Signal transduction in the wound response of tomato plants.

The wound response of tomato plants has been extensively studied, and provides a useful model to understand signal transduction events leading from injury to marker gene expression. The principal markers that have been used in these studies are genes encoding proteinase inhibitor (pin) proteins. Activation of pin genes occurs in the wounded leaf and in distant unwounded leaves of the plant. This paper reviews current understanding of signalling pathways in the wounded leaf, and in the systemically responding unwounded leaves. First, the nature of known elicitors and their potential roles in planta are discussed, in particular, oligogalacturonides, jasmonates and the peptide signal, systemin. Inhibitors of wound-induced proteinase inhibitor (pin) expression are also reviewed, with particular reference to phenolics, sulphydryl reagents and fusicoccin. In each section, results obtained from the bioassay are considered within the wider context of data from mutants and from transgenic plants with altered levels of putative signalling components. Following this introduction, current models for pin gene regulation are described and discussed, together with a summary for the involvement of phosphorylation-dephosphorylation in wound signalling. Finally, a new model for wound-induced pin gene expression is presented, arising from recent data from the author''s laboratory.     

Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis

Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDalpha, PLDbeta, and PLDgamma differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDalpha, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDalpha attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDalpha-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid (JA) in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase (LOX)-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves.LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid (NDGA), an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13(S)-H(P)ODE) was confirmed to be the main product of LOX throughout the experimental time. In addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from Its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides)

In order to study condensed tannin synthesis and its induction by herbivory, a dihydroflavonol reductase (DFR) cDNA was isolated from trembling aspen (Populus tremuloides). Bacterial overexpression demonstrated that this cDNA encodes a functional DFR enzyme, and Southern analysis revealed that DFR likely is a single-copy gene in the aspen genome. Aspen plants that were mechanically wounded showed a dramatic increase in DFR expression after 24 h in both wounded leaves and unwounded leaves on wounded trees. Feeding by forest tent caterpillar (Malacosoma disstria) and satin moth (Leucoma salicis) larvae, and treatment with methyl jasmonate, all strongly induced DFR expression. DFR enzyme activity was also induced in wounded aspen leaves, and phytochemical assays revealed that condensed tannin concentrations significantly increased in wounded and systemic leaves. The expression of other genes involved in the phenylpropanoid pathway were also induced by wounding. Our findings suggest that the induction of condensed tannins, compounds known to be important for defense against herbivores, is mediated by increased expression of DFR and other phenylpropanoid genes.     

In vivo substrates and the contribution of the common phospholipase D, PLDalpha, to wound-induced metabolism of lipids in Arabidopsis

The common plant phospholipase D (PLD), PLDalpha, has been proposed to be involved in wound-induced production of jasmonic acid. To better understand the role(s) of PLDalpha in the wound response, detailed lipid analysis was carried out to determine the in vivo substrates and the contribution of PLDalpha in wound-induced lipid metabolism in Arabidopsis thaliana. Mechanical wounding of Arabidopsis leaves resulted in significantly less hydrolysis of phosphatidylcholine (PC) in PLDalpha-deficient than in wild-type plants. Hydrolysis of phosphatidylethanolamine, phosphatidylglycerol (PG), and phosphatidylinositol within 30 min of wounding was not significantly different in PLDalpha-deficient and wild-type leaves. Phosphatidic acid (PA) levels increased rapidly in wild-type and, to a lesser extent, in PLDalpha-deficient plants. The acyl composition of the PA generated by wounding suggests that the major in vivo substrate of PLD in wild-type leaves was PC, and that PG hydrolysis accounted for 10-15% of the wound-induced PA in wild-type leaves. Comparison of the acyl compositions of the wound-induced PA of wild-type and PLDalpha-deficient leaves indicated that PLDalpha hydrolyzed PG more readily than other PLD isoforms did. Wounding produced substantial increases in free linoleic and linolenic acids in wild-type plants, whereas PLDalpha-deficient plants showed only a slight increase in linoleic acid and no significant increase in linolenic acid. These results demonstrate that PLDalpha and at least one other PLD isoform, as well as other hydrolytic enzymes, are active in mechanically wounded Arabidopsis leaves, and PLDalpha is involved in wound-induced metabolism of polyunsaturated fatty acids.     

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.     

In vivo substrates and the contribution of the common phospholipase D,PLDα, to wound-induced metabolism of lipids in Arabidopsis

The common plant phospholipase D (PLD), PLDα, has been proposed to be involved in wound-induced production of jasmonic acid. To better understand the role(s) of PLDα in the wound response, detailed lipid analysis was carried out to determine the in vivo substrates and the contribution of PLDα in wound-induced lipid metabolism in Arabidopsis thaliana. Mechanical wounding of Arabidopsis leaves resulted in significantly less hydrolysis of phosphatidylcholine (PC) in PLDα-deficient than in wild-type plants. Hydrolysis of phosphatidylethanolamine, phosphatidylglycerol (PG), and phosphatidylinositol within 30 min of wounding was not significantly different in PLDα-deficient and wild-type leaves. Phosphatidic acid (PA) levels increased rapidly in wild-type and, to a lesser extent, in PLDα-deficient plants. The acyl composition of the PA generated by wounding suggests that the major in vivo substrate of PLD in wild-type leaves was PC, and that PG hydrolysis accounted for 10–15% of the wound-induced PA in wild-type leaves. Comparison of the acyl compositions of the wound-induced PA of wild-type and PLDα-deficient leaves indicated that PLDα hydrolyzed PG more readily than other PLD isoforms did. Wounding produced substantial increases in free linoleic and linolenic acids in wild-type plants, whereas PLDα-deficient plants showed only a slight increase in linoleic acid and no significant increase in linolenic acid. These results demonstrate that PLDα and at least one other PLD isoform, as well as other hydrolytic enzymes, are active in mechanically wounded Arabidopsis leaves, and PLDα is involved in wound-induced metabolism of polyunsaturated fatty acids.     

Induction of ASCORBATE PEROXIDASE 2 expression in wounded Arabidopsis leaves does not involve known wound-signalling pathways but is associated with changes in photosynthesis

ASCORBATE PEROXIDASE 2 (APX2) encodes a key enzyme of the antioxidant network. In excess light-stressed Arabidopsis leaves, photosynthetic electron transport (PET), hydrogen peroxide (H(2)O(2)) and abscisic acid (ABA) regulate APX2 expression. Wounded leaves showed low induction of APX2 expression, and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid (JA), chitosan and ABA were not involved in the wound-induced expression of APX2, but were shown to require PET and were preceded by a depressed rate of CO(2) fixation. This led to an accumulation of H(2)O(2) in veinal tissue. Diphenyl iodonium (DPI), which has been shown previously to be a potent inhibitor of H(2)O(2) accumulation in the veins of wounded leaves, prevented induction of APX2 expression probably by inhibition of PET. Thus, the weak induction of APX2 expression in wounded leaves may require H(2)O(2) and PET only. As in other environmental stresses, wounding of leaves resulted in decreased photosynthesis leading to increased reactive oxygen species (ROS) production. This may signal the induction of many 'wound-responsive' genes not regulated by JA-dependent or other known JA-independent pathways.     

Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding

A new metabolite profiling approach combined with an ultrarapid sample preparation procedure was used to study the temporal and spatial dynamics of the wound-induced accumulation of jasmonic acid (JA) and its oxygenated derivatives in Arabidopsis thaliana. In addition to well known jasmonates, including hydroxyjasmonates (HOJAs), jasmonoyl-isoleucine (JA-Ile), and its 12-hydroxy derivative (12-HOJA-Ile), a new wound-induced dicarboxyjasmonate, 12-carboxyjasmonoyl-l-isoleucine (12-HOOCJA-Ile) was discovered. HOJAs and 12-HOOCJA-Ile were enriched in the midveins of wounded leaves, strongly differentiating them from the other jasmonate metabolites studied. The polarity of these oxylipins at physiological pH correlated with their appearance in midveins. When the time points of accumulation of different jasmonates were determined, JA levels were found to increase within 2-5 min of wounding. Remarkably, these changes occurred throughout the plant and were not restricted to wounded leaves. The speed of the stimulus leading to JA accumulation in leaves distal to a wound is at least 3 cm/min. The data give new insights into the spatial and temporal accumulation of jasmonates and have implications in the understanding of long-distance wound signaling in plants.