Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis

Abstract

Wounding in multicellular eukaryotes results in marked changes in gene expression that contribute to tissue defense and repair. Using a cDNA microarray technique, we analyzed the timing, dynamics, and regulation of the expression of 150 genes in mechanically wounded leaves of Arabidopsis. Temporal accumulation of a group of transcripts was correlated with the appearance of oxylipin signals of the jasmonate family. Analysis of the coronatine-insensitive coi1-1 Arabidopsis mutant that is also insensitive to jasmonate allowed us to identify a large number of COI1-dependent and COI1-independent wound-inducible genes. Water stress was found to contribute to the regulation of an unexpectedly large fraction of these genes. Comparing the results of mechanical wounding with damage by feeding larvae of the cabbage butterfly (Pieris rapae) resulted in very different transcript profiles. One gene was specifically induced by insect feeding but not by wounding; moreover, there was a relative lack of water stress-induced gene expression during insect feeding. These results help reveal a feeding strategy of P. rapae that may minimize the activation of a subset of water stress-inducible, defense-related genes.

Figure 1.

cDNA Microarray Analysis of Gene Expression after Mechanical Wounding. A fluorescently labeled cDNA probe was prepared from mRNA isolated from control Arabidopsis leaves by reverse transcription in the presence of Cy3-dCTP. A second probe, labeled with Cy5-dCTP, was prepared from leaves that were mechanically wounded (60 min). After the simultaneous hybridization of both probes with a cDNA microarray containing 150 defense-related Arabidopsis ESTs and scanning of the array, a pseudocolor image was generated. Genes induced or repressed after mechanical wounding are represented as red or green signals, respectively. Genes expressed at approximately equal levels between treatments appear as yellow spots. The intensity of each spot corresponds to the absolute amount of expression of each gene. The actual size of the array is 8 × 8 mm. Control genes are in the first row of top left, top right, and bottom left quadrants.

Figure 2.

Clustered Display of Data from the Time Course of Mechanical Wounding. A time course of wound-inducible gene expression in Arabidopsis leaves was constructed using cDNA microarrays. For simplicity, only those genes for which the transcript levels changed substantially as a result of wounding are included. Genes were ordered using a clustering program (see Methods) so that those with similar expression patterns would be grouped together. Each gene is represented by a single row of colored boxes, and each time point is represented by a single column. Induction (or repression) ranges from pale to saturated red (or green). The numbers of independent experiments were as follows: 15 min, 2; 30 min, 1; 60 min, 2; 90 min, 9; 3 hr, 3; 6 hr, 2; 9 hr, 1; and 24 hr, 1.

Figure 3.

Reproducibility of cDNA Microarray Experiments. mRNA samples (2 μg) from Arabidopsis leaves harvested 90 min after wounding or from control Arabidopsis leaves were labeled with Cy5 or Cy3, respectively, and hybridized with a cDNA microarray. After scanning each fluor separately, the fluorescent signal intensity was integrated and corrected for local area background. Expression ratios between treated and control samples were calculated. Results are shown for a set of representative wound-inducible genes. Values ±se represent the average of nine independent experiments. Genes shown in duplicate (FAD2 and ACX1) are represented by two different ESTs on the microarray, which show highly similar expression ratios.

Figure 4.

Comparison between the Expression of a Subset of Genes and the Levels of Jasmonate Family Members after Mechanical Wounding. (A) The average expression profile of a cluster of genes showing a similar temporal expression profile is represented. Dashed lines indicate standard deviation. For experimental details, see Figure 2. (B) Arabidopsis leaves were extracted at different times, and the tissues were analyzed for JA (circles), OPDA (squares), and dinor OPDA (dnOPDA, triangles) content in both wounded (solid lines) and control (dashed lines) plants. Mean values ±se were calculated for three plants. FW, fresh weight.

Figure 5.

Contribution of Jasmonates and Ethylene to Wound-Inducible Gene Expression. Relative changes in Arabidopsis gene expression after wounding of leaves for 90 min were studied in mutants. Expression ratios calculated from experiments comparing unwounded with wounded wild-type plants are plotted against expression ratios from experiments comparing unwounded with wounded mutant plants ([A] and [C]). Black dots represent genes that did not substantially change expression after wounding in both the wild type and mutants (based on the threshold of a twofold change). Blue dots represent genes that were induced (or repressed) in both wild-type and mutant plants. Red dots represent genes that were induced only in wild-type plants. Green dots represent genes (NPR1 and MPK3) that were induced only in mutant plants. (A) Jasmonate-insensitive mutant coi1-1. (B) RNA gel blot analysis of MPK3 mRNA accumulation 90 min after wounding of wild-type (WT) or coi1-1 plants. A chlorophyll a/b binding protein probe (CAB) was used as a control for equal RNA loading. U, unwounded; W, wounded. (C) Ethylene-insensitive mutant ein2-1. Each scatter plot represents the mean of two independent experiments.

Figure 6.

Comparison between Effects of Mechanical Wounding and Insect Feeding. (A) Relative changes in gene expression were measured 3 hr after wounding Arabidopsis leaves and after challenging leaves with P. rapae larvae for 3 hr. Expression ratios calculated from experiments comparing unwounded with wounded plants are plotted against expression ratios from experiments comparing unchallenged with insect-challenged plants. Black dots represent genes that showed no marked change in expression after wounding or insect challenge (based on the threshold of a twofold change). Blue dots represent genes that were induced in both treatments. The red dot represents a gene (HEL) that was induced only in insect-challenged plants. Green dots represent genes that were induced only after mechanical wounding. (B) RNA gel blot analysis of HEL mRNA accumulation in leaves challenged for 3 hr with P. rapae larvae. A chlorophyll a/b binding protein probe (CAB) was used as a control to assess equal RNA loading. C, unchallenged; P, P. rapae.

Figure 7.

Transcript Signatures for Mechanical Wounding, Dehydration, and Insect Feeding. Arabidopsis leaves were mechanically wounded (90 min), dehydrated (120 min), or challenged with P. rapae larvae (180 min). Cy3- or Cy5-labeled cDNA probes were prepared with mRNA samples from control (open columns) or treated (filled columns) plants, respectively, and were hybridized with a cDNA microarray. After scanning each fluor separately, the fluorescent signal intensity was integrated and corrected for local area background. Results are shown for a set of genes illustrating typical patterns of expression. Genes marked with an arrowhead were induced in only one treatment. (A) Mechanical wounding. (B) Dehydration. (C) P. rapae.