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Hua Zhang and Paul H. Jennings

The effects of heat shock duration and persistence on the induction of chilling tolerance in cucumber roots were studied using total root growth, electrolyte leakage, and membrane peroxidation as injury indices after chilling. Heat shock reduced the chilling induced electrolyte leakage, decreased membrane peroxidation as measured by MDA content, and resulted in a greater total root growth after chilling compared to the control. Heat shocks at 40°C, applied to 36 hr germinated seedlings for time periods from 1 to 15 hr, all resulted in an increase in chilling tolerance in a time-dependent manner. The heat shock induction of chilling tolerance is most effective when heat shock was imposed immediately before chilling, but the effect is persistent even 32 hr after heat shock when seedlings are held at 25°C before chilling. The possible mechanism of heat shock effect and its persistence will be discussed in relation to heat shock proteins and antioxidant enzyme systems.

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Allan B. Woolf, Asya Wexler, Dov Prusky, Elana Kobiler, and Susan Lurie

Effect of direct sunlight on the postharvest behavior of five avocado (Persea americana Mill.) cultivars (Ettinger, Fuerte, Hass, Horshim and Pinkerton) was examined. Probes placed 6 to 7 mm under the peel showed that the temperature an the side exposed to the sun could be as much as 15 to 20 °C higher than the temperature of shade fruit, while the nonexposed side of the fruit was ≈5 °C higher than the shade fruit. With the exception of `Ettinger', sun fruit, and especially the exposed side, were found to be most tolerant to postharvest 50 and 55 °C hot water treatments. Similarly, storage of fruit at 0 °C for between 3 to 6 weeks caused severe chilling injury to shade fruit, with less effect on sun fruit. Furthermore, there was little or no damage on the exposed side of the sun fruit. During postharvest ripening at 20 °C, sun fruit showed a delay of between 2 to 5 days in their ethylene peak compared with shade fruit. The exposed side of the sun fruit was generally firmer than the nonexposed side, and the average firmness was higher than that of shade fruit. Activities of polygalacturonase and cellulase were similar in shade and sun fruit of similar firmness. After inoculation with Colletotrichum gloeosporioides (Penz.) Penz@sacc., the appearance of lesions on sun fruit occurred 2 to 3 days after shade fruit. Levels of heat-shock proteins were examined using western blotting with antibodies for Class I and II cytoplasmic heat-shock proteins. Class I reacted with proteins from the exposed side of sun fruit and Class II with proteins from both sides of sun fruit. Thus, it is clear that preharvest exposure of fruit to the sun can result in a wide range of postharvest responses.

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L. V. Gusta

Plants acclimate to abiotic stresses, e.g. heat, freezing drought and salinity, in response to environmental cues such as temperature, daylength and water. Plants can respond within minutes to the cue e.g. heat tolerance or within hours or days, e.g. drought and freezing tolerance. Heat shock proteins are measurable within 20 to 30 minutes of a heat stress and the plants aclimate almost immediately. In contrast, proteins related to freezing tolerance are measurable within hours but days are required before a measurable increase in freezing tolerance can be detected. In almost all stresses it appears that the environmental cue effects the water status of the plant which in turn affects the level of endogenous abscisic acid (ABA). ABA has been implicated to ameliorate the stress by inducing genes to produce stress proteins. There is a certain degree of commodity between stresses in ragards to stress proteins, however each stress has their own unique set of stress proteins. For example heat shock proteins did not confer stress tolerance. Proteins involved in water and osmotic stress tolerance share a high degree of commonality. I” all stresses a unique class of proteins are synthesized which are classified as heat or boiling stable (do not coagulate at 100°). These proteins are suggested to be involved in the stress response. Many of these heat stable proteins are induced by ABA alone or in combination with jasmonic acid (JA). Analogs of ABA which are either slowly converted to ABA or are degraded slowly or taken up at a faster rate than ABA have been tested for the efficacy in inducing the stress responses. Analogs have also been identified which inhibit the ABA induced response. How these analogs may have practical significance will be discussed.

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Elizabeth Vierling

When plants experience high temperature stress, they respond by synthesizing a discrete set of proteins called heat shock proteins (HSPs). This response is not unique to plants, but is observed in all other eukaryotes. It is now known that the HSPs are evolutionarily conserved proteins, and furthermore, that HSPs function not only during stress, but also during normal growth and development. My laboratory has characterized several of the major groups of HSPs in higher plants. We have cloned genes encoding plant HSP70 proteins and low molecular weight (LMW) HSPs (17-23 kDa). Using this information we have investigated the expression of HSPs both in the field, and under laboratory conditions which mimic field situations. We have determined the temperature limits for expression of HSPs in vegetative tissues, and have also found that HSPs are frequently produced in plant reproductive structures, even in the absence of stress. As a first step toward understanding HSP function, we have characterized the intracellular localization of HSPs. Results show that there are unique HSPs in the cytoplasm, chloroplast and endomembrane system. These ubiquitous proteins appear to play essential roles in many cellular processes.

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Sharon J. Keeler, John J. Frett, and Sherry L. Kitto

Heat stress on field grown Phaseolus lunatus (lima bean) can have a significant influence on yield. Lima bean crops grown in Delaware typically yield less pounds per acre than the same cultivars grown in California. Part of this effect may be due to extreme heat conditions or fluctuations during Delaware's summers, which can affect blossom and pod set. Our purpose was to analyze the heat tolerance of various cultivars of P. lunatus using quick bioassays and to establish a relationship to yield in greenhouse temperature trials. Two assays were used. The first, a hypocotyl extension assay, consisted of a treatment of germinated seedlings at 25, 35, or 42 for 2 h and observations of hypocotyl extension at 72 and 96 h posttemperature treatments. Three cultivars [`Fordhook' 1072 (heat-sensitive), `Jackson Wonder' (heat-tolerant), and `Early Thorogreen' (heat tolerant)] were analyzed. Initial results indicated that `Jackson Wonder' and `Early Thorogreen' are capable of surviving the 42C heat shock, but `Fordhook 1072' is not. In the second assay, we measured electrical conductivity of a solution containing hypocotyl sections following incubation at various temperatures (R1). Tissue samples then were boiled and conductivity was measured again (R2). The ratios of R1/R2 × 100 were determined as percent injury. Preliminary data suggests that `Jackson Wonder' is more heat-tolerant in this assay than `Fordhook 1072'. Subsequent experiments will analyze the induction of specific heat shock proteins as a function of cultivar-specific heat tolerance.

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Suping Zhou, Roger Sauve, Tara Fish, and Theodore W. Thannhauser

Tomato (Solanum lycopersicum cv. Money Maker) seedlings at the two-leaf stage were grown in one-half strength Hoagland solution supplemented with 50 mm NaCl for 4 days, with 100 mm NaCl for 4 days, with 150 mm NaCl for 4 days, and with a final concentration 200 mm NaCl for 2 days. Solutions were refreshed every 2 days for treated and untreated seedlings. Non-treated plants were grown in nonamended one-half strength Hoagland solution. Three biological replicates (BR) were included for treated and control experiments. At the end of treatments, the uppermost three newly expanded leaves from all 12 plants in each BR were collected and bulked to extract total protein. Proteomic analysis resulted in the identification of several salt-induced and salt-suppressed proteins. Salt-induced proteins were: vacuolar H+-ATPase A1 subunit isoform (1.6-fold), germin-like protein (1.5-fold), ferredoxin-NADP (+) reductase (1.2-fold), quinone oxidoreductase-like protein (4.4-fold), heat-shock protein (4.9-fold), and pyrophosphorylase (1.7-fold). Salt-suppressed proteins were: ATPase alpha subunit (−1.5-fold) and rubisco activase (−1.4-fold). Proteins identified in this study affect cellular activities for antioxidant, stress protection, carbon fixation, and carbohydrate partitioning in young tomato leaves under salt stress.

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Susan Lurie and Joshua D. Klein

Mature-green tomato (Lycopersicon esculentum Mill.) fruit, when kept for 3 days at 36, 38, or 40C before being kept at 2C for 3 weeks, did not develop chilling injury, while unheated fruit placed at 2C immediately after harvest did. When removed from 2 to 20C, the heated tomatoes had lower levels of K+ leakage and a higher phospholipid content than unheated fruit. Sterol levels were similar in heated and unheated fruit while malonaldehyde concentration was higher in heated fruit at transfer to 20C. The unheated tomatoes remained green, and brown areas developed under the peel; their rate of CO2 evolution was high and decreased sharply, while ethylene evolution was low and increased at 20C. In contrast, the heat-treated tomatoes ripened normally although more slowly than freshly harvested tomatoes: color developed normally, chlorophyll disappeared, and lycopene content increased, CO2, and ethylene evolution increased to a climacteric peak and K+ leakage increased with time. During prestorage heating, heat-stress ethylene production was inhibited, protein synthesis was depressed, and heat-shock proteins accumulated. There appears to be a relationship between the “heat shock response” and the protection of tomato fruit from low-temperature injury.

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Sunao Hisada, Tomoya Akihama, Tomoko Endo, Takaya Moriguchi, and Mitsuo Omura

A cDNA library was constructed from satsuma mandarin (Citrus unshiu Marc.) fruit tissues during the rapid cell enlargement phase. A total of 950 individual cDNA clones was partially sequenced and compared with GenBank databases for characterizing the gene repertoire expressed during this developmental phase. Among these, 426 cDNA clones (44.8%) showed sequence identity with previously characterized genes with optimized (OPT) scores of ≥200, while 524 clones (55.2%) resulted in low OPT scores (<200) and did not show any significant sequence identity with previously published genes. Based on nucleotide sequence, most clones with OPT scores of ≥200 were assumed to be transcription-, translation-, cell-wall-metabolism-, and stress-response-related genes. Other clones showed homology with published sequences related to housekeeping and stress-response-related genes, including metallothionein-like proteins, late-embryogenesis-abundant (LEA) proteins, and heat-shock proteins. These results suggested that Citrus fruit during rapid cell enlargement were metabolically active and expanding in response to biotic and abiotic stress. For clones with low nucleotide sequence homology, the recurrence was evaluated by aligning nucleotide sequences of each clone and generating contig maps. Expressed sequence tags (ESTs) of 162 clones with OPT scores <200 have not been reported for any other organism. Collectively, randomly sequenced cDNA clones described in this study provided information on types of genes expressed during the rapid cell enlargement phase in Citrus fruit. These genes should be used as candidates for Citrus genome mapping projects.

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Abdur Rab and Mikal E. Saltveit

Chilling sensitivity increased as the radicle of germinating corn (Zea mays L. `Jubilee' hybrid), cucumber (Cucumis sativus L. `Poinsett 76'), mung bean (Phaseolus aureus Roxb. `Berkin'), and tomato (Lycopersicon esculentum Mill. `Rio Grande') seeds increased in length from 1 to 7 mm. In contrast, radicles of germinating okra (Hibiscus esculentus L. `Clemson' spineless) seeds exhibited similar levels of chilling sensitivity at all radicle lengths. The degree of chilling sensitivity varied among the species in relation to time required to elicit a significant response and the magnitude of the elicited response. Based on subsequent radicle elongation, okra and cucumber were the most sensitive species to chilling at 2.5C for 96 h; tomato and corn were relatively less sensitive, and mung bean was the least sensitive. This pattern of sensitivities changed when other criteria were used to evaluate chilling sensitivity. The development of lateral roots decreased with prolonged chilling in all species, except for corn in which the apical tip remained viable even after 192 h of chilling. Heat shock (0 to 10 min at 45C) induced chilling tolerance in all species, except okra. In okra, neither increasing the heat shock temperature nor decreasing the severity of chilling (i.e., temperature and duration of exposure) resulted in a significant reduction in chilling injury. The differential induction of heat shock proteins in okra and the other species is discussed.

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Janet Slovin

Our lab has initiated a project to determine if specific proteins expressed by strawberry function as part of the thermotolerance system. We have developed tools for investigating the role of heat shock proteins (HSPs) and other gene products in strawberry thermotolerance. These tools include an inbred diploid testing system, EST sequence data, and molecular markers for heat tolerance. We developed an inbred line, 5AF7, of the diploid strawberry Fragariavesca for testing gene function because the diploid genome is small (164Mbp), the life cycle of the plant is short (about 4 months), the plant size is small (a plant will produce fruit in a 4-inch pot), some genetic work is already done, F. vesca is transformable with Agrobacteriumtumefaciens, and results should be transferable to the commercial octoploid varieties. A cDNA library was constructed in the pCMVsport 6.1 vector using combined RNA from batches of aseptically grown F. vesca seedlings treated to various elevated temperature regimes. Over 1500 EST sequences from the library have been deposited in GenBank and are available annotated at the ESTAP Fragaria database ( HSP101 affects thermotolerance in Arabidopsis(Queitsch et al., 2000, Plant Cell 12:479). A PCR fragment for HSP101 was generated from F. vesca with degenerate primers, and used to obtain a full-length cDNA clone from the library. Primers spanning an intron were designed for RTPCR from the sequence of the cDNA. Semi-quantitative RTPCR indicates that HSP101 is expressed constitutively in young leaves at 25 °C and is not induced at moderately higher temperatures (32 °C) even after 5 hours. Induction occurs within 1 hour at 37 °C.