LeHC T2 is 155 amino acids long with a predicted
molecular mass of 17.4kD and a pI of 7.24. The LeHCT? was ﬁlrther designated as

LeHSPI 7. 4.

106

The LeHSP17 .4 has 92% similarity at the amino acid level with LeHSP17 .6
indicating that is another member of the tomato cytosolic class H smHSP family. Table I
indicates that sequence identities ranged between 54% (Arabidopsis thaliana HSP17.6b)
and 95% (Lycopersicum peruvianum HSP17.4). A comparison of amino acids sequence
of the LeHSPl7.4 and the cytosolic class H smHSPs from other higher plants is shown on
Figure 2. The cytosolic class H smHSP have several motifs, including the putative
bipartite nuclear localization signal RmKRXmRMERRm4 (marked in purple) suggesting
that these proteins may be targeted to the nucleus (Garcia-Bustos et al., 1991; Robbins et
al., 1991; Silver, 1991; Raikhel, 1992), the putative cAMP protein kinase
phosphorylation R"°XXS site (blue) which is unique to tomato homologues and the
polyproline motif (red) which is similar to known SH3-binding sites (Ren et al., 1993;

Pawson, 1995; Rickles et al., 1995).

107

GNGGCCGCTNCGACACGGCTGCGAGAAGACGACAGAAGGGGGACAGAATAAAATT 55
CCATTTTCAAACACGATAGAAGAATGGATTTGAGGTTGATGGGTATTGATAACAC 1 1 0
MDLRLMGIDNTll
ACCACTCTTCCACACTCTTCAGCATATGATGGAAGCTGCTGGTGAAGATTCCGTG 165
PLFHTLQHMMEAAGEDSV 29
AATGCACCACCAAAGAAGTATGTTCGTGATGCTAAGGCAATGGCTGCGACACCAG 22o
NAPPKKYVRDAKAMAATP 47
TGGACGTGAAAGAGTATCCTGATTCATATGTTTTCGTTGTGGATATGCCAGGGTT 275
VDVKEYPDSYVFVVDMPGLGS
GAAATCTGGAGATATCAAAGTGCAGGTAGAAGAAGACAATGTGCTGTTGATTAGT 33o
KSGDIKVQVEEDNVLLIS 84
GGTGAAAGGAAGAGGGAAGAAGAGAAAGAAGGTGTAAAGTTTAT TAGAATGGAGA 3 8 5
GERKREEEKEGVKFIRME 102
GAAGGGTTGGGAAATTCATGAGGAAGTTTAGTCTGCCGGAGAATGCGAATACTGA 44o
RRVGKFMRKFSLPENANTDlZl
TGCAATTTCTGCAGTTTGTCAAGATGGAGTTCTGACTGTTACTGTTCAGAAGCTG 495
AISAVCQDGVLTVTVQKLlBQ
CCTCCTCCTGAGCCAAAGAAGTCCAAAACCATTCAGGTCAAAGTTGCTTGAAAAT 550
PPPEPKKSKTIQVKVA. 155
ATAAAGTTACTCTGTTTTCTTGCTCTGTTTTGATG’IE‘IEGCAATTGCTGCTC 605
TAGAT TACCATATTTTGATGCATCCAAGGAT TAACAAAATACAAATTTTAATGCA 6 6O
TGTATCTTGTTTGATAAAGAATCGAATTTTAATTACTTTTTGCCTCATCTCCTTG 715
ATTGTGTGTTATAACTGTTTCACGAAAAGCCATTTACTTTAATCAAAAAAAAAAA 770
AAAAAAAAAAAAAAAAA 787

Figure 1. Nucleotide and deduced amino acid sequences of the LeHSPI 7.4
(LeHCT2) cDNA. Nucleotides and amino acids are represented as normal and bold
letters, respectively. The putative polyadenylation signal is boxed.

108

Table I. Amino acid sequence identity of LeHSPI 7.4 to those of the cytosolic class II
smHSPs from various species

 

 

Species Protein Sequence Accession Reference
Identity Number
%
Arabidopsis thaliana AtHSP17.6a 63 X63443 Bartling et al. (1992)
Arabidopsis thaliana AtHSPl7.6b 59 Yl4070 Prandl et al. (1998)
Arabidopsis thaliana AtHSPl7.0 54 X89504 Grellet et al. (1995)
Picea glaucum PgHSP17.0 64 L47717 Dong and Dunstan
(1996)
Picea glaucum PgHSP17.l 63 L47740 Dong and Dunstan
(1996)
Picea abies PaHSP16.9 64 X99346 Schubert et al. (1997)
Zea mays ZmHSP17.5 57 X54075 Goping et al. (1991)
Zea mays ZmHSP18.1 60 $59777 Atkinson et al. (1993)
Zea mays ZmHSP17.8 61 X54076 Goping et al. (1991)
T rin'cum aestivum TaHSP17.3 60 X58279 Weng et al. (1991)
Pisum sativum PsHSP17.7 74 M33901 Lauzon et al. (1990)
Glycine max GmHSP17.9 75 X07159 Raschke et al. (1988)
Petroselinum PcHSP l 7.9 74 X95716 Eckey-Kaltenbach et
crispum al. (1997)
Medicago sativa MSHSP17 .0 73 X98617 Kelemen et al. (1996)
Lycopersicum LeHSP17.6 92 U723 96 Kadyrzhanova et al.
esculentum (1998)
Lycopersicum LpHSP17 .4 95 AJZZSO49 Forreiter and Loew
peruvianurn (1998)
Helianthus annuus HaHSP17.9 71 229554 Coca et al. (1994)
Pharbiﬁs (Ipomoea) PnHSPl7.2 71 M99429 Krishna et al. (1992)
nil
Pharbitis (Ipomoea) PnHSPl8.8 62 M99430 Krishna et al. (1992)

nil

109

Figure 2. Amino acid sequence comparison among the cytosolic class II smHSPs.
Consensus amino acid sequence is showed in bold face. The secondary structure of the
protein is shown as predicted by the PHDsec (Proﬁle network prediction HeiDelberg)
method (www.embl-heidelberg.de/predictprotein/predictprotein.html; Rost and Sander,
1994). Helices are indicated with red box while the strands are presented as green arrows.
The putative bipartite nuclear localization signal is in purple, the putative polyproline
motif is in red and the putative cAMP protein kinase phosphorylation site of tomato
homologues is in unbolded blue. The consensus I and H domains are indicated in blue.
Gaps within the alignment were introduced to optimize the alignment. The nucleotide
and amino acid sequences of the cytosolic class H small heat-shockproteins were
obtained from the GeneBank and the Accession Numbers are listed in Table H. Amino
acid sequences were aligned using CLUSTAL V method in DNASTAR.

110

 

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8.8Hmmxna
m.enmm:cn
o.mﬁmmzsm
H.mﬁmmx8m
o.mﬁmmxsm
m.mﬁdm:od
H.8Hmmzed
o.mammxem
o.eﬁmm:n<
e.esmm:n<
m.eﬁmmmn<

wDWCQWCOU
o.mammzcm
0.8Hmmmcm
m.esmm:om
N.ommmznq
e.enmm:uq
6.8H8mxuq
0.8Hmmxoz
m.eﬁmm:om
m.eﬁmmzse
8.8Hmmznm
m.eﬂmm:oe
o.mﬁmmmsm
H.8Hmmxsm
o.mﬁmmxam
m.eﬁnm:om
H.8Hmmmem
0.8Hmmmem
c.8Hmmxn<
6.8Hmmxh<
c.8Hmmmom

lll

Differential Display, Cloning and Sequence of Heat-Induced Chilling Tolerance-
Related HSPs.

Using differential display of mRN A, we cloned a cytosolic class H smHSP
(LeHSPI 7. 6) that was heat induced and related to chilling tolerance of tomato fruit
(Kadyrzhanova et al. 1998). To test the hypothesis that other HSPs may also be related to
cold tolerance, a modiﬁcation of differential display of mRN A was used where the
arbitrary primers are replaced with gene-speciﬁc primers for other HSP encoding genes.
Based on sequence information we designed primers speciﬁc for smHSPs and high
molecular weight HSPs. Gene-speciﬁc differential display fragments that were induced
during heat shock (H) and subsequently at cold temperatures (HC) were selected, cloned
into the pCRH plasmid vector and sequenced.

The characteristics of the heat-inducible chilling tolerance cDNAs isolated by
gene speciﬁc differential display are summarized in the table H. Three members of
cytosolic class I smHSPs (LeHCT 3, LeHCT 4 and LeHCT8), a chloroplastic smHSP
(LeHCT 5), a mitochondrial smHSP (LeHC T 6) and a cytosolic HSP90 (LeHCT7) were
cloned. The nucleotide and the deduced amino acid sequence of the LeHCT 3 is presented
in Figure 3. The differential display fragment LeHCT 3 is 324 bp long and contained the
sequence of the cytosolic class I smHSP speciﬁc primer. This piece is identical to
pTOM66 (Fray et al., 1990). The 3’UTR, however, is longer than the pTOM66,
suggesting that the LeHCT 3 has a complete 3’ end, which contains a putative poly(A)
signal. LD-PCR screening of the HC cDNA library, with a LeHCT 3 3’-end primer

derived from a sequence near the polyadenylation signal and of the 5’-PCR primer

112

supplied from CapFindermPCR cDNA library construction kit, resulted in isolation of
two full-length cDNAs; the LeHCT 3 and LeHCT 8, respectively. The nucleotide
sequence of the LeHCT 3 is 724 bp long and contains an ORF ﬁom 62 to 526, a 5’-UTR
of 61 nucleotides and a 3’-UTR of 198 nucleotides (Figure 3). A putative poly A signal
(AATAA) is found at the nucleotide position 701. The deduced amino acid sequence of
the LeHCT 3 corresponds to a polypeptide of 154 amino acids with a predicted molecular
weight of 17.7 kD and a pI of 5.9. LeHCT 3 was further designated as LeHSPI7. 7 and is
identical to the tomato cytosolic class I smHSP, pTOM66 (Fray et al., 1990) with one
substitution in the amino acid sequence; the P57 is an A”.

The nucleotide sequence of the LeHCT 8 is 742-bp long and includes an 86-bp 5’-
leader sequence, followed by a 467-bp sequence of an ORF and a 189-bp of an 3’ UTR
(Figure 4). The deduced amino acid sequence of the LeHCT8 corresponds to a 17.8 kD
protein with a pI of 5.6. The LeHCT8 was further named as LeHSP17.8 and has 98%
similarity in the coding region with LeHSPI 7. 7.

In addition to LeHC T 3, the same combination of primers generated another
differential display fragment designated as LeHCT 4. The LeHCT 4 is 320-bp in length
and has 97% similarity in the coding region with LeHCT 3, but the 3’ UTR is different,
indicating that it is another member of cytosolic class I smHSP family (Figure 5).
Analogous to the LeHCT 3, the LeHCT 4 contains a putative polyadenylation signal 137-
bp before the poly(A) tail. A gene-speciﬁc primer before the poly A tail of the LeHCT 4
was designed to screen the (HC) cDNA library using the LD-PCR approach using this
primer in combination with the 5’-PCR primer provided from CapFinderTMPCR cDNA

library construction kit. This screening provided the full-length cDNA LeHC T 4.

113

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114

GATCAAAATCGAAAGCAAGCAAGCAAAAAAACGTAGAAAATTCTCAAAAAGTTCA 5 5
CTGAAAATGTCTCTGATCCCAAGAATTTTCGGCGATCGACGAAGCAGCAGCATGT l 1 0
MSLIPRIFGDRRSSSM 16
TCGATCCATTTTCAATTGACGTATTTGATCCATTCAGGGAATTAGGCTTCCCAAG 165
FDPFSIDVFDPFRELGFPS35
TACCAATTCAGGGGAGAGCTCTGCATTTGCCAACACACGAATAGACTGGAAGGAA 2 2 O
TNSGESSAFANTRIDWKES3
ACTCCAGAAGCTCATGTGTTCAAGGTTGATCTTCCAGGGCTTAAGAAGGAGGAAG 275
TPEAHVFKVDLPGLKKEE 71
TCAAAGTGGAAGTCGAGGAGGATAGGGTTCTTCAGATCAGCGGAGAGAGGAACGT 3 3 O
VKVEVEEDRVLQISGERNVQO
GGAGAAGGAAGATAAGAATGATAAGTGGCATCGCATGGAGCGAAGCAGCGGGAAA 3 8 5
EKEDKNDKWHRMERSSGKIOB
TTCATGAGGAGATTTAGACTTCCGGAGAATGCAAAGATGGAT CAAGTTAAGGCGT 4 4 0
FMRRFRLPENAKMDQVKAIZS
CTATGGAGAATGGAGTGCTTACTGTTACTGTTCCAAAGGAAGAGGTGAAGAAGCC 495
SMENGVLTVTVPKEEVKKP145
TGAGGTCAAGTCCATTGAGATCTCTGGTTAAATGCTCTGGTTGGGAACAAACCTG 550
EVKSIEISG. 154
TAGTATTAAGTCAAGTGTGTACTGTCGAAGATTTTGAGTTTACTTATTTTCTGTC 605
TGTGTCTTGTGCGCTGAGTCGTTTTACTAGTTGGTTGTTATCTGTTTGATGTATT 660
TTCCTTGAGAACTCTTATGTGTGAAAGGATGTAT TAC TAC'IEEEEAGTATTTC 7 l 5
TGGTGCCAT 724

 

 

 

 

 

 

Figure 3. Nucleotide and deduced amino acid sequences of the cytosolic class I
LeHSPI 7. 7 (LeHCT 3) cDNA. Nucleotides and amino acids are represented as normal
and bold letters, respectively. The putative polyadenylation signal is boxed. The
differential display LeHC T 3 nucleotide sequence is underlined.

115

AC GGC TNC CAGAAGAC GACAGAAGAGGCAT CAAAATCGAAAG CAAGCAAGCAAAA 5 5
AAACGTAGAAAATTCTCAAAAAGTTCACTGAAAATGTCTCTGATCCCAAGAATTT 110
M s L I P R I 7
TCGGCGATCGACGAAGCAGCAGCATGTTCGATCCATTTTCAATTGACGTATTTGA 165
FGDRRSSSMFDPFSIDVFDZS
TCCATTCAGGGAATTAGGCTTCCCAAGTACCAATTCAGGGGAGAGCTCTGCATTT 220
PFRELGFPSTNSGESSAF 44
GCCAACACACGAATAGACTGGAAGGAAACTCCAGAAGCTCATGTGTTCAAGGTTG 275
ANTRIDWKETPEAHVFKV 62
ATCTTCCAGGGCTTAAGAAGGAGGAAGTCAAAGAGGAAGTCGAGGAGGATAGGGT 33o
DLPGLKKEEVKEEVEEDRVBI
TCTTCAGAT CAGCGGAGAGAGGAACGTGGAGAAGGAAGATAAGAAT GATAAGTGG 3 8 5
LQISGERNVEKEDKNDKW 99
CATCGCATGGAGCGAAGCAGCGGGAAATTCATGAGGAGATTTAGACTTCCGGAGA 4 4 o
HRMERSSGKFMRRFRLPE 117
ATGCAAAGATGGATCAAGTTAAGGCGTCTATGGAGAATGGAGTGCTTACTGTTAC 495
NAKMDQVKASMENGVLTVT136
TGTTCCAAAGGAAGAGGTGAAGAAGCCTGAGGTCAAGCCCATTGAGATCTCTGGT 550
VPKEEVKKPEVKPIEISGISA
TAAATGCTCTGGTTGGGAACAAACCTGTAGTATTAAGTCAAGTGTGACTGTCGAA 605
GATTTTGAGTTTACTTATTTTCTGTCATGGCTTGGGCCCTGAGTCGTTTACTAGT 660
TGGTTGGTATCTGTTTGATGTATTTTCCTTGAGAACTCTTGAGAACTCTTATGTG 700
TGAAAGGATGTATTACTACMAGTATTTCTGGTGCCAT 742

Figure 4. Nucleotide and deduced amino acid sequences of the cytosolic class I
LeHSPI 7. 8 (LeHCT 8) cDNA. Nucleotides and amino acids are represented as normal
and bold letters, respectively. The putative polyadenylation signal is boxed.

116

GAT CAAAATCGAAAGCAAGCAAGCAAAAAAACGTAGAAAAT TCTCAAAAAAGT TC 5 5
ACTGAAAATGTCTCTGATCCCAAGAATTTTCGGCGATCGACGAAGCAGCAGCATG 1 1 O
MSLIPRIFGDRRSSSMlG
TTCGATCCATTTTCAATCGACGTATTTGATCCATTCAGGGAATTAGGCTTCCCAG 1 65
FDPFSIDVFDPFRELGFP34
GTACCAATTCAGGGGAGAGCTCTGCATTTGCCAACACACGAATAGACTGGAAGGA 2 2 0
GTNSGESSAFANTRIDWKES3
AACTCCAGAAGCTCATGTGTTCAAGGCTGATCTTCCAGGGCTTAAGAAGGAGGAA 2 7 5
TPEAHVFKADLPGLKKEE71
GTCAAAGTGGAAGTCGAGGAGGATAGGGTTCTTCAGATCAGCGGAGAGAGGAACG 3 3 O
VKVEVEEDRVLQISGERN 89
TGGAGAAGGAAGATAAGAATGACAAGTGGCATCGCGTGGAGCGAAGCAGCGGGAA 3 8 5
VEKEDKNDKWHRVERSSGKIOB
ATTCAT GAGGAGAT TTAGACTTCCGGAGAATGCAAAGATGGAT CAAGTTAAGGCT 4 4 0
FMRRFRLPENAKMDQVKAIZS
TCAATGGAGAACGGAGTGCTTACTGTTACTGTTCCAAAAGAAGAGGTGAAGAAGC 495
SMENGVLTVTVPKEEVKK144
CTGAGGTCAAGTCCATTGAGATCTCTGGTTAAAAATACATTTGTGAATTAAGTTG 550
PEVKSIEISG. 154
ATGTGTATGGTCEETAAATﬂTGAGTTGTTGTGTCTGTTGAAGGTTTGAAGTT 605
GCTCTGTTTTTCTATCGAAAGTCTTGAGTCGGCTCTGTTTCTCACCTAATGGCGT 660
AGTTGATGTACTTGCTGTAAAATTTCATGTTGAAAGATGTAATAGTAGTGTTGTA 715
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 745

 

 

 

 

 

 

 

Figure 5. Nucleotide and deduced amino acid sequences of the cytosolic class I
LeHSPI 7. 6 (LeH CT 4) cDNA. Nucleotides and amino acids are represented as normal
and bold letters, respectively. The putative polyadenylation signal is boxed. The
differential display LeHCT 4 nucleotide sequence is underlined.

117

The length of the LeHCT 4 is 745 bp. It contains a 62-bp sequence of the 5’-untraslated
region, followed by 466-hp sequence of an ORF and a 217-bp 3’-untranslated region,
including the 31 A residues of the poly(A) tail. The translation product is 154 amino
acids long with a predicted molecular weight of 17.6kD and p1 of 5.9. The LeHCT 4 was
designated as cytosolic class I smHSP LeHSPI 7. 6.

The derived amino acid sequences of the three tomato cytosolic class I smHSP are
compared with the other sequences present in the GeneBank databases in Figure 6. Table
III shows that the sequence similarities among the tomato and the other higher plant
cytosolic class I smHSPs ranged from 60% (Hordeum vulgare HSP] 7. 0b and Agrostis
stolomfera HSP16.5) to 95% (Lycopersicum peruvianum HSP! 7. 8).

At the N-tenninus, the tomato cytosolic class I smHSPs contain a putative cAMP
protein kinase phosphorylation site (RUXXS), which is also observed in the
Lycopersicum peruvianum (F orreiter and Loew, 1998) and Douglas ﬁr (P. menziesiz)
(Dong and Dunstan, 1996) homologues shown in blue (Figure 6). These proteins also
share a distinct region of conserved amino acids near the N-terminus
(F 17DPFXXDX‘XDPF) followed by a large variable region (10 to 28 amino acids), and a
large region of conservation to the C-terminus. The C-terminus region contains the
conserved “heat shock domain”, which consists of two subdomains, the consensus I and
II separated by a variable length domain (Vierling, 1991). The subdomain consensus
region 11, which contains the putative substrate-binding region (Lee et al., 1997), is
highlighted in red. Secondary structure prediction as well as the crystal structure of the

Methanoccocusjannaschii HSP16.5 (Kim et al., 1998) suggests that this region is a loop

118

between BB and B4 (Figure 6). In this region, the hydrophobic residues are involved in
dimer interaction.

Another differential display fragment isolated was the LeHC T 5 (Figure 7). The
partial cDNA LeHCT 5 is 327 bp long and is identical to the pT GM] 1 1 (chloroplastic
smHSP LeHSP21) isolated by Lawrence et al. (1997) and Sabehat et al. (1998a). In
parallel with chloroplastic smHSP, a mitochondrial related smHSP differential display
fragment LeHCT 6 was isolated. LeHCT 6 is 660 bp long, containing 113-hp 3’ end of an
ORF and a large 3’-untranslated region of 547 nucleotides, with a poly (A) signal 66-bp
before the polyadenylation tail (Figure 8). The ORF encodes a predicted polypeptide of
36 amino acids and has 50% similarity with the other mitochondrial smHSP from plants.
To isolate a heat-inducible chilling tolerance HSP90 homologue, a primer was designed
from the conserved C-terminus domain of the HSP90 family. Using the differential
display approach a fragment of 447 nucleotide designated as LeHCT7 was isolated
(Figure 9). LeHCI7 is 447 bp long, containing a 406-bp 3’ untranslational region. There
was no consensus eukaryotic polyadenylation motif (AATAAA), which is not unusual for
the HSP90 family. For instance, the LeHSC80 did not contain a polyadenylation signal
(Koning et al., 1992). To obtain a larger clone, we used a primer designed from 5’
terminal portion of the LeHC T7 to screen the (HC) cDNA library by LD-PCR as
described for the cytosolic class I smHSPs. The longest cDNA clone found was about
1.61kb long. This fragment was designated as LeHSP90. Efforts to get a full-length

cDNA clone by LD-PCR were unsuccessful.

119

Table III. cytosolic class I smHSP similarity to LeHSPl7.7

 

 

Species Protein Sequence Accession Reference
Similarity Number
% .
Pennicetum HSP16.9 68 X94192 Caven et al. (1996)
glaucum
Pennicetum HSP17.0 68 X94191 Caven et al. (1996)
glaucum
Zea mays HSP17.2 67 X65725 Jorgensen and Nguyen
(1994)
00'20 saliva HSP16.9a 72 M80938 Tzeng et al. (1992)
Oryza sativa HSP16.9b 72 M80939 Tzeng et al. (1992)
Olyza sativa HSP16.9c 71 U81385 Huang et al. (1996)
T riticum aesn'vum HSP16.9a 65 X64618 Weng et al. (1995)
Hordeum vulgare HSP17.0a 67 Y07844 Slocombe et al. (1996)
Hordeum vulgare HSP17.0b 60 X64560 Marmiroli et al. (1993)
T riticum aestivum HSP16.9b 67 P12810 McElwain and Spiker
(1989)
Agrostis stolonifera HSP16.5 60 AF 007762 Park and Luthe (1998)
var. palustris
Chenopodium HSP18.3 64 Q05832 Knack et al. (1992)
rubrum
Helianthus rmnuus HSP17.7 72 U46545 Coca et a1. (1996)
Heliamhus annuas HSP17.6 69 X59701 Almoguera and Jordano
(1992)
Dancus carota HSP17.8 72 X53851 Darwish et al. (1991)
Dancus carota HSP17.9 70 X53852 Darwish et a1. (1991)
Glycine max HSP17.5a 80 M11318 Nagao et al. (1985)
Glycine max HSP17.6 76 M11317 Nagao et a1. (1985)
Glycine max HSP18.5 74 X07160 Raschke et al. (1988)
Glycine max HSP17.5b 77 P04794 Czarnecka et al. (1985)
Glycine max HSP17.3 78 P02519 Schofﬂ et al. (1984)
Medicago sativa HSP18.2 78 X58711 Gyorgyey et al. (1991)
Pisum sativum HSP18.1 78 M33899 Lauzon et al. (1990)
Lycopersicum HSP17.7 100 X56138 Fray et al. (1990)
esculentum
Lycopersicum HSP17.8 98 This study
esculentum
Lycopersicum HSP17 .6 98 This study
esculentum
Lyc0persr’cum HSP17 .8 94 AJZZSO47 Forreiter and Loew
peruvianum (1998)
Lycopersicum HSP17.6 95 AJ225048 Forreiter and Loew
peruvianum (1998)
Lycopersicum HSP17.7 92 AJZZSO46 Forreiter and Loew
peruvianum (1998)

 

120

Table III (cont’d)
Oryza sativa
Oryza sativa
Olyza sativa
Oryza sativa
Oryza sativa
OIyza saliva
Pennisetum

glaucum

Helianthus annuus

Nicotiana tabaccum
Cuscutajaponica
Arabidopsis
thaliana
Brassica rapa
Arabidopsis
thaliana
Arabidopsis
thaliana

F ragaria ananassa

Pseudotsuga
menziesii
Pseudotsuga
menziesii

HSP17.3b

HSP17.8
HSP18.0

HSP17.3a

HSP17.4
HSP17.7
HSP17.9

HSP18.6
HSP18.0
HSP17.6
HSP17.4

HSP17.6
HSP17.6

HSP18.2

HSP17.4

HSP18.2a

HSP18.2b

65
62
65
67
67
65
69

69
72
73
68

69
71

72

70

67

121

M80186
X75616
U83670
U83669
D12635
U83671
X94193

U46544
X70688

ABOl7273

X17293

AF 022217

X16076

X17295

U6363 1

X92983

X92984

Tseng et al. (1992)
Lee et al. (1995b)
Guan et al. (1998)
Guan et al. (1998)
Nishi et al. (1992)
Guan et al. (1998)
Caven et al. (1996)

Coca et al. (1996)
Zarsky et al (1995)
Yamada (1998)
Takahashi and Komeda
(1989)

Kim and Jo (1997)
Helm and Vierling
(1989)
Takahashi and Komeda
(1989)
Medina-Escobar et a1.
(1998)
Kaukinen et al. (1996)

Kaukinen et al. (1996)

Figure 6. Amino acid sequence comparison among the cytosolic class I smHSPs.
Consensus amino acid sequence is showed in bold face. The secondary structure of the
protein is indicated as predicted by the PHDsec method (www.cmbl-
heidelberg.de/predictprotein/predictprotein.html; Rost and Sander, 1994). In addition,
the secondary structure of a small heat shock protein ﬁ'om Methanococcus jannaschii, a
hyperthermophilic archaeon, is showed based on its crystal structure (Kim et al., 1998).
Helices are indicated with red box while the B-strands are presented as green arrows. The
putative cAMP protein kinase phosphorylation site of tomato homologues is underlined
in blue. In addition the consensus I and H domains are underlined. The amino acids in
the consensus domain 11 responsible for substrate recognition are in red. Gaps within the
alignment were introduced to optimize the alignment. The DNA and amino acid
sequences of the cytosolic class I small heat-shock proteins were obtained from the
GeneBank. Accession Numbers are listed in Table III. Amino acid sequences were
aligned using CLUSTAL V method in DNASTAR

122

PgHSP16.9
PgHSP17.0
ZmHSP17.2
OsHSP16.9
OsHSP16.9
OsHSP16.9
TaHSP16.9
HVHSP17.0
HVHSP17.0
TaHSP16.9
AsHSP16.5
C2HSP18.3
HaHSP17.7
HhHSP17.6
DcHSP17.8
DcﬂSPl7.9
GMHSP17.5
GMHSP17.6
GmHSP18.5
GMHSP17.S
GmHSP17.3
.MSHSP18.2
PsHSP18.1
LeHSPl7.7
LeHSP17.8
LeHSP17.6
LpHSP19.9
LpHSP20.1
LpHSP20.0
OsHSP17.3
OsHSP17.8
OSHSP18.0
OsHSP17.3
OsHSP17.4
OsHSP17.7
PgHSP17.9
HBHSP18.6
NtHSP18.0
CjHSP17.6
AtHSP17.4
BcﬂSP17.6
AtHSP17.6
AtHSP18.2
FBHSP17.4
RmHSP18.2
RMHSP18.2
Conacnlus

I“!

 

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MSLV ........ RRGN.VFDPFSMD.LW.DPFDNM.FRS ....... IVPS...SSSSDTAAEANARIDW
MSLV ........ SRSS.VFDPFSMD.LW.DPFDSM.FRS.......IVQSAG.SPDSDTAAFAAARIDW
MSLV........RRSN.VFDPFSMD.LW.DPFDTM.FRS.......IVPSAI.STNSETAAFASARIDW
MSLV ........ RRSN.VFDPFSLD.LW.DPFDSV.FRS.......VVPA...TSDNDTAAFANARIDW
MSLV ........ RRSN.VFDPFSLD.LW.DPFDSV.FRS.......VVPA...TSDNDTAAFANARIDW
MSLV........RRSN.VFDPFA.D.FW.DPFDGV.LRS.......LVPA...TSDRDTAAFANARVDW
MSIV........RRTN.VFDPFA.D.LWADPFDT..FRS.......IVPAIS.GGGSETAAFANARMDW

MSIV........RRSN.AFDPFA.D.LWADPFDT..FRS.......IVPAFS.GN.SETAAEANARVDW
MSIV........RRSN.VLDPEA.D.LWADPLDT..FRS.......IFPAIS.GGNSETAV.RERRMDW
MSIV......,.RRSN.VFDPEA.D.LWADPFDT..FRS.......IVPAIS.GGSSETAAEANARVDW
MSIV........RWSN.VFDPFSLD.LWADPFDA..FRS.......ILPA:A.SGNHDTAAFVNARMDW
MSLIPNNWFNTGRRSN.IFDPFSLDEIW.DPFFGL....PSTL..STVPRSET..AAETAAFANARIDW
MSIIP..SFFTGNGSN.IFDPFSSE.IW.DPFQG....LSSVI..NNLPESSR....ETTAIANTRIDW
MSIIP..SFFTSKRSN.IFDPFSLD.TW.DPFQG....IIST.......EPAR....ETAAIVNARIDW
MSIIP..SFFGGRRSN.VFDPFSLD.VW.DPFKDFPL...VTSSA...SEFGK....ETAAFVNTHIDW
MSIIP..SFFGSRRSN.VLNPFSLD.IW.DPFQDYPL...ITSSGT.SSEFGK....ETAAFANTHIDW
MSLIP..SIFGGRRSN.VFDPFSLD.VW.DPFKDFHF..PTSL.SA ....... .....NSAFVNTRVDW
MSLIP..SIFGGPRSN.VFDPFSLD.MW.DPFKDFHV..PTSSVSA...........ENSAFVNTRVDW
MSLIP..NFFGGRRNN.VFDPFSLD.VW.DPFKDFPF..PNTLSSAS...FPEFSR.ENSAFVSTRVDW
MSLIP..GFFGGRRSN{VFDPFSLD.MW.DPFKDFHV..PTSSVSA...........ENSAFVSTRVDW
MSLIP..SFFGGRRSS{VFDPFSLD.VW.DPFKDFPF..PSSL.SA...........ENSAFVSTRVDW
MSLIP..SFFGGRRSN.VFDPFSLD.VW.DPFKDFPF.NNSAL.SAS...FPR....ENSAFVSTRVDW
MSLIP..SFFSGRRSN.VFDPFSLD.VW}DPLKDFPF.SNSSP.SAS...FPR....ENPAFVSTRVDW
MSLIP..RIFGDRRSSSMFDPFSID.VF.DPFRELGFPSTNSG..............ESSAEANTRIDW
MSLIP..RIFGDRRSSSMFDPFSID.VF.DPFRELGFPSTNSG..............ESSAEANTRIDW
MSLIP..RIFGDRRSSSMFDPFSID.VF.DPFRELGFPGTNSG.... ...... ....ESSAFANTRIDW
MSLIP..RIFGDRRSSSMFDPFSID.VF.DPFRELGFPGTNSG..............BTSAEANTRIDW
MSLIP..RIFGDRRSSSMFDPFSID.VF.DPFRELGFPGTNSR..............ETSAEANTRIDW
MSLIP..RIFGDRRSTSVFDPFSID.VF.DPFKELGFTVSNSG..............ETSAFANTRIDW

MSMI... ..... RRSN.VFDPFSLD.LW.DPFDGFPFGSG......SGSLFPRANS.DAAAEAGARIDW
MSLI. ..... ..RRSN.VFDPFSLD.LW.DPFDGFPFGSGSRSSGTIFPSFPRGTSSETAAEAGARIDW
MSLI ........ RRSN.VFDPFSLD.LW.DPFDGFPFGSGSRSSGTIFPSFPRGTSSETAAFAGARIDW
MSMI ........ RRSN.VFDPFSLD.LW.DPFDGFPFGSG ...... SGSLFPRANS.DAAAFAGARIDW
MSMI ........ RRSN.VFDPFSLD.LW.DPFDGFPFGSG ...... SGSLFPRANS.DAAAFAGARIDW
MSLI ....... .RRGN.AFDPFSLD.LW.DPVDGFPFGSGGSSSS.SGSLFPRANS.DAAAEAGARIDW

MSLI........RRSN.VFDPFSLD.EW.DPFEGFPFGSGSNS.GSLFPSFPRTSS.ETAAEAGARIDW
MSIIP..NFFGRRRTN.CFDPFSLD.VW.DPFEGFPFNNNNF..GSLSDQV.R.SSSETSSFVNANVDW
MAMIP..SFFGGRRSN.IFDPFSLD.IF.DPFEGFPFSGTV........ANVPSSARETSAFANARIDW
MSLIP..SFFEGRRSN.AFDPFSLE.EW.DPFFSNTVANLSG..........SSSAREASAFANARIDW
MSEVP..SFFGGRRTN.VFDPFSLD.VW.DPFEGFLTP.G.LTNAPAK.........DVAAFTNAKVDW
MSLIP..SFFGGRRTN.VFDPFSLD.LY.DPFEGFLTPSG.MTNAISK.........DVAAFTNAKVDW
MSLIP..SIFGGRRTN.VFDPFSLD.VF.DPFEGFLTPSG.LANAPAM.........DVAAFTNAKVDW
MSLIP..SIFGGRRSN.VFDPFSQD.LW.DPFEGFFTPSSALANA........STARDVAAFTNARVDW
MAL.S..LFGNSRRSN.VFDPFSLD.TW.DPFQGFGPLMN...........SSSTAGDTSAFAQTRIUW
MSIIP..SFFG.RRSSSAFDPFSLD.VW.DPFRAFTDLSGGGPSGQFVN........EASAMANTQIDW
MSIIP..SFFG.RRSSSAFDPFSLD{VW.DPFRAFTDLAAGGPSGQFVN........EASAIANTQIDW
I! EDP? D DP DI

 

123

PgHSP16.9
PgHSP17.0
ZMHSP17.2
OsHSP16.9
OsHSP16.9
OsHSP16.9
TaHSP16.9
HVHSP17.0
HVHSP17.0
TBHSP16.9
AsHSP16.5
CrHSP18.3
HaHSP17.7
HaHSP17.6
DcHSP17.8
DcHSP17.9
GmHSP17.5
GmHSP17.6
GMHSP18.5
GMHSP17.S
GmHSP17.3
.MBHSP18.2
PsHSP18.1
LeHSPl7.7
LeHSP17.8
LeHSP17.6
LpHSP19.9
LpHSP20.1
LpHSP20.0
OsHSP17.3
OsHSP17.8
OsHSP18.0
OsHSP17.3
OsHSP17.4
OsHSPl7.7
PgHSP17.9
HaHSP18.6
NtHSP18.0
CjHSP17.6
AtHSP17.4
BcHSP17.6
AtHSP17.6
AtHSP18.2
FBHSP17.4
RmHSP18.2
PmH3P18.2
Conscnlua

 

+H u
+—> -—>—> '-> —>

KETP.EVHVFKADLPGVKKEEVKVEVEDG.NVLVISGQRSKEKEDKNDRWHRVERSSGQFVRRFRLPE
KETP.EAHVFKADLPGVKKEEVKVEVEDG.NVLVISGQRSKEKEDKNDRWHRVERSSGQFMRRFRLPG
KETP.EAHVFKADLPGVKKEEVKVEVEDG.NVLVISGQRSREKEDKDDKWHRVERSSGQFIRRFRLPD
KETP.ESHVFKADLPGVKKEEVKVEVEEG.NVLVISGQRSKEKEDKNDKWHRVERSSGQFMRRFRLPE
KETP.ESHVFKADLPGVKKEEVKVEVEEG.NVLVISGQRSKEKEDKNDKWHRVERSSGQFMRRFRLPE
KETP.ESHVFKADLPGVKKEEVKVEVEEG.NVLVISGQRSKEKEDKNDKWHRVERSSGQFMRRFRLPE
KETP.EAHVFKADLPGVKKEEVKVEVEDG.NVLVVSGERTKEKEDKNDKWHRVERSSGKFVRRFRLLE
KETP.EAHVFKADLPGVKKEEVKVEVEDG.NVLVVSGERTKEKEDKNDKWHRVERSSGKFVRRFRLPE
KGRRLEAHVFKADLPGVKKEEVKVEVEDG.NVLIVSGERTKEKEDKNDKWHRVERRSGKFVRPFRLPE
KETP.EAHVFKVDLPGVKKEEVKVEVEDG.NVLVVSGERSREKEDKNDKWHRVERSSGKFVRRFRLPE
KETP.EAHVFKADLPGVKKEEVKVEVEGG.NVLVVSGER.KGEGGQERQVATLERSSGKFVRRFRLPE
KETP.EAHVFKADLPGVKKEEVKVEVEDG.NVLRISGQRAREKEEKNDTWHRVERSSGQFMRKFRLPE
KETP.EAHVFKADLPGLKKEEVKVEVEEG.RVLQISGERSRENVEKNDKWHRMERSSGKFLRRFRLPE
KETP.EAHVLKADLPGMKKEEVKVEVEDG.RVLQISGERCREQEEKDDTWHRVERSSGKFIRRFRLPE
KETP.QAHVFKADLPGLKKEEVKVELEEG.KVLQISGERNKEKEEKNDKWHRVERSSGKFLRRFRLPE
KBTP.QAHVFKADLPGLKKEEVKVEVEEG.KVLQISGERNKEKEEKNNKWHRVEFSSGKFLRRFRLPE
KETP.EAHVFEADIPGLKKEEVKVQIEDD.RVLQISGERNLEKEDKNDTWHRVERSSGNFMRRFRLPE
KETQ.EAHVLKADIPGLKKEEVKVQIEDD.RVLQISGERNVEKEDKNDTWHRVDRSSGKFMRRFRLPE
KETP.EAHVFKADIPGLKKEEVKVQIEDD.KVLQISGERNVEKEDKNDTWHRVERSSGKFMRRFRLPE
KETP.EAHVFKADIPGLKKEEVKVQIEDD.RVLQISGERNVEKEDKNDTWHRVERSSGKFTRRFRLPE
KETP.EAHVFKADIPGLKKEEVKLEIQDG.RVLQISGERNVEKEDKNDTWHRVERSSGKLVRRFRLPE
KETP.EAHVFKADLPGMKKEEVKVEIEDD.RVLQISGERSVEKEDKNDQWHRLERSSGKFMRRFRLPE
KETP.EAHVFKADLPGLKKEEVKVEVEDD.RVLQISGERSVEKEDKNDEWHRVERSSGKFLRRFRLPE
KETP.EAHVFKVDLPGLKKEEVKVEVEED.RVLQISGERNVEKEDKNDKWHRMERSSGKFMRRFRLPE
KETP.EAHVFKVDLPGLKKEEVKEEVEED.RVLQISGERNVEKEDKNDKWHRMERSSGKFMRRFRLPE
KETP.EAHVFKADLPGLKKEEVKVEVEED.RVLQISGERNVEKEDKNDKWHRVERSSGKFMRRFRLPE
KETP.EAHVFKADLPGLKLEEVKVEVEED.RVLQISGERNMEKEDKNDKWQRVERSSGKFMRRFRLPE
KETP.EAHVFKADLPGLKKEEVKVEIEED.RVLQISGERNVEKEDKNDTWHRVERSSGKFMRRFRLPE
KETP.EAHVFKADLPGLKKEEVKVEVEED.RVLQISGERNVEKEDKNDTWHRVERSSGKFMRRFRLPE
KETP.EAHVFKADVPGLKKEEVKVEVEDG.NVSRSAGERIKEQEEKTDKWHRVERSSGKFLRRFRLPE
KETP.E.HVFKADVPGLKKEEVKVEVEDG.NVSRSAGEASKEQEEKTDKWHRVEASSGKFLRRFRLPE
KETP.E.HVFKADVPGLKKEEVKVEVEDG.NVLQISGERSKEQEEKTDKWHRVERSSGKFLRRFRLPE
KETP.EAHVFKADVPGLKKEEVKVEVEDG.NVLQISGERIKEQEEKTDKWHRVERSSGKFLRRFRLPE
KETP.EAHVFKADVPGLKKEEVKVEVEDG.NVLQISGERIKEQEEKTDKWHRVERSSGKFLRRFRLPE
KETP.EVHVFKADVPGLKKEEVKVEVDDG.NILQISGERSREQEEKSDKWHRVERSSGKFLRRFRLPE
KETP.EAHVFKADVPALKKEEVKVEVEDG.NVLQISGERNKEQEEKTDTWHRVERSSGKFMRRFRLPE
RETN.DAHVFKADVPGLKKEEVKVEVEDD.RVLQISGERNKESEEKGDTWHRVERSSGKFVRRFRLPE
KETP.DSHIFKMDVPGIKKEEVKVEVEEG.RVLQISGERSREQEEKNDTWHRMERSSGKFMRRFRLPG
KETP.EAHIFKADVPGLKKEEVKVEVEEG.KVLQISGERSKEKEEKNDTWHRVERSSGKFLRSFRLPE
RETP.EAHVFKADVPGLKKEEVKVEVEDG.NILQISGERSSENEEKSDTWHRVERSSGKFMRRFRLPB
RETP.EAHVFKADLPGLKKEEVKVEVEDG.NILQISGERSSENEEKSDRWHRVERSSGKFMRRFKLPE
RETP.EAHVFKADLPGLRKEEVKVEVEDG.NILQISGERSNENEEKNDKWHRVERSSGKFTRRFRLPE
KETP.EAHVFKADLPGLKKEEVKVEVEDK.NVLQISGERSKENEEKNDKWHRVERASGKFMRRFRLPE
KETP.EAHVFKADLPGLKKEEVKVELEEG.NVLQISGERSKEQEEKNDKWHRVERSSGKFVRRFRLPD
KETP.EAHIFKADLPGLKKEEVKIELEEGQRILQISGERSKEEEQKNDKWHRIERSRGKFLRRFRLPD
KETP.EAHIFKADLPGLKKEEVKIELEEGQRILQISGBRSKEEEQKNNKWHRIERSRGKFLRRFRLPD
Ir a rx.n PG KKEEVK E g; as n. n a x 'lnn.znsaa r n.tnng

CONSENSUS 839108111

124

PgHSP16.9
PgHSP17.0
ZmHSP17.2
OsHSP16.9
OsHSP16.9
OsHSP16.9
TBHSP16.9
HVHSP17.0
HVHSP17.0
TaHSP16.9
AsHSP16.S
CTHSP18.3
HaHSP17.7
H2HSP17.6
DdHSP17.8
DCHSP17.9
GmHSP17.5
GMHSPI7.6
GMHSPIB.S
GMHSP17.5
GMHSP17.3
.MSHSP18.2
PsHSP18.1
LeHSPl7.7
LeHSP17.8
LeHSP17.6
LpHSP19.9
LpHSP20.1
LpHSP20.0
OsHSP17.3
OsHSP17.8
OsHSP18.0
OsHSP17.3
OsHSP17.4
05HSP17.7
PgHSP17.9
HaHSP18.6
Ntl-ISP18. 0
CjHSPl7.6
AtHSP17.4
BcHSP17.6
AtHSP17.6
AtHSPlB.2
FBHSP17.4
PnHSP18.2
RMHSP18.2
Cons-nan:

ﬂ—H
-—> —>-->

DAKTDQVNAGLENGVLTVTVPKAEG.KKPEVKAIEISG
NAKVDQVKAGLENGVLTVTVPKAEE.KKPEVKAIEISG
DAKVDQVKAGLENGVLTVTVPKAEE.KKPEVKAIEISG
NAKVDQVKAGLENGVLTVTVPKAEV.KKPEVKAIEISG
NAKVDQVKAGMENGVLTVTVPKAEV.KKPEVKAIEISG
NAKVDQVKASMENGVLTVTVPKAEV.NKPEVKAIEISG
DAKVEEVKAGLENGVLTVTVPKAEV.KKPEVKAIQISG

DAKVEEVKAGLENGVLTVTVPKTEV.KKPEVKAIEISGI

DGKVDEVKAGLENGVLTVTVPKAEV.KKPEVKAIEISG
DAKVEEVKAGLENGVLTVTVPKAEV}KKPEVKAIEISG
NAKVEEVKAGLENGVLTVTVPKAEV.KKPEVKAIEISG
NAKVDQVKAGMENGVLTVTVPKNEA.PKPQVKAINVYE
NAKMDQVKAAMENGVLTVTVPKAEV.KKPEVKAIDIS

NAKMDEVKAMMENGVLTVVVPKEEEEKKPMVKAIDISG
NAKVDEVKAAMANGVVTVTVPKVEI.KKPEVKAIDISG
NANVDEVKAGMENGVLTVTVPKVEM.KKPEVKSIHISG
NAKVEQVKASMENGVLTVTVPKEEV.KKPDVKAIEISG
NAKVEQVKACMENGVLTVTIPKEEV.KKSDVKPIEISG
NAKVEQVKASMENGVLTVTVPKEEV.KKPDVKAIEISG
NAKVNEVKASMENGVLTVTVPKEEV.KKPDVKAIEISG
NAKVDQVKASMENGVLTVTVPKEEI.KKPDVKAIDISG
NAKMDQVKAAMENGVLTVTVPKEEV.KKPEVKTIDISG
NAKMDKVKASMENGVLTVTVPKEEI.KKAEVKSIEISG
NAKMDQVKASMENGVLTVTVPKEEV.KKPEVKSIEISG
NAKMDQVKASMENGVLTVTVPKEEVLKKPEVKPIEISG
NAKMDQVKASMENGVLTVTVPKEEV.KKPEVKSIEISG
NAKMDQVKASMENGVLTVTVPKEEM.KKPDVKSIEISG
NAKMDQVKASMENGVLTVTVPKEEV.KKPDVKSIEISG

NAKMDQVKASMENGVLTVTVPKEEV.NNPDVKSIEISGA

NTKPEQIKASMENGVLTVTVPKEEP.KKPDVKSIQITG
NTKPEQIKASMENGVLTVTVPKEEP.KKPDVKSIQVTG
NTKPEQIKASMENGVLTVTVPKEEP.KKPDVKSIQVTG
NTKPEQIKASMENGVLTVTVPKEEP.KKPDVKSIQITG
DTKPEQIKASMENGVLTVTVPKEEP.KKPDVKSIQITG
NTKPEQIKASMENGVLTVTVPKEEP.KKPDVKSIQISG
NAKTDQIRASMENGVLTVTVPKEEV.KKPEVKSIQISG
NAKVDQVKAAMENGVLTVTVPKVEV.KKPDVKSIQISG
NAKMEEIKAAMENGVLTVTVPKEEE.KKSEVKAIDISG
NAKVDQVKAAMENGVLTVTVPKVEE.KKAEVKSIQISG
NAKVEEVKASMENGVLSVTVPKVQE.SKPEVKSIDISG
NAKVDEVKASMENGVLSVTVPKMAE.RKPEVKSIDISG
NAKMEEIKASMENGVLSVTVPKVPE.KKPEVKSIDIS

NAKMEEVKATMENGVLTVVVPKAPE.KKPQVKSIDISGAN
NAKVDQVKAAMENGVLTVTVPKAPE.PKPQVKSIDISGA

NAKVEEIKAAMENGVLTVTVPKQPEPQPPQPKSIEISG
NAKVEEIKAAMENGVLTVTVPKQPEPQPPQPKSIEISG
K KI. ENGVL'V'VPKZI K I

CONSENSUS REGION I

125

151
153
153
151
151
150
152
152
152
152
151
163
157
154
158
160
154
155
162
155
154
159
159
154
154
154
154
154
155
155
161
161
155
155
160
160
164
160
158
156
155
155
159
154
159
159

GGGGGAACACAAAAAGGAAGAAGACGGAAGAGATAAACACTCATGGGGTAGGAAT 55
G a H K K E a D G R D K H s W’ G R. N 18
TATAGCTCTTACGACACTCGTTTAAGTCTCCCAGATAATGTTGTGAAGGATAAAA 110
Y s s Y D T R L s L P n N V' V’ K n K 37
TCAAAGCGGAACTGAAGAATGGAGTTCTTTTCATCTCGATTCCAAAGACTGAAGT 165
I K .A. a L K N G ‘v L F I s I P K T a V’ 56
GGAGAAAAAGGTGATTGATGTCCAAATTAACTAACATTCAGAATCGCCATGCTTT 220
a K. K ‘v I n V' Q I N . 66
TGTTGTTCTAGGTTCATTTGTAACCTTGTGTAAAATGTATGTTCGCATAT- 275
EEEEEFAAGTAGTGCATCTTTGATAAAAAAAAAAAAAAAAAAAAAAAAAAAA 327

Figure 7. Nucleotide and deduced amino acid sequences of the LeHSP21.0 cDNA
(LeHCT5). Nucleotides and amino acids are represented as normal and bold letters,
respectively. The putative polyadenylation signal is boxed.

GATCAAAGGTCCCAGGGTGGTCAGCATTGTCATCAGGGATATCTTGGGTCGTCAC 55
I K G p R ‘v ‘v s I V’ I R D I L G R a 18
CTAATAGGTACGGAGACAACGGTAGGCCGAAATCTGGAGGTGGGTATGATCGAAT 110
L I G T E T T 'V’ G R. N L s ‘v G :M I a 36
GAGGAGAGGATTTAGTGAAGCTGATTCTGTGAACTTTGTGAACCGGCCTAGTTCT 165
ANCGGAGCTCGGCCCAACTCTAGTGGATGATGGCATCATGATCCTGATGGAGCTT 220
AGCTCCCCTTTGCCGATGAGGATGGAGAGGTCTTTTCTGAGGCAAATAGGCTTGC 275
AAGATCAGCTATGGATGGATGTTAATGGTTGTTGAAGTCATGGAAGTAGGATTAT 330
GACCAGGTTGCTCCTTTTGTCTGCAGTAGTCCAGTAACCAGATTATGAATTTGCT 385
GGTTTTAAACCGAGACNTCCAAGGATCCAAGATCNATCAAATTGTTCTTTCATTT 44o
ACCGCCTTCTAATTTTCTTTGCTTTCTACCTTCTGTACTCTCTCAGGAAGAGAGA 495
AGACTTGGATGAAATGTAANGTATTGTATGTTNTACTGTATGTTATACTGTGCAA 550
CTACCAIAAATTGGTGTATTANNAAE§§::§§FACCACCATCACCAIATTGTATT 605
TCCAGACTTATCTGTATCACAGTGCATACTAATTTTCTGTTTTCAAAAAAAAAAA 660

Figure 8. Nucleotide and deduced amino acid sequences of the putative LeHSP22.0
cDNA (LeHCT6). Nucleotides and amino acids are represented as normal and bold
letters, respectively. The putative polyadenylation signal is boxed.

126

The nucleotide and deduced amino acid sequence of the LeHSP90 are shown on
Figure 9. This partial cDNA of LeHSP90 contain an ORF of 1202 bp encoding a
predicted polypeptide of 406 amino acids. Comparison of the predicted amino acid
sequence between LeHSP90 and the other higher plants HSP903 reveals sequence
identities ranging ﬁ'om 82% (LeHSC80) to 92% (Nicotiana tabacum HSP82) (Table IV).
The amino acid alignment of LeHSP90 with the some of the cytosolic HSP90s is
presented in Figure 10.

The eukaryotic HSP90s were dissected by proteolysis into three independently
folded domains, the N-terminus domain 1-236, the middle domain 272-615 and the C-
terrninal domain 621-730 (Nemoto et al., 1997; Stebbins et al., 1997). LeHSP90 is
lacking the N-terminal domain and part of the middle domain. The ~25-kDa N-terminal
domain (underlined in Figure 10) was identiﬁed as the binding site for the benzoquinoid
ansarnycin drug geldanamycin (Stebbins et al., 1997) and for ATP (Grenert et al., 1997;
Prodromou et al., 1997). The putative ATP-binding sites GXXGXG are marked blue.
This domain was shown to have a chaperone activity by binding to unfolded polypeptides
and preventing aggregation in vitro (Young et al., 1997; Scheibel et al., 1998). The
middle domain is located between the two highly charged domains of the protein (marked
green). In this domain the LeHSP90 contains a putative leucine zipper motif (marked
red), which may be involved in the speciﬁcity and stability of hetero— or homodirners.
The 12kD C-terminal domain (double underlined in Figure 10) has been shown to bind to
partially folded proteins in an ATP-independent way potentially regulated by
cochaperones (Young et al., 1997; Scheibel et al., 1998). In addition, the ~200 C-

terminal residues of HSP90, including the C-terminal domain and part of the middle

127

Figure 9. Nucleotide and deduced amino acid sequences of the LeHSP90 cDNA.
Nucleotides and amino acids are represented as normal and bold letters, respectively. The
differential display LeHCI7 nucleotide sequence is underlined.

128

ATACCTTGCAGTCAAACACTTCTCTGTTGAGGGGCAACTTGAATTCAAGGCAAT
Y L .A. V' K H F S V’ E G Q L E F K .A I
CCTCTTTGTACCTAAGAGGGCTCCATTTGATCTATTTGACACCCGCAAGAAGAT
L F ‘V P K R .A. P F D L F D T R K. KC.M
GAACAACATCAAACTTTATGTCAGGAGGGTGTTCATCATGGACAACTGTGAGGA
N N I K L Y V' R R ‘V F I IN D N C E E
ACTTATCCCTGAGTACCTTGGATTCGTGAAGGGTGTTGTTGACTCTGATGATTT
L I P E Y L G F ‘V K G 'V 'V D S D D L
GCCCCTCAATATCTCCCGTGAAATGCTGCAGCAGAACAAGATTCTCAAGGTCAT
P L N I S R. E MI L Q Q N K I L K ‘V I
TAGGAAGAACCTCGTGAAGAAATGTATTGAGATGTTCAATGAGATTGCAGAGAA
R K N L 'V K K C I E MC F N E I A. E N
CAAGGAGGACTACAACAAGTTCTACGAGGCTTTCTCAAAGAACTTGAAGCTGGG
K E D Y N K F Y E A. F S K N L K L G
CATTCATGAAGATAGCCAGAACAGGGCTAAGTTGGCTGACTTCCTTCGATATCA
I H E D S Q N R .A K L .A. D F L R. Y Q
GTCAACCCAAGAGTGTGATGAGCTGACAAGTTTGAAAGATTATGTAACCAGGAT
S T Q E C D E L T S L K D Y V' T R 1M
GAAGGAGGTTCAGAAAGACATCTACTACATCACTGGAGAGAGCAAAAAGGCAGT
K E V’ Q K D I Y Y I T G E S K KC A.'V
TGAAAATTCACCATTCTTGGAACGCCTAAAGAAGAAAGGATATGAAGTACTCTT
E N S P F L E R L K K K G Y E 'V L F
CATGGTTGATGCCATTGATGAATATGCTATTGGGCAACTGAAGGAATATGATGG
MC V' D A. I D E Y A. I G Q L K E Y D G
TAAGAAACTGGTTTCTGTTACAAAGGAGGGACTGAAGCTCGATGACGAGAGCGA
K K L 'V S 'V T K E G L K L D D E S E
AGAAGAAAAGAAGAAAAAGGAAGAGAAAAAACAATCCTTTGAGAGCCTTTGCAA
E E K K K K E E K K Q S F E S L C K
GGTCATCAAGGACATTCTTGGAGACAAAGTTGAGAAGGTTGTAGTCTCTGATAG
V' I K D I L G D K ‘V E K ‘V 'V ‘V S D R
GATTGTTGATTCTCCATGTTGCTTAGTGACAGGTGAGTATGGTTGGACAGCTAA
I V' D S P C C L 'V T G E Y G 'W' T A. N
CATGGAAAGGATCATGAAAGCTCAAGCTTTGAAGGACAATAGCATGAGCTCTTA
IM E R I IM K .A. Q .A L K D N S 3M 8 S Y
CATGTCTAGCGAGAAGACAATGGAAATCAACCCTGATAATGGCATTGTGGAGGA
Mi 8 S E K T 1M E I N P D N G I 'V E E
GTTGAGGAAGAGAGCTGAAGTTGACAAGAATGACAAGTCGGTGAAAGATCTTGT
L R K R .A. E V' D K ‘N D K S 'V K D L 'V
GCTGCTGCTGTTTGAGACAGCTTTGCTAACATCTGGTTTTAGTCTTGATGACCC
L L L F E T .A. L L T S G F S L D D P
GAATACATTTGCTGCAAGAATTCATAGAATGCTGAAGTTGGGTTTGAGCATTGA
N T F .A..A. R I H R. M L K L G L S I D
CGAAGAAGAGGAAGCTGGTGTGGATGTTGATGATATGCCTCCTCTGGAGGATGT
E E E E A. G 'V D 'V' D D M: P P L E D ‘V
TGGTGAGGAAAGCAAGATGGAAGAAGTGGACTAATCATTGAAATCAGTTGAGAC
G E E S K 1M E E V’ D .
TTTTGAGATGCGTGAAAATACAATTTGAGCGTGTTGCTTTTTTTCTCATCTTGT

 

129

53
18
106
36
159
54
212
72
265
90
318
108
371
126
424
144
477
162
530
180
583
198
636
216
689
234
742
252
795
270
848
288
901
306
954
324
1007
342
1060
360
1113
378
1169
396
1222
406
1275

GTCTAGTATAGTTTTTTTTTTTAGGCAAGAAAAGCTGTTCAATCAAAATGATCA
ATTAACAAAGGTTGGCATATTACATTGAAAGTAACTTTAGTTTCATGGGTGTCA
ACTGGGACTTTGGACCAAAGCCTTATGCAACTAACCCTTCACAACAACCTGAAG
TACTCACAATTACAATTACAACATAGTGTTGATAGACAAAAGGTAAAGAAGTGA
TAAAACAATTATCCAACTGTATATTGGAGAATTGCTTAGTTGCTGCCACCGCCG
AAGAGATAACCCAAAGAAGATCCACCTCCAGGATTTGCCATAGG

B0

1328
1381
1434
1487
1540
1610

Table IV. HSP90 Accession Numbers

 

 

HSP90 Protein Sequence Accession Reference
(organisms) Similarity Number
%
Arabidopsis AtHSP81 88 P27323 Yabe et al. (1994)
thaliana
Arabidopsis AtHSP82 88 D00710 Takahashi and Komeda
thaliana (1991)
Arabidopsis AtHSP83 88 M62984 Conner et al. (1990)
thaliana
Arabidopsis AtHSP90 86 Y07613 Milioni and Hatzopoulos
thaliana (1997)
Lycopersicum LeHSP90 100 This study
esculentum -
Nicotiana tabacum NtHSP82 92 X63195 Severin et a1. (1991)
Oryza sativa OsHSP82 84 211920 Van Breusegem et al.
(1994)
Pharbitisﬂpomoea) PnHSP90 91 M99431 Felsheim and Das (1992)
nil
Zea mays ZmHSP82 86 $59780 Mars et al. (1993)
Lycopersicum LeHSC80 82 M96549 Koning et al. (1992)

esculentum

 

131

Figure 10. Amino acid sequence comparison among the HSP90s. Consensus amino
acid sequence is shown in bold face. The ATP and geldanamacin-binding domain are
underlined and the ATP-binding region is in blue. The leucine zipper motif is shown in
red. The C-terminal domain responsible for oligomerization activity is double
underlined. In green are the K/E rich regions. Gaps within the alignment were introduced
to optimize the alignment. The DNA and amino acid sequences of the HSP90 were
obtained from the GeneBank. Accession Numbers are listed in Table IV. Amino acid
sequences were aligned using CLUSTAL V method in DNASTAR

132

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSPBZ
OsHSP82
PnHSP9O
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSP82
OsHSP82
PhHSPQO
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP9O
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

MA..DVQMA...DAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSD
MA .......... DAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSD
MA..DVQMA...DAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSD
MA..DVQMA...DAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSD

MAS ......... ETETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSD
MA..DVQMA...EAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNASD
MASADVHMAGGAETETFAFQAEINQLLSLIINTFYSNKEIFLRELISNASD
MS .......... DVETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSD

E TFEE INQLLSLI INTFYSNKF. IFLRELISN SD

ALDKIRFESLTDKSKLDGQPELFIRLVPDKSNKTLSIIDSGIGMTKADLVN
ALDKIRFESLTDKSKLDGQPELFIHIIPDKTNNTLTIIDSGIGMTKADLVN
ALDKIRFESLTDKSKLDGQPELFIRLVPDKANKTLSIIDSGIGMTKADLVN
ALDKIRFESLTDKSKLDGQPELFIRLVPDKPNKTLSIIDSGIGMTKADLVN

ALDKIRFESLTDKSKLDAQPELFIHIVPDKASNTLSIIDSGIGMTKSDLVN
ALDKIRFESLTDKSKLDAQPELFIRLVPDKTNKTLSIIDSGVGMAKADLVN
ALDKIRFESLTDKSKLDAQPELFIRLVPDKASKTLSIIDSGVGMTKSDLVN
ALDKIRFESLTDKSKLDGQPELFIHIIPDKANNTLTIIDSGIGMTKADLVN
ALDKIRFESLTDKSKLDQPELFI PDK TL IIDSG GM K DLVN

NLGTIARSGTKEFMEALQAGA.DVSMIGQFGVGFYSAYLVAEKVVVTTKHN
NLGTIARSGTKEFMEALAAGA.DVSMIGQFGVGFYSAYLVADKVVVTTKHN
NLGTIARSGTKEFMEALQAGAJDVSMIGQFGVGFYSAYLVAEKVVVTTKHN
NLGTIARSGTKEFMEALQAGA.DVSMIGQFGVGFYSAYLVAEKVVVTTKHN

NLGTIARSGTKEFMEALAAGA.DVSMIGQFGVGFYSAYLVAERVVVTTKHN
NLGTIARSGTKEFMEALQAGA.DVSMIGQFGVGFYSAYLVAEKVIVTTKHN
NLGTIARSGTKEFMEALAAGATDVSMIGQFGVGFYSAYLVADRVMVTTKHN
NLGTIARSGTKEFMEALAAGA.DVSMIGQFGVGFYSAYLVAEKVVVTTKHN
NLGTIARSGTKEFMEAL.AGA.DVSMIGQFGVGFYSAYLVA 'V'VTTKHN

DDEQYVWESQAGGSFTVTRDVDGEPLGRGTKITLFLKDDQLEYLEERRLKD
DDEQYVWESQAGGSFTVTRDTSGETLGRGTKMVLYLKEDQLEYLEERRLKD
DDEQYVWESQAGGSFTVTRDVDGEPLGRGTKISLFLKDDQLEYLEERRLKD
DDEQYVWESQAGGSFTVTRDVDGEPLGRGTKISLFLKDDQLEYLEERRLKD
......................................... EY........
DDEQYVWESQAGGSFTVTRDTSGEQLGRGTKITLYLKDDQLEYLEERRLKD
DDEQYIWESQAGGSFTVTRDVDGEQLGRGTKITLFLKEDQLEYLEERRIKD
DDEQYVWESQAGGSFTVTHDTTGEQLGRGTKITLFLKDDQLEYLEERRLKD
DDEQYVWESQAGGSFTVTRDTSGENLGRGTKMVLYLKEDQLEYLEERRLKD
Egggggggggaccsrrern GE LGRGTK L LR 0922 EERRLKD

KB

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSP82
OsHSP82
PnHSP9O
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSP82
OsHSP82
PhHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP9O
LeHSP9O
NtHSP82
OsHSP82
PnHSP9O
ZmHSP82
LeHSC80

LVKKHSEFISYPIYLWIEKTTEKEISDDEDEDEPKKENEGEVEEVDEEKEK
LVKKHSEFISYPISLWIEKTIEKEISDDEEEEEKKD.EEGKVEEVDEEKEK
LVKKHSEFISYPIYLWTEKTTEKEISDDEDEDEPKKENEGEVEEVDEEKEK
LVKKHSEFISYPIYLWTEKTTEKEISDDEDEDEPKKENEGEVEEVDEKKEK

......EFISYPIYLWTEKTTEKEISDDED. DEPKKDEEGAVEEVDEDKEK
LIKKHSEFISYPISLWTEKTTEKEISDDEDEEEKKDAEEGKVEDVDEEKEE
LVKKHSEFISYPIYLWTEKTTEKEISDDED.DEPKKEEEGDIEEVDEDKEK
LVKKHSEFISYPIYLWTEKTTEKEISDDEEEEDNKKEEEGDVEEVDDEDKD
LIKKHSEFISYPISLWVEKTIEKEISDDEEEEEKKD.EEGKVEEVDEEKEK
L KKHSEFISYPI LN EKT EKEISDDE K EG E VD

D...GKKKKKIKEVSHEWELINKQKPIWLRKPEEITKEEYAAFYKSLTNDW
E ..EKKKKKIKEVSHEWDLVNKQKPIWMRKPEEINKEEYAAFYKSLSNDW
D...GKKKKKIKEVSHEWELINKQKPIWLRKPEEITKEESAAFYKSLTNDW
D ..GKKKKKIKEVSHEWELINKQKPIWLRKPEEITKEEYAAFYKSLTNDW

E...KGKKKKIKEVSHEWQLINKQKPIWLRKPEEITKDEYASFYKSLTNDW
K...EKKKKKIKEVSHEWSLVNKQKPIWMRKPEEITKEEYAAFYKSLTNDW
E...GKKKKKIKEVSHEWQLINKQKPIWLRKPEEITKEEYASFYKSLTNDW
TKDKSKKKKKVKEVSHEWVQINKQKPIWLRKPEEITRDEYASFYKSLTNDW
E...EKKKKKVKEVSNEWSLVNKQKPIWMRKPEEITKEEYAAFYKSLTNDW

KKKKK KEVS EW' NKQKPIW’RKPEEI E.A.FYKSL NDW’

EDHLAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKLNNIKLYVRRVFIM
EEHLAVKHFSVEGQLEFKAILFVPKRAPFDLFDTKKKPNNIKLYVRRVFIM
EDHLAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKLNNIKLYVRRVFIM
EDHLAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKLNNIKLYVRRVFIM
...LAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKMNNIKLYVRRVFIM
EEHLAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKMNNIKLYVRRVFIM
EEHLAVKHFSVEGQLEFKAVLFVPKRAPFDLFDTRKKLNNIKLYVRRVFIM
EDHLAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKMNNIKLYVRRVFIM
EDHLAVKHFSVEGQLEFKAILFVPRRAPFDLFDTRKKLNNIKLYVRRVFIM
EEHLAVKHFSVEGQLEFKAVLFVPKRAPFDLFDTKKKPNNIKLYVRRVFIM
E HLAVKHFSVEGQLEFKA LFVPKRAPFDLFDT KK NNIKLXVRRVFIM

DNCEELIPEYLSFVKGVVDSDDLPLNISRETLQQNKILKVIRKNLVKKCIE
DNCEDIIPEYLGFVKGIVDSEDLPLNISRETLQQNKILKVIRKNLVKKCLE
DNCEELIPEYLSFVKGVVDSDDLPLNISRETLQQNKILKVIRKNLVKKCIE
DNCEELIPEYLSFVKGVVDSDDLPLNISRETLQQNKILKVIRKNLVKKCIE
DNCEELIPEYLGFVKGVVDSDDLPLNISREMLQQNKILKVIRKNLVKKCIE
DNCEELIPEYLGFVKGVVDSDDLPLNISREMLQQNKILKVIRKNLVKKCIE
DNCEELIPEWLSFVKGIVDSEDLPLNISREMLQQNKILKVIRKNLVKKCVE
DNCEELIPEYLGFVKGVVDSDDLPLNISREMLQQNKILKVIRKNLVKKCIE
DNCEELIPEWLGFVKGVVDSDDLPLNISRETLQQNKILKVIRKNLVKKCIE
DNCDELIPEYLSFVKGIVDSEDLPLNISRETLQQNKILKVIRKNLVKKCVE
DNC LIFE L FVKG VDS DLPLNISRE LQQNKILKVIRKNLVKKC

KM

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PhHSPQO
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

MFNEIAENKEDYTKFYEAFSKNLKLGIHEDSQNRGKIADLLRYHSTKSGDE
LFFEIAENKEDYNKFYEAFSKNLKLGIHEDSQNRTKIAELLRYHSTKSGDE
MFNEIAENKEDYTKFYEAFSKNLKLGIHEDSQNRGKIADLLRYHSTKSGDE
MFNEIAENKEDYTKFYEAFSKNLKLGIHEDSQNRGKIADLLRYHSTKSGDE
MFNEIAENKEDYNKFYEAFSKNLKLGIHEDSQNRAKLADFLRYQSTQECDE
MFNEIAENKEDYNKFYEAFSKNLKLGIHEDSQNRAKLADLLRYHSTKSGDE
LFFEIAENKEDYNKFYEAFSKNLKLGIHEDSTNRNKIAELLRYHSTKSGDE
MFNEIAENKDDYNKFYEAFSKNLKLGIHEDSQNRAKLADLLRYYSTKSGDE
MFFEIAENKDDYAKFYDAFSKNIKLGIHEDSQNRAKLADLLRYHSTKSGDE
LFFEIAENKEDYNKFYEAFSKNLKLGIHEDSQNRAKFAELLRYHSTKSGDE

F EIAENK DY KFYEAFSKNLKLGIHEDS NR K.A LRY ST DE

MTSFKDYVTRMKEGQKDIFYITGESKKAVENSPFLERLKKRGYEVLYMVDA
LTSLKDYVTRMKEGQNDIFYITGESKKAVENSPFLEKLKKKGIEVLYMVDA
MTSFKDYVTRMKEGQKDIFYITGESKKAVENSPFLERLKKRGYEVLYMVDA
MTSFKDYVTRMKEGQKDIFYITGESKKAVENS.FLERLKKRGYEVLYMVDA
LTSLKDYVTRMKEVQKDIYYITGESKKAVENSPFLERLKKKGYEVLFMVDA
MTSLKDYVTRMKEGQKDIYYITGESKKAVENSPFLERLKKKGYEVLYMVDA
LTSLKDYVTRMKEGQNDIYYITGESKKAVENSPFLEKLKKKGYEVLYMVDA
LTSLKDYVTRMKEGQKDIYYITGESKKAVENSPFLERLKKKGYEVLEMVDA
TTSLKDYVTRMKEGQKDIYYITGESRKAVENSPFLERLKKKGYEVLFMVDA
MTSLKDYVTRMKEGQNDIYYITGESKKAVENSPFLEKLKKKGYEVLYMVDA

S KDYVTRMKE Q DI YITGES KAVENSPFLE LKKKG EVLIMVDA

IDEYAVGQLKEYDGKKLVSATKEGLKLEDETEEE.KKKREEKKKSFENLCK
IDEYAIGQLKEFEGKKLVSATKEGLKLDETEDE..KKKKEELKEKFEGLCK
IDEYAVGQLKEYDGKKLVSATKEGLKLEDETEEE.KKKREEKKKSFENLCK
IDEYAVGQLKEYDGKKLVSATKEGLKLEDETEEE.KKKREEKKKSFENLCK
IDEYAIGQLKEYDGKKLVSVTKEGLKLDDESEEE.KKKKEEKKQSFESLCK
IDEYAVGQLKEYDGKKLVSATKEGLKLDDDSEEE.KKKKEEKKKSFENLCK
IDEYAVGQLKEFEGKKLVSATKEGLKLDESEDE..KKRKEELKEKFEGLCK
IDEYAVGQLKEYDGKKLVSATKEGLKLEDDDEEE.KKKREEKKKSFENLCK
IDEYAVGQLKEYDGKKLVSATKEGLKLDDEDDEEAKKRREERKKRFEELCK
IDEYSIGQLKEFEGKKLVSATKEGLKLDESEDE..KKKQEELKEKFEGLCK
IDEY GQLKE GKKLVSATKEGLKL E KK EB K FE LCK

TIKEILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
VIKDVLGDKVEKVIVSDRVVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
TIKEILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
TIKEILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
VIKDILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALKDNSM
IIKDILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
VIKEVLGDKVEKVVVSDRVVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
IIKDILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
VIKDILGDRVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDSSM
VMKDVLGDKVEKVIVSDRVVDSPCCLVTGEYGWTANMERIMKAQALRDSSM

K LGD VEKV VSDR VDSPCCLVTWW

135

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

AtHSP81
AtHSP82
AtHSP83
AtHSP90
LeHSP90
NtHSP82
OsHSP82
PnHSP90
ZmHSP82
LeHSC80

SGYMSSKKTMEINPDNGIMEELRKRAEADKNDKSVKDLVMLLYETALLTS
AGYMSSKKTMEINPENSIMDELRKRADADKNDKSVKDLVLLLFETALLTS
SGYMSSKKTMEINPDNGIMEELRKRAEADKNDKSVKDLVMLLYETALLTS
SGYMSSKKTMEINPDNGIMEDLRKRAEADKNDKSVKDLVMLLYETALLTS
SSYMSSEKTMEINPDNGIVEELRKRAEVDKNDKSVKDLVLLLFETALLTS
SSYMSSKKTMEINPDNGIMEELRKRAEADKNDKSVKDLVLLLFETALLTS
AGYMSSKKTMEINPENAIMEELRKRADADKNDKSVKDLVLLLFETALLTS
SSYMSSKKTMEINPDNGIMEELRKRAEADKNDKSVKDLVLLLFETALLTS
SAYMSSKKTMEINPDNGIMEELRKRAEADRNDKSVKDLVLLLFETALLTS
AGYMSSKKTMEINPENSIMDELRKRADADKNDKSVKDLVLLLFETALLTS
he: 21 ' SJ" ' ' 110W 1 ,T‘ ..

 

GFSLDEPNTFAARIHRMLKLGLSIDEDENVE.EDGDMPELEE..DAAEES
GFSLDEPNTFGSRIHRMLKLGLSIDDDDAVEAD.AEMPPLED.DADAEGS
GFSLDEPNTFAARIHRMLKLGLSIDEDENVE.EDGDMPELEE..DAAEES
GFSLDEPNTFAARIHRMLKLGLSIDEDENVE.EDGDMPELEE..DAAEES
GFSLDDPNTFAARIHRMLKLGLSIDEEEEAGVDVDDMPPLED...VGEES
GFSLDDPNTFAARIHRMLKLGLSIDEEEE.AVEDADMPALEE...TGEES
GFSLDDPNTFGSRIHRMLKLGLSIDEDETAEAD.TDMPPLED...DAGES
GFSLDDPNTFGARIHRMLKLGLSIDEEEA.G.DDADMPALEE..EAGEES
GFSLDDPNTFAARIHRMLKLGLNIDEDAAAD.EDADMPALDE..GAAEES
GFSLEEPNTFGNRIHRMLKLGLSIDEESG.DAD.ADMPALEDPEADAEGS

W 12212.1. g

 

KMEEVD
KMEEVD
KMEEVD
KMEEVD
KMEEVD
KMEEVD
KMEEVD
KMEEVD
KMEEVD
KMEEVD

KMEEVD

B6

domain, is involved in HSP90 dimerization (Minarni et al., 1994; Nemoto et al., 1995;
Wearch and Nicchita, 1996). Recently, the C-terrninal domain of the HSP90 was
demonstrated to be necessary and sufficient for interaction with the tetratricopeptide
repeat (TPR)-containing cofactor proteins, including the immunophilins FKBP52 and
CyP-40 (Young et al., 1998). Finally, a typical carboxyl-terminal pentapeptide MEEVD,
common in cytosolic HSP90 homologues from both plant and animals, was observed in

the LeHSP90.

Expression Patterns of the smHSPs, HSP90s and Ripening Related Genes.

Mature green tomato fruits heat-treated at 42°C for 2 days were transferred to 2°C for 14
days, and then were subsequently raised to 20°C. Heat shock at 42°C for 2 days caused
an increase in the expression of the cytosolic smHSPs members but had little or no effect
on the expression of the chloroplastic smHSP (LeHCT 5) and the LeHSP90 (Figure 11).
Interestingly, the developmentally regulated LeHSC 80 transcript and the fruit ripening
LeE8 transcript were decreased to low or undetectable levels, respectively. Storing the
heat-treated fruits at 2°C for 2 weeks caused a strong induction of the cytosolic class I
smHSPs, the chloroplastic smHSP, the LeHSP90 as well as the LeHSC80 transcripts;
mRNA levels of the cytosolic class II smHSPs were unaffected. In contrast, the mRN A
levels of LeE8 were increased slowly indicating that low temperature stimulated its
expression. Increasing the temperature from 2 to 20°C resulted in a decrease of steady-
state levels of the smHSP (cytosolic class I and II as well as chloroplastic) and the

LeHSP90 aﬁer 3 days. However, the levels of the LeHSC80 were slightly decreased

137

Q
s s g:
2°C-14 n é

MG 42°C-2 D 2°C-14 D

Cytosolic class II smHSP (LeHSP 1 7.6)
Cytosolic class II smHSP (LeHSP I 7.4)

Cytosolic class I smHSP (LeHSP] 7. 7)

Cytosolic class I smHSP (LeHSP] 7.6)
Chloroplastic smHSP (LeHCT5)
LeHSP90
LeHSC80
LeE8

 

LeUBI3 (Ubiquitin)

Figure 11. The expression patterns of the tomato smHSPs, HSP90s and E8 are
affected by heat treatment and low temperature storage of the fruits. RNA was
extracted from fruits harvested at mature green (MG) stage. These fruits were heat-
treated at 42°C for 2 days and then the temperature was lowered to 2°C for 14 days.
Aﬁer the low temperature storage, the fruits were transferred to 20°C for 3 or 9 days.
Another set of mature green fruits was transferred directly to 2°C for 14 days and then to
20°C for 3 days. Dr. Comai (University of Washington, Seattle) and Dr. Fischer
(University of California, Berkley) kindly provided the LeHSC80 and the LeE8 speciﬁc
probes, respectively.

138

after 3 days at 20°C but recovered after 9 days. The steady-state levels of LeE8
increased when the fruits were transferred to a permissive ripening temperature and aﬁer
9 days at 20°C and were higher than those at the mature green stage indicating that the
ripening process had recovered from the imposed stress.

When the mature green tomato fruits were transferred to 2°C, the expression of
the smHSPs differed from that of the heat-treated fruits. For instance, low temperature
did not stimulate the expression of the cytosolic class I smHSP and the chloroplastic
smHSP, whereas it slightly increased the levels of cytosolic class II smHSP.
Interestingly, low temperature elevated the expression of the HSP903 members.
However, the low temperature induced expression of the LeHSP90 mRN A was lower
than the expression of the same gene in the heat-treated tomatoes stored at the same
temperature and period. The E8 expression was down-regulated by low temperatures, but
at a lower rate than observed in ﬁuits from the combination of heat- and low temperature
treatments.

The sustained strong induction of the smHSPs and HSP90s at low temperature
after the heat treatment strongly suggests that chaperone participation is needed to

prevent chilling injury at reduced temperatures.

139

 

Immunodetection of the smHSP and HSP90 in Heat-Treated Cold Stored Tomato
Fruit.

To test whether the cytosolic class I and H smHSP as well as the HSP90 proteins
accumulated at low temperatures after the heat treatment, we used antibodies for
cytosolic class I HSP17.6 from Arabidopsis thaliana (Helm and Vierling, 1989), for
cytosolic class II HSP17 .8 from wheat (Vierling, unpublished) and for HSP83 from
Pharbitis nil (Krishna et al., 1997). Proteins were extracted from mature green ﬁuits that
have been heated at 42°C for 2 days, from heat and nonheated fruits stored at 2°C for 2
weeks or transferred at 20°C for 3 days and from fruits kept at 20°C for 9 days.

Immunodetection analyses indicate that the cytosolic class I and II smHSP as well
as the HSP90 proteins are present in the heat-treated fruits at low temperatures (Figure
12). More importantly, these proteins were still detectable when the fruits were
transferred to room temperature for 3 days after the cold period. Low temperatures had
no eﬂ‘ect on the expression of the smHSP and the HSP90. During fruit ripening the
expression of these were low or undetectable.

The cytosolic class I antiserum ﬁom Arabidopsis thaliana cross-reacted with at
least two tomato homologues in one-dimensional SDS-PAGE. The size was determined
to be 18.3 and 17.6kD, respectively. The cytosolic class 11 antibody from wheat
recognized a tomato protein with molecular mass of 17.8kD, but the cross-reactivity was
poor, suggesting that an antibody raised for the LeHSP17.6 is necessary to accurately
determine the expression of this protein. The antibody raised against the C-terminus of
the PnHSP83a (F elsheirn and Das, 1992) detected a tomato homologue with expected

size of 83.5kD.

140

 

4%" *9
_ U

2°C-14 D 'é
MG 42°C-2 D 2°C-14 D 20°C-9 D

 

Cytosolic class I smHSP Anti-AtHSPl7.6

Cytosolic class II smHSP Anti-TaHSP17.8

Anti-PnHSP ﬂ

 

 

 

Figure 12. Immunodetection of cytosolic small HSPs and HSP90 protein levels in
heat-treated/cold stored tomato fruits. Total soluble proteins were extracted from
mature green (MG) tomato fruits heat-treated at 42°C for 2 days. The temperature was
then lowered to 2°C and fruits were held for 14 days. Aﬁer the low temperature storage,
the fruits were transferred to 20°C for 3 days. Another set of mature green fruits was
transferred directly to 2°C for 14 days and then to 20°C for 3 days. Fruits kept at 20°C for
9 days were the ripening controls. Western blots were performed using the cytosolic
class I and II smHSP antibodies ﬁ'om Arabidopsis and wheat, respectively, as well as the
Pharbitis nil HSP83 antiserun. AtHSP17.6 and TaHSP17.8 antibodies were kindly
provided by Dr. Vierling (University of Arizona, Tuscon) and the PnHSP83 antibody was
provided by Dr. Krishna (University of Western Ontario, London, Canada).

141

Expression Patterns of the Tomato smHSPs and HSP90: Genes under Different
Heat treatment Regimes.

To examine further the heat-shock proteins transcript accumulation during
different regimes of heat shock and subsequent low temperature storage, mature green
ﬁuits were kept at 42°C for 12, 24, 36 and 48 h. The fruits were then transferred to 2°C
for 14 days or room temperature for 1 to 3 days. RNA was extracted at the speciﬁc times
and analyzed for the heat-shock proteins transcript accumulation.

Northern analyses indicate that the cytosolic class I, cytosolic class II, the
LeHSP90 and the LeHSC80 mRNA are rapidly induced after 12 h exposure to 42°C
(Figure 13). Similarly, the chloroplastic smHSP is induced by heat shock (Figure 14B).
The RNA levels for the cytosolic smHSPs declined slowly as the heat shock continued.
In contrast, the HSP90s and the chloroplastic smHSP mRN A decreased rapidly to almost
undetectable levels after 36 h at 42°C. The differentiation of the cytosolic smHSP versus
the HSP90 mRN A accumulation during heat shock suggests that the cytosolic smHSPs
mRN A is more stable at prolonged high temperatures than the chloroplastic smHSP and
the HSP903 mRN As.

After holding fruits at 20°C for 1 or 3 days after heat shock, the cytosolic class H
and the LeHSP90 mRN A levels declined rapidly to insigniﬁcant levels regardless the heat
shock period. The cytosolic class I smHSP transcript decreased slowly and it was present
after 3 days at 20°C. The decline of the cytosolic class I transcript at 20°C was more
rapid for the moderate heat shock than for severe heat shock, indicating that the mRN A

was more stable under prolonged heat shock. Contrary to the LeHSP90 pattern, the

142

developmentally regulated LeHSC80 mRN A recovered from the heat shock and reached
the same levels as the control fruits after 3 days at 20°C. The recovery of the LeHSC80
mRN A was independent from the duration of the heat shock. Surprisingly, the putative
mitochondrial smHSP (HCT 6) was constitutively expressed at very low levels. The fruit

ripening related gene E8 mRN A virtually disappeared after 36 h of high temperatures

143

20°C-1D zo°cen
E— __4___2°c 42°C 42°C
2 12h 24h 36h 48h 12h 24h 36h 48h 12h 24h 36h48h

VI?
xﬂﬂucula

 

   
   
  

Cytosolic class I smHSP LeHSP I 7. 7

Cytosolic class II smHSP LeHSPl 7.4
Mitochondrial smHSP LeHSPZZ
LeHSP 90

LeHSC 80

   

 

Figure 13. The effect of heat shock on the level of transcripts for the smHSPs and
HSP903 in tomato fruits. Mature green fruits were heat-treated at 42°C for 12, 24, 36
and 48 h and then were transferred to 20°C for 1 or 3 days. Mature green tomatoes were
also kept at 20°C for 3 days. Total RNA was prepared from the pericarp tissue and the
transcript level for cytosolic class I LeHSP17. 7, cytosolic class H LeHSPI 7. 4,
mitochondrial LeHSP22, LeHSP90 and LeHSC80 were analyzed as described in the text.

Figure 14. The effect of heat treatment on smHSPs, HSP90 and E8 mRNA levels in
tomato fruits subsequently stored at 2°C for 14 days. A Mature-green (MG) fruits
were heat-treated at 42°C for 12, 24, 36 and 48 h. The 24 and 48 h heat-treated and the
nonheated ﬁuits were transferred to 2°C for 14 days. Total RNA was extracted and
analyzed for the cytosolic class I LeHSP17. 7, cytosolic class H LeHSPI 7. 4,
mitochondrial LeHC T 6, LeHSP90 and LeHSC80 gene expression. B. Mature green
fruits were kept at 20°C for 3, 6, and 9 days or heat-treated at 42°C for 12, 24, 36 and 48
h. The heat-treated and the nonheated ﬁuits were transferred to 2°C for 14 days. After
the low temperature storage, the heat-treated and nonheated fruits were shifted to 20°C
for 3 days. Total RNA was extracted and analyzed for cytosolic class I smHSP
(LeHSP17. 7), chloroplastic smHSP (HC T 5), LeHSP90 and LeE8 gene expression.

145

 

2°C-14D
Q 42°C 42°C
2 12h 24h 36h 48h 24h 48h

 

 

 

2°C-14D

    
    

Cytosolic class I smHSP LeHSP 17. 7

Cytosolic class H smHSP LeHSP 1 7.4
Mitochondrial smHSP LeHSP 22

 

LeHSP 9o

LeHSC so

B

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.'-' A‘Il .

Cytosolic class I smHSP LeHSP17. 7

Chloroplastic smHSP Le HSP 21
Le HSP 90

LeE8

(Figure 143), suggesting that the fruit ripening and especially the ethylene-dependent
responses were down-regulated.

In contrast to fruits held at ambient temperature, when the heat-treated fruits were
exposed to low temperatures, the mRN A for the cytosolic classes I and H smHSPs,
LeHSP90, LeHSC80 (Figure 14A) and chloroplastic smHSP (Figure 148) were
reinduced. However, low temperature exposure of nonheated ﬁuit, had no eﬁ‘ects on the
expression of the cytosolic class I smHSP (Figure 14A) and chloroplastic smHSP (Figure
14B) mRNA while the cytosolic class H smHSP was slightly induced. Interestingly, low
temperatures regulated the putative mitochondrial smHSP (Figure 14A).

Collectively, the results suggest that some of the HSP genes, which are not cold
inducible, are regulated by low temperatures only if they have been induced by heat
shock. Surprisingly, the reinduction of the smHSP mRN As at low temperatures was
dependent on the length of the heat shock prior to cold storage. For instance, fruits that
received 36 h or 48 h heat shock accumulate more smHSPs mRN A than fruits that
received high temperatures for 12 or 24 h.

Although the LeHSP90 mRNA was initially induced by heat shock (Figure 13),
the LeHSP90 was also regulated by low temperatures (Figure 14A and 14B).
Interestingly, the heat-treated ﬁuits had a higher LeHSP90 transcript level than the
nonheated ﬁuits at low temperature storage. Similarly to LeHSP90, low temperatures

stimulated LeHSC80 mRNA accumulation. The cold-induced expression of HSP genes

147

in the heat-treated fruits raises the possibility that both high- and low molecular weight
HSPs may play critical roles in resistance to chilling stress.

In addition, we examined whether exposure of heat-treated ﬁuits to ambient temperature
affects the subsequent accumulation of the HSP transcripts under low temperature.
Mature green fruits were exposed to 42°C for 12, 24, 36 or 48 hours, transferred to 20°C
for 1 or 3 days, then shiﬁed to 2°C for 14 days and moved back to ambient temperatures
for 3 days. Unexpectedly, exposure of heat-treated fruits for l or 3 days at 20°C did not
prevent the reinduction of HSPs at low temperature (Figure 15A and B). Nevertheless,
these ﬁuits developed chilling injury. This was particularly the case for the ﬁuits which
were kept at ambient temperature for 3 days. These data suggest that heat—shock proteins
are not the only factors that contribute to the chilling tolerant phenotype of tomatoes.
Holding the tomatoes at 20°C for 3 days aﬁer heat shock and before storage allowed
ripening to recover and proceed. During the recovery period, the ﬁuit produced ethylene
(Lurie and Klein, 1992; Whitaker, 1994) and the ripening related genes, like LeE8, were

restored (Figure 15A and B).

148

Figure 15. The effect of heat treatment and post-treatment at 20°C on smHSPs,
HSP90 and E8 mRNA levels in tomato fruits subsequently stored at 2°C for 14 days.
A. Heat-treated ﬁuits at 42°C for 12, 24, and 36 h kept at 20°C for 1 day and then
transferred to 2°C for 14 days. The ﬁuits were shifted to 20°C for 3 days after the low
temperature storage. Total RNA was extracted and analyzed for the cytosolic class H
LeHSPI 7. 4, LeHSP90 and LeE8 gene expression. B. Heat-treated fruits at 42°C for 12,
24, 36, and 48 h were kept at 20°C for 3 days and then transferred to 2°C for 14 days.
After the low temperature storage the ﬁuits were shifted to 20°C for 3 days. Total RNA
was extracted and analyzed for the cytosolic class I LeHSP17. 7, LeHSP90, LeHSC80 and
LeE8 gene expression.

149

 

20°C-3D

 

2°C-14D
42°C -> 20°C-l D

Cytosolic class II smHSP Le HSP] 7.4

LeHSP90

 

LeE8

20°C-3D

2°C-14D
42°C -> 20°C- 31)

. Cytosolic class IsmﬂSPLeHSPIZ7
Le HSP90

Le HSC80

Le EB

 

 

150

DISCUSSION

We hypothesized that heat-shock proteins (HSP) may be involved in increasing
tolerance to chilling injury following heat treatment. This was based on using differential
display of mRN A, to clone and characterize a cytosolic class H small heat-shock protein
(LeHSP17.6) (Kadyrzhanova et al., 1998). To‘ elucidate if other heat-shock proteins are
induced by heat shock and persist during sub sequent storage at low temperatures, we
cloned a second member of the cytosolic class H smHSP, three cDNAs encoding a
cytosolic class I smHSP and partial cDNA clones for a chloroplastic smHSP, a
mitochondrial smHSP and a heat-inducible HSP90 member.

LeHSPl7.4 is similar to the previous cytosolic class H HSP17.6 isolated from
tomato fruit (Kadyrzhanova et al., 1998). There was no difference in the expression
pattern of the two cytosolic class H smHSPs in Northern analysis conducted with gene-
speciﬁc probes. Both genes are induced by heat shock and the mRN As are more stable
as the heat persists. Interestingly, the transcripts were detected after the heat-treated
ﬁuits were stored at 2°C for 2 weeks. However, when the fruits were chilled at 2°C
without prior heat treatment, the mRN A accumulation was low but still detectable,
indicating a weak stimulation by cold. Cytosolic class H smHSPs have been induced in
response to several conditions including, ozone (Eckey-Kaltenbach, et al., 1997), gamma
irradiation and H202 application (Banzet et al., 1998), different light regimes (Krishna et
al., 1992), water stress (Almoguera et al., 1993), treatment with amino acid analogues
(Lee et al., 1996), heavy-metal stress (Czamecka et al., 1984), embryogenesis (Coca et

al., 1994; DeRocher and Vierling, 1994; Dong and Dunstan, 1996) and pollen

151

development (Bouchard, 1990, Dietrich et al., 1991; Atkinson et al., 1993; Kobayashi et
al., 1994). Fruit ripening appears to have no role in the expression of cytosolic class H
smHSP.

Polyclonal antibodies raised against T aHSP17.8 recognized tomato cytosolic class
II homologues. The pattern of the protein expression followed the mRN A patterns and
the proteins were still detectable at 20°C 3 days after the cessation of the low temperature
storage. This suggests that these proteins may be important in the repair of damage
incurred during the heat stress or subsequent cold stress. The pea cytosolic class H
smHSP are components of large complexes in vitro and in vivo, and are larger than those
of the class I smHSP complexes (Helm et al., 1997). It has been suggested that the
quaternary structure of class H complexes are different from those of the class I
complexes (Helm et al., 1997). Secondary structure protein prediction suggests that the
two classes might have similar structure with a few differences. Although it has been
proposed that cytosolic class H functions as a molecular chaperone (Lee et al., 1995a;
Helm et al., 1997), we do not have any evidence of chaperone activity in vivo or in vitro,
particularly at low temperatures.

In addition to the cytosolic class H smHSP, we have isolated and characterized
three cDNAs that encode members of the cytosolic class I smHSP family. The ﬁrst gene
(LeHSP17. 7) was identical to the pT OM66 gene previously isolated as a fruit ripening
related gene (Fray et al., 1990; Sabehat et al., 1998a). The other two have 97% similarity
at the amino acid level. The mRN A levels of the LeHSP17. 7 and LeHSP17.6 were
induced by high temperature and the transcripts were detected in ﬁuit during subsequent

low temperature storage. Interestingly, the expression of these genes was not detected in

152

fruits exposed to low temperatures only. These data are in agreement with the results
reported by (Sabehat et al., 1996; 1998a) where the induction of HSP17 mRNA during
the heat treatment and translation of HSP17 protein persisted during subsequent storage
of the ﬁuit at 2°C.

The expression of the cytosolic class I smHSPs were distinguished from the
cytosolic class H smHSPs only in the fact that they are induced during ﬁuit ripening in
tomatoes (Picton and Grierson, 1988; Rothan et al., 1997; Sabehat et al., 1998a) and
strawberries (Medina-Escobar et al., 1998). Cytosolic class I smHSP was also induced
in the COg-treated tomato fruit (Rothan et al., 1997) and perhaps may be required to
protect proteins against cellular acidosis induced by C02 solubilization. In many plants,
the cytosolic class I smHSP is expressed during development. For example, cytosolic
class I mRN A and its protein accumulated during embryo development (Vierling and
Sun, 1989; Almoguera and Jordano, 1992; Coca et al., 1994; DeRocher and Vierling,
1994; zurNieden et al., 1995; Wehmeyer et al., 1996; Carrango et al., 1997), seed
germination (Vierling and Sun, 1989; Kaukinen et al., 1996; Wehmeyer et al., 1996),
somatic embryogenesis (Gyorgyey et al., 1991; Zarky et al., 1995).

Western analysis indicates that the AtI-ISP17.6 polyclonal antibody cross-react
with at least two cytosolic class I smHSP from tomato fruit. The expression pattern of
proteins mimics the transcript pattern and the results obtained with cytosolic class H
homologues. Interestingly the protein presence in fruits at 20°C aﬁer cold storage
suggesting that the class I smHSP may protect other proteins during recovery from the
stress. Cytosolic class I smHSP from pea has been shown to have chaperone activity in

vitro (Lee et al., 1995a; Lee et al., 1997) by protecting citrate synthase against thermal

153

aggregation in an ATP-independent fashion. Interestingly, F orreiter et al. (1997), using a
stable transformed Arabidopsis cell suspension culture overexpressing luciferase as a
reporter gene, demonstrated that the cytosolic class I AtHSP17 .6 showed chaperone
activity in vivo by preventing the thermal inactivation of the reporter protein. Moreover
the crystal structure of the M]HSP16.5 has been reported (Kim et al., 1998) and it forms a
hollow spherical complex, where certain protein or RNAs critical for the cell survival
may reside in under stress conditions. Several observations, suggests that the consensus
region H of the “heat shock domain” are not necessary only for oligomerization but also
for chaperone activity (Merck et al., 1993; Plater et al., 1996; Leroux et al., 1997; Yeh et
al., 1997).

Another cDNA isolated was a chloroplastic smHSP identical to pTOMl 11
(Lawrence et al., 1997; Sabehat et al., 1998a). The mRNA accumulation proﬁle was
similar to cytosolic class I smHSP. In addition, similar results by Sabehat et al. (1998a)
indicate that the protein was detected at low temperatures only in the heat-treated ﬁuit.

In parallel with the identiﬁcation of small HSPs, which were induced by heat
shock and persisted at low temperatures, we isolated a partial cDNA that encodes a
member of HSP90 family. The HSP90 family is composed of genes encoding
structurally related proteins ranging from 80 to 90kD (Scheibel and Buchner, 1997).
HSP90 genes have been isolated from several plant species, including Arabidopsis
(Conner et al., 1990; Yabe et al., 1994, Milioni and Hatzopoulos, 1997), barley (Walther-
Larsen et al., 1993), Brassica napus (Krishna et al., 1995), Catharantlms roseus
(Schroder et al., 1993), maize (Marrs et al., 1993), Pharbitis nil (Felsheim and Das,

1992), rye (Schmitz et al., 1996), and tomato (Koning et al., 1992). LeHSP90 is

154

 

predicted to be in the cytosol and has 82% similarity with the LeHSC80 at the amino acid
level. The LeHSC80 is expressed constitutively at high levels at physiological
temperatures (Koning et al., 1992) and induced only slightly by heat shock in tomato
fruits. The LeHSP90 however is not expressed at ambient temperatures, but the
expression is enhanced strongly by heat shock. The induction of both genes appears to
be transient. The maximum level of HSP90 mRNA is achieved after 12 h of HS followed
by a gradual decline, a phenomenon called autoregulation of the heat shock response
(Schofﬂ and Key, 1982; Gurley and Key, 1991). The inhibition of the HSP90 expression
by prolonged heat shock is distinguished from smHSP autoregulation; it appears that
HSP90 mRNAs are less stable at high temperatures (Gallic et al., 1995). Another
explanation of the negative regulation of the HSP90s is the observation that HSP90 is a
major repressor of the HSF in mammalians cells (Ali et al., 1998; Zou et al., 1998).

In addition, we found that both HSP903 are induced by low temperatures. Similar
results have been reported by Krishna et al. (1995). Using a polyclonal antibody raised
against PnHSP83 (Krishna et al., 1997) we detected a cross-reacting protein ﬁom tomato.
The protein expression pattern indicates that the HSP905 are detectable after 2 days at
42°C. The level of the protein in the heat-treated fruit is high at low temperatures and
continues to be expressed at 20°C for 3 days. In contrast, cold had little effect in HSP90
expression of nonheated fruit. The role of the HSP90 at low temperatures is unknown.

HSP90 is the most abundant chaperone (Jakob and Buchner, 1994). HSP90 is
referred to as “molecular glue” in the cytoplasm of mammalian cells (Csermely et al.,
1998). HSP90 binds to a wide range of proteins including, kinases (Miyata and Yahara,

1992), phosphatases (Chen et al., 1996), nuclear hormone receptors (Pratt, 1997), actin

155

(Czar et al., 1997), tubulin (Fostinis et al., 1992), calmodulin (Minami ct al., 1993),
proteasome (Tsubuki et al., 1994) and heat shock transcription factor (N adeau et al.,
1993). HSP90 also forms a large cytosolic complex, named as foldosome, with other
molecular chaperones such as HSC70, immunophilins, CDC37 and p23 (Pratt, 1993).

Recent studies (Young et al., 1997; Scheibel et al., 1998) demonstrated that
HSP90 has two independent chaperone sites, the N-terminal and the C—terininal site. The
N-terminal chaperone site can be inhibited by geldanamycin (Whiteshell et al., 1994).
HSP90 displays a heat-induced chaperone activity at 46°C (Y onehara et al., 1996).
However, recent studies in yeast suggest that HSP90 is required for a speciﬁc subset of
proteins having difﬁculties reaching their native conformation (Nathan et al., 1997).
Moreover, under stress conditions, HSP90 enhances the rate at which a heat.damaged
protein is reactivated (Forreiter et al., 1997; Nathan et al., 1997) and does not protect the
proteins from thermal inactivation.

One of the possible models for the mechanism by which heat treatment attenuates
chilling injury may be attributed to molecular chaperone activities of HSPs (Boston et al.,
1996). Molecular chaperones are a group of intracellular proteins that ensures correct
folding, oligomeric assembly, transport across membranes or disposal by degradation of
other conformer unstable proteins by binding to them and releasing them. Additionally,
molecular chaperones prevent incorrect interaction within and between non-native
polypeptides which result in their irreversible aggregation (Hartl, 1996). The cytosolic
class I smHSP prevent thermal aggregation by selectively binding to non-native proteins
forming soluble high molecular weight complexes (Lee et al., 1997). These complexes

were stable even after storage at 4°C (Lee et al., 1997). However, we do not know if the

156

complexes are formed at low temperatures. If so, expression of smHSP during low
temperature will be beneﬁcial to chilling-sensitive plants like tomatoes. Therefore,
demonstration of chaperone activity of heat-shock proteins at low temperatures is
necessary.

The combination of the heat treatment and low temperature storage revealed an
interesting observation. The expression of the smHSPs mainly was induced at low
temperatures only if the ﬁuits have been exposed to heat shock. The reinduction of the
transcripts in the cold was more favorable after prolonged exposure to high temperatures.
To our knowledge similar phenomena have not been reported in plant or other organisms.
Perhaps the HSFs are able to bind to the promoter regulatory elements and switch on the
transcription of the smHSP in the cold. This is consistent with the observation that the
Drosophila heat shock factor had little trimer dissociation and loss of DNA binding
activity when incubated at 4°C after a heat shock period (Zhong et al., 1998).
Furthermore, exposure to low temperature may activate new cold-inducible transcription
factors that reactivate smHSP gene transcription. Transcription factors that bind to the
putative STRE element (Marchler et al., 1993 ), found in the promoter of the cytosolic
class H HSP17 .6, might be a good candidates.

The heat shock induced protection against chilling injury was transient. Fruits
transferred to ripening temperatures after exposure to heat shock and before chilling lost
the heat shock induced protection within several days (Lurie and Sabehat, I997).
Unpredictably, exposure of heat-treated ﬁuits for 1 or 3 days at 20°C did not prevent the
reinduction of HSPs at low temperature. Collectively, these data suggest that heat-shock

proteins are not the only factors that contribute to the chilling tolerant phenotype of

157

tomatoes. In agreement, Guy et al. (1986) suggested that the heat-induced freezing
tolerance of Neurospora crassa spores did not involve synthesis of heat-shock proteins,
since the presence of cycloheximide during heat shock did not diminish the protection
that heat shock afforded against freezing injury.

Indeed, heat treatments induce the synthesis of heat-shock proteins and interrupts
or down-regulates the expression of genes involved in ﬁuit ripening metabolism. The
latter is likely to be responsible for the cross-protection, whereas the former is mainly
involved in thermotolerance. The difference in the behavior to chilling sensitivity
between heat-treated fruit and fruit that recovered from heat shock lies on the ability of
the latter to synthesize and perceive ethylene the ripening hormone (Theologis, 1992).
The notion that heat-induced chilling tolerance is due to discontinuation of the ﬁuit
ripening metabolism is supported by the following observation. Antisense-ACC oxidase
melon fruits were resistant to chilling injury (JC Pech, personal communication). When
the same fruits were treated with ethylene, the susceptibility to chilling injury was
restored indicating that ethylene insensitivity is important for chilling tolerance. Thus,
in order to promote chilling insensitivity not only heat-shock proteins but also ethylene

insensitivity may be required.

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170

 

CHAPTER IV

IDENTIFICATION OF THE GENOMIC SEQUENCE OF LeHSPI 7.6 AND ITS

CIS-ACTIN G ELEMENTS

ABSTRACT

The LeHSP17.6 gene encodes a cytosolic class H smHSP of tomato. LeHSP17.6
is accumulated massively in tomato ﬁuits exposed to heat shock and slightly by low
temperatures. The combination of the heat treatment and low temperature storage
revealed an interesting observation. The expression of the LeHSP17. 6 was induced at
low temperatures only if the fruits have been exposed to heat shock. The reinduction of
the transcripts in the cold was more favorable after continued exposure to high
temperatures. We have cloned an extended 5’ ﬂanking region of the LeHSP17.6 in order
to identify cis—acting elements involved in the regulation of this gene, particularly during
the reinduction in the cold after the heat shock treatment. A detailed analysis of the
LeHSP17.6 promoter region revealed multiple heat shock elements, stress response
elements, GATA elements and long tracts of repetitive sequences with high AT content.

The possible function of these sequences is discussed.

17]

 

 

INTRODUCTION

Plants are bound to their habitat, they can not run away from many threatening
environmental and anthropogenic stressors, and therefore need special mechanism to
avoid stress or to adapt to stress (Lichtenthaler, 1998). Cells pre-exposed to mild non-
damaging stress conditions will induce resistance/tolerance not only to the factor used,
but also tolerance against severe stress caused by other agents. This is referred as the
cross-protection or cross-resistance mechanism. For example, a mild heat shock will
protect the plants against severe heat shock, a process called thermotolerance (N over,
1991; Vierling, 1991). In addition, it will induce resistance against other environmental
stresses (Bonham-Smith et al, 1987; Orzeck and Burke, 1988; Kuznetsov et al., 1997;
Sabehat et al., 1998b). The induction of the heat shock response can protect cells against
a variety of other toxic insults, such as ethanol and hydrogen peroxide (Ruis and Shuller,
1995; Storz and Polla, 1996).

Heat and anoxia stress treatments are being used in postharvest to induce
resistance to chilling injury (Lurie and Klein, 1991; Pesis et al., 1994). Protection of
tomatoes from chilling injury afforded by prestorage heat treatment has been correlated
with the induction of transcription of smHSPs mRNA during the heat treatment and
translation of the smHSPs which persisted during subsequent storage of the fruit at 2°C
(Sabehat et al., 1996; 1998a; Kadyrzhanova et al., 1998). Using differential display of
mRNA we cloned a full-length HCTl cDNA that encodes a 17.6 kD cytosolic class H
smHSP (Kadyrzhanova et al., 1998). This gene is expressed in tomato ﬁuits exposed to

high temperatures. Further, this gene is expressed in massive amounts at low

172

A‘w

 

“Wu. LL. 11':-

temperatures only if the ﬁuits have been exposed to heat shock (see chapter HI). The
same expression pattern has been reported for the other tomato smHSP (Sabehat et al.,
1998a; Chapter ID).

The purpose of this study was to isolate and characterize the genomic DNA
sequence of the cytosolic class H smHSP LeHSP17.6 and investigate the role of the

upstream sequence element in the heat required cold expression of this gene.

MATERIAL AND METHODS

Genomic Library Screening

The tomato genomic library, provided by Dr. J. Giovanonni of Texas A&M
Univ., was used. This library harbors approximately ten genomic equivalents and was
constructed in lambda DASHII (Stratagene) ﬁ'om L. esculentum (cv. Alisa Craig)
genomic DNA. Approximately 5x105 phages from a tomato genomic library were
screened with a 32P-labeled probe corresponding to the LeHSP17. 6 coding region using
standard procedures (Sambrook et al., 1989). Single hybridizing phages containing a 7-

kb tomato DNA insert were isolated, subcloned and approximately 3kb was sequenced.

DNA Sequencing
DNA sequence determination was performed on Applied Biosystems 373A
(Foster City, CA) at the MSU Instrumentation Facility. Nucleotide sequence data were

assembled and analyzed using DNA STAR (DNA STAR, Madison, WI).

173

Promoter Analysis

Analysis of transcription factor-binding sites on the LeHSP17. 6 promoter was
performed using the TESS transcription element search software of J. Schug and QC.
Overton (http://agave.humgen.upenn.edu/tess/index.htnrl, University of Pennsylvania,
Philadelphia) or by screening the databases on transcriptional regulation TRANSFAC,

TRRD, and COMPEL (Heinemeyer et al., 1998).

RESULTS

The tomato genomic library, was screened to obtain the genomic sequence of
LeHSP17. 6. A single hybridizing phage containing a 7kb pair tomato DNA insert was
isolated. The DNA sequence of the ~3kb from the genomic clone was determined. The
genomic clone of the LeHSP17. 6 exactly matches the sequence of LeHSP17. 6 cDNA,
indicating that is an intronless gene. In addition, it contains a 1.095kb fragment of the
promoter region as well as 1.0kb of the 3’ end region.

The promoter sequence of LeHSPI 7. 6 is presented in Figure l. The transcription-
starting site was predicted to be 78 nucleotide upstream of the start codon ATG. The
sequence at this site, CTCACTG closely resembles the consensus plant transcription
initiation sequence, CTCATCA (Joshi, 1987). A putative TATA box, TATATAA, about
30 bp upstream from this site (position -110). Table I summarizes the potential

transcription binding sites and cis-acting elements in the LeHSP17.6 promoter. Several

174

copies of the HSE (heat shock element), AGAAn or nTTCT, are found at position -120 to
-129, -153 to -174, -279 to -287, -301 to -317, -880 to -891 and -905 to -915. In addition
there are three copies of the putative STRE (stress response element), CCCCT, at
positions -92 to 96, -254 to 259, and -615 to 620. Moreover there are six NIT2 motifs,
TTGATA or its complement TATCAA, at the positions -257 to -263, -271 to ~277, -326
to -332, -337 to -343, -722 to -728 and -985 to -991. Furthermore, the promoter contains
a G-box (-181 to -185), a Cd responsive element (-693 to -698), a salicylic acid inducible
element (-487 to -495), a GAGA element (-174 to -178), a CCAAT or Y-box element (-
291 to -297) and two REa element, the ﬁrst overlaps with the Y-box at position -294 to -
299 and the second is located in the 5’ UTR of the gene at position -56 to -62. In
addition, the 5’ ﬂanking region of the tomato cytosolic class H smHSP include runs of
simple sequences, (A),,, (T), and (TA)n preceeding the HSEs and an AT-rich upstream

region.

175

'1‘ “’3' -.—— ——'-

 

CACAGATTCTCTTCATTTAATTGAACTTAATAAATTAGTTTTAAATTTATA -1043
ATTTTTACTTTTAATTTTTCAAAAAAATTATTTAGGAGTATATATGACTCT -993
TCTATCAAAGTTAAAGGTATATTTTAATTTTTTTCATACATAAATTATTTT -943
GATA
TTGACTTCTTTTATTATAATTACTTGAGTTTCTTATTCTTATTTTAATTTT -893
TTTCTTTCATTCTTTAGTTTAGAGAAAAAAATTTTAAACTATTTTGTCTAT -843
ATTGTAATTTGATTTTTGTATTCGAAGAAAAAAATTTGTCGTCTACAAGTT -793
TTACAAGAATATTAGTGAAACATAAATAAATTTGATTATCAAAATAATAAT -743
GATA
TCTAAATTAGTCATTGAAACAAAAAAAAGTCAAAAAAATATATGTTTGACG -693
CdRE
AGGATTAAATTTACTCATATGAGATTATATTTTTTAAAAAATAATAATAAA -643
AATTTAATTTAATTTTTTTTATTTCCCCTTAQAQGAAAAGGGTATATGTGA —593
STRE SARE
TTAATTTGTTTATAAATAGATCCCTTATATGAGTCATACTCATAACAATGG -543
CTATGTCAGCTCCAAATTACTAAGTTGAGGATATATCAGATCTTTTTGTCC -493
CTAAATTAATATATATATATATGTATATATATATATGTATATATAIATATA. —443
TATATATATATATATATAIATATATATAAAGTAATATTAATATTAATAATG —393
33xTA
TATTCGAAATTACATTATTATATTTTATTAATATATTTAGTATAGTTAATI -343
CGATATTTTAATTTCATATCAAAAATAAGAAAATTGACTTTCTAAACCAAT -293

 

 

 

 

 

GATA GATA HSE REa/Y-box
CAT GATTGAATT TTC TTGATAAATAAATATATCAACCCCTTAAAAAATAGA — 2 4 3
HSE GATA GATA STRE

AACAAAAAATAAAAATAAAAAGGAACAATCTGCGAGTTTCTAGACCAGTGT -l93
AGAATCAACGTGATGAAGAGAAGAAGCTCCTAGAAACTTTCTTCATTCTTC -143

 

 

 

GJhx GAGA HSE HSE
TTTGATCATCCTCCAGAACTTTCCACTTTTCCAOm CCTAACCCC —93
HSE STRE
ETTACCCCATTCTCégTGCAATTACAAATCAAACCAAAATTGACAAATTTC -43
"‘REE'

ACGCACAAAATCACAATATCCAAAAATTTCTCAATACTGAAAATG

 

Figure 1. Nucleotide sequence of tomato cytosolic class II smHSP LeHSP17.6 gene.
Upstream sequences ﬁom the start codon are assigned negative numbers. The TATA
sequence is bold boxed. Important sequences are bold underlined: HSE-like promoter
sequences (in red), STRE (in blue), GATA-1 like elements (in magenta), G-box (in
green), TA-runs, REa-like elements, CCAAT or Y-box and the GAGA factor. The start
of the 5’ untranslated region is double underlined.

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177

DISCUSSION

The genomic sequence of the cytosolic class II smHSP LeHSP17.6 has been
determined; it is an intronless gene. One common feature that the cytosolic small HSPs
genes have, is the absence of an intron within the protein-coding region (Gurley and Key,
1991). These heat shock genes may have lost the introns in the optimization of
expression under temperature stress conditions. A brief and severe heat shock has been
shown to interrupt the splicing and translation of Drosophila hsp83 mRN A (Yost and
Lindquist, 1986), suggesting a selective advantage for intronless heat shock genes under
extreme temperature stress conditions (Lindquist, 1986).

Promoter regions of cytosolic class II smHSPs genes have been reported ﬁ'om
maize (Dietrich et al., 1991), Pharbitis nil (Krishna et al., 1992) and soybean (Raschke et
al., 1988). The conserved features within the cytosolic class II smHSP gene promoters
are the occurrence and the relatively strict spacing of the TATA box (20 to 30 bps from
the transcription start site), the transcription start site and the ﬁrst proximal HSE. Next, I

discuss the most important elements observed in the LeHSP17. 6 promoter.

HSE (Heat Shock Element)

The expression of the heat shock genes in plants is regulated primarily at the
transcription level. Induction of the heat shock genes requires a conserved cis-acting
element, termed as heat shock element (HSE), located within the promoter region of

these genes (Pelham, 1982). HSEs consists of repeated units of the S-bp sequence

178

nGAAn arranged in alternating orientation (Amin et a1, 1988; Xiao and Lis, 1988) and
recently reﬁned as AGAAn (Fernandes et al., 1994). The number of pentameric units in
an HSE can vary from three to eight (F ernandes et al., 1994). Furthermore, the degree of
homology of each pentameric unit to the consensus AGAAn motif can vary (Bonner et
al., 1994). The nature of the initial pentamer, GAA or its complement TTC, is also
varied. The nTTCT pentamer displayed higher biological activity in yeast and it was
capable of binding two HSF trimers instead of one (Bonner et al., 1994). In addition,
HSE can tolerate 5 bp insertion between repeating units, provided that the spacing and
the orientation of the pentameric units are maintained (Amin et al., 1988).

The ﬁrst demonstration that the plants shared the highly conserved mode of
promoter activation was seen in the heat-inducible transcription of the Drosophila HSP70
promoter in transgenic tobacco callus and plants (Spena et al., 1985; Spena and Schell,
1987). In plants the optimal HSE core consensus was shown to be 5’-aGAAg-3’ (Barros
et al., 1992). The promoter regions of the small heat-shock proteins genes have multiple
COpies of HSE-like elements within 150 bp upstream from the TATA box sequence
(Schoﬂl et al., 1984; Nagao et al., 1985; Raschke et al., 1988). Analyses of 5’ and 3’
promoter deletions of the cytosolic class I GmHSPI 7-3B in transgenic tobacco
demonstrated the ﬁmctional signiﬁcance of HSEs for the heat shock dependent
transcription of the native genes or ﬁised reporter genes (Baumann et al., 1987; Schofﬂ et
al., 1989). Similar results have been observed by deletion mutagenesis of the cytosolic
class I Gmhsp17.5-E promoter indicating that the TATA-proximal HSEs contribute to the

abundance of the thermal induction (Nagao et al., 1986; Czarnecka et al., 1989).

179

HSEs are the binding sites for the trans-active heat shock factor (HSF), and the
efﬁcient binding requires at least three pentameric units (Perisic et al., 1989; Sorger and
Nelson, 1989). Tomato HSFs are able to trimerize and translocate to the nucleus (Lyck et
al., 1997, Scharf et al., 1998). The Arabidopsis thaliana HSF] has been shown to bind
consensus tripartite HSE sequences and the HSE-containing region of Drosophila HSP70
promoter (Hubel and Schofﬂ, 1994; Hubel et al., 1995).

Prior to a heat signal the promoter of the heat shock proteins are in open
conﬁguration as evidenced by nuclease hypersensitivity experiments (Wu, 1980). At
least three different transcription factors are bound to the uninduced promoter, the GAGA
factor, the CCAAT factor and the TBP (TATA binding protein) (Wu, 1984; Giardina et
al., 1992; Landsberger and Wolffe, 1995). The GAGA factor, which occupies the GAGA
elements, act at the early step of the assembly of the promoter by keeping the HSE clear
from repressive nucleosomes (Lis and Wu, 1994). The potentiation of heat shock
promoters in vivo is characterized by an initiated, paused polymerase, waiting to receive
an activating signal from HSF in order to resume transcription (Sandaltzopoulos and
Berger, 1998). Recently, TBP was found to interact with the HSF at the Drosophila
hsp70 promoter (Mason and Lis, 1997) and with AtHSFl in vitro and in viva (Reindl and
Schofﬂ, 1998). The HSF have been reported to increase transcription from heat shock
promoter by affecting the rate of reinitiation (Sandaltzopoulos and Becker, 1998).

When cells are stressed, HSF homotrimerizes, acquires DNA-binding activity,
translocates to the nucleus, is hyperphosphorylated and becomes transcriptional
competent (Morimoto et al., 1996; Voellmy, 1996). Prolonged heat shock or recovery

from heat shock results in attenuation of the heat shock response. It has been suggested

180

that the attenuation of the heat shock response is autoregulated by heat shock proteins,
particularly the HSP70 and the HSP90 (DiDomenico et al., 1982; Abravaya et al., 1991;
Baler et al., 1996; Shi et al., 1998; Zou et al., 1998). In plants, certain HSFs in subclass
B (Nover et al., 1996) may work as repressors that counteract transcriptional activation
(Czarnecka-Verner et al., 1998). Negative regulation of the HSFs also occurs by
phosphorylation (Kline and Morimoto, 1997; Schoffl et al., 1998). However, when
amino acids analogs are incorporated into the naked polypeptides, the heat-shock proteins
are constitutively up-regulated and attenuation does not occur (DiDomenico et al., 1982,

Mosser et al., 1988).

AT-rich Promoter Element

The 5’ ﬂanking region of the LeHSP17.6 is comprised of 33 contiguous A-T bp
positioned 300 bp upstream of the TATA box (Figure 1). The smHSPs genes often have
blocks of AT-rich sequences located within the promoter region (Nagao et al., 1985;
Baumann et al., 1987; Raschke et al., 1988; Czamecka et al., 1989; Krishna et al., 1992).
Deletion of an AT-rich region from the Gmhspl 7. 3B promoter resulted in a substantial
loss of transcriptional activity, suggesting that this region may function as enhancer-like
elements (Baumann et al., 1987). The best evidence that the AT-rich sequences may act
as enhancer-like elements is the observation that synthetic AT-rich oligonucleotide are
able to stimulate transcription when placed prior to a truncated maize A6011 promoter
(Czamecka et al., 1992). However, the function of the AT-rich domains depends on

additional cis-elements (Rieping and Schoﬁl, 1992).

181

The AT-rich blocks ﬁ'om the Gmhspl 7. 5E promoter were able to bind nuclear
proteins in vitro (Czamecka et al., 1990; 1992). Proteins that bind with high affinity to
AT-rich sequences belong to the HMGs (high mobility group proteins) family (Pedersen
et al., 1991; Czamecka et al., 1992). HMG may help to maintain a chromatin structure
accessible to speciﬁc transcription factors. AT-rich region are very similar to scaffold or
matrix attachment regions reported in Drosophila (Jackson, 1986). A scaffold
attachment sequence in the 3’ ﬂanking region of the GmHSP17. 6-L, mediated enhanced
and gene-dosage expression of a chimeric heat shock gene in transgenic tobacco plants

(Schoffl et al., 1993).

CCAAT (Y -box) and RE! Elements

In addition to the preceeding elements (HSE and AT -rich), a perfect CCAAT box
is located 188 bp upstream the TATA box (Figure 1). The CCAAT boxes act as
functional cis elements in the soybean hsp17. 3-B promoter (Rieping and Schofﬂ, 1992).
The CCAAT boxes and the HSE act synergisticly but not additively to increase heat
shock inducibity (Rieping and Schoﬁl, 1992). In other organisms, CCAAT (or Y-box)
elements are required for oocyte expression of the Xenopus HSP70 (Bienz, 1986). The
CCAAT-binding protein may interact with HSE to enhance its binding to HSF, especially
in the absence of heat stress (Bienz, 1986). In the Xenopus HSP 70 promoter, the Y-box
elements are important for disrupting local chromatin structure and for facilitating the
association of HSF and transcriptional activation in chromatin (Landsberger and Wolffe,

1995). Recently, it has been reported that the CCAAT-binding protein, in the Xenopus

182

HSP 70 promoter, establishes pre-set chromatin architecture that can modulate
transcription by recruiting the p300 acetyltransferase (Li et al., 1998).

The CCAAT element found in the LeHSP17.6 promoter is overlapped by another
element, the AACCAA (designated as REa), which is required for phytochrome
regulation of the Lemna gibba Lhcb2 *1 (Degenhardt and Tobin, 1996). This element
appears also in 5’ UTR region. The REa element was proposed to ﬁmction as a repressor

of transcription in the dark (Degenhardt and Tobin, 1996).

STRE (Stress Response Element)

The 5’ ﬂanking region of the LeHSP17.6 contains three motifs that closely
resemble the stress response element (STRE) present in the promoter of stress-induced
genes in yeast. STREs have never been reported in plant gene promoters. However, the
cytosolic class II smHSP gene promoter from maize has two STREs, one near the TATA
box and the other 320 nucleotides upstream of the transcription site (Dietrich et al.,
1991). The cytosolic class H smHSP gene promoter from Pharbitis nil and ﬁ'om soybean
does not have this. The only plant promoters that have the same number and orientation
pattern of the STREs, observed in the LeHSP17.6 promoter, are the HVA22 gene, an
ABA-induced gene (Shen et al., 1993) and HVAI (Straub et al., 1994) a ABA, cold, salt
and heat-induced gene. The STREs are not the essential elements for the ABA-induction
of the H VA22 (Shen and Ho, 1995). Therefore, the role of the STREs in plants is
unknown. Below I summarized the role of the STREs in yeast stress-inducible genes.

In yeast there are at least three positive transcriptional elements activated by

stresses (Attﬁeld, 1997). These are heat shock elements (HSEs), stress-response

183

elements (STREs) and AP-l responsive elements (ARES). Kobayashi and McEntree
(1990) have demonstrated that a nonpalidromic sequence from DDR2 promoter was able
to confer heat shock inducibity of a reporter gene, independent of HSF. This element
(also called STRE), which has the consensus core sequence CCCCT or AGGGG, has
been reported to mediate stress induction (Marchler et al., 1993).

STRE-dependent response involves diverse genes such as C IT 1 , a cytosolic
catalase T (Wieser et al., 1991), DDR2, a DNA damage repair protein (Kobayashi and
McEntree, 1990; 1993), HSP12 (Praekelt and Meacock, 1990; Varela et al., 1995),
HSP26 (Petko and Lindquist, 1986), HSP104 (Mager and De Kruijff, 1995), SSA 1, SSA4
(HSP 70), UBI4 (Kobayashi and McEntree, 1993), GPDI, a glycerol-3-phosphate
dehydrogenase (Blomberg and Adler, 1989), PIPZ (Mager and De Kruijff, 1995), GAC 1,
a phosphatase type-1 subunit (Francois et al., 1992), YPSI (trehalose-6-phosphate
synthase), IPSZ, a trehalose phosphate phosphatase (Gounalaki and Thireos, 1994),
C YC 7 (iso-Z-cytochrome C), ALD2 (cytosolic aldehyde dehydrogenase), GSYZ, a
glycogen synthase (Ni and LaPorte, 1995) and SODZ (Flattery-O’Brein et al., 1997). The
diversity of these gene functions indicates the generality of the STRE-dependent response
(Attﬁeld, 1997). Genes with STREs are crucial to survival of cells that have been
exposed to severe (lethal) stress (Ruis and Schuller, 1995).

Basal and induced levels of STRE-dependent transcription are enhanced in rasz
mutants with low protein kinase A activity and dramatically reduced in bcyl mutants
with high constitutively protein kinase A (Belazzi et al., 1991; Boorstein and Craig, 1990;
Marchler et al., 1993; Engelberg et al., 1994). It appears that protein kinase A represses

the activity of STRE elements (Ruis and Schuller, 1995). Moreover, Schuller et al.

184

(1994) identiﬁed STREs as speciﬁc targets of the HOG (high osmolarity glycerol)
pathway. Defects in this pathway completely abolished the transcription of a STRE-lacZ
reporter gene construct by osmotic stress. Therefore, two signaling pathways identiﬁed
as STRE mediators, the HOG pathway (3 MAP kinase cascade) and the RAS-cAMP
pathway as a negative regulator.

The transcription factors that recognize the STRE have been identiﬁed as
multicopy suppressors of the Snflp protein kinase mutant (Estruch and Carlson, 1993).
STRE-transcriptional factors (designated as Msn2 and Msn4) are Csz zinc ﬁnger DNA-
binding proteins and ﬁinction as transcription activators (Estruch and Carlson, 1993;
Schmitt and McEntee, 1996). MSNZ and MSN4 have been shown to bind to STRE in the
gel mobility-shift assays (Martinez-Pastor et al., 1996; Schmitt and McEntee, 1996).
Moreover, yeast strains with deleted Msn2/4 ﬁrnction are hypersensitive to carbon
starvation, DNA damage and other stresses such as heat, oxidative and osmotic stress
(Martinez-Pastor et al., 1996; Schmitt and McEntee, 1996). The stress sensitivity of those
mutants was restricted to severe stress. Expression of C TT 1 , HSPIZ and DDR2 induced
by heat shock, high salt concentration, sorbic acid or ethanol was abolished in the double
msn2msn4 mutant (Martinez-Pastor et al., 1996; Schmitt and McEntee, 1996). In
addition the H’SZ, encoding trehalose phosphate phosphatase, is regulated through the
MSNZ/STRE pathway (Schmitt and McEntee, 1996).

MSN2/4p controls a lot of genes induced at the diauxic transition (Boy-Marcotte
et al., 1998), including alcohol dehydrogenase-like (ALD3/5), transketolase (IKLZ),
hexokinase 1, HSP70, trehalose-6-phosphate synthase (IPSI), glutamate dehydrogenase,

glycerol phosphate phosphatase (GPP), aldehyde dehydrogenase-like, HSP104,

185

glucokinase(GLK1), phosphoglucomutase (PGMZ), and SODZ. Diauxic transition is the
transient growth arrest of yeast grown on glucose media, when the glucose is exhausted.
Then, the cells are grown on ethanol as a carbon source. In addition, STRE-driven IacZ
reporter gene was induced during diauxic transition and cAMP had a negative effect on
this induction (Boy-Marcotte et al., 1998).

Genes like YPSI and PGMZ are involved in carbohydrate storage which is a
cellular response to various stresses (Parrou, 1997). Interestingly, a lot of genes encoding
metabolic enzymes are dependent on Msn2/4 STRE response (DeRisi et al., 1997; Boy-
Marcotte et al., 1998; Moskvina et al., 1998).

In yeast ﬁve genes encoding HSPs (HSP12, HSP26, HSP42, HSP78, HSP104)
were identiﬁed to have several STREs in their promoter (Moskvina et al., 1998; Treger et
al., 1998). The promoters of these genes contain HSEs, which have been shown to be
required for heat shock activation. Treger et al. (1998) evaluated the contribution of the
STREs in the heat shock regulation of these genes. The HSC 70 showed no requirement
for Msn2/4p. The HSP12 was predominadly regulated by the STRE, while the HSP26
expression occurred mainly via HSF/HSE pathway. Both transcripts were abolished after
heat shock exposure in the triple mutant (mmZ/msn4/hsf1). Similarly the HSP78 and the
HSP104 expression were regulated by the two pathways. Surprisingly, the level of
HSP82 gene was slightly affected by inactivation of both pathways, suggesting that a
third independent stress regulatory pathway might occur for the heat shock induced
expression of these genes. The role of the STRE element in the regulation of LeHSPI 7. 6

upon heat shock and heat-required cold induction it remains to be elucidated.

186

NIT2 or GATA Core Elements

The promoter sequence of LeHSPI 7.6 contains six NIT2 consensus core
sequences, TTGATA or its complement TATCAA. The arrangement of the NIT2
elements is analogous to cis-regulatory sequences found in the promoters of nitrate-
inducible genes in Neurospora crassa, regulated by the NIT2 Zn-ﬁnger protein (F u and
Marzluf, 1990; Chiang and Marzluf, 1994). The NIT2 binding sites contain two GATA
elements, which have varied spacing but must be less than 30 bp of each other (Chiang
and Marzluf, 1994). In Neurospora the NIT2, a member of the GATA family of
transcription factors, is a globally acting nitrogen regulatory protein that positively
activates the expression of many nitrogen catabolic genes, including nitrate reductase
(Marzluf, 1997). In spinach, nitrite reductase, the second enzyme in the nitrate
assimilation pathway reducing nitrite to ammonium, is induced upon addition of nitrate.
This expression was mediated by nitrate-speciﬁc binding of the NIT2 factor to a
TTGATA sequence, observed in the promoter of the nitrite reductase (Rastogi et al.,
1997). NIT2-like protein (NTLl) has been cloned and characterized from tobacco
(Daniel-Vedele and Caboche, 1993). Although the promoter of the tomato cytosolic class
11 small HSP17.6 has 6 NIT2-related cis-acting elements, there is no report that this gene
is involved in nitrate assimilation pathway or during nitrogen limitation.

The core sequence of the NIT2 element is the “GATA” which is recognized by
other GATA factors. This motif, called GATA box or I-box, is present in the light
regulated CAB genes of different species (Gidoni et al., 1989). Therefore, the I-box has
been suggested to be involved in light-regulated gene expression of photosynthetic genes

(Manzara et al., 1991; Terzaghi and Cashore, 1995). The Pharbitis nil cytosolic class II

187

 

smHSP shp-I is induced by photoperiod and by light treatment of dark grown seedling
(Krishna et al., 1992). However, the light induction may be mediated by GT-l-like
elements found in the promoter of the shsp-I (Krishna et al., 1992). These GATA factors
regulate distinct metabolic pathways. How these factors speciﬁcally regulate their own
target genes is unknown. Perhaps, the ﬂanking region of the GATA core binding sites
might determine the DNA-binding speciﬁcity of each GATA factor (Ravagnani et al.,
1997)

In conclusion, detailed analyses of the LeHSP17.6 promoter region revealed
multiple heat shock elements, stress response elements, GATA elements and long tracts
of repetitive sequences with high AT content. The LeHSP17.6 is accumulated in tomato
fruits exposed to heat shock and slightly by low temperatures. The combination of the
heat treatment and low temperature storage revealed an interesting observation. The
expression of the LeHSP17.6 was induced at low temperatures only if the fruits have
been exposed to heat shock. The reinduction of the transcripts in the cold was more
favorable after continued exposure to high temperatures. These cis-acting elements may
be useful to ﬁrrther understand the novel expression pattern of the LeHSP17.6 during the

combination of the high and low temperatures.

188

 

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\

CONCLUSIONS AND FUTURE RESEARCH

The speciﬁc goals of this study were a) to develop, optimize and characterize a
commercially applicable postharvest heat treatment for cold storage of tomato and b) to
investigate the involvement of heat shock genes and heat-shock proteins in heat
treatment-induced resistance to chilling injury.

To achieve the ﬁrst goal, heat treatments (warm air) were investigated. Treatment
of tomatoes in 42°C in humidiﬁed air for 36 h or 48 h before storage at chilling temperatures
(2°C) for 14 days reduced CI and decay during subsequent ripening at 20°C. This
protection was lost if the tomatoes were transferred from 42°C to 20°C for 1 to 3 days
before low temperature storage, however the CI was less than that of control ﬁ'uit.
Ripening of the heat-treated ﬁ'uits was normal although slower than that of the control ﬁuits.
The next step is to evaluate the effectiveness of heat treatments on a commercial scale. Hot
water application appears to be promising as part of the packinghouse line. Thus, in the
ﬁxture this work may have very practical application to postharvest treatment of fruits and
vegetables. Heat treatments can decrease fungal rots, both by killing the fungal spores and
by activating defense mechanism in the fruit tissue. Further research on the defense
mechanisms activated by heat shock may yield beneﬁts to agriculture; heat treatment may
slow ripening, decrease sensitivity to chilling injury, and decrease fungal infections.

The second goal, to investigate the involvement of HSP in the resistance to chilling
injury, has been well documented in this study. Using differential display of mRNA, a

full-length cDNA that encodes a cytosolic class II smHSP (LeHSP17.6). To elucidate if

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other heat-shock proteins are induced by heat shock and persist during subsequent storage
at low temperatures, we cloned a second member of the cytosolic class H smHSP, three
cDNAs encoding a cytosolic class I smHSP and partial cDNA clones for a chloroplastic
smHSP, a mitochondrial smHSP and a heat-inducible HSP90 member.

The cytosolic class I and H smHSPs and the chloroplastic smHSP were up
regulated by heat treatment, and this was slightly increased during subsequent storage at
cold temperatures. Non-heated fruits stored at chilling temperatures displayed low levels
of cytosolic class H smHSP mRNA On the other hand, although the HSP90 mRNA was
induced during heat treatment, its expression was regulated by cold; the heat-treated
ﬁ'uits had a higher transcript level when stored cold than non-heated fruits. Cytosolic
class I and II smHSPs transcripts were more stable than those of LeHSP90s and
chloroplastic smHSP if heat shock was prolonged. The expression pattern of proteins
encoded by mRNAs for the smHSPs and HSP90s followed a similar trend. More
importantly, the cytosolic class I and II smHSP as well as the HSP90 were still detectable
when the fruits were transferred to 20°C for 3 days.

Collectively, correlation of accumulation of HSPs with resistance of the tissue to
chilling injury was established. The cold-inducible expression of HSP genes in the heat-
treated ﬁ'uits raised the possibility that both high- and low-molecular-weight HSPs may
play a critical role in resistance to chilling stress. Therefore, it would be interesting to
test the in viva role of the HSP in the attenuation of chilling injury in tomatoes by using
transgenic plants that over- or underexpress each gene (cytosolic class I or H or HSP90)
under control by CM 358 promoter, a ﬁuit speciﬁc promoter(s) and a cold- inducible

promoter. It would also be interesting to study the role of HSPs on chilling tolerance,

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fruit development and ripening by expressing the tomato HSFs in sense and antisense
orientation under control of a ﬁuit-speciﬁc promoter (E8 or 2A11) or a cold-inducible
promoter such as corlSa. Perhaps the induction of the whole battery of HSPs will be
more beneﬁcial than the overexpression of a single gene. In addition, demonstration that
the smHSP have chaperone activity at low temperatures in vitro and in viva is needed.
Interestingly, the carboxyl end of the LeHSP17.6 cytosolic class II smHSP
contains a polyproline motif PPPEPKKP. The PxxP motif is known to be involved in
protein/protein interactions among proteins with SH3 (Src homology)-binding domain.
The SH3 -domain is important for intra- as well as intermolecular interaction that
regulates Src catalytic activity, localization and recruitment of substrates. The SH3-
binding pocket has two hydrophobic grooves that contact the core xPxxP sequence.
However, in plants there is no report on SH3 domain—containing proteins. To test the
possibility that the polyproline motif of the LeHSP17 .6 cytosolic class II smHSP binds to
an SH3 domain, a yeast two-hybrid analysis is recommended utilizing the SH3 domain
from known proteins. If the LeHSP17.6 interacts with the SH3 domain proteins, then the
next step is to screen a tomato yeast two-hybrid library to ﬁnd proteins with a SH3
domain from plants. It would be interesting to determine the biological signiﬁcance of
SH3 domain-containing proteins in plant development and responses to the environment.
The combination of the heat treatment followed by low temperature storage
enhanced the expression of the smHSPs and this was correlated with chilling injury
tolerance. An intervening return to 20°C prior to returning the ﬁuits to a chilling
temperature re-induced expression of the smHSPs but the hits were subject to chilling

injury. The re-induction of the transcripts in the cold was more favorable after continued

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exposure to high temperatures. To our knowledge similar phenomena have not been
reported in plants or other organisms. Detailed analyses of the LeHSP17.6 promoter
region revealed multiple heat shock elements (HSE), stress response elements (STRE),
GATA elements and long tracts of repetitive sequences with high AT content. The
expression of the LeHSP17.6 was mainly induced at low temperatures only if the fruits
had been exposed to heat shock. These cis-acting elements may be useful to further
investigate the novel expression pattern of the cytosolic smHSPs employing high and low
temperature regimes.

The heat shock induced protection against CI was lost if the fruits were
transferred to 20°C after heat shock and before cold storage. Surprisingly, the re-
induction of the HSPs at low temperatures was not prevented. These data suggest that
heat-shock proteins are not the only factor(s) that contribute to the chilling-tolerant
phenotype of tomatoes. The data suggests that ethylene insensitivity as a consequence of
the prolonged heat treatment may be important. Thus, transgenic plants that overexpress
smHSPs and in parallel are ethylene insensitive will increase the chilling tolerance of

tomatoes and other subtropical and tropical ﬁuits.

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