Article Figures & Data
Figures
- 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.
Tables
- Table 1.
Relative Transcript Abundance after Wounding, Dehydration, or Insect (Pieris rapae) Feedinga
Wounding Dehydration P. rapae Gene Descriptionb Wild Type coi1-1 Wild Type Wild Type COI1-dependent genesc ACO1 Aminocyclopropane–carboxylic acid oxidase 2.2 1.8 1.2 1.5 ASA1 Anthranilate synthase (α subunit) 3.1 1.0 2.7 1.7 ASB Anthranilate synthase (β subunit) 2.3 1.2 2.5 1.7 AOS Allene oxide synthase 4.8 0.6 4.8 2.1 AWI31 Unknown 2.7 1.7 2.2 1.8 CCR Cinnamoyl–coA reductase 2.1 1.2 2.4 1.3 CHS Chalcone synthase 2.0 1.0 3.0 2.1 4CL 4-Coumarate:coA ligase 2.8 1.5 2.0 1.3 COMT O-methyltransferase 4.7 1.6 3.6 1.4 CYP83B1 Cytochrome P450 2.9 1.0 4.4 1.8 FAD7 Fatty acid desaturase 4.7 1.2 2.7 1.5 HEL Hevein-like protein 1.1 1.2 0.7 2.8 HPL Hydroperoxide lyase 4.8 1.0 7.1 2.2 JIP Jasmonate-inducible protein 2.9 1.6 2.5 3.1 JR3 Aminohydrolase 9.3 1.9 8.9 3.6 LOX2 Lipoxygenase 3.2 0.7 4.9 2.3 MBP Myrosinase binding protein 2.6 1.0 2.2 2.1 MPK3 Mitogen-activated protein kinase 0.9 2.2 1.1 1.5 MT1 Metallothionein 2.2 1.5 1.9 2.1 NPR1 Transcription factor inhibitor 1.2 2.4 0.9 1.0 PAL2 Phenylalanine ammonia-lyase 5.6 1.4 1.1 0.3 PR-2 β-1-3-Glucanase 3.0 1.5 3.8 1.6 SAHH S-adenosyl-l-homocysteine hydrolase 2.2 1.4 1.2 1.0 TSA Tryptophan synthase (α subunit) 6.3 1.0 3.7 2.0 TSB Tryptophan synthase (β subunit) 2.7 1.3 2.6 1.8 COI1-independent genesc ACX1 Acyl-coA oxidase 8.3 2.4 12.6 1.7 CM1 Chorismate mutase 2.1 2.1 2.6 1.7 DBP Oligogalacturonide binding protein homolog 2.7 2.6 2.3 1.4 ER5 Late embryogenesis abundant-like protein 6.6 13.8 11.2 2.4 ERF4 Ethylene-responding factor 4.0 3.1 4.6 2.2 GPX2 Glutathione peroxidase 2.0 2.3 4.9 1.7 GST1 Glutathione S-transferase 9.2 12.0 2.2 2.1 GST5 Glutathione S-transferase 6.2 2.7 11.5 2.9 OEC Oxygen-evolving protein 3.0 2.5 2.1 1.2 OPR1 OPDA reductase 9.3 7.7 1.5 1.7 PAL1 Phenylalanine ammonia-lyase 3.8 2.1 3.5 1.6 PGIP Polygalacturonase-inhibiting protein 2.9 7.3 4.5 1.8 PME1 Pectin methyl esterase 2.0 2.2 0.7 1.1 PR3AIV Chitinase 2.9 7.6 0.7 1.8 PRODH Proline dehydrogenase 11.6 10.4 0.03 2.4 TCH1 Calmodulin 2.4 5.2 1.4 1.6 TCH2 Calmodulin-related protein 2.4 4.5 0.8 1.3 TCH3 Calmodulin-related protein 2.4 4.6 0.3 1.3 TCH4 Endotransglycosylase 3.7 3.7 1.0 1.9 RNS1 RNase 64.6 52.4 22.1 2.3 XERO2 Dehydrin-like protein 23.8 83.4 13.9 3.1 -
↵a Samples from wounded (90 min), dehydrated (120 min), or insect-challenged (180 min) Arabidopsis leaves were fluorescently labeled with Cy5-dCTP, and respective control samples (untreated) were labeled with Cy3-dCTP. After hybridization with a cDNA microarray and scanning, expression ratios were calculated. Ratios correspond to fluorescent values from treated plants relative to untreated plants.
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↵b For further details, see http://www.unil.ch/ibpv.
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↵c Genes induced (having a ratio >2.0) in wild-type plants after wounding as well as the P. rapae–inducible HEL and two genes (MPK3, NPR1) only induced in the wounded coi1-1 mutant are included. Genes that are induced after wounding in both wild-type and coi1-1 plants are considered independent of the COI1 pathway. Our classification of COI1-dependent or -independent genes simplifies data analysis; we do not imply that this simplification exists in nature.
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