from Alan Bennett (University of California, Davis), and. HSP17 was from Lutz Nover (Goethe University, Frank- furt, Germany). Total RNA was isolated from 10 g ...
Plant Physiol. (1 996) 1 1 0: 1207-1 21 4
Reversible lnhibition of Tomato Fruit Gene Expression at High Temperature' Effects on Tomato Fruit Ripening Susan Lurie*, Alex Handros, Elazar Fallik, and Roni Shapira
Department of Postharvest Science, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250 Israel (S.L., A.H., E.F.); and Department of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University, Rehovot 761 00, Israel (R.S.)
caused by alterations in gene expression (Mansson et al., 1985). %ter et al. (1985) identified 19 different mRNA families that were present in ripe fruit but were undetectable or present at a much reduced level in unripe fruit. A number of cDNA clones prepared from these "As have been identified and thereby linked to ripening-related physiological changes. pTOM13 has been identified as a cDNA clone encoding ACC oxidase (Hamilton et al., 1990), an enzyme that catalyzes the last step in the ethylene synthesis pathway. pTOM6 encodes for PG, an enzyme that depolymerizes pectins in the fruit cell wall (Grierson et 1986)' Phytoene 'ynthase, an enzyme in the pathway Of lycopene synthesist has been identified with pToM5 (Fray and Grierson, 1993). These clones have Provided a useful means to examine different factors that influence the ripening process. Short exposures to temperatures above 35°C have been found to inhibit fruit ripening reversibly (Biggs et al., 1988; Lurie and Klein, 1990), whereas fruit held for extended periods at high temperature ripen abnormally (ogura et al., 1975). rn tomatoes, elevated temperatures inhibit ethylene synthesis (Biggs et al,, 1988) and PG accumulation (Yoshida et al., 1984) and interfere with lycopene synthesis (Ogura et al., 1975).Some of these effects have been attributed to the temperature disruption Of enzyme (Ogura et al.r 1975)r but and Grierson (1988) demonstrated that inhibition also occurred at the level of gene expression. If the inhibitory effect of high temperature is reversible, then fruit subjected to a heat stress might ripen normally but more slowly than unheated fruit. This was confirmed in tomatoes heated to 38°C for 3 d, which ripened more slowly than unheated fruit ( ~ and ~Klein,~ 1991).However, the molecular events occurring in the fruit were not investigated. Tomato fruit are chilling sensitive and have reduced storage life and decreased ability to ripen when stored at temperatures below 10°C; the degree of damage is related to the length of time at a particular temperature. Chilling injury could be reduced in temperature-sensitive tissues by modifying the pattern of temperature exposure, e.g. periodically warming the chilled tissue above the chill-
The reversible inhibition of three ripening-related processes by high-temperature treatment (38°C) was examined i n tomato (Lycopersicon esculentum 1. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20°C or fruit were first held at chilling temperature (2OC) after heating and then daced at 2ooc* Ethylene production and colar development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSPl7 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSp17 protein disappeared from fruit tissue after 3 d at 20°C but remained when fruit were held at 2°C. The persistence of heat-shock proteins at low temperature may be relevant t o the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1 -carboxylic acid oxidase and polygalacturonase ako did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished.
In tomato (Lycopeysicon esculentum L.) the ripening process involves a complex and coordinated series of changes in pigmentationT flavorf texturef and aroma resulting from physiological and biochemical activity. These changes are
'
This research was partially funded by the United States-Israel Binational Agricultural Research Foundation, the U.S. Cooperative Development Foundation, and the Ministry for Economic Cooperation of the Federal Republic of Germany under the GermanIsraeli Agricultural Research Agreement for the Benefit of the Tlurd World. This is a contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel No. 1531-E, 1995. * Corresponding author; e-mail vtfrst8volcani.agri.gov.il; fax 972-3-9683622.
Abbreviations: HSP, heat-shock protein; PG, polygalacturonase. 1207
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ing temperature (Cabrera and Saltveit, 1990), or holding the tissue near the chilling temperature or above 35°C before chilling (Lafuente et al., 1991; Lurie and Klein, 1991; Saltveit, 1991). Temperature acclimation at low temperature has been found to correlate with the accumulation of a number of unique proteins (Guy et al., 1985; Mohapatra et al., 1989), and at elevated temperature HSPs accumulated (Lafuente et al., 1991; Lurie and Klein, 1991). The appearance of the HSPs coincided with increased chilling resistance. Therefore, the mechanism whereby heat stress before a chilling treatment slows ripening and prevents chilling injury may be a modulation of gene expression by reversible inhibition of ripening-related genes and enhanced expression of HSP genes. In this study we investigated the effect of a heat stress on ripening-related processes of harvested mature green tomatoes. Ethylene production, fruit softening, and color development were examined on both the physiological and molecular levels. MATERIALS A N D METHODS
Mature green tomatoes (Lycopersicon esculentum L. cv Daniella) of uniform size and color were obtained directly from a greenhouse and divided into four lots. The first lot was placed immediately at 2"C, the second was placed at 20°C, and the third and fourth lots were heated for 3 d at 38"C, after which one lot was transferred to 2°C and the other to 20°C. During the heat treatment the fruit reached 38°C internally within 4 h and maintained that temperature. RH was maintained at 90 to 95%. Color development was measured as the "a" value on a colorimeter (Minolta, Tokyo, Japan). A negative value was obtained on green fruit, values close to O were given to turning fruit, and the higher the positive value the redder the fruit color. Firmness of each fruit was obtained using a Durometer (Shore Manufacturing, Jamaica, NY). Values above 55 were assigned to very firm fruit, values between 40 and 55 indicated that the fruit were firm, and values below 40 indicated that the fruit were soft. These measurements were periodically performed on 10 fruit from each treatment for the duration of the experiment. Ethylene was measured by placing five fruit from each treatment into individual 0.5-L jars and closing them for 1 h each day. Gas samples were withdrawn with a syringe through a septum in the jar lid and injected into a gas chromatograph equipped with a flame ionization detector and a 1-m alumina-packed column. The jars were left open and ventilated between measurements. At each time assayed, the pericarp of three fruit from each treatment was chopped, frozen in liquid N,, and held at -80°C until extraction. For protein extraction, 2 g of tissue were ground in liquid N,, extracted with Tris-buffered phenol, pH 7.0, and precipitated by 0.1 M ammonium acetate in methanol (Hurkman and Tanaka, 1986). The precipitate was washed two times with 0.1 M ammonium acetate in methanol and twice with 80% acetone. The pellet was dried under N, and solubilized in Laemmli sample buffer (Laemmli, 1970). Protein concentration was determined by the method of Marousky and Harbaugh (1979).
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Twenty micrograms of protein of each sample were separated on a 12% polyacrylamide gel (Protean ][I, Bio-Rad) and transferred to a nitrocellulose filter using a gel blotter (Bio-Rad). After transfer the nitrocellulose filter was washed with mild (1%)fat and incubated overnight at 4°C with the primary antibody. Binding of the antibody to the filter was revealed using the alkaline phosphatase reaction. The antibodies for ACC oxidase were received from Dave Dilley (Michigan State University, East Lansing), PG was from Alan Bennett (University of California, Davis), and HSP17 was from Lutz Nover (Goethe University, Frankfurt, Germany). Total RNA was isolated from 10 g of frozen tissue by the method of Zheng et al. (1992). Ten grams of fi-ozen tissue were ground to a powder in liquid N2 with a mortar and pestle and homogenized with a Polytron in 20 mL of lysis buffer (8 M guanidine hydrochloride, 10 mM EDTA, 300 miv Tris-HC1, pH 7.6, 8% P-mercaptoethanol). After the sample was centrifuged at 3,0008 for 10 min, the supernatant was filtered through Miracloth and extracted twice with pheno1:chloroform (1:1)and once with chloroform. RNA was precipitated in 33 mM sodium acetate and 80% ethanol and centrifuged at 15,000 rpm for 20 rriin at 4°C in a microfuge, and the resulting pellet was washed with 3.0 M sodium acetate, pH 5.5, to remove polysaccharide contaminants. Following a wash with 70% ethanol, the resulting pellet was dissolved in 10 mM Tris, pH 7.6, 1 mM EDTA, 1%SDS and precipitated overnight at 4°C with 2.5 M LiC1. After the sample was centrifuged at 13,OOOg for 20 min the precipitate was redissolved, reprecipi tated in sodium acetate and ethanol, and finally redissolved in 10 mM Tris-HCI, pH 7.6, 1 mM EDTA, 0.1% SDS. Total RNA was used for slot blot analysis. Each RNA sample was checked for integrity by northern hybridization to one of the probes (Sambrook et al., 1989) prior to slot blot analysis. RNA slot blot analysis was performed with 10 pg of total RNA extracted from tomato fruit from the different temperature regimes. The RNA samples were denatured at 65°C for 10 min in 200 p L of a solution coritaining 1 M NaC1, 6% formaldehyde, and 20 mM sodium phosphate, pH 7.0. Samples were chilled on ice and transferred to a nylon membrane (Hybond-N, Amersham) in a BioDot SF microfiltration apparatus (Bio-Rad) following the manufacturer's instructions. The ACC oxidase, phytoene synthase, and PG probes contained 1400-, 1700-, and 1800-bp PstI fragments of the clones pTOM13, pTOM5, and pTOM6, respectively (Slater et al., 1985). The probe for the heat-shock 17-kD protein contained a 490-bp PstI fragment isolated from the pBSK+ hspl7 (Schoffl et al., 1984), and that for the heat-shock 70-kD protein was a 2100-bp EcoRI fragment isolated from pUC19+ hsp70 (Winter et al., 1988). The ribosomal-probe used as an interna1 standard was a 1000-bp BamHI fragment isolated from the pUC18 plasmid (provided by E. Lifschitz, Haifa University, Israel). All inserts were isolated by digestion with the appropriate restriction enzymes followed by electrophoresis in a 1% agarose gel in Tris-acetate-EDTA buffer. After staining the inserts were excised from the agarose and
Reversible lnhibition by Heat of Tomato Ripening-Gene Expression purified by the QIAEX gel extraction kit (Qiagen, Chatsworth, CA) following the manufacturer's instructions. Purified DNA fragments were labeled with [cy-32P]dATP (Amersham, 6000 Ci/mmol) using the random priming kit (Boehringer Mannheim). The Hybond-N filters were prehybridized and hybridized at 42°C according to standard protocols (Sambrook et al., 1989). Filters were washed in 0.1X SSC, 0.1% SDS at 65°C. The hybridization intensity was quantified by exposing the filter in a Bio Imaging Analyzer (model BAS1000; Fujix, Tokyo, Japan). Each filter was hybridized to the four probes sequentially and stripped between hybridizations. Following stripping the filter was always checked for complete remova1 of the previous probe before a new hybridization. After the fourth probe was stripped the filter was hybridized to the ribosomal probe PT3 and the intensity of this hybridization was used to normalize and if necessary to correct the intensity of the other probes.
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RESULTS Ethylene Production, ACC Oxidase mRNA, and Protein
The mature green tomatoes at harvest were already producing 3 nL g-' h-' ethylene (Fig. 1A). When allowed to ripen at 20°C the fruit showed a typical pattern of ethylene production, with the maximum attained after 4 d. If the fruit were placed at either high (38°C) or low (2°C) temperature, ethylene production decreased during the time the fruit were held at these temperatures (Fig. 1). When the heated fruit were removed to 2O"C, ethylene production recovered (Fig. 1A). The peak production of ethylene from the heated fruit was after 5 d at 20°C and was 2.5 times higher than the peak production of control fruit (data not shown). Ethylene production from heated fruit that were held at 2°C remained low until the tomatoes were removed to 20°C, after which production increased. In contrast, the unheated fruit that were chilled for 14 d lagged for 7 d after return to 20°C before ethylene production began to increase (Fig. 1B). The level of mRNA for ACC oxidase in control fruit that had been held at 20°C increased with time, even after the tomatoes had passed the peak of their ethylene evolution (Fig. 1A). The message level in heated fruit was barely detectable after 0.5 d of heating (Fig. 1A). When the heated fruit were placed at 2O"C, ACC oxidase mRNA again accumulated, similarly to fruit placed at 20°C after harvest. During chilling, ACC oxidase mRNA also increased in both heated and unheated fruit (Fig. 1B). After 14 d the message in heated fruit was at a level similar to that in climacteric fruit that had been held at 20°C for 6 d, whereas in unheated fruit it increased to a smaller extent (Fig. 1, cf. A and B). This increase was not reflected by high ethylene production in these fruit. When chilled fruit, both heated and control, were transferred to 20°C the mRNA level did not change appreciably in heated fruit but increased in unheated fruit. Protein levels of ACC oxidase as determined by western blot followed the level of mRNA when fruit were held at 20°C. ACC oxidase protein was present at harvest (Fig. 2a, lane A), and the amount of protein increased after 3 d at 20°C (Fig. 2a, lane B). The amount of ACC oxidase protein decreased from the level at harvest after 3 d at 38°C (Fig.
Days after Harvest Figure 1. ACC oxidase mRNA (pTOM13) abundance and ethylene production of mature green tomatoes. A: O, Ethylene from fruit at 2OOC; O, ethylene from fruit held at 38°C for 3 d and then transferred to 20°C. B: O, Ethylene from fruit held for 14 d at 2°C and then transferred to 20°C; +, ethylene from heated fruit held for 14 d at 2°C and then transferred to 20°C. SD is indicated. In both A and B open bars are mRNA levels of ACC oxidase from unheated fruit and shaded bars are mRNA levels of ACC oxidase from heated fruit. The shaded arrow indicates when fruit were transferred from 38 to 20°C (A) or 2°C (B), and the solid arrow indicates when fruit were transferred from 2 to 2 O T .
2a, lane 1).However, 3 d at 20°C following heat treatment led to enhanced ACC oxidase protein levels (Fig. 2a, lane 2), which paralleled enhanced ethylene production in these tomatoes (Fig. 1).The protein levels in both heated and unheated tomatoes increased similarly when fruit were transferred to 20°C after 14 d of chilling (Fig. 2a, lanes D, E, 4, and 5). This is in contrast to the lag seen in ethylene production in unheated fruit after chilling (Fig. 1B) and the accumulation of message while heated and unheated fruit were held at 2°C (Fig. lB), which is not reflected in increased protein at 2°C (Fig. 2, lanes C and 3). Fruit Color Development and Phytoene Synthase mRNA
Unheated fruit developed red color normally when held at 20°C and reached a pink color (a positive colorimeter "a"
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3A). Holding either unheated or heated fruit at 2°C led to a slow increase in message level in fruit of both treatments. When the chilled fruits were removed to 20°C, the message accumulated to higher levels in heated fruit than in unheated fruit, just as color development was better in heated fruit than in unheated fruit after chilling (Fig. 3B).
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Fruit Softening, PC mRNA, and Protein
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The firmness of the tomato fruit decreased as they ripened at 20°C, whether the fruits were heated before being placed at 20°C or placed at 20°C directly after harvest (Fig. 4A). If fruits were chilled after harvest there was only a small decrease in firmness during the period they were held at 2°C (Fig. 4B).
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value) after 4 d, which corresponded with peak ethylene production (Fig. 3A). The heated fruit showed no change in color during the heat treatment, but when placed at 20°C they developed red color normally, reaching the "a" values of unheated fruit by d 11 after harvest (data not shown). No change in fruit color occurred during the time when the fruit were chilled at 2°C, but upon transfer to 20°C, the unheated fruit developed red color more slowly than fruit that had been held at 38°C before chilling (Fig. 3B). The phytoene synthase mRNA levels paralleled quite closely color development of the fruit. The message was present at a low level in mature green fruit at harvest but increased rapidly in fruit held at 20°C (Fig. 3A). During heating at 38°C there was no change in message level, but following the heat treatment there was a sharp increase in message level 3 d after the fruits were placed at 20°C (Fig.
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Days after Harvest Figure 3. Phytoene synthase mRNA (pTOMS) abundance and color development of mature green tomatoes held under various temperature regimes. A: O, Color change of fruit at 20°C; •, color change from fruit held at 38°C for 3 d and then transferred to 20°C. B: 0, Color change in fruit held for 14 d at 2°C and then transferred to 20°C; *, color change in heated fruit held for 14 d at 2°C and then transferred to 20°C. so is indicated. In both A and B open bars are mRNA levels of phytoene synthase from unheated fruit and shaded bars are mRNA levels of phytoene synthase from heated fruit. The shaded arrow indicates when fruit were transferred from 38 to 20°C (A) or 2°C (B), and the solid arrow indicates when fruit were transferred from 2 to 20°C.
Reversible lnhibition by Heat of Tomato Ripening-Gene Expression 70
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and when placed at 20°C the mRNA of unheated fruit increased to a greater extent than heated fruit (Fig. 4B). This correlated with fruit softening, which was greater in control fruit than in heated fruit after chilling (Fig. 4B). PG protein was not detectable by western blots in fruit at harvest (Fig. 2b, lane A). However, by 3 d at 20°C it had accumulated to high levels, but during 3 d at 38°C there was no accumulation (Fig. 2b, lanes B and 1).Following the heat treatment the protein accumulation was similar to control tomatoes when the heated fruit were transferred to 20°C (Fig. 2b, lane 2). During chilling no PG protein could be detected in fruit from either treatment (Fig. 2b, lanes C and 3). However, at 20°C following chilling, protein was barely apparent after 5 d, although by 11 d high levels were found in both heated and unheated fruit (Fig. 2b, lanes D, E, 4, and 5 ) .
Days after Harvest Heat-Shock mRNA and Protein
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Days after Harvest Figure 4. PG mRNA (pTOM6) abundance and fruit softening of mature green tomatoes held under various temperature regimes. A: O, Firmness fruit at 2 0 T ; @, firmness of fruit held at 38°C for 3 d and then transferred to 2OOC. B: O, Firmness of fruit held for 14 d at 2°C and then transferred to 20°C; e , firmness of heated fruit held for 14 d at 2 O C and then transferred to 20°C. SD is indicated. In both A and B open bars are mRNA levels of PC from unheated fruit and shaded bars are mRNA levels of PG from heated fruit. The shaded arrow indicates when fruit were transferred from 38 to 20°C (A) or 2°C (B), and the solid arrow indicates when fruit were transferred from 2 to
20°C.
When removed to 20°C, however, the rate of softening was similar to that of fruit placed directly at 20°C. The heated fruit softened slowly during holding at 2°C but when removed to 20°C did not show a difference in their rate of softening. As a consequence, heated fruit after chilling and after a period of ripening at 20°C were firmer than unheated fruit. The level of PG mRNA in control and heated but unchilled fruit followed the same pattern as mRNAs for ACC oxidase and phytoene synthase (Fig. 4A). In fruit held at 20°C after harvest the level of message increased, whereas in fruit during heat treatment the mRNA level was low and then increased when the fruit were transferred to 20°C. In control or heated fruit that were chilled the mRNA levels remained low,
HSP70 is both constitutively expressed and induced by high temperatures. Tomato RNA from both unheated and heated fruit hybridized strongly to the HSP70 probe, and no differences were discerned (data not shown). In contrast, the message for HSP17 was very low in abundance at harvest and increased dramatically within 0.25 d of heating the tomatoes (Fig. 5A). The level peaked after 1 d of heat treatment and then began to decline. By the 3rd d of heat treatment the level of mRNA was similar to that of 0.25 d of heat, and by 1 d after remova1 from heat (i.e. d 4) the heated fruit had similar levels of HSP17 mRNA as unheated fruit, whether fruit were transferred to 20°C (Fig. 5A) or 2°C (Fig. 58). The protein for HSP17 as measured by western blot was absent at harvest but began to appear after 0.25 d of heating and accumulated to high levels after 1 d of heat treatment (Fig. 2c, lanes 1-3). The protein remained present during the heat treatment and for 2 d after transfer to 20°C, but by 4 d it had disappeared (Fig. 2c, lanes 4-6). If, however, the heated fruit were transferred to 2°C the protein persisted during the period of chilling and disappeared only when the fruit were rewarmed (Fig. 2c, lanes 8-10). This is in contrast to the mRNA, which declined to basal levels after the heat treatment (Fig. 5). Unheated fruit held for 2 weeks at 2°C had no HSP17 protein detectable by western blot (Fig. 2c, lane 7). DISCUSSION
A heat treatment of 3 d at 38°C inhibited ripening of mature green tomatoes. Recovery from heat-induced inhibition occurred when the fruit were transferred to 20"C, either directly or after being chilled at 2°C for 14 d. Unheated tomatoes held at 2°C did not ripen normally after transfer to 20°C, as seen by impaired ethylene production (Fig. 1) and poor color development (Fig. 2), whereas heated tomatoes ripened slowly but normally. High temperatures inhibit ethylene production in tomatoes and other fruit. The inhibition and recovery we noted in this work was similar to the response observed in apples (Lurie and Klein, 1990). Atta-Aly (1992) observed that in
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Days after Harvest Figure 5. H S P l 7 mRNA (pBSK+ hspl7) abundance in mature green tomatoes held under various temperature regimes. In both A and B open bars are mRNA levels of HSP 1 7 from unheated fruit and shaded bars are mRNA levels of H S P l 7 from heated fruit. The shaded arrowhead indicates when fruit were transferred from 38 to 20°C (A) or 2°C (B) and the solid arrowhead indicates when fruit were transferred from 2 to 20°C.
tomatoes held at 35°C the ACC leve1 sharply increased while ethylene production decreased, indicating inactivation of ACC oxidase. The present study showed that high temperature also affected ACC oxidase synthesis by decreasing the abundance of mRNA (Fig. 1). The increase in ACC oxidase mRNA at low temperature (Fig. 1) had been observed previously, even before the pTOM13 cDNA was identified as encoding ACC oxidase (Watkins et al., 1990). This accumulation of mRNA was not accompanied by increased ethylene evolution (Fig. 1B; Watkins et al., 1990) or by increased levels of ACC oxidase protein (Fig. 2a). It is interesting that, although ACC oxi-
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dase mRNA accumulated in both heated and unheated tomatoes during chilling, and ACC oxidase protein increased in fruit from both treatments upon rewarming, the unheated tomatoes had a 7 d delay before their ethylene production rate increased, whereas the heated tomatoes did not display this delay. The implication is that another step in ethylene synthesis was impaired by chilling the unheated tomatoes, possibly ACC synthase, the rate-limiting step in ethylene production (Boller et al., 1979). Lycopene synthesis is inhibited by high temperatures (Cheng et al., 1988). In heated tomatoes color development resumed after the temperature stress was removed (Fig. 2; Cheng et al., 1988; Lurie and Klein, 1992). As well as being sensitive to elevated temperatures, lycopene synthesis could be permanently impaired by low temperatures, as shown by the failure of unheated tomatoes to turn red after chilling (Fig. 2b; Lurie and Klein, 1992). This low temperature inhibition was alleviated by heating, and heated tomatoes did develop red color after chilling (Fig. 2b). Lycopene is one component of the carotenoid biosynthetic pathway. Phytoene, the first carotenoid in the pathway, is synthesized from two molecules of geranylgeranyl diphosphate by phytoene synthase. Control points of the pathway are unclear, but as the first dedicated enzyme of the pathway, phytoene synthase plays an important role. The mRNA abundance of phytoene synthase paralleled the development of red peel color in unchilled tomatoes. Fruit softening was not particularly affected by heat treatment if the fruit were not chilled (Fig. 3); however, during ripening after chilling the heated fruit remained firmer than unheated fruit. Mitcham and McDonald (1992) found that after heating of mature green tomatoes the rate of cell-wall degradation was reduced, whereas the synthesis of polyuronide components continued, and a s a consequence, heated fruit were firmer than unheated fruit. For many years PG was thought to be the primary enzyme responsible for tomato fruit softening. Purified PG protein caused breakdown of the middle lamella of tomato fruit cells in vitro (Crooks and Grierson, 1983).The enzyme was synthesized de novo during the onset of rip'ening and increased dramatically as ripening proceeded (Tucker and Grierson, 1982). However, recent work with transgenic plants has introduced doubt as to the exact association between cell-wall degradation caused by PG artd tomato fruit softening (Smith et al., 1988; Giovannoni et al., 1989). The results of the present study support these doubts. The levels of PG mRNA did not correlate well with the firmness measurements of the whole tomatoes (Fig. 3),, and PG protein was present in high amounts in both heated and unheated fruit upon rewarming after chilling, although the heated fruit were firmer than unheated fruit (Fig. 3). Therefore, although heat treatment inhibited expression of PG, this may not be the definitive cause of the treatment's effect on softening. In summary, heat treatment of mature green tomatoes for 3 d at 38°C inhibited a11 three ripening-related processes, ethylene production, fruit color development, and softening, and this was reversed upon transferrin,g the fruit to 20"C, indicating a common response to high tempera-
Reversible lnhibition by Heat of Tomato Ripening-Gene Expression
ture. H i g h temperature causes a general decrease i n protein synthesis (Vierling, 1991), a n d this may be due t o a decrease i n either transcription o r translation of messages. Since t h e m R N A expression studied i n this work decreased d u r i n g heat treatment, an inhibition of transcription appeared to b e a general response of t h e ripening-related processes. Whether t h e down-regulation of the ripeningrelated genes w a s due t o nuclear factors normally present being removed, or factors present causing inhibition of transcription, o r t o other mechanisms, cannot be determ i n e d f r o m this study. However, it is intriguing t o speculate that the mechanism is a c o m m o n one for a11 of t h e ripening-related processes. High-temperature stress induces t h e expression of HSPs. Plant tissue heat-shock response is characterized b y t h e synthesis of multiple, a b u n d a n t low-molecular-weight HSPs (Vierling, 1991). HSP17 mRNA, i n contrast t o t h e ripening-related mRNAs, increased greatly during t h e h e a t treatment. The m a x i m u m level occurred after 1 d of heat treatment, a n d the message level declined thereafter (Fig. 5). This behavior of heat-shock messages has been observed in other tissues (Vierling, 1991). The protein of HSP17, however, remained present longer t h a n t h e m R N A . Although t h e protein disappeared within 3 d a t 20°C after the heat treatment, it remained present i n t h e tissue for 1 4 d if t h e fruit w e r e held a t 2°C (Fig. 2c). Plant low-molecular-weight HSPs h a v e recently been shown t o protect proteins from denaturation d u r i n g high-temperature stress (Lee e t al., 1995). The persistence of HSP17 protein a t low temperature m a y indicate that it protects t h e tissue against t h e consequences of chilling, similar t o its postulated role a t h i g h temperatures. This finding has practical importance for subtropical fruit that a r e unable t o ripen after exposure t o low temperature. Manipulation of prestorage temperature t o enhance resistance t o chilling injury m a y allow longer storage of these fruit. Since h o t air a n d water treatments a r e u n d e r development for control of insects and fungi on fruit a n d vegetables (Paull, 1990), it m a y b e possible t o modulate these treatments to b o t h eliminate pests a n d induce resistance t o l o w temperature. Received August 25, 1995; accepted December 29, 1995. Copyright Clearance Center: 0032-0889 / 96/ 110/ 1207/ 08.
LITERATURE ClTED
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