Microarray analysis of tomato's early and late wound response reveals new regulatory targets for Leucine aminopeptidase A

Abstract

Wounding due to mechanical injury or insect feeding causes a wide array of damage to plant cells including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum) is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late) and spatial (locally and systemically) responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI) and wild-type (WT) leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2) and two dehydrin (TAS14 and Dhn3) genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A's role in modulation of wound responses extends beyond the well described late-wound gene core.

Figure 1. Gene expression patterns in WT plants at 1 and 8 hr after wounding.

Genes that were differentially regulated in wounded (Local, Lo) and apical, non-wounded (Systemic, Sys) WT leaves at 1 and 8 hr after wounding were identified by analysis of the potato 10-K cDNA arrays (Materials and Methods). Differentially expressed genes (DEGs) were defined as those with p <0.05, |FC| ≥ 0.8.

Figure 2. Changes in phenylpropanoid synthesis and catabolism gene RNAs after wounding.

Many genes involved in phenylpropanoid metabolism were wound-regulated DEGs. For each biochemical step the number of genes regulated out of total number of clones representing those genes on the TIGR 10-K (version 3) potato cDNA microarray is indicated as a single or cluster of blocks. The colored blocks represent DEGs regulated at 1 hr (grey) and 8 hr (black) or both (checkered). Up-regulated DEGs are indicated by solid arrows and down-regulated DEGs are represented by dotted arrows. For some biochemical steps, enzymes were not represented on the array; no boxes appear at these steps. For complete pathway see Plant Metabolic Network (PMN; pmn.plantcyc.org). PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; C4L, 4-coumarate-CoA ligase; CCR, cinnamoyl-CoA reductase; CHI, chalcone isomerase; LDOX, Leucoanthocyanidin dioxygenase; CHS, Chalcone synthase; MAT, Malonyltransferase; ASA, Anthranilate synthase alpha; SK, Shikimate kinase; CS, Chorismate synthase; GltS, Glutamate synthase; THT, Tyramine N-hydroxycinnamoyl transferase; C3H, P-coumaroyl shikimate 3'-hydroxylase; CMT, Caffeoyl-CoA O-methyltransferase.

Figure 3. Changes in polyamine, SAM, and ethylene synthesis and catabolism gene RNAs after wounding.

Many genes involved in polyamine, SAM, and ethylene metabolism were wound-induced DEGs in tomato. DEG expression patterns are represented as described in Figure 2. SAM, S-adenosylmethionine synthetase; SAMDC, S-adenosylmethionine decarboxylase; SPDS, Spermidine synthase; ADC, Arginine decarboxylase; AIH, Agmatine iminohydrolase; ODC, Ornithine decarboxylase; NAD, N-acetylornithine deacetylase; GABA-T, GABA Transaminase.

Figure 4. Relative RNA fold change in WT vs.

LapA-SI lines 1 and 8h after wounding. Correlation of relative RNA fold change (FC) between WT and LapA-SI lines after wounding. RNAs accumulated to similar levels in WT and LapA-SI leaves are indicated by closed diamonds. The genes differentially regulated between in LapA-SI and WT leaves are indicated by open circles (genotype DEGS (gDEGs); p<0.05, |FC| ≥0.8]). LapA RNAs are indicated by open triangles.

Figure 5. Genes differentially regulated in LapA-SI before wounding (putative 0-hr gDEGs).

Genes with RNAs at similar levels in LapA-SI and WT leaves before wounding (0 hr) are indicated with closed diamonds (log₂ fold change (FC) <1.5). Putative 0-hr gDEGs are indicated with open circles (p<0.05, |FC| ≥0.8). LapA RNAs are indicated by open triangles.

Figure 6. Quantitative RT-PCR analysis of selected mRNAs in leaves after wounding.

Relative expression of LEA (TAS14, Dhn3, Dhn2, ER5) and PR (PR-1a2, PR-1c) transcripts were determined 0, 1, 8, and 24 hr after wounding in WT (white), LapA-SI (grey) and LapA-OX (black) leaves (n=3). Significant differences between transcript accumulation was determined [ANOVA, Tukey post-hoc test (p<0.05)].