Vacuolar localization of wound-induced carboxypeptidase inhibitor in potato leaves

The wound-induced carboxypeptidase inhibitor in potato leaves was shown to be localized in the central vacuoles of the cells. The inhibitor was quantified by immunological assays (ELISA) in protoplasts and vacuoles isolated from upper unwounded leaves of 5- to 6-week old potato plants that had been wounded on their lower leaves 48 hours earlier to induce the accumulation of the carboxypeptidase inhibitor. The regulation of the synthesis and compartmentation of the inhibitor is similar to that of wound-induced serine proteinase Inhibitors I and II in potato and tomato leaves and appears to be part of an induced defense response against attacking pests.     

Tobacco proteinase inhibitor I genes are locally,but not systemically induced by stress

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.     

Fast upward propagation of the wound signal that systemically elevates phosphatidic acid

Phosphatidic acid (PA) increases in response to wounding at the neighboring unwounded leaf as well as at the wounded leaf of many plants (Lee et al., 1997). This indicates that a signal propagates from the wounded leaf to its neighboring leaves. In this paper, we report the speed and direction of propagation for a systemic wound signal that elevates PA. When a leaf of a soybean (Glycine max) seedling at the 2-leaf-stage was wounded, the PA level of the neighboring leaf did not change within the first min, but did increase significantly in 2 min, returning to the control level after 15 min. This implies that the systemic wound signal was generated at least within 2 min of wounding, and was propagated at a speed of at least 10–16 mm/min. When we wounded individual leaves of soybean and tobacco (Nicotiana tabaccum) seedlings that had 3 or 4 leaves, PA levels were elevated only in the younger leaves located above the wounded leaf, but not in the older, lower leaves. Thus, the PA-elevating wound signal preferentially moves upward in these plants.     

Long distance run in the wound response--jasmonic acid is pulling ahead

When plants are wounded, a long-distance signal is produced that activates the wound response in unwounded leaves. Recent work by Gregg Howe's group suggests that jasmonic acid is the long-distance wound signal. Previously, many data indicated that the signaling peptide systemin might function as the systemic wound signal. According to the new data, systemin is crucial for the production of the systemic wound signal in the wounded leaf, but might not travel long distances.     

Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression

The proteinase inhibitor II (pin2) gene family exhibits two different modes of expression. It is, on the one hand, constitutively expressed in flowers of potato and tomato plants. and in potato tubers. On the other hand, its expression is induced in the plant foliage by mechanical wounding. To define cis-regulatory elements involved in pin2 promoter activity, deletion analysis of a potato pin2 promoter has been performed in stably and transiently transformed potato and tobacco plants. Two different elements, a quantitative enhancer and a regulatory element, are required for promoter activity. While functional promoter elements required for pin2 activity in tubers and wounded leaves could not be separated, its expression in flowers is mediated by different cis-acting sequences. Induction of pin2 expression in leaves by treatment with the plant growth regulators abscisic acid and jasmonic acid, and the general metabolite sucrose, depends on the presence of the regulatory element involved in expression in tubers and wounded leaves. Thus, pin2 expression in tubers and wounded leaves apparently results from the action of similar hormonal signals on closely linked promoter elements, while a different signal pathway leads to its constitutive expression in flowers.     

A Sulfhydryl Reagent Modulates Systemic Signaling for Wound-Induced and Systemin-Induced Proteinase Inhibitor Synthesis

The sulfhydryl group reagent p-chloromecuribenzene sulfonic acid (PCMBS), an established inhibitor of active apoplastic phloem loading of sucrose in several plant species, is shown to be a powerful inhibitor of wound-induced and systemin-induced activation of proteinase inhibitor synthesis and accumulation in leaves of tomato plants (Lycopersicon esculentum cv Castlemart). PCMBS, supplied to young tomato plants through their cut stems, blocks accumulation of proteinase inhibitors in leaves in response to wounding. The application of systemin directly to fresh wounds enhances systemic accumulation of proteinase inhibitors to levels higher than wounding alone. Placed on fresh wounds, PCMBS severely inhibits systemic induction of proteinase inhibitors, in both the presence and absence of exogenous systemin. PCMBS inhibition can be reversed by cysteine, dithiothreitol, and glutathione. Radiolabeled systemin placed on fresh wounds is readily transported from the wounded leaves to upper leaves. However, in the presence of PCMBS, radiolabeled systemin is not transported away from wound sites. Induction of proteinase inhibitor I synthesis by oligouronides (degree of polymerization [almost equal to] 20), linolenic acid, or methyl jasmonate was not inhibited by PCMBS. The cumulative data support a possible role for sulfhydryl groups in mediating the translocation of systemin from wound sites to distal receptor sites in tomato plants and further support a role for systemin as a systemic wound signal.     

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.     

Imaging photosynthesis in wounded leaves of Arabidopsis thaliana

Chlorophyll fluorescence imaging provides a non-invasive and non-destructive means with which to measure photosynthesis. This technique has been used, in combination with 14CO2 feeding, to study the spatial and temporal changes in source-sink relationships which occur in mechanically wounded leaves of Arabidopsis thaliana. Twenty-four hours after wounding, cells proximal to the wound margin showed a rapid induction of PhiII upon illumination (a measure of the efficiency of photosystem II photochemistry) whilst cells more distal to the wound margin exhibited a much slower induction of PhiII and a large, transient increase in NPQ (a measure of the rate constant for non-photochemical energy dissipation within the light-harvesting antenna). These results are indicative of an increase in sink strength in the vicinity of the wound and this was confirmed by the retention of 14C photosynthate in this region. It has been hypothesized that wound-induced cell wall (apoplastic) invertase (cwINV) activity plays a central role in generating localized increases in sink strength in stressed plant tissue and that hexose sugars generated by the sucrolytic activity of cwINV may act as a signal regulating gene expression. Enzyme activity measurements, quantitative RT-PCR, and T-DNA insertional mutagenesis have been used to determine that expression of AtcwINV1 is responsible for all induced cwINV activity in mechanically wounded leaves. Whilst inactivation of this gene abolished wound-induced cwINV activity, it did not affect localized alterations in source-sink relationships of wounded leaves or wound-regulated gene expression. The signals that may regulate source-sink relationships and signalling in wounded leaves are discussed.     

Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris

The effect of regurgitant from Leptinotarsa decemlineata Say larvae on wound-induced responses was studied using two plant species, Solanum tuberosum L. and Phaseolus vulgaris L. Wounding of one leaf of intact S. tuberosum plants differentially affected ethylene production and activities of peroxidase and polyphenol oxidase. Only polyphenol oxidase activity was stimulated by wounding in both wounded and systemic leaves. Peroxidase activity was not affected by wounding. Wounding caused only a transient increase of ethylene production from wounded leaves. The application of regurgitant to wound surfaces stimulated ethylene production as well as activities of peroxidase and polyphenol oxidase in both wounded and systemic leaves. Wounding significantly enhanced ethylene production and polyphenol oxidase activity in wounded and systemic leaves of P. vulgaris . The application of regurgitant caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants. Several substances were tested for their role as possible endogenous signals in P. vulgaris . Hydrogen peroxide and methyl jasmonate appeared as potential local and systemic signals of ethylene formation in wounded bean plants. Local ethylene production in leaf discs was differentially affected by the regurgitant application in potato versus bean plants. While all tested concentrations of regurgitant caused stimulation of ethylene formation from potato leaf discs, ethylene production was completely inhibited by increasing concentrations of the regurgitant in bean leaf discs. Our data present evidence that ethylene may play an important role in the interaction between plants and herbivores at the level of recognition of a particular herbivore leading to specific induction of signalling cascades.     

Systemic signaling in tomato plants for defense against herbivores. Isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene

An 18-amino acid peptide in tomato leaves called systemin is a primary signal released at wound sites in response to herbivory that systemically signals the activation of defense genes throughout the plants. We report here the isolation of three hydroxyproline-rich glycopeptides from tomato leaves, of 20, 18, and 15 amino acids in length, that signal the activation of defense genes, similar to the activity of the systemin peptide. The three new peptides cause an alkalinization of suspension-cultured cells and induce the synthesis of defensive proteinase inhibitor proteins when supplied at fmol levels to young tomato plants through their cut stems. This suggests that they are part of the wound signaling of tomato plants that activates defense against herbivores and pathogens. Isolation of cDNAs coding for the tomato peptides revealed that they are all derived from the same pre-proprotein precursor that is systemically wound-inducible. The peptides are considered members of the functionally characterized systemin family of defense signals from plants that are synthesized both in wounded leaves and in distal, unwounded leaves in response to herbivory or other mechanical wounding. The precursor deduced from the cDNA exhibits a leader sequence, indicating that it is synthesized through the secretory pathway, where it is hydroxylated and glycosylated. The amino acid sequence of the precursor exhibited weak identity to the precursor of two hydroxyproline-rich defense signals recently found in tobacco, suggesting that the two pre-protein precursors have evolved from a common ancestral protein. The identification of hydroxyproline-rich glycoprotein systemins in tomato indicates that the initiation of wound signaling is more complex than previously thought and appears to involve multiple peptide signals.     

Cross-talk between wound signalling pathways determines local versus systemic gene expression in Arabidopsis thaliana

Plants react to mechanical damage by activating a set of genes, the products of which are thought to serve defensive functions. In solanaceous plants, cell wall-derived oligosaccharides and the plant hormones jasmonic acid and ethylene participate in the signalling network for wound-induced expression of proteinase inhibitors and other defence-related genes, both in the locally damaged and in the systemic non-damaged leaves. Here we show that in Arabidopsis thaliana, these signalling components interact in novel ways to activate distinct responses. In damaged tissues, oligosaccharides induce the expression of a specific set of wound-responsive genes while repressing jasmonic acid-responsive genes that are activated in the systemic tissues. The oligosaccharide-mediated repression of the jasmonic acid-dependent signalling pathway is exerted through the production and perception of ethylene in the locally damaged tissue. This cross-talk between separate wound signalling pathways thus allows the set up of different responses in the damaged and the systemic tissues of plants reacting to injury.     

Phosphatidic acid activates a wound-activated MAPK in Glycine max

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound-activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n-butanol, an inhibitor of phospholipase D, the wound-induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension-cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA-induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound-induced MAPK signaling in plants.     

Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ-specific signal transduction pathways

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound-inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root-specific pin2 homologue accumulated in the JA-treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA-induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA-induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA-mediated gene induction has enabled identification of a JA-independent pathway for ABA induction of pin2 and the other wound-inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound-inducible genes. Our data suggest the presence of a complex, organ-specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.     

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.     

Both wound-inducible and tuber-specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family

A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.