Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce

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 1000 µl kg⁻¹ FW), or as an aqueous spray or dip (0.01–100 µ M ) at 5 or 10°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.     

Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce

Mechanical wounding by cuts or punctures caused a brief increase in ethylene production by iceberg lettuce ( Lactuca sativa L.) leaf tissue. Wounding increased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, which was a function of the degree of injury. Wound-induced PAL activity appeared after 4 h and reached maximum activity in about 24 h before slowly declining to normal levels in about a week. A signal for PAL induction was transmitted at about 0.5 cm h⁻¹ from the site of injury to cells up to 2.5 cm away. Treatment with 100 μ2-aminoethoxyvinylglycine prevented wound-induced ethylene production but did not affect induced PAL activity. Injury increased the concentration of several soluble phenolic compounds that were easily oxidized to brown substances by polyphenol oxidase (EC 1.10.3.2) isolated from lettuce tissue. Wounding also increased peroxidase (EC 1.11.1.7) activity and lignin content, with cell wall lignification localized in wounded and adjacent cells. Although wounding alone did not induce russet spotting, it did greatly increase susceptibility to ethylene-induced russet spot development. In the presence of 3 μ1⁻¹ ethylene, the russet spot score increased as the degree of injury increased.     

Mono-carboxylic acids and their salts inhibit wound-induced phenolic accumulation in excised lettuce (Lactuca sativa) leaf tissue

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 m M sodium acetate (2 carbons, C2) and 91 ± 4% for 20 m M 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 m M 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 m M ) 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.     

Differential expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii

The heat-shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. In contrast, heat-shock-related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70,000-Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation.     

Evidence for a senescence-associated gene induced by darkness

Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H₂O₂ via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Gene expression during leaf development in Lolium temulentum: Patterns of protein synthesis in response to heat-shock and cold-shock

At an optimal growth temperature of 20°C, expending 4th leaves of Lolium temulentum L. synthesised a broad spectrum of polypeptides which altered with the maturity of the leaf tissue. Elevation of the temperature to 35°C, or above, induced synthesis of heat-shock proteins (hsp), and all parts of the 4th leaf were capable of this response. The threshold temperature for induction of hsp synthesis was little affected by the growth temperature (5 or 20°C). In contrast, a sudden 15–18°C decrease in temperature did not result in a marked alteration of protein synthesis patterns. It is concluded that in this species adaptation to rapid temperature reduction is not mediated by stress protein synthesis.     

Involvement of components of the phospholipid-signaling pathway in wound-induced phenylpropanoid metabolism in lettuce (Lactuca sativa) leaf tissue

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.     

Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection

Physical injury inflicted on living tissue makes it vulnerable to invasion by pathogens. Wounding of Arabidopsis thaliana leaves, however, does not conform to this concept and leads to immunity to Botrytis cinerea , the causal agent of grey mould. In wounded leaves, hyphal growth was strongly inhibited compared to unwounded controls. Wound-induced resistance was not associated with salicylic acid-, jasmonic acid- or ethylene-dependent defence responses. The phytoalexin camalexin was found to be involved in this defence response as camalexin-deficient mutants were not protected after wounding and the B. cinerea strains used here were sensitive to this compound. Wounding alone did not lead to camalexin production but primed its accumulation after inoculation with B. cinerea , further supporting the role of camalexin in wound-induced resistance. In parallel with increased camalexin production, genes involved in the biosynthesis of camalexin were induced faster in wounded and infected plants in comparison with unwounded and infected plants. Glutathione was also found to be required for resistance, as mutants deficient in γ-glutamylcysteine synthetase showed susceptibility to B. cinerea after wounding, indicating that wild-type basal levels of glutathione are required for the wound-induced resistance. Furthermore, expression of the gene encoding glutathione- S -transferase 1 was primed by wounding in leaves inoculated with B. cinerea . In addition, the priming of MAP kinase activity was observed after inoculation of wounded leaves with B . cinerea compared to unwounded inoculated controls. Our results demonstrate how abiotic stress can induce immunity to virulent strains of B. cinerea , a process that involves camalexin and glutathione.     

Regulation of heat-shock protein synthesis in chicken muscle culture during recovery from heat shock

Exposure of chick myotube cultures to a temperature (45 degrees C) higher than their normal growing temperature (37 degrees C) caused extensive synthesis of three major polypeptides of Mr = 25 000, 65 000 and 81 000 referred to as 'heat-shock polypeptides' (hsps). When these cells were allowed to recover from heat-shock treatment at 37 degrees C for 6-8 h, the rate of accumulation of isotope into the 65 000-Mr and 81 000-Mr hsps declined to levels comparable to those in control cultures maintained at 37 degrees C. However, incorporation of isotope in the 25 000-Mr hsp continued at an elevated rate for a longer period than the 65 000-Mr and 81 000-Mr hsps. When heat-shocked cells were allowed to recover at 37 degrees C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.     

A Complex Array of Proteins Related to the Multimeric Leucine Aminopeptidase of Tomato

Leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding, pathogen infection, and insect infestation (V. Pautot, F.M. Holzer, B. Reisch, L.L. Walling [1993] Proc Natl Acad Sci USA 90: 9906-9910). Polyclonal antibodies to a glutathione S-transferase-LAP fusion protein and affinity-purified antibodies recognizing LAP antigenic determinants detected four classes of polypeptides in tomato (Lycopersicon esculentum) leaves. All four classes had multiple polypeptides in two-dimensional polyacrylamide gel electrophoresis immunoblots. Although antigenically related to the wound-induced tomato LAP proteins, the 77- and 66-kD LAP-like proteins accumulated in both healthy and wounded leaves. Two classes of 55-kD polypeptides with distinctive isoelectric points were designated as plant LAPs; only the acidic LAP proteins accumulated to high levels after mechanical wounding or Pseudomonas syringae pv tomato infection of tomato leaves. The temporal accumulation of LAP mRNAs was correlated with the increase in acidic LAP protein subunits. A slow-migrating LAP activity was detected using a native gel assay after wounding. The molecular mass of the native wound-induced LAP enzyme was 353 kD. The 55-kD acidic LAP proteins were associated with induced LAP activity, whereas the neutral LAPs and the LAP-like proteins were not associated with this exopeptidase. A second, fast-migrating aminopeptidase was detected in both healthy and wounded tomato leaves. Cell fractionation experiments revealed that wound-induced LAP is a soluble enzyme.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Archaebacterial heat-shock proteins

The response to heat shock was examined in seven archaebacterial strains from the genus Halobacterium. Upon heat shock each strain preferentially synthesized a limited number of proteins which fell into three narrow mol. wt. ranges. Further examination of the heat-shock response in H. volcanii revealed that heat-shock protein (hsp) synthesis was greatest at 60°C. Synthesis of hsps at this induction temperature was both rapid and transient. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. H. volcanii cells also responded with a `heat shock-like' response to salt dilution, a natural environmental stress for these organisms. These results indicate that the heat shock or stress response which is charactertistic of eukaryotic and eubacterial cells is also present among members of the archaebacterial genus Halobacterium.     

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.