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 PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock 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 45°C 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 (R² > 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 45°C 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.     

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.     

Changes of phenolic metabolism and oxidative status in nitrogen-deficient Matricaria chamomilla plants

The effects of nitrogen deficiency on selected physiological attributes, phenylalanine ammonia-lyase (PAL, EC. 4.3.1.5) activity, phenolic contents, peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities, lipid peroxidation status and H₂O₂ accumulation were studied in N-deficient Matricaria chamomilla (L.) over 12 days. N deficiency enhanced root growth and inhibited shoot growth. Chlorophyll composition and F _v/F _m were not affected by N stress, but nitrogen and soluble proteins decreased in both the rosettes and the roots. PAL activity, expressed per mg protein, was enhanced in N-deficient rosettes and tended to decrease by the end of the experiment, while in the roots PAL activity was maintained. Total phenolic contents increased in both rosettes and roots. Peroxidase and catalase activities in N-deficient rosettes tended to decrease by the end of the experiment, while in the roots they increased on the 12th day of deficiency. Furthermore, lipid peroxidation status increased in N-deficient roots on the 12th day, indicating that antioxidative protection was insufficient to scavenge reactive oxygen species being generated. Surprisingly, H₂O₂ content was even lower in N-deficient roots by the end of the experiment, while in the leaves increased. This observation in correlation to lipid peroxidation and H₂O₂ degradation is discussed. The importance of PAL activity and phenolic metabolites in combination with antioxidative enzymes for plant protection against oxidative stress and the significance of PAL activity for the mobilization of N availability in N-deficient tissue are also discussed in view of existing information.     

Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants

Jasmonic acid (JA) and salicylic acid (SA) have both been implicated as important signal molecules mediating induced defenses of Nicotiana tabacum L. against herbivores and pathogens. Since the application of SA to a wound site can inhibit both wound-induced JA and a defense response that it elicits, namely nicotine production, we determined if tobacco mosaic virus (TMV) inoculation, with its associated endogenous systemic increase in SA, reduces a plant's ability to increase JA and nicotine levels in response to mechanical damage, and evaluated the consequences of these interactions for the amount of tissue removed by a nicotine-tolerant herbivore, Manduca sexta. Additionally, we determined whether the release of volatile methyl salicylic acid (MeSA) from inoculated plants can reduce wound-induced JA and nicotine responses in uninoculated plants sharing the same chamber. The TMV-inoculated plants, though capable of inducing nicotine normally in response to methyl jasmonate applications, had attenuated wound-induced JA and nicotine responses. Moreover, larvae consumed 1.7- to 2.7-times more leaf tissue from TMV-inoculated plants than from mock-inoculated plants. Uninoculated plants growing in chambers downwind of either TMV-inoculated plants or vials releasing MeSA at 83- to 643-times the amount TMV-inoculated plants release, exhibited normal wound-induced responses. We conclude that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation. The release of volatile MeSA from inoculated plants is not sufficient to influence the wound-induced responses of neighboring plants. Received: 6 January 1999 / Accepted: 11 January 1999     

Salicylic acid and methyl jasmonate improve chilling tolerance in cold-stored lemon fruit (Citrus limon)

Chilling injury (CI) is associated with the degradation of membrane integrity which can be aligned to phenolic oxidation activated by polyphenol oxidase (PPO) and peroxidase (POD), enzymes responsible for tissue browning. Phenylalanine ammonia-lyase (PAL) is a further enzyme prominent in the phenolic metabolism that is involved in acclimation against chilling stress. It was hypothesized that treatment with methyl jasmonate (MJ) and salicylic acid (SA) may enhance chilling tolerance in lemon fruit by increasing the synthesis of total phenolics and PAL by activating the key enzyme regulating the shikimic acid pathway whilst inhibiting the activity of POD and PPO. Lemon fruit were treated with 10 μM MJ, 2 mM SA or 10 μM MJ plus 2 mM SA, waxed, stored at −0.5, 2 or 4.5 °C for up to 28 days plus 7 days at 23 °C. Membrane integrity was studied by investigating membrane permeability and the degree of membrane lipid peroxidation in lemon flavedo following cold storage. The 10 μM MJ plus 2 mM SA treatment was most effective in enhancing chilling tolerance of lemon fruit, significantly reducing chilling-induced membrane permeability and membrane lipid peroxidation of lemon flavedo tissue. This treatment also increased total phenolics and PAL activity in such tissue while inhibiting POD activity, the latter possibly contributing to the delay of CI manifestation. PPO activity was found to be a poor biochemical marker of CI. Treatment with 10 μM MJ plus 2 mM SA resulted in an alteration of the phenolic metabolism, enhancing chilling tolerance, possibly through increased production of total phenolics and the activation of PAL and inhibition of POD.     

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.     

Wound-response regulation of the sweet potato sporamin gene promoter region

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a -glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

Salicylic acid-induced changes to growth and phenolic metabolism in <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> plants

The influence of salicylic acid (SA) doses of 50 and 250 μM, for a period of up to 7 days, on selected physiological aspects and the phenolic metabolism of Matricaria chamomilla plants was studied. SA exhibited both growth-promoting (50 μM) and growth-inhibiting (250 μM) properties, the latter being correlated with decrease of chlorophylls, water content and soluble proteins. In terms of phenolic metabolism, it seems that the higher SA dose has a toxic effect, based on the sharp increase in phenylalanine ammonia-lyase (PAL) activity (24 h after application), which is followed by an increase in total soluble phenolics, lignin accumulation and the majority of the 11 detected phenolic acids. Guaiacol-peroxidase activity was elevated throughout the experiment in 250 μM SA-treated plants. In turn, some responses can be explained by mechanisms associated with oxidative stress tolerance; these mitigate acute SA stress (which is indicated by an increase in malondialdehyde content). However, PAL activity decreased with prolonged exposure to SA, indicating its inhibition. Accumulation of coumarin-related compounds (umbelliferone and herniarin) was not affected by SA treatments, while (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids increased in the 250 μM SA-treated rosettes. Free SA content in the rosettes increased significantly only in the 250 μM SA treatment, with levels tending to decrease towards the end of the experiment and the opposite trend was observed in the roots.     

Distribution of Phenylalanine Ammonia Lyase and Chalcone Synthase within Trunks of Robinia pseudoacacia L.

During heartwood formation, a kind of apoptosis in the inner parts of woody axes, phenolic substances are accumulated by in situ biosynthesis. In Robinia pseudoacacia L, these compounds are mainly flavonoids. In the present work, we performed a study to show if there is a correlation between measurable activities and detectable protein levels of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and chalcone synthase (CHS; EC 2.3.1.74), key enzymes of general phenylpropanoid metabolism and flavonoid biosynthesis, respectively. After separation of total protein extracts by one-dimensional micro-gel electrophoresis, newly emerging polypeptides were detectable within the sapwood-heartwood transition zone, pointing toward a transient activation of metabolism shortly before cell death occurs. Most prominent was a polypeptide around 46 kDa. By immunoblotting, this band was identified as a CHS subunit. Thus, the exclusive presence of both enzyme protein and extractable enzyme activity of CHS in the heartwood bordering tissue was shown. In contrast, levels of PAL protein were similar in all xylem tissues which contain living cells. PAL activity, however, was measurable only in the differentiating xylem and the sapwood-heartwood transition zone. From these results we conclude that during heartwood formation, CHS and PAL differ in their mode of regulation. It seems likely that CHS activity is regulated at the level of enzyme protein while PAL regulation is most probably post-translational.     

Wound-induced superoxide production and PAL activity decline with potato tuber age and wound healing ability

Wound healing of potato tubers involves the concerted action of several enzymes that facilitate polymerization of phenolics into suberin at the wound site. A decline in the efficiency of healing and resistance to pathogens with advancing tuber age was associated with reduced ability of older tubers to produce superoxide radicals (FRs) in response to wounding. Autophotographs of luminol‐treated longitudinal sections of tissue from 6‐, 18‐ and 30‐month‐old tubers revealed a substantial decline in superoxide production at the wound surface with advancing age. Older tubers were less able to respond to wounding by increasing phenylalanine ammonia lyase (PAL) activity. This enzyme produces t‐cinnamic acid, which constitutes a component of the phenolic domain of suberin, and is normally induced by wounding and/or ethylene. Interestingly, the ability of wounded tissue to oxidize exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) to C₂H₄ also decreased with advancing tuber age. The oxidation of ACC was inhibited by the FR scavenger, n‐propyl gallate (PG), and inhibition was greatest in tissue from younger tubers, reflecting their greater ability to produce superoxide radicals upon wounding. Regardless of tuber age, 1‐aminocyclobutane‐1‐carboxylic acid, an ACC oxidase inhibitor, did not inhibit C₂H₄ generation from exogenous ACC. Hence, C₂H₄ production from ACC by wounded tuber tissue is largely non‐enzymatic and FR‐driven, and thus serves as an indicator of the ability of wounded tissue to produce superoxide. Age‐induced reduction in PAL activity and FR production at the wound surface probably limited the oxidative polymerization of phenolics into suberin during wound periderm formation. The age‐induced loss in ability of wounded tissue to heal and resist pathogens is thus consistent with reduced synthesis and polymerization of phenolic adducts into suberin, a consequence of reduced FR and PAL activity at the wound surface.     

Altered Kinetic Properties of Tyrosine-183 to Cysteine Mutation in Glutamine Synthetase of <Emphasis Type="Italic">Anabaena variabilis</Emphasis> Strain SA1 Is Responsible for Excretion of Ammonium Ion Produced by Nitrogenase

A L-methionine-D,L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N₂ reduction. In order to determine the biochemical basis for the NH₄ ⁺-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn⁺² for optimum activity, while SA0-GS was Mg⁺² dependent. SA0-GS had the following apparent K _m values at pH 7.5: glutamate, 1.7 mM; NH₄ ⁺, 0.015 mM; ATP, 0.13 mM. The apparent K _m for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 mM; NH₄ ⁺, 12.4 mM; ATP, 0.17 mM). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH₄ ⁺-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K _m values for NH₄ ⁺ and glutamate, and thus limited the assimilation of NH₄ ⁺ generated by N₂ reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH₄ ⁺ binding and metal ion coordination. Received: 3 July 2002 / Accepted: 29 July 2002     

Influence of natural substances of phenolic character and diethyldithiocarbamate on the metabolism of L-tryptophan in cabbage,maize and pea

The effect of phenolic substances isolated earlier from cabbage, maize and pea on L-tryptophan-3-¹⁴C (L-Try-¹⁴C) metabolism in those plants was investigated. For the sake of comparison the effect of diethyldithiocarbamate (DIECA) on cabbage was also observed. A phenolic substance of unknown structure isolated from cabbage was utilised in a 0.5 mg/ml concentration, p-coumaric acid (PCA) from maize in 0.7 mg/ml concentration and quercetinglucosyl-coumarate (QGC) found in pea in 8 mg/ml concentration were used. The chosen concentrations were on the limit of their inhibitory effect on the growth of the respective plant apical segments. The effect of DIECA was investigated at 0.1 mg/ml concentration. Using non-labelled L-Try as substrate (5×10^?3 m) biological tests have shown that QGC and DIECA have a weaker but distinct effect on the increase in auxin level (+17% and +15%, respectively). All phenolic compounds, as well as DIECA increase the intake of L-Try-¹⁴C from the incubation solutions. Phenolic substances decrease the conversion of L-Try-¹⁴C to its metabolites. The changes were studied after a 20 hour period of L-Try-¹⁴C metabolism. On chromatograms changes in the proportion of L-Try-¹⁴C metabolites took place, especially in the areas of substances of complex character from which IAA splits off easily,i.e. in the areas of 3-indolylacetylglucose and 3-indolylacetamide. The radioactivity of these areas is generally decreased (e.g. the decrease of radioactivity in the 3-indolylacetylglucose area under the influence of the phenolic substance ofBrassica plants is ?7,4%, under the influence of PCA in maize it is ?8.9% and under the influence of QGC in pea it is ?17.1%). DIECA also decreased the radioactivity of this zone, by ?10,5%. In cabbage a large part of L-Try-¹⁴C was transformed to glucobrassicin; its biosynthesis increases in the presence of the phenolic substance of cabbage by +3%, in the presence of DIECA by +27%. The results are discussed in a working hypothesis based on the key formation of IAA, accompanied by its oxidation and detoxication.     

Phenylalanine ammonia-lyase,chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid walnut tree

Summary During the first growth phase of walnut (Juglans sp.) stump shoots, the concentrations of the two major phenolic compounds are not correlated with an increasing rate of shoot growth. The concentration of hydrojuglone glucoside (naphtoquinone) decreases as shoot growth rate increases, whereas the concentration of myricitrin (flavonol) remains constant. In contrast, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity is proportional to the growth rate of shoots. Rejuvenation, which induces a higher growth rate and vegetative propagation ability, results in an increase of both PAL and chalcone synthase (CHS, EC 2.3.1.74) activities and hydrojuglone glucoside/myricitrin ratio. Moreover, physiological ageing is characterized by an accelerated functioning of polyphenolic metabolism. Fluctuations in PAL activity are associated with changes in shoot growth rate and with rejuvenation, but PAL does not directly control the accumulation of flavonoid compounds during rejuvenation. On the contrary, mathematical correlation of CHS activity and flavonoid accumulation during annual shoot growth of both adult and rejuvenated trees, indicates that CHS is the rate-limiting enzyme of the pathway.