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.

Exposure to a chilling temperature of 2.5degreesC for 96 h inhibited the subsequent growth of cucumber seedling radicles at 25degreesC by 92%. Exposing seedling with 5 +/- 1 mm long radicles to acetaldehyde vapour (275 mul l(-1)) or to an aqueous ethanol solution (0.6 M) for 2 h, or to 45degreesC 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 [S-35]-methionine) by about 70%. In contrast, treatment for 2 h with an aqueous arsenite solution (100 muM) had no effect on chilling sensitivity or the incorporation of [S-35]-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.

Chilling at 6degreesC 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 20degreesC, 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 45degreesC 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 45degreesC, but slowly recovered to normal levels after about 30 min Chloroplasts aggregation was prevented by a 1 or 2 min 45degreesC 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).

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.

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, 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.

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.

Cold soil and air temperatures reduce germination, seedling growth, and stand establishment of many important agronomic and horticultural crops. Chilling tolerance of many seeds is lost as they germinate and grow into young seedlings. Cucumber (Cucumis sativus cv. Poinsett 76) seedling radicles lose chilling tolerance as they emerge and elongate. Chilling at 2.5 degrees C for 72 It reduced subsequent elongation at 259 degrees C by 12,41, and 77% for 1-, 10-, and 20-mm-long radicles, respectively. Radicle elongation followed an exponential decline with increasing mannitol concentration (r(2) > 0.9) for radicles initially 1 or 10 mm in length. Chilling 1-mm radicles treated for 24 h in 0.3 or 0.6 M mannitol inhibited elongation 90 or 40%, respectively. During mannitol treatment, 0.3 and 0.6 M treated radicles increased 12.5 and 0.7 mm in length. Chilling 10-mm radicles treated for 24 h in 0.3 or 0.6 M mannitol inhibited elongation 99 or 33%, respectively. During mannitol treatment, 0.3 and 0.6 M treated radicles increased 23.8 and 1.1 nun in length. The percentage inhibition of chilling-induced radicle elongation was related to the initial radicle length when chilled by a curve of the form C-L = C-o X (1 - e(-KL)) that describes many biological reactions where the rate of change is constant and proportional to the amount of reactants present The increase in chilling-induced inhibition of radicle elongation with increasing radicle length was consistent with the progressive loss of protective compounds, possibly through dilution as the tissue expanded in volume during radicle elongation.