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11 pages, 8 figures., In plant tissue, a wound signal is produced at the site of injury and propagates or migrates into adjacent tissue where it induces increased phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity and phenylpropanoid metabolism. We used excised mid-rib leaf tissue from Romaine lettuce (Lactuca sativa L., Longifolia) as a model system to examine the involvement of components of the phospholipid-signaling pathway in wound-induced phenolic metabolism. Exposure to 1-butanol vapors or solutions inhibited wound-induced increase in PAL activity and phenolic metabolism. Phospholipases D (EC 3.1.4.4), an enzyme involved in the phospholipid-signaling pathway is specifically inhibited by 1-butanol. Re-wounding tissue, in which an effective 1-butanol concentration had declined below active levels by evaporation, did not elicit the normal wound response. It appears the 1-butanol-treated tissue developed resistance to wound-induced increases in phenylpropanoid metabolism that persisted even when active levels of 1-butanol were no longer present. However, a metabolic product of 1-butanol, rather than 1-butanol itself, may be the active compound eliciting persistence resistance. Inhibiting a subsequent enzyme in the phospholipid-signaling pathway, lipoxygenase (LOX; EC 1.13.11.12) with 1-phenyl-3-pyrazolidinone (1P3P) or reducing the product of LOX activity with diethyldithio-carbamic acid (DIECA) also inhibited wound-induced PAL activity and phenolic accumulation. The effectiveness of 1-butanol, DIECA, and 1P3P declined as the beginning of the 1-h immersion period was delayed from 0 to 4 h after excision. This decline in effectiveness is consistent with involvement of the inhibitors in the production or propagation of a wound signal. The wound signal in lettuce moves into adjacent tissue at 0.5 cm h−1, so delaying application would allow the signal to move into and induce the wound response in adjacent tissue before the delayed application inhibited synthesis of the signal. Salicylic acid (SA) inhibits allene oxide synthase (AOS, EC 4.2.1.92), another enzyme in the phospholipid-signaling pathway. Exposure to 1 or 10 mM SA for 60 min reduced wound-induced phenolic accumulation by 26 or 56%, respectively. However, 1 mM SA lost its effectiveness if applied 3 h after excision, while 10 mM SA remained effective even when applied 4 h after excision. At 1 mM, SA may be perturbing the wound signal through inhibition of AOS, while at 10 mM it appears to have some generally inhibitory effect on subsequent phenolic metabolism. These data further implicate the phospholipid-signaling pathway in the generation of a wound signal that induces phenolic metabolism in wounded leaf tissue.

Cucumber seedling radicles decrease in chilling tolerance as they increase in length or decrease in vigor. The protein content of the apical 5 mm of the radicle decreased with decreases in chilling tolerance (R-2 = 0.92). This general reduction in protein content was reflected in a decrease of six dehydrin-like proteins with apparent molecular weights of 13.0, 15.0, 16.8, 23.0, 26.8, and 33.5 kDa. The disappearance of naturally occurring dehydrin-like proteins in cucumber seedling radicles as they elongate or lose vigor was correlated with a loss of chilling tolerance. Exposure to an osmotic (0.6 M mannitol) or heat (2 min at 45 degrees C) stress enhanced chilling tolerance. The osmotic-shock treatment induced both chilling tolerance and the appearance or strengthening of dehydrin-like proteins previously present in radicles. The heat-shock treatment also induced high levels of chilling tolerance and protein(s) that reacted with a 23 and 70 kDa antibody. However, these heat-shock protein (HSPs) did not cross react with the probe for dehydrin-like proteins. When organized into high, medium, and low chilling tolerance groups, radicle that were chilling tolerant contained either the 13.0 and 16.8 kDa dehydrin-like proteins, or the 15.0 and 23.0 kDa dehydrin-like proteins, or the 23 or 70 kDa HSP.

Wounding lettuce (Lactuca sativa L., var. Longifolia) leaves induced an eight-fold increase in the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), and the subsequent accumulation of phenolic compounds and tissue browning at 10degreesC. PAL is a key enzyme in the synthesis of phenolic compounds. A PAL cDNA was previously isolated by reverse-transcription PCR using total RNA from wounded lettuce leaves. RNA gel blots showed that maximum accumulation of both PAL mRNA and PAL enzyme activity occurred 24 h after wounding. A 2-min heat shock at 45degreesC administered within 5 min of wounding delayed the wound-induced increase in PAL activity, but did not delay the increase in wound-induced PAL mRNA. Changes in the content of PAL protein were also followed by immunoblot using anti-PAL antibody raised against the bacterially expressed protein from the cDNA. Immunoblots showed that the level of PAL protein in wounded lettuce tissue was significantly reduced by the heat shock treatment. These data suggest that heat shock reduces the rise in wound-induced PAL enzyme activity by reducing the translation of wound-induced PAL mRNA, or by increasing the turnover of the induced PAL protein.

Phenylalanine ammonia-lyase (PAL) catalyses the first step controlling the rate of phenylpropanoid metabolism. Wounding is a ubiquitous stress in nature and in the harvesting and preparation of fruits and vegetables that induces an increase in PAL activity, an accumulation of phenolic compounds and subsequent tissue browning. A wound-inducible PAL gene (LsPAL1) was isolated from Romaine lettuce by RT-PCR. The putative protein encoded by LsPAL1 is similar to predictive polypeptides sequences for other PALs. The kinetics of PAL mRNA accumulation is similar to those of induced PAL enzyme activity, with enzyme activity following mRNA accumulation by 12 h. Wound-induced PAL transcripts accumulated in cells close to the wound sites. Tissue printing showed that PAL mRNA was associated with tissue next to the epidermis and vascular bundles. A heterologous PAL protein was expressed in E. coli and was found to show significant PAL activity.

Exposure to a chilling temperature of 2.5 degrees C for 96 h inhibited the subsequent growth of cucumber seedling radicles at 25 degrees C by 92%. Exposing seedling with 5 +/- 1 mm long radicles to acetaldehyde vapour (275 micro l l(-1)) or to an aqueous ethanol solution (0.6 M) for 2 h, or to 45 degrees C for 10 min before chilling, increased chilling tolerance so that the chilling treatment reduced growth by only 47, 39 or 36%, respectively. All of these effective treatments induced the synthesis of a number of proteins, and suppressed de novo protein synthesis (i.e. the incorporation of [(35)S]-methionine) by about 70%. In contrast, treatment for 2 h with an aqueous arsenite solution (100 micro M) had no effect on chilling sensitivity or the incorporation of [(35)S]-methionine, yet it induced the synthesis of a complement of proteins that were similar to that induced by the effective heat-shock treatment. A unique protein or set of proteins may be responsible for heat-shock-induced chilling tolerance, but none was detected. The ability of various abiotic stresses to suppress protein synthesis may be more important in increasing tolerance to chilling injury than their ability to induce the synthesis of specific proteins.

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45degreesC reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R-2 > 0.90) in tissue developing the normal wound response and in tissue subjected to 0-180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45degreesC for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.

Ethylene production was measured from excised 10-mm apical and subapical root segments from 50 cultivars in 19 species of 7 families. Monocotyledonous species tended to have much lower rates of ethylene production than dicotyledonous species. Ethylene production was generally higher in apical root segments than in subapical segments within 1 h of wounding. However, cultivars of Cucumis melo, C. sativus, Helianthus annuus, Hibiscus esculentus, and Zea mays had higher rates of ethylene production from subapical segments. In apical root segments, Phaseolus aureus cv. Berken had the highest ethylene production rate (76.7 etal g(-1) h(-1)), while Zea mays cv. Silver Queen had the lowest rate (0.6 etal g(-1) h(-1)). In subapical root segments, Cucumis sativus cv. Armenian had the highest rate (55.7 etal g(-1) h(-1)), while Zea mays cv. Silver Queen again had the lowest rate (0.6 etal g(-1) h(-1)). The many different responses in magnitude and kinetics of wound-induced ethylene production among the species, cultivars and tissues should provide interesting and useful systems with which to study wound responses and induced ethylene production.

Chilling whole cucumber seedlings that had 10-mm long radicles for 4 days at 2.5°C significantly inhibited subsequent radicle growth both by increasing the time it took the seedlings to recover from chilling and attain a linear rate of radicle growth, and by decreasing the subsequent rate of linear growth. Exposing cucumber seedlings to 45°C for up to 20 min had no effect on subsequent radicle growth, while longer exposures produced reductions in growth. A heat shock at 45°C for 10 min induced the optimal protection to 4 days of chilling at 2.5°C by reducing chilling inhibition from 60 to 42%. Two hours after being chilled, heat shocked or heat shocked and then chilled, there was no difference in protein content of the apical 1 cm of the seedling radicle among these treatments and the non-heat shocked, non-chilled control. Two days after treatment, the protein content was still similar in tissue that had been heat shocked or heat shocked and chilled, while it was significantly reduced in tissue that had been chilled. In general, 2 h after treatment, the activity of the 5 antioxidant enzymes examined in this study [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2)] were reduced by chilling and unaffected or increased by heat shock. When heat shock was followed by chilling, there was a consistent effect of the heat shock treatment on preventing the loss of enzyme activity following chilling. This protective effect of the heat shock treatment was even more pronounced after 2 days of recovery at 25°C for SOD, CAT and APX. In contrast, the activity of GR and GPX was substantially higher in chilled tissue than in tissue that had been heat shocked before being chilled. Elevated levels of GR and GPX therefore appear to be correlated with the development of chilling injury, while elevated levels of SOD, CAT and APX appear to be correlated with the development of heat shock-induced chilling tolerance.

Wounds and injuries incurred during preparation of fresh-cut lettuce (Lactuca sativa L.) stimulate phenolic metabolism, which leads to tissue browning. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) is the first committed enzyme in the phenylpropanoid pathway and regulates its overall activity. Maximum activity of wound-induced PAL occurred sooner as the storage temperature increased from 0 to 25 °C, but the maximums were lower. A heat shock at 50 °C for 90 seconds protected fresh-cut lettuce tissue against browning, helped retain greenness, and decreased subsequent production of phenolics when applied either after or before wounding. Browning was reduced when the heat shock was applied up to 36 hours after wounding, while the maximum effect occurs around 6 hours before cutting. Like the heat-shock treatment, the protein synthesis inhibitor cycloheximide reduced wound-induced PAL activity, but it did not prevent tissue browning. When cycloheximide was applied in combination with heat-shock treatments, browning did not occur. Heat shocks may control tissue browning by more than just interfering with protein synthesis.

Chilling at 2.5°C reduced the subsequent growth of cucumber (Cucumis sativus L.) radicles at 25°C. The reduction in radicle growth was linear for 1–3 days of chilling at ≈10% per day of treatment, but then it increased in a non-linear pattern until subsequent radicle growth was all but eliminated by 6 days of chilling. A heat shock of 40°C for 4–12 min increased chilling tolerance such that 4 days of chilling caused only a 36% decrease in radicle growth, compared to 66% for seedlings not heat shocked. Heat shocks were only able to protect that part of radicle growth that was in excess of the linear decrease in radicle growth projected from 0–3 days. There appear to be two effects of chilling on radicle growth. The first inhibition of subsequent growth was linear and was not affected by heat shocks. The second inhibition was much more severe; it appeared after 3 days of chilling and could be prevented by heat shock. Seeds classified with different levels of vigor (i.e., different initial rates of growth) did not respond significantly different to chilling stresses following heat-shock treatments.

Seeds of cotton (Gossypium hirsutum L.), kenaf (Hibiscus cannabinus L.), okra [Abelmoschus esculentus (L.) Moench. `Clemson Spineless' (syn. Hibiscus esculentus L.)], rice (Oryza sativa L.), and wheat (Triticum sativum (L.) Lam.) were germinated and grown at 25 °C until their radicles reached 10 mm in length. They were then exposed to chilling temperatures for 0 to 5 days followed by 3 days at 25 °C. Radicle length was measured periodically and inhibition of elongation was used as an indicator of the severity of chilling injury. Exposure to chilling reduced radicle elongation in all species except chilling insensitive wheat. When seedlings were heat-shocked at 45 °C for 1 to 12 min before being chilled, radicles of the chilling sensitive okra, kenaf, cotton, and rice seedlings elongated more than seedlings not heat-shocked before chilling. The method of heat-shock application and the stringency (i.e., time× temperature) of the heat-shock and chilling treatments all affected the response of the tissue. In comparison to nonheat-shocked wheat seedlings, the radicles of chilling insensitive wheat seedlings did not elongate more than seedlings in which the heat shocks were applied before chilling. A brief heat shock ameliorates chilling injury in these chilling sensitive species.

An in vitro assay was used to determine the effect of AA and pH on the enzymatic and nonenzymatic production of ethylene (C2H4) from ACC. We were interested in the effect of AA on C2H4 production from ACC because aldehydes, primarily AA, can accumulate in tissue as the result of ripening, storage under modified atmospheres, packaging, and stress. Using crude extracts of ACC oxidase from tomato (Lycopersicon esculentum Mill. `Castlemart') and apple (Malus ×domestica Borkh. `Golden delicious'), C2H4 production from ACC was shown to increase in response to an increase in pH above 7.2 and inclusion of 0.2 to 2 mm AA. Nonenzymatic C2H4 production from ACC also increased linearly with increasing AA concentrations in all the buffers tested. Removal of ascorbate or O2 suppressed AA-induced nonenzymatic C2H4 production. Nonenzymatic AA-induced production of C2H4 from ACC appeared to be an ascorbate dependent oxidation, which was augmented by O2 and was sensitive to minor pH fluctuation. The nonenzymatic AA-stimulated conversion of AEC to 1-butene lacked stereospecificity. Formaldehyde and propionaldehyde also stimulated C2H4 production from ACC. These data indicate that ACC oxidase assays or C2H4 measurements assessing physiological status can be seriously affected by the presence of aldehydes, such as AA. Chemical names used: AA, acetaldehyde; ACC, 1-aminocyclopropane-1-carboxylic acid; AEC, 1-amino-2-ethylcyclopropanecarboxylic acid; ADH, alcohol dehydrogenase; EtOH, ethanol.

Wounding during minimal processing of lettuce (Lactuca sativa, L.) induces alterations in phenolic metabolism that promote browning and the loss of quality. The activity of phenylalanine ammonia-lyase (PAL; the first committed enzyme in phenylpropanoid metabolism) and the concentration of phenolic compounds (e.g., chlorogenic acid, dicaffeoyl tartaric acid, and isochlorogenic acid) increase in excised iceberg lettuce midrib segments after wounding. The effect of short heat-shock treatments on browning and phenolic metabolism in excised midrib segments of iceberg lettuce was studied. As the heat-shock temperature increased from 20 to 70 °C, there was a decrease in the subsequent increase in PAL activity and the accumulation of phenolic compounds in excised midrib segments. Treatments of 45 °C for 120 s, 50 °C for 60 s, or 55 °C for 30 s significantly reduced the increase in PAL activity and subsequent browning seen in control tissue after wounding. Exposure to 45 °C for 480 s, 50 °C for 60 s, or 55 °C for 45 s prevented PAL activity from rising above initial levels. Phenolic compounds remained at initial levels for 3 days in excised midribs exposed to 50 °C for 90 s or to 55 °C for 60 s. However, 55 °C damaged the tissue, as indicated by a* and L* Hunter color values. The synthesis of chlorogenic acid, dicaffeoyl tartaric acid, and isochlorogenic acid was greatly reduced by these heat-shock treatments. These treatments also decreased polyphenol oxidase activity and, to a lesser extent, peroxidase activity.

`Castlemart' tomato (Lycopersicon esculentum Mill.) pericarp discs were used to study the physiological effects of acetaldehyde and ethanol on fruit ripening. Short-term exposure of discs from mature-green fruit to acetaldehyde vapors on a fresh mass basis (≤500 μg·g-1) or ethanol vapors (≤3 mg·g-1) promoted ripening, while higher concentrations inhibited ripening. Discs from mature-green fruit absorbed greater amounts of ethanol and produced significantly higher concentrations of acetaldehyde than discs from breaker fruit. Ripening was promoted by ethanol when the discs were unable to retain or produce a certain level of acetaldehyde. Inhibition of ripening by 4 hours of exposure to ethanol (6 mg·g-1) was almost completely abolished by hypobaric treatments (18 kPa for 24 hours). However, acetaldehyde-induced ripening inhibition (2 days exposure to 180 μg·g-1) was only slightly reduced by vacuum. Concentrations of acetaldehyde and ethanol that inhibited ripening reduced C2H4 production, whereas lower concentrations of acetaldehyde and ethanol that promoted ripening increased C2H4 production. Application of 4-methylpyrazole, an alcohol dehydrogenase inhibitor, enhanced acetaldehyde-induced ripening inhibition and reduced ethanol-induced ripening inhibition or promotion at all concentrations of acetaldehyde and ethanol tested. The inhibition or promotion of ripening of excised tomato pericarp discs by ethanol and acetaldehyde depended on initial fruit maturity, applied volatile concentration, and duration of exposure.

The phenolic composition of whole heads and excised midrib sections of iceberg, butter leaf, and romaine lettuce (Lactuca sativa L.) was followed at 5 and 10 °C during the first 3 days after wounding or during continuous exposure to 10 μL·L-1 ethylene in air. After 3 days of storage at 5 and 10 °C, only 5-caffeoylquinic acid (chlorogenic acid), 3,5-dicaffeoylquinic acid (isochlorogenic acid), caffeoyltartaric acid, and dicaffeoyltartaric acid were detected in wounded lettuce midribs. Of these four compounds, chlorogenic acid accumulated to the highest level in all three lettuce types. The content of caffeic acid derivatives increased 3- and 6-fold after 72 hours of storage at 5 and 10 °C, respectively. The synthesis of caffeoyltartaric acid was not induced by wounding in iceberg lettuce, while chlorogenic acid increased 5-fold at 5 °C and 10-fold at 10 °C. Similar relative phenolic compositions were detected in the three lettuce types studied, although at different concentrations. Changes observed in the content of individual phenolic compounds during the first 3 days of ethylene exposure seemed to follow the same pattern observed during wound induction of the synthesis of phenolic compounds. Chlorogenic acid increased 5-fold and isochlorogenic acid increased 10-fold, while the content of caffeoyltartaric derivatives were not significantly altered by ethylene treatment. Isochlorogenic acid, which was only present in low amounts in the control, was synthesized in the later steps of wound and ethylene induction. Similar kinetics for the induction of phenolic compounds were observed in the three lettuce types studied, suggesting that the mechanisms by which wounding induces phenylpropanoid synthesis are common for the different lettuce types.

Inhibition of tomato (Lycopersicon esculentum Mill.) fruit ripening by exogenously applied ethanol was shown to be caused by elevated endogenous levels of acetaldehyde (AA). Exposure of excised pericarp discs of mature-green tomato fruit to ethanol or AA vapors produced elevated levels of both compounds in the tissue, but only the levels of AA were associated with ripening inhibition. Ripening inhibition was dependent on both the applied concentration and the duration of exposure. Discs treated with inhibitory levels of AA had levels of ethanol that were elevated but below that associated with inhibition of ripening. The in vivo activity of alcohol dehydrogenase was inhibited 40 to 60% by 4-methylpyrazole (4-MP), a competitive inhibitor of this enzyme. The inhibitory effect of ethanol on ripening was reduced by the simultaneous application of 4-MP. Tissue treated with 4-MP plus AA vapors had higher endogenous levels of AA and ripening was inhibited longer than in tissue without 4-MP. The tissue AA level resulting from ethanol or AA application appears to be the critical determinant of ripening inhibition.

Germinating `Poinsett 76' cucumber (Cucumis sativus) seeds are chilling sensitive, and subsequent radicle elongation is inhibited by exposure to nonfreezing temperatures below 10 °C. Reorienting germinated seeds with 5-mm-long radicles from a vertical to a horizontal position induced gravitropic curvature within 2 hours at 10 to 25 °C without significantly affecting the rate of radicle elongation. However, neither elongation nor curvature occurred in similar seeds held at 2.5 or 5 °C. Chilling seeds with 5-mm-long radicles at 2.5 °C for 18 hours significantly reduced the subsequent rate of radicle elongation at 25 °C by 47% compared with nonchilled control, while gravitropic curvature was reduced by only 34%. After 36 hours of recovery at 25 °C, the difference was even more pronounced; elongation was reduced by 26% while curvature was reduced by only 6%. Clearly, gravitropic curvature was less chilling sensitive than radicle elongation, despite the fact that differential elongation of cells in the radicle are needed to produce curvature. Exposing seeds with 5-mm-long radicles to a heat shock of 45 °C for 4 to 10 minutes significantly diminished the chilling-induced reduction in radicle elongation and gravitropic curvature.

Exposing head lettuce (Lactuca sativa L., crisphead or Iceberg type) leaf tissue to hormonal levels of ethylene (10 μl l−1) at 5°C promotes the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and an increase in its activity. It also promotes the appearance of the postharvest physiological disorder called russet spotting (RS). Discontinuing ethylene exposure after 4 days resulted in a rapid decline in PAL activity which was delayed by treating excised midrib leaf tissue with actinornycin D or cycloheximide at 5°C. Only cycloheximide delayed the loss of PAL activity in tissue that was transferred from 5 to 15°C. Activity of PAL from Rhodolorula glutinis was. slowly lost during incubation in buffer alone, but there was a logarithmic decline in its activity over time when it was incubated with aliquots of the resuspended 10000 g pellet from homogenized, lettuce tissue affected with RS. The in vitro loss in PAL activity was 9–fold higher in extracts from lettuce showing RS symptoms than from control lettuce, boiled samples or the buffer control. The PAL-inactivating factor isolated from lettuce affected with RS had a pH optimum around 8.0. It appears that the rapid loss in PAL activity after the discontinuation of exposure to ethylene is dependent on the de novo synthesis of a PAL-inactivating factor.

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.

This study was undertaken to determine if endogenous IAA content and axillary bud development correlate with phenylalanine ammonia-lyase (PAL) induction and russet spotting (RS) susceptibility among RS susceptible and resistant cultivars of Iceberg lettuce (Lactuca sativa L.). Final levels of ethylene-induced PAL activity and RS development were highly correlated among cultivars, field conditions, and harvest dates. Harvested Iceberg lettuce midribs contained relatively low amounts of free IAA (maximum of 5.2 ng·g-1 fresh weight). There was poor correlation between free IAA content in lettuce leaf midribs and final RS development among all cultivars, growing conditions, and harvest dates. Axillary bud development, as measured by the number of visible buds per head, bud weight, or bud length, were not significantly correlated with final RS development or midrib IAA content. Cultivars with higher initial free IAA content lost much of their IAA after 8 days storage at 5C in air ± ethylene.

Cucumber (Cucumis sativus L. cv. Poinsett 76) seeds were chilled at 2.5°C in a study of the chilling sensitivity and recovery of radicle tissue. The effect of chilling on radicle growth and the production of carbon dioxide and ethylene was measured. Chilling sensitivity of radicles increased as they grew from 1 to 7 mm in length. The length, not the age of the radicles, determined the level of chilling sensitivity. Apical tissue was most sensitive to chilling and slowest to recover from chilling, followed by subapical and basal tissue. Our data demonstrate that the chilling sensitivity of young seedling radicles differs along their length and that the rapid chilling-induced inhibition of elongation is probably due to an inability of meristematic cells to remain viable and active when chilled.

The density of excised 2-cm celery (Apium graveolens L.) petiole segments was highly correlated with a subjective evaluation of pithiness. Loss of density and the appearance of pithiness was stimulated by lengthening the duration of storage, raising the storage temperatures above 0C, and excising petiole segments. Segments excised from the upper two- thirds of the petiole lost less density during storage than segments excised from the bottom third of the petiole. Segments with initial high densities lost slightly less density during storage at 5C for 5 weeks than segments that were initially less dense. The extent of pithiness development varied significantly among six cultivars held at 5C for 2 weeks. Treating whole petioles with 1 µM abscisic acid for 4 days significantly increased density loss. Exposing petiole segments to up to 100 µl·liter -1 ethylene in humidified air for up to 2 weeks at 5C did not significantly change density over air controls. The loss of density and the development of pithiness in lightly processed celery petioles could be reduced by selecting resistant cultivars, monitoring water stress during growth, using only segments excised from the upper two-thirds of the petiole, and selecting segments with initial high densities. Air spaces that form in the cortex of many plant tissues such as celery petioles, corn roots, tomato stems, and roots of wetland species are collectively called aerenchyma. Aerenchyma develops through the lysis of parenchymatou s cells of the cortex and is stimulated by a variety of stresses, such as deficiencies in water, oxygen, and minerals, and is thought to enhance oxygen transport within the plant since it is associated with the tolerance of some crops to anaerobic soils (Drew et al., 1979). When aerenchyma forms in tomato stems and celery petioles, it is often referred to as pithiness. Pithiness is characterized by the appearance of whitish regions and air spaces within the tissue, and is a major source of quality loss and decreased shelf life in celery. Several climatic and storage factors have been associated with the development of pithiness in field-grown celery plants, in harvested stalks and processed petioles. Abscisic acid (ABA) may have a role in aerenchyma development since an increase in endogenous levels of ABA followed water stress and the develop- ment of aerenchyma (Livne and Vaadia, 1972). Water stress during growth favors development of pithiness in celery (Aloni and Pressman, 1979). They found that the level of free ABA in water stressed celery increased before the onset of pithiness and was related to the development of pithiness in celery petioles. Withholding water increased the level of ABA 7-fold after 24 h, with a rapid increase in pithiness occurring after 3 days. Also, exogenously applied ABA induced pithiness in detached petioles, with the degree of pithiness increasing with ABA concentration. They hypothesized that ABA production induced by water stress stimulated the development of pithiness. Aloni and Pressman (198 1) subsequently investigated the relationship between water stress in the field and ABA content on the development of pithiness in tomato stems. The degree of pithiness was greater in drought- stressed plants treated with 20 µ M ABA than in water-stresse d plants alone. Nonstressed plants treated with ABA exhibited more pithiness than controls, but less than water-stressed plants. Aloni

Roots of 24-h-old germinated cucumber (Cucumis sativus cv. Poinsett 76) seeds were subjected to thermal and chemical stresses, equilibrated at 25°C for 2 h and chilled at 2.5°C for 96 h. The germinated seeds were then held at 25°C for 72 h after they were chilled and the elongation of the primary root was used as a measure of chilling tolerance. Control roots elongated from an initial length of 0.2 cm to a final length of 6.3 cm at the end of 72 h. while chilled roots elongated to a final length of only 0.4 to 0.6 cm. Exposure to 0.4 M ethanol for 4 h or to 40°C for 1 h induced substantial chilling tolerance and the roots had a final length of 4.1 and 3.1 cm. respectively. Exposure to 7.5°C for 3 h conferred less chilling tolerance (elongation to 1.4 cm). while exposure to other chemicals (i.e. aqueous solutions of Ca(NO3)2, mannitol. methanol and NaCl) produced less, though still significant increases in chilling tolerance. A more severe chilling treatment of 144 h at 2.5°C was required to consistently induce elevated rates of ion leakage. Only the heat and the ethanol shock treatments significantly reduced chilling-induced ion leakage. Inclusion of the protein synthesis inhibitor cycloheximide negated the protective effects of these shock treatments. It appears that de novo protein synthesis is required for induction of chilling tolerance by a variety of chemical and thermal shock treatments.

Unlike horticulturally mature fruit of 'Dasher II' and 'Poinsett 76' cucumbers (Cucumis sativus L.), two cultivars that differ significantly in their level of chilling tolerance, imbibing and germinating seeds of these two cultivars responded similarly to chilling temperatures (e.g., increases in fresh weight, time to radicle emergence, and root growth). 'Dasher II' and 'Poinsett 76' seeds were imbibed and germinated at 10 to 30C, and seeds germinated at 25C for 24 h were chilled at 2.5C for various durations. In comparison, seeds from an aged lot of 'Poinsett 76' seed (1989) responded very differently from the 1992 seed lots in all experiments. The chilling tolerance level of germinating 'Poinsett 76' seed varied with the seedling age as measured by resumption of root growth. Our results suggest that some factor that confers chilling tolerance is gradually lost during the early stages of germination following imbibition. Uniform germination and seedling establishment is crucial for the commercial growth of many agricultural crops. Species that originated in the tropics or subtropics are especially sensitive to chilling (i.e., physiological injury caused by exposure to nonfreez- ing temperatures below ≈10C) that often occurs under early season temperate conditions (Bennett et al., 1992; Bradow, 1990; Christiansen, 1968; Harrington and Kihara, 1960; Herner, 1990; Nelson and Sharples, 1980; Pollock and Toole, 1966; Sachs, 1977). Although low soil temperatures can be avoided in commer- cial practice by delaying planting, there is an economic incentive to achieve early season harvest or extend the growing season by planting under less than ideal conditions early in the season. The delay in planting to await more favorable temperature conditions can be compensated for by using transplants, but transplants of chilling sensitive crops are also susceptible to injury when exposed to chilling temperatures during production or after planting.

Freshly harvested asparagus (Asparagus officinalis L.) spears were exposed to an anaerobic N2 atmosphere for 0, 2, 4, or 6 hours at 2.5 or 20C and then returned to 2.5C. Carbon dioxide production was measured each day, starting 3 days before and ending 11 days after the treatment. Significant increases in CO2 production relative to 0-hour controls were found within 1 day of treatment at 20C and were directly proportional to the duration of the anaerobic exposure. At 2.5C, CO2 production relative to the 0-hour control was stimulated by the 2- and 4-hour treatments and depressed by the 6-hour treatment, with the relative rate of production inversely proportional to the duration of the anaerobic treatment. A decrease in CO2 production occurred 7 days after N2 treatment, regardless of temperature. A sensory panel evaluated effects of treatments on appearance quality 7 and 15 days after treatment and on taste quality 4 days after treatment. Judges could not detect any significant differences between anaerobic treatments and control. No significant difference was found in the percent of decayed asparagus among treatments as detected by visual evaluation 6 days after treatment. It appears that exposure to an anaerobic N2 atmosphere for up to 6 hours was not detrimental to the storability or quality of harvested asparagus spears. These results indicate that cooling with vaporized liquid N2, during which an anaerobic atmosphere could be produced before the spears were significantly cooled, would not reduce subsequent quality or storability.

The carbon dioxide and ethylene concentrations in tomato fruit (Lycopersicon esculentum cv. Castelmart) and their stage of ripeness (characteristic external color changes) were periodically measured in fruit attached to and detached from the plant. An external collection apparatus was attached to the surface of individual tomato fruit to permit non-destructive sampling of internal gases. The concentration of carbon dioxide and ethylene in the collection apparatus reached 95% of the concentration in the fruit after 8 h. Gas samples were collected every 24 h. A characteristic climacteric surge in carbon dioxide (2-fold) and ethylene (10-fold) concentration occurred coincident with ripening of detached tomato fruit. Fruit attached to the plant exhibited a climacteric rise in ethylene (20-fold) concentration during ripening, but only a linear increase in carbon dioxide concentration. The carbon dioxide concentration increases in attached fruit during ripening, but the increase is a continuation of the linear increase seen in both attached and detached fruit before ripening and does not exhibit the characteristic pattern normally associated with ripening climacteric fruit. In tomato fruit, it appears that a respiratory climacteric per se, which has been considered intrinsic to the ripening of certain fruit, may not be necessary for the ripening of “climacteric” fruit at all, but instead may be an artifact of using harvested fruit.

The effects of heat shock on the chilling tolerance of mung bean [Vigna radiata (L.) Wilczek] seedling tissue were studied by using two measurements of chilling injury: increased 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and solute leakage. ACC oxidase activity (measured as ACC-induced ethylene production) of freshly excised mung bean hypocotyl segments was highly dependent on the temperature at which the seedlings were grown. However, this highly temperature-dependent level of ACC oxidase activity was probably a wound response since it was almost entirely eliminated by incubating the excised segments at 20°C for 3 h. In contrast, heating of excised segments to 40°C for up to 4 h resulted in a time-dependent increase in ACC oxidase activity which was sensitive to cycloheximide, indicating rapid protein synthesis during the heat treatment. ACC oxidase activity fell sharply during subsequent chilling at 2. 5°C. After 3 days of chilling, all treated segments, regardless of their initial ACC oxidase activity, showed a decline to the same low activity level and ACC oxidase activity continued to fall slowly for up to 9 days at 2. 5°C. Hypocotyl segments excised from seedlings held at 15°C showed no change in solute leakage, but leakage increased rapidly when seedlings were either chilled at 2. 5°C or heated to 32°C (just below the heat shock temperature). Chill-induced leakage from non-heat-shocked segments increased steadily with chilling duration and was unaffected by cycloheximide concentration up to day 6. Within the elevated rate of leakage on day 9, however, leakage was lower from segments exposed to 10 and 50 μM cycloheximide. Solute leakage was markedly reduced for up to 9 days when segments were heat shocked at 40°C for 3 or 4 h with or without 10 M cycloheximide, but the presence of 50 μM cycloheximide caused an initial doubling of solute leakage and a 3-fold increase after 3 days of chilling. Cycloheximide prevented formation of heat shock protection against chilling from the start at 50 μM and after 9 days at 10 μM. These results indicate that the protection afforded by heat shock against chilling damage is quantitative and probably involves protein synthesis.

The physiological responses associated with chilling of horticultural ly mature cucumber (Cucumis sativus L.) fruit were examined using 13 lines that differ in chilling sensitivity. The low correlation coefficient between pitting and decay suggested that these two early manifestations of chilling injury are not significantly related. Likewise, fruit pitting and decay were not highly correlated with the tolerance of seedlings to chilling, suggesting that fruit and seedlings of the same line may have dissimilar sensitivity to chilling temperatures. Exudates from fruit cut in half transversely were collected on filter paper. The amount of exudate showed a significant correlation with pitting, decay, and percent ion leakage after 10 days of chilling. The fresh and dry weight of the exudates from fruit kept for 8 days at 12.5C ranged from 141- to 346-mg fresh weight and from 15 to 47-mg dry weight, respectively. Cucumber lines that were more sensitive to chilling had watery exudate, as indicated by their lower dry weight and percent solids. The conductivity of exudates from sensitive lines was higher (60 µsiemen/cm) than from chilling resistant lines (30 µsiemen/cm). Chilling-induced ethylene production was higher in sensitive than in resistant lines, and chilling caused a greater loss of ethylene forming enzyme activity in resistant lines than from chilling sensitive lines. Chilling injury is an economically important postharvest problem that reduces the overall quality and marketability of many harvested fruits and vegetables indigenous to the tropics and subtropics (Couey, 1982; Lyons, 1973; Saltveit and Mor- ris, 1990). Sensitivity to chilling injury varies among species of Passiflora (Patterson et al., 1976) and Lycopersicon (Paull, 1981), among cultivars of avocados (Persea americana Mill.; Hatton et al., 1965), cucumbers (Jasim and Marr, 1989), pa- payas (Carica papaya L. ; Chen and Paull, 1986), tomatoes (Lycopersicon esculentum Mill.; Burgis, 1970; King and Ludford, 1983), and watermelons (Citrullus lunatus L.; Risse et al., 1990). Chilled cucumbers exhibit reduced shelf life characterized by accelerated water loss, surface pitting, and increased sus- ceptibility to disease (Eaks and Morris, 1956, 1957; Ryall and Lipton, 1979). A significant increase in solute leakage was observed from mesocarp tissue excised from cucumbers exposed to chilling temperatures (Cabrera and Saltveit, 1990; Tatsumi and Murata, 1978; Tatsumi et al., 1981). Jasim and Marr (1989) observed that leakage of Mg+ and K+ was greater in sensitive than in resistant cultivars of cucumbers after 20 days at 4.4C. Surface pitting developed on 'Poinsett 76' fruit that were exposed to 2.5C for > 3 days (Cabrera and Saltveit, 1990). Similar results were observed by Jasim and Marr (1989) in resistant cultivars, (e.g., 'Dasher II' and 'Universal'), but not in sensitive cultivars, (e.g., 'Monarch' and 'Sweet Slice'), which showed slight pitting after the fruit were exposed to 4.4C for 3 days. The objectives of this present study were to characterize the physiological responses of fruit from various cucumber cultivars to chilling temperatures, and to correlate these changes with subjective evaluations of chilling injury in those fruit.

Tomato fruit (Lycopersicon esculentum Mill., cv. Castelmart) were harvested at various degrees of ripeness and exposed to ethanol vapor at 0, 2, or 4, ml·kg-1 in a 20-liter jar for 0, 2, 4, or 6 hours at 20C. Ripening was measured as changes in subjective color and in firmness and production of CO2 and ethylene. The soluble solids concentration (percent), titratable acidity (percent), and pH were measured at the end of the storage period when the fruit were red-ripe. Ethanol's inhibition of ripening was not confined to mature-green fruit, but also inhibited reddening of breaker, turning, and pink fruits. Storage of mature-green fruit at 20, 15, or 12C after treatment with 0 or 2 ml ethanol/kg at 20C prolonged the delay in ripening for 5, 6, and 7 days, respectively, compared with controls. There was no reduction in the quality of these fruit when they were red-ripe, even though there was an 11-day difference between the time the 20C control and the 12C-treated fruit became red-ripe. An informal panel did not detect any differences in flavor between these control and ethanol-treated fruit that were red-ripe. Increasing the duration of exposure to ethanol vapors from 2 to 6 hours had a pronounced effect on ethylene and CO2 production, but it did not significantly prolong the inhibition of ripening of mature-green fruit nor did it change their rate of softening during ripening. Increasing the temperature during exposure increased the effectiveness of ethanol, with the same level of inhibition produced by 6 hours at 20C, 4 hours at 25C, or 2 hours at 30C. Postharvest use of ethanol vapor to retard ripening may be a useful technique to extend the market life of tomato fruit.

Etiolated pea (Pisum sativum cv Alaska) seedlings growing against a horizontal barrier in the soil will assume a horizontal orientation and continue to grow for prolonged periods of time. With removal of the barrier or after seedlings grow out from underneath the obstruction, seedlings immediately return to normal vertical growth. Ethylene production increased several hours after the seedlings began to grow horizontally and not at the first contact with a barrier. Increases in ethylene production from horizontally growing seedlings were associated with decreased rates of elongation and increased stem diameter. The data suggest that increased ethylene production does not play a mediating role in the horizontal growth of pea seedlings when obstructed during emergence. We conclude that seedlings follow a path of least resistance when they grow against a barrier in the soil.

Additional index words. CO2, climacteric, color, C zH4, firmness, respiration, soluble solids "Abstract. External color, length, diameter, fresh weight, C0 2 production, internal C2HA concentration, flesh firm- ness, soluble solids concentration (SSC), flesh color, and seed cavity diameter were measured during fruit growth and maturation of seven melon cultivars (Cucumis melo L., Inodorus Group, Naud. CV. 'Amarelo', 'Golden Beauty Casaba', 'Honey Dew', 'Honey Loupe', 'Juan Canary', 'Paceco', and 'Santa Claus Casaba') of known age. There was no increase in C0 2 production either during ripening (e.g., loss of firmness and increased SSC) or with increasing C2H4 levels in fruit from any of the seven cultivars. There was a significant decline in respiration only at the second sampling date, which ranged from 14 to 18 days after anthesis. Respiration measured 1 week later was substantially higher and was followed by a general decline. This post 14- to 18-day rise in respiration was not a climacteric since it occurred well in advance of other ripening characteristics, e.g., loss of firmness, increase in SSC, or rise in internal C2H4. The increase in internal C2H4. coincided with or followed attainment of full fruit size, while flesh softening and the rapid rise in SSC preceded the rise in internal C 2H4, concentration. Respiration declined from 67 to 18 ml CO 2/ kg per hour by day 43 in all cultivars, except 'Honey Dew' and 'Honey Loupe'. Respiration in 'Honey Loupe' remained above 23 ml CO2/kg per hour and showed a rise to 32 ml/kg per hour on day 53. Respiration in 'Honey Dew' did not fall below 18 ml CO2/kg per hour until day 53. As with internal C 2H4 levels, there was no correlation between changes in and any marked change in the other signs of ripening that were measured. Morphological and physiological changes during fruit growth, maturation, and ripening of many melon cultivars have not been studied extensively, even in countries where the fruit are grown commercially and are very popular. Many diverse types of mel- ons are marketed; they differ in shape, external and internal color, surface netting, sweetness, flavor, and storability. While muskmelons have long been popular in the United States, ca- sabas are only now becoming as popular with American con- sumers as they are with consumers in other countries. The best flavor for melons depends on sweetness and on various volatile compounds that are not yet fully identified (Ya

Mikal E. Saltveit, Jr.Saitveit, M, E,, Jr, 1991, Prior temperature exposure affects subsequent chillingsensitivity, - Physiol, Plant, 82: 529-536,The chilling sensitivity of small discs or segments of tissue excised from chilling-sensitive species was significantly altered by prior temperature exposure subsequentto holding the tissue at chilling temperatures as measured by a number of physiolog-ical processes sensitive to chilling. This temperature conditioning was reversible by anadditional temperature exposure before chilling, and mature-green and red-ripetomato tissue exhibit similar chilling sensitivities.Exposing pericarp discs excised from tomato fruit (Lycopersicon escuientum Mill, cv,Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h beforechilhng the discs at 2,5°C for 4 days significantly altered the rate of ion leakage fromthe discs, but had no effect on the rate of ion leakage before chiUing and only aminimal effect on discs held at a non-chilling temperature of 12°C, Exposing chilling-sensitive tissue to temperatures below that required to induce heat-shock proteins butabove 20°C significantly increased chilling sensitivity as compared to tissue exposedto temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at2,5°C were higher from fruit and vegetative tissue of chilling-sensitive species {Cucu-mis sativus L, cv, Poinsett 76, and Cucurbita pepo L, cv. Young Beauty) that werepreviously exposed for 6 h to 32°C than from similar tissue exposed to 12°C, Exposureto 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chillingtolerant species {Malus domestica Borkh, cv. Golden Delicious, Pyrus communis L,cv, Bartlett), Ethylene and CO2 production were higher and iycopene synthesis waslower in chilled tomato pericarp discs that were previously exposed for 6 h to 32''Cthan the values from tissue exposed to 12°C for 6 h before chilling. Increased chillingsensitivity induced by a 6 h exposure to 32''C could be reversed by subsequentexposure to 12°C for 6 h.Key words — Apple, carbon dioxide, cucumber, Cucumis sativus, Cucurbita pepo,ethylene, ion leakage, Iycopene, Lycopersicon escuientum, Malus domestica, pear,Pyrus communis, squash, tomato,Mikal E, Saltveit, Jr,, Mann Laboratory, Dept of Vegetable Crops, Univ, of Cali-fornia, Davis, CA 95616, USA,IntroductionMany plants of tropical and sub-tropical origin, andsome tissues from plants of temperate origin are injuredby expostire to non-freezing temperattires below 12°C(Bramlage 1982, Couey 1982, Lyons 1973, Saltveit andMorris 1990), Chilling-sensitive piants include avoca-dos, bananas, cucumbers, peppers and totnatoes, whilechilling-resistant plants include apples and pears. To-mato fhiit are particularly stisceptible to chilling injuryat the mattire-green stage when they are noenally har-vested and shipped. The response of tomato fruit toReceived 19 November, 1990; revised 30 March, 1991

Rates of CO2 production and O2 consumption from aged disks of carrot (Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O2 in gas mixtures. A transient peak in the rate of CO2 production started 5 to 7 min after transfer to 2% or lower O2 mixtures and peaked at 50 min. After the peaks in CO2 production from the 0, 1 and 2% O2 treatments and after the stable production from the 4 and 8% O2 treatments, the rate of CO2 production from all low O2 treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O2 controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O2. Production of CO2 started to increase 5 to 7 min after transfer to 0, 1 and 2% O2, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g−1 h−1 from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g−1 h−1 from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO2 production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.

Symptoms of chilling injury were reduced by intermittently warming cucumber fruit (Cucumis sativus L. cv. Poinsett 76) from 2.5 to 12.5C for 18 hr every 3 days. Fruit continuously held at 2.5C for 13 days developed severe pitting and decay after 6 days at 20C, while fruit continuously held at 12.5C or intermittently warmed showed no pitting or decay during subsequent holding at 20C. The increased rate of C2H4 production during the first warming period, from 12 nl·(kg·hr)-1 at 2.5C to 201 nl·(kg·hr)-1 at 12.5C, was significantly greater than that during the second or third warming periods, i.e., 53 to 98 and 53 to 55 nl C2H4/(kg·hr), respectively. Respiration increased 3-fold during the initial warming period, but only 2-fold during subsequent warming periods. Leakage of cellular ions from excised disks of mesocarp tissue was around 6% and 10% of the total ion content of the tissue for control and intermittently warmed fruit, respectively, but increased to 17% for fruit that were continuously held at 2.5C for 10 days. After 320 hr (three cycles) of chilling and warming, chilled fruit showed significantIy lower ethylene-forming enzyme activity than the control or intermittently warmed fruit. Fruit held at 12.5C contained 0.09 to 0.34 nmol·g-1 of ACC. ACC levels were 6.23 nmol·g-1 in fruit exposed to 2.5C for 320 hr. In contrast, intermittently warmed fruit only showed 30% and 27% increases in ACC content during the first and second warming periods, respectively. Periodic warming appears to allow chilled fruit to acclimate to subsequent periods of chilling. Chemical names used: 1-aminocyclopropane-1-carboxylic acid (ACC).

10 pages, 11 figures., Wounding, as during excision and preparation of lettuce (Lactuca sativa L.) leaf tissue for salads, induces the synthesis and accumulation of phenolic compounds that participate in subsequent reactions that cause tissue browning. Exposure of excised 5-mm mid-rib segments of romaine lettuce leaf tissue to vapors of mono-carboxylic acids or aqueous solutions of mono-carboxylic acids or their salts inhibited wound-induced phenolic accumulation (WIPA) and subsequent tissue browning. The decline in phenolic content followed a quadratic curve with increasing concentration, reaching a maximum inhibition after 60 min of 74 ± 8% for 50 mM sodium acetate (2 carbons, C2) and 91 ± 4% for 20 mM sodium decanoate (capric acid, C10). Respiration (i.e. carbon dioxide production) was unaffected by concentrations of formic, acetic, or propionic acids that reduced wound-induced phenolic content or that increase ion leakage from the tissue into an isotonic mannitol solution. However, WIPA was suppressed up to 70% at concentrations (20 mM acetate) that did not increase ion leakage over that of water controls. Various acetate salts (i.e. ammonium, calcium, magnesium, and sodium) all produced the same level of inhibition. The effectiveness of the compounds increased with increasing number of carbons in the molecule from 1 to 10, and was unaffected by whether the carbons were a straight chain or branched or whether the treatment was delayed by up to 6 h. The effectiveness of butyrate (C4) in reducing WIPA (27% reduction at 20 mM) was less than that predicted from the response of the two adjacent mono-carboxylates similarly applied: propionate (C3) (62%) and valerate (C5) (73%). It appears that, unlike the n-alcohols, mono-carboxylates are not interfering with the synthesis or propagation of a wound signal but are interfering with subsequent steps in the production and accumulation of wound-induced phenolic compounds.

Chilling at 6 degrees C caused an immediate cessation of protoplasmic streaming in trichomes from African violets (Saintpaulia ionantha), and a slower aggregation of chloroplasts in the cells. Streaming slowly recovered upon warming to 20 degrees C, reaching fairly stable rates after 4, 15, 25 and 35 min for tissue chilled for 2 min and for 2, 14 and 24 h, respectively. The rate of ion leakage from excised petioles into an isotonic 0.2 M mannitol solution increased after 12 h of chilling and reached a maximum after 3 days of chilling. A heat shock at 45 degrees C for 6 min reduced chilling-induced rates of ion leakage from excised 1-cm petiole segments by over 50%, namely to levels near that from non-chilled control tissue. Heat-shock treatments themselves had no effect on the rate of ion leakage from non-chilled petiole segments. Protoplasmic streaming was stopped by 1 min of heat shock at 45 degrees C, but slowly recovered to normal levels after about 30 min Chloroplasts aggregation was prevented by a 1 or 2 min 45 degrees C heat-shock treatment administered 1.5 h before chilling, but heat-shock treatments up to 6 min only slightly delayed the reduction in protoplasmic streaming caused by chilling. Tradescantia virginiana did not exhibit symptoms associated with chilling injury in sensitive species (i.e. cessation of protoplasmic streaming in stamen hairs and increased ion leakage from leaf tissue).

Salicylic acid (SA) is one component of a complex signalling pathway that is induced by a number of biotic and abiotic stresses. Exposing seedling radicles to aqueous solutions of 0.5 mM salicylic acid for 24 h before chilling at 2.5 degrees C for 1-4 days reduced the chilling-induced increase in electrolyte leakage from maize and rice leaves, and cucumber hypocotyls, but not from their radicles. The SA treatments that induced chilling tolerance in the aerial portion of the seedlings did not induce chilling tolerance in the radicles, even though the SA treatments were applied to the radicles. A comparison of activity among five antioxidant enzymes showed that SA did not alter enzyme activities in the radicles, but that chilling tolerance induced by SA in the aerial portions of maize and cucumber plants was associated with an increase in the activity of glutathione reductase and guaiacol peroxidase.

Cucumber seedling radicles become more chilling sensitive as they elongate. Chilling seedlings with radicles 20 mm long for 48 h at 2.5 degrees C inhibited subsequent growth by 36%, while it reduced the growth of 70 mm-long radicles by 63%. Although the growth rate of non-chilled cucumber radicles at 25 degrees C is constant from 20 to 80 mm, tissue viability [i.e. reduction of TTC (2,3,5-triphenyltetrazolium chloride) to formazan] and DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) radical scavenging activity of apical tissue declines as radicles elongate from 20 to 80 mm in length. TTC reduction, DPPH-radical scavenging activity and protein content of apical tissue were higher in 20 than in 70 mm radicles immediately after chilling and after an additional 48 h of growth at 25 degrees C. Catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in the apical tissue of 20 than in 70 mm radicles before chilling. Immediately after chilling and after an additional 48 h at 25 degrees C, superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.6.4.2), and guaiacol peroxidase (GPX; EC 1.11.1.7) activity increased more rapidly in 70 mm radicles than in 20 mm radicles (SOD, GR, and GPX activity in 70 mm radicles was 1.5-, 1.9- and 8.6-fold higher, respectively, than in 20 mm radicles). However, APX and CAT activity in 20 mm radicles were always higher than in 70 mm radicles. Growth after chilling enhanced the activity of all antioxidant enzymes compared to that found in non-chilled tissue; however, CAT activity in 70 mm radicles did not recover to levels found in non-chilled tissue. Higher levels of CAT, APX and DPPH-radical scavenging activity are correlated with higher chilling tolerance of 20 mm-long cucumber radicles compared to 70 mm-long radicles.

Cutting leaves of Romaine lettuce (Lactuca sativa L. cv. Longifolia) produces a wound signal that induces the synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and the accumulation of phenolic compounds in cells up to 2 cm from the site of injury, and tissue browning near the site of injury. The response of leaves within a head of Romaine lettuce to putative chemical wound signals [abscisic acid (ABA), jasmonate (JA) and methyl jasmonate (MeJA)] differed significantly with leaf age. Exposure of harvested heads of lettuce to ABA, JA, MeJA, or salicylic acid (SA) did not induce changes in PAL activity, the concentration of phenolic compounds or browning in mature leaf tissue that was similar to the level induced by wounding. Methyl jasmonate applied as vapour (10, 100 or 1000mgr;l kg-1 FW), or as an aqueous spray or dip (0.01-100mgr;M) at 5 or 10 degrees C did not produce an effect on PAL activity or browning that differed significantly from the untreated controls. In contrast, JA, MeJA and SA did induce elevated levels of PAL activity in younger leaves. However, the levels induced were far lower than those induced by wounding. Wound induced phenolic metabolism in mature leaves appears to be induced by different signals than those functioning in young leaves.

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37 degrees C for 6 hours followed by 4 days at 2.5 or 12.5 degrees C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5 degrees C, but not to 12.5 degrees C, a nonchilling temperature. Exposure to 37 degrees C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5 degrees C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37 degrees C and held at the nonchilling temperature of 12.5 degrees C. There was no relationship between abscisic acid content following exposure to 12.5 or 37 degrees C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42 degrees C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42 degrees C.

The rate of wound ethylene synthesis was reduced by more than 85% when 9-millimeter subapical sections of etiolated 7-day-old Pisum sativum L., cv. Alaska seedlings were incubated in water during the 26-minute induction period prior to wound ethylene synthesis, but the rate of synthesis was unaffected if sections were incubated in water during the actual synthesis of wound ethylene. The characteristic timing of the wound response was unaffected by either treatment. The ability of various chemical solutions and aqueous plant extracts to alter the rate of wound ethylene synthesis was studied by first incubating subapical pea stem sections in solutions under anaerobic conditions (anaerobiosis delays the induction and synthesis of wound ethylene; Plant Physiol 61: 675-679), and then measuring wound ethylene synthesis after the tissue was transferred to air. Solutions of several reported precursors of ethylene synthesis, such as methionine, homoserine, or propanal, did not reverse the water-caused reduction of wound ethylene synthesis. A water-soluble, heat-stable factor in extracts from pea seedlings, and solutions of 23 nanomolar triacontanol, 10 micromolar kinetin, or 10 micromolar benzyladenine prevented the reduction of wound ethylene synthesis, but were ineffective if administered after an initial 15-minute anaerobic water incubation. This suggested that the active solutions may have only prevented the loss of some ephemeral, though necessary factor, rather than actually containing the substrate or inducer of wound ethylene synthesis. Attempts to isolate and characterize the active fraction from aqueous tissue extracts were unsuccessful. Free radical quenchers, inhibitors of protein synthesis, and rhizobitoxine, an inhibitor of ethylene synthesis from methionine, all reduced wound ethylene synthesis when administered in solutions which previously had maintained wound ethylene synthesis.

Periodic shaking increased the resistance of potted poinsettia (Euphorbia pulcherrima Klotzsch ex. Willd., cv. Annette Hegg Diva) plants to mechanically induced leaf epinasty, but this resistance was lost within 24 hours of discontinuing the shaking. Bending petioles, not their reorientation in the gravitational field, caused increased ethylene production by excised petioles. Foliar sprays containing 40 μm cycloheximide, which were applied 24 hours before the plants were sleeved for 24 hours, completely prevented mechanically induced leaf epinasty, but were phytotoxic to the bracts.

A 1.5% O2 atmosphere, relative to normal air, dramatically inhibited ethylene-induced russet spot development, PAL, and ionically bound POD, and ionicaliy bound IAA oxidase activities and reduced soluble phenolic content in stored iceberg lettuce (Lactuca sativa L.). Low O2 also inhibited eythylene production and respiration. Polyphenol oxidase activity was slightly inhibited by low O2. The results suggest that low O2 inhibition of ethylene action and attendant effects on phenolic metabolism and IAA oxidase activity may be responsible for inhibition of russet spotting by 1.5% O2. Chemical names used: indole-3-acetic acid (IAA); phenylalanine ammonia-lyase (PAL); peroxidase (POD); polyphenol oxidase (PPO).

A 10-minute soak in 1.0 mm vitamin K5 reduced ethylene production over 90%, while doubling carbon dioxide production by cortical tissue from pre-climacteric apples (Malus domestica Borkh.). Reduced ethylene production persisted for at least 4 hours, while carbon dioxide production declined to rates not significantly different from the controls. Vitamin K5 also reduced ethylene production by 50% from quartered fruit of tomato (Lycopersicon esculentum Mill.) at different stages of maturity, and from cortex tissue from apples at or near their climacteric peak of ethylene production.

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 +/- 2 degrees C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (-)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.

Ag(I), applied in aqueous solution as AgNO3, inhibited ethylene synthesis and ripening of mature green banana fruit slices and pericarp discs of mature green tomato fruit. It also inhibited ethylene production by cortical tissue from post-climacteric apples. Concentrations of Ag(I) which reduced ethylene synthesis had an inconsistent effect on CO2 production: no effect on banana slices (1.0 mm Ag+), stimulation in apple tissue (0.1 mm Ag+), and inhibition in tomato tissue (0.3 mm Ag+). This was accompanied by a slight amount of tissue necrosis at these concentrations. Of the 7 metallic salts tested, only Ag(I) inhibited ripening and ethylene synthesis at observed non-phytotoxic levels. Inhibition of ripening and ethylene synthesis by Ag(I) was evident in tissue treated with sufficient exogenous ethylene to elicit both responses in control tissue. The inability of applied ethylene to overcome the inhibitory effect of Ag(I) suggests that the silver ion may interfere with the primary action of ethylene in the tissue.

Basipetal auxin transport occurred in intact and excised Dracaena marginata stem sections at an apparent velocity of about 5 mm/hr. Application of 20 µl of 0.1 mm 2,3,5-triiodobenzoic acid (TIBA) to the basal cut end of a 3-cm stem segment excised 20 cm from the apex of 1-m-tall plants reduced basipetal auxin transport by 60%. Similar application to the apical end had no effect on acropetal auxin movement. Lateral auxin movement was observed 24 hours after application of radioactive auxin to decapitated stems which had grown horizontally for at least 30 days.

Growth patterns of cucumbers (Cucumis sativus L.) were studied by photographing developing fruit at 2- or 3-day intervals over a 30-day period beginning with pollination (day 0). Nine cultivars were studied: ‘Chinese Long Green’, ‘Sprint 440’, ‘Marketmore 76’, and ‘Minisol’ (all fresh-market types); ‘Riesenschal’ (a schälgurken type); and ‘Marbel’, ‘Kobus’, ‘Calypso’, and ‘Wisconsin SMR-18’ (all pickling types). Analysis of the photographs showed that all sections of the fruit grew in length at a constant rate during the 30-day period. The pattern of growth was fairly uniform, except that there was slightly more growth in the center section than at the ends, and slightly more growth at the blossom end than at the peduncle end of the fruit. Fresh-market and schälgurken types had the longest fruit over the 30-day period of growth, but pickling types had the highest percent change in length. Pickling types produced shorter fruit because they stopped growing earlier than the two other types (12 vs. 14 days, respectively).

Ethanol concentrations that were induced in pericarp discs of mature-green tomato fruit (Lycopersicon esculentum Mill, cv Castlemart) either by anaerobic metabolism or by exposure to ethanol vapor inhibited ripening without increasing the rate of ion leakage. Inhibition of ripening (i.e. lycopene synthesis) of excised tomato pericarp tissue by ethanol vapor was reversed by increasing concentrations of the plant hormone ethylene. A Lineweaver-Burk plot indicated noncompetitive interaction between ethanol and ethylene. Methanol and n-propanol also inhibited lycopene synthesis without significantly increasing ion leakage. The similar inhibitory effects of methanol, ethanol, and n-propanol at concentrations which did not stimulate ion leakage, and the relationship between activity and lipophilia of the alcohols suggest that their mode of action was through disruption of membranes associated with ethylene action.

The ethanol content of ‘Harrison’ and ‘Jersey’ highbush (Vaccinium corymbosum L.) and ‘Premier’ rabbiteye blueberries (V. ashei Reade) increased linearly with time (1 to 4 hours) and exponentially with temperature (0° to 30°C) during exposure to an atmosphere of pure N2 or CO2. CO2 production was depressed about 20% under N2 gas. Berries which were placed in air after 4 hours in CO2 lost half the CO2 they contained every 30 minutes. After 3 hours, CO2 evolution from treated and control fruit was the same. Fruit quality was unaffected by exposure at 0° to 30° to pure N2 or CO2 gas for up to 4 hours. However, 22 hours in a CO2 atmosphere at either 0° or 30° reduced fruit quality. An equation was derived to calculate ethanol accumulation under different cooling rates.