Wound-responsive Genes Research Articles

CDNA microarray analysis of fusicoccin-induced changes in gene expression in tomato plants.

The effects of the fungal toxin fusicoccin (FC) on the tomato (Lycopersicon esculentum Mill.) transcriptome were analyzed in the context of defense-related genes using a spotted microarray of 235 cDNAs. Pronounced changes in transcript abundance were observed for 64 (27%) of the represented genes. FC appears to have an antagonistic effect on wound and pathogen defense responses, in that it causes the induction of pathogenesis-related and the down-regulation of wound response genes. The transcripts for many proteins involved in photosynthesis and carbohydrate metabolism were strongly repressed. Genes related to the biosynthesis of jasmonic acid and aromatic amino acids, on the other hand, were found to be up-regulated. In addition to these expression changes, which occurred rather late after FC treatment, rapid and transient induction kinetics were observed for a small group of genes encoding a calcium-dependent protein kinase, two mitogen-activated protein kinases, a matrix metalloproteinase and a homologue of the respiratory burst oxidase. These genes have not been described previously in tomato, nor has their regulation by FC been reported. Salicylic acid was shown not to be required for the induction of these transcripts and a function for the respective proteins in the FC-induced, salicylic acid-independent activation of pathogenesis-related genes is discussed.

Transcriptional Profiling Reveals Novel Interactions between Wounding, Pathogen, Abiotic Stress, and Hormonal Responses in Arabidopsis,

Mechanical wounding not only damages plant tissues, but also provides pathways for pathogen invasion. To understand plant responses to wounding at a genomic level, we have surveyed the transcriptional response of 8,200 genes in Arabidopsis plants. Approximately 8% of these genes were altered by wounding at steady-state mRNA levels. Studies of expression patterns of these genes provide new information on the interactions between wounding and other signals, including pathogen attack, abiotic stress factors, and plant hormones. For example, a number of wound-responsive genes encode proteins involved in pathogen response. These include signaling molecules for the pathogen resistance pathway and enzymes required for cell wall modification and secondary metabolism. Many osmotic stress- and heat shock-regulated genes were highly responsive to wounding. Although a number of genes involved in ethylene, jasmonic acid, and abscisic acid pathways were activated, many in auxin responses were suppressed by wounding. These results further dissected the nature of mechanical wounding as a stress signal and identified new genes that may play a role in wounding and other signal transduction pathways.

Isolation of wound inducible genes from Castanea sativa stems and expression analysis in the bark tissue

In vitro shoot cultures of chestnut ( Castanea sativa Mill.) were used to identify wound-responsive genes. cDNA fragments of genes induced 3 and 24 h after wounding were isolated from stem tissue by the differential mRNA display method. Corresponding partial and full-length clones were isolated from a cDNA library of wounded stems. Putative wound-responsive signalling genes (serine–threonine protein kinase, two calmodulin genes), a novel wound-responsive putative chaperon gene (Csp13.9), and a new family of proline-rich proteins were identified. Northern analysis of bark tissue from 14-month-old seedlings revealed strong induction of these genes upon wounding in a temporal manner. Therefore we conclude that these early wound-inducible genes are involved in the wound response of bark tissue.

A wound-inducible tobacco peroxidase gene expresses preferentially in the vascular system.

A tobacco peroxidase gene tpoxN1 was reported to be expressed within 1 h after wounding in leaves [Hiraga et al. (2000a) Plant Cell Physiol. 41: 165]. We describe here further results on the wound-induced tpoxN1 expression. The quick tpoxN1 induction occurred preferentially in stems and petioles, but was negligible in leaf blades even 8 h after wounding. Induced GUS activity was also detected rapidly after wounding in the stem of transgenic tobacco plants carrying the tpoxN1 promoter::GUS fusion gene, localized mainly in the vascular systems where it was maintained this level for 14 d or more. Strong GUS activity was also found in the petiole and veinlet as well as the epidermal tissue in the stem. Treatment of known inducers for wound-responsive genes such as jasmonate, 1-aminocyclopropane-1-carboxylate, spermine, phytohormones and other stress treatments did not enhance wound-induced tpoxN1 gene expression in stems at all, but rather repressed it in some cases. Studies using metabolic inhibitors suggested that phosphorylation and dephosphorylation of proteins together with de novo protein synthesis are likely to be involved in the wound-induced tpoxN1 expression as well as some other wound-responsive genes. Thus, tpoxN1 is a unique wound-inducible and possible wound-healing gene which is rapidly expressed being maintained for a long time in veins via an unknown wound-signaling pathway(s).

Mapping wound-response genes involved in post-harvest physiological deterioration (PPD) of cassava (Manihot esculenta Crantz)

The genome locations of the wound-response genes that were expressedduring the post-harvest physiological deterioration (PPD) of cassava, suchas phenylalanine ammonia lyase, β-1.3 glucanase, hydroxyprolinerich glycoprotein, catalase, 1-aminocyclopropane 1-carboxylate, cysteineprotease inhibitor, aspartic protease, a partial cDNA for serine/threonineprotein kinase and peroxidase, have been identified on the frameworkmolecular genetic map of cassava. Also, molecular markers linked toputative quantitative trait loci (QTLs) influencing PPD of cassava weremapped using an F1mapping population derived from elite parentallines (TMS 30572 × cm 2177-2). A molecular linkage map previouslyconstructed based on the segregation of 240 RFLP, 100 RAPD, 85microsatellite and five isoenzyme markers on 144 F1 individuals wasused for the QTL mapping.A set of 10 molecular markers with a significant association with putativeQTLs for PPD were identified based on probability values < 0.005in order to minimize the detection of false positives. Based on single-markerregression, eight putative QTLs located on the linkage groups G, P, L, U,and X of the female-derived framework map were found to explain between 5–12% of the phenotypic variance of the PPD. In the male-derived frameworkmap, two putative QTLs on linkage groups C and L explained 13% and11% of this variance, respectively. This study thus identified the majorgenome regions of cassava related to physiological post-harvestdeterioration, thereby providing tools for the identification of gene(s)controlling this trait.

The induction of tomato leucine aminopeptidase genes (LapA) after Pseudomonas syringae pv. tomato infection is primarily a wound response triggered by coronatine.

Tomato plants constitutively express a neutral leucine aminopeptidase (LAP-N) and an acidic LAP (LAP-A) during floral development and in leaves in response to insect infestation, wounding, and Pseudomonas syringae pv. tomato infection. To assess the physiological roles of LAP-A, a LapA-antisense construct (35S:asLapA1) was introduced into tomato. The 35S:asLapA1 plants had greatly reduced or showed undetectable levels of LAP-A and LAP-N proteins in healthy and wounded leaves and during floral development. Despite the loss of these aminopeptidases, no global changes in protein profiles were noted. The 35S:asLapA1 plants also exhibited no significant alteration in floral development and did not impact the growth and development of Manduca sexta and P. syringae pv. tomato growth rates during compatible or incompatible infections. To investigate the mechanism underlying the strong induction of LapA upon P. syringae pv. tomato infection, LapA expression was monitored after infection with coronatine-producing and -deficient P. syringae pv. tomato strains. LapA RNA and activity were detected only with the coronatine-producing P. syringae pv. tomato strain. Coronatine treatment of excised shoots caused increases in RNAs for jasmonic acid (JA)-regulated wound-response genes (LapA and pin2) but did not influence expression of a JA-regulated pathogenesis-related protein gene (PR-1). These results indicated that coronatine mimicked the wound response but was insufficient to activate JA-regulated PR genes.

Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor.

Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, prosystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively. Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity.

Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase.

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C(18) fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.

Screening of Wound-Responsive Genes Identifies an Immediate-Early Expressed Gene Encoding a Highly Charged Protein in Mechanically Wounded Tobacco Plants

In order to identify genes that are temporally and spatially regulated during wound response, a cDNA population in mechanically wounded tobacco leaves was screened by the fluorescence differential display method. Of 28 clones initially identified to have altered levels of transcripts within 3 h of wounding, eight were characterized. Although each clone showed a unique pattern of transcript accumulation, one distinct clone was further characterized because of its immediate-early response. Its transcripts began to accumulate 10 min after wounding, reached a maximum level within 1 h and disappeared after 2 h. The response, which occurred repeatably and systemically, was observed by the treatment with propionic acid or erythrosin B, indicating that cytosolic acidification could be one of the signals for immediate-early response of this gene. The cDNA encodes a polypeptide of 513 amino acids with a relative molecular mass of 60,952. The putative polypeptide is rich in lysine (K), glutamic acid (E) and aspartic acid (D), which constitute up to 70% of total amino acids, and was therefore designated as KED. The KED polypeptide is composed of a highly hydrophilic N-terminal region and a relatively hydrophobic C-terminal region, suggesting that KED may function through electrostatic interactions with cellular components.

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.

Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants.

Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells. A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity. Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L. peruvianum cell cultures and to induce wound response genes in whole tomato plants. Conversely, an activator of the H+-ATPase (fusicoccin) acidified the growth medium of L. peruvianum cell cultures and suppressed systemin-induced medium alkalinization. Likewise, in fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed. However, fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes. Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane. In addition, alkalinization of the L. peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase. Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes. The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed.

Jasmonic acid-dependent and -independent wound signal transduction pathways are differentially regulated by Ca2+/calmodulin in Arabidopsis thaliana.

We have used wound- and jasmonic acid (JA)-responsive genes as molecular markers to elucidate the pathway(s) of wound signal transduction in Arabidopsis thaliana. The JA-responsive (JR) genes JR1, JR2, and JR3 are strongly induced by wounding and by JA, while the wound-responsive (WR) genes WR3 and acyl CoA oxidase (ACO) are induced by wounding only. Accumulation of JR transcripts upon wounding was blocked by indomethacin. However, indomethacin did not affect either induction of these genes by JA or wound-induced expression of WR genes, suggesting that JA synthesis is only needed for wound-dependent induction of JR genes, and also that separate JA-dependent and -independent wound signal transduction pathways exist in Arabidopsis. The two pathways are differentially regulated by Ca2+ and calmodulin. Mobilization of intracellular Ca2+ pools blocked induction of JR genes by both wounding and JA, but not the induction of WR genes by wounding, but this effect could not be reproduced by increasing intracellular Ca2+ levels using ionophores. In addition, calmodulin antagonists blocked the expression of JR genes and up-regulated WR gene expression. Ca2+ and calmodulin seem to act downstream of both JA and the COI1 gene in the JA-dependent pathway, and downstream of reversible phosphorylation events that differentially regulate JA-dependent and JA-independent wound signal transduction pathways.

Transcriptional activation of the tobacco retrotransposon Tto1 by wounding and methyl jasmonate.

The tobacco retrotransposon Tto1, one of a few active retrotransposons of plants, has been shown to be activated by tissue culture. Its transposition is regulated mainly at the transcriptional level. It is shown here that expression of Tto1 can be induced in leaves of tobacco by wounding stress. Exogenous supply of methyl jasmonate, which is known to be a potent inducer of certain wound-responsive genes in plants, also induces Tto1 RNA expression. Tto1 RNA was detected within 2 to 4 h after wounding/cutting treatment, and increased levels of Tto1 RNA were observed during subsequent incubation periods for 48 h. Expression of Tto1 RNA after cutting treatment was induced more significantly in young expanding leaves rather than in older mature leaves, suggesting that developmental or physiological factors may be required for the strong response to Tto1 transcription to wounding stimuli. Experiments with transgenic tobacco plants carrying the Tto1-LTR: beta-glucuronidase fusion gene (LTR:GUS) revealed that Tto1 actually contains cis-regulatory regions in response to wounding and methyl jasmonate. These findings are discussed in relation to the mechanism of transcriptional activation and the evolutionary role played by retrotransposons.

Jasmonate is essential for insect defense in Arabidopsis.

The signaling pathways that allow plants to mount defenses against chewing insects are known to be complex. To investigate the role of jasmonate in wound signaling in Arabidopsis and to test whether parallel or redundant pathways exist for insect defense, we have studied a mutant (fad3-2 fad7-2 fad8) that is deficient in the jasmonate precursor linolenic acid. Mutant plants contained negligible levels of jasmonate and showed extremely high mortality ( approximately 80%) from attack by larvae of a common saprophagous fungal gnat, Bradysia impatiens (Diptera: Sciaridae), even though neighboring wild-type plants were largely unaffected. Application of exogenous methyl jasmonate substantially protected the mutant plants and reduced mortality to approximately 12%. These experiments precisely define the role of jasmonate as being essential for the induction of biologically effective defense in this plant-insect interaction. The transcripts of three wound-responsive genes were shown not to be induced by wounding of mutant plants but the same transcripts could be induced by application of methyl jasmonate. By contrast, measurements of transcript levels for a gene encoding glutathione S-transferase demonstrated that wound induction of this gene is independent of jasmonate synthesis. These results indicate that the mutant will be a good genetic model for testing the practical effectiveness of candidate defense genes.

Ethylene as a Signal Mediating the Wound Response of Tomato Plants

Plants respond to physical injury, such as that caused by foraging insects, by synthesizing proteins that function in general defense and tissue repair. In tomato plants, one class of wound-responsive genes encodes proteinase inhibitor (pin) proteins shown to block insect feeding. Application of many different factors will induce or inhibit pin gene expression. Ethylene is required in the transduction pathway leading from injury, and ethylene and jasmonates act together to regulate pin gene expression during the wound response.

Induction of Wound Response Genes in Tomato Leaves by Bestatin, an Inhibitor of Aminopeptidases

Induction of Wound Response Genes in Tomato Leaves by Bestatin, an Inhibitor of Aminopeptidases

Coordinated activation of corneal wound response genes in vivo as observed by in situ hybridization

We used subtractive screening of a cDNA library prepared from corneoscleral rims after cauterizing rat corneas. We identified 76 clones whose corresponding mRNA increased during the wound healing process in an in vivo model of injury which damages the corneal epithelium, stroma, and endothelium. Of these clones, 31 sequences encode known proteins. Another 45 clones are novel sequences based on comparison with the GenBank/EMBL databases. Changes in the level of expression of the novel genes, and a selected number of the known genes, were examined by in situ hybridization 22 and 72 hr after corneal injury. The majority produced a ‘wound pattern’ of expression such that the mRNAs were highly induced in all cell types adjacent to the wound site at 22 hr post injury. This signal decreased in intensity with distance from the wound site. In a subset of corneoscleral rims examined by in situ hybridization, the mRNAs for these genes were also highly induced in the limbal epithelium, where the progenitor corneal epithelial stem cells reside. By 72 hr, when acute tissue damage had been repaired, the induced mRNA was only faintly present in the thickened epithelium. Our results provide a useful framework for further studies defining the pathophysiological roles of the known and novel proteins encoded by the isolated cDNA clones.

Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines.

Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.

Wound-responsive gene expression in poplars

Plants have evolved elaborate biochemical defense mechanisms to protect themselves from pathogen invasion and insect herbivory. Often these biochemicals are synthesized only when the plant is under attack, and may accumulate systemically (throughout the plant) in response to a local injury. Some of the defensive molecules are low molecular weight phenolic or terpenoid compounds; others are proteins harmful to the invading pathogen or grazing herbivore. The de novo synthesis of defensive proteins is often preceded by a coordinate increase in transcription of the genes that code for these proteins. Hybrid poplars ( Populus trichocarpa T. and G. × P. deltoides Marsh) respond to mechanical wounding (simulating pathogen invasion or insect chewing) by systemically accumulating specific transcripts in the unwounded upper leaves of trees whose lower leaves have been damaged. Some of the induced poplar leaf mRNAs code for chitinases, enzymes which may play a role in degrading the cell walls of pathogenic fungi and bacteria. Other wound-regulated transcripts encode proteins with amino acid sequence similarity to a family of trypsin inhibitors. Proteinase inhibitors protect plants by disrupting the digestive process in the gut of a grazing insect. By understanding the response of trees to wounding at the molecular level, it may be possible to improve disease and insect resistance by augmenting the tree's natural defenses through selective breeding and genetic engineering. Trees that possess increased levels of chitinases or proteinase inhibitors may be superior in detering pests. Genetic variability in the levels of these defensive proteins could be used to design breeding strategies for enhanced pest resistance. Additionally, pest control genes from other sources, such as the insecticidal toxin genes from Bacillus thuringiensis, could be introduced into poplar trees by genetic engineering. Transcription of the bacterial toxin genes could be regulated in an appropriately wound-inducible manner by fusing the bacterial genes to transcriptional promoters from wound-responsive poplar genes.