Arachidonic acid: an evolutionarily conserved signaling molecule modulates plant stress signaling networks

Abstract

Fatty acid structure affects cellular activities through changes in membrane lipid composition and the generation of a diversity of bioactive derivatives. Eicosapolyenoic acids are released into plants upon infection by oomycete pathogens, suggesting they may elicit plant defenses. We exploited transgenic Arabidopsis thaliana plants (designated EP) producing eicosadienoic, eicosatrienoic, and arachidonic acid (AA), aimed at mimicking pathogen release of these compounds. We also examined their effect on biotic stress resistance by challenging EP plants with fungal, oomycete, and bacterial pathogens and an insect pest. EP plants exhibited enhanced resistance to all biotic challenges, except they were more susceptible to bacteria than the wild type. Levels of jasmonic acid (JA) were elevated and levels of salicylic acid (SA) were reduced in EP plants. Altered expression of JA and SA pathway genes in EP plants shows that eicosapolyenoic acids effectively modulate stress-responsive transcriptional networks. Exogenous application of various fatty acids to wild-type and JA-deficient mutants confirmed AA as the signaling molecule. Moreover, AA treatment elicited heightened expression of general stress-responsive genes. Importantly, tomato (Solanum lycopersicum) leaves treated with AA exhibited reduced susceptibility to Botrytis cinerea infection, confirming AA signaling in other plants. These studies support the role of AA, an ancient metazoan signaling molecule, in eliciting plant stress and defense signaling networks.

Figure 1.

EP Plants Exhibit Altered Tolerance to a Range of Biotic Challengers. (A) Choice bioassays performed on pairs of wild-type and EP plants that were caged. A single female aphid (M. persicae) was released in each cage. The initial nymph deposition preference was determined within 2 d of aphid release, in four independent experiments. Bar graphs represent the actual numbers of aphids. One-tailed binomial tests were used to determine significance (P = 0.0012). (B) Visual symptoms 3 d after spot inoculation with conidia of B. cinerea (grape isolate). Lesion size determined 3 d after inoculation. Data are means of 48 independent biological replicates ± se. Asterisks denote a significant difference from the wild type (P < 0.005) as determined by t tests. (C) and (D) Hydroponically growing plants were inoculated with zoospores of P. capsici. Disease development was monitored at 48 h after inoculation by sporangia counts on roots in 10 microscope fields (C) and by colonization as measured by real-time quantitative PCR analysis of P. capsici DNA (D). Asterisks denote a significant difference from the wild type (χ² = 5.45, P = 0.002 for sporangial counts; χ² = 14.4, P = 0.0002 for colonization) as determined by Wilcoxon rank sums test. Data are the means of 40 independent biological replicates ± se. (E) Bacterial growth in wild-type and EP1 plants inoculated with Pst. The bioluminescence was recorded 2 d after inoculation as photon counts per second (Cps). Asterisks denote a significant difference from the wild type (P = 0.001) as determined by t test. Data are means of four independent experiments ± se. [See online article for color version of this figure.]

Figure 2.

Levels of JA Pathway Transcripts and Metabolites Are Enhanced in EP Plants. (A) Measurements of JA metabolite levels in the wild type (white bars) and EP1 (black bars) before (NW) and 90 min after wounding (W) show enhanced basal- and wound-induced levels of JA in EP plants. Data are means of six independent experiments ± sd. (B) Simplified representation of JA-pathway genes. (C) Total RNA was extracted from 4-week-old rosette leaves before (NW) and 90 min after wounding (W) and subjected to real-time quantitative PCR analysis. The transcript levels of each gene (DGL, LOX1 through 6, AOS, and VSP2) were normalized to At4g34270 (T1P41-like family protein) and At4g26410 (M3E9) measured in the same samples. Data are means of three biological replicates ± se.

Figure 3.

Expression of SA Pathway Genes and SA Metabolite Levels Are Reduced in EP Plants. (A) Measurements of SA metabolite levels in unwounded wild type (white bars) and EP1 (black bars) show reduced SA levels in EP1 plants. Data are means of six independent experiments ± sd. (B) Simplified representation of SA pathway genes. (C) Total RNA was extracted from 4-week-old rosette leaves and subjected to real-time quantitative PCR analysis. The transcript levels of each gene, EDS5, PAD4, ICS1, NPR1, WRKY70, and PR1, were normalized to At4g34270 (T1P41-like family protein) and At4g26410 (M3E9) measured in the same samples. Data are means of three biological replicates ± se.

Figure 4.

AA Coordinates Events Underlying Alteration in Expression of JA and SA Pathway Genes and Metabolites. Measurements were performed on leaves exogenously treated with 10 μM AA (black bars) or mock treated with 0.02% ethanol (white bars). (A) and (C) Measurements of JA and SA metabolite levels in AA- and mock-treated plants. (B) and (D) Total RNA was extracted from AA- or mock-treated 4-week-old rosette leaves and subjected to real-time quantitative PCR analysis. The transcript levels of each gene, AOS, VSP2, PAD4, ICS1, WRKY70, and PR1, were normalized to At4g34270 (T1P41-like family protein) and At4g26410 (M3E9) measured in the same samples. Data are means of three biological replicates ± se.

Figure 5.

Alteration of SA Pathway Transcripts and Metabolites in Response to AA is JA Dependent. (A) Measurements of SA metabolite levels in mock-treated (0.02% ethanol; white bars) and AA-treated (black bars) aos plants. Data are the means of six biological replicates ± sd. (B) Total RNA was extracted from mock- or AA-treated 4-week-old rosette leaves of aos plants and subjected to real-time quantitative PCR analysis. The transcript levels of each gene, PAD4, WRKY70, and PR1, were normalized to At4g34270 (T1P41-like family protein) and At4g26410 (M3E9) measured in the same samples. Data are means of three biological replicates ± se.

Figure 6.

JA Is Required for AA-Mediated Enhanced Tolerance to Botrytis. (A) Visual symptoms 3 d after spot inoculation with conidia of B. cinerea. Lesion size determined 3 d after inoculation. Data are means of 45 independent biological replicates ± se. Asterisks denote a significant difference in AA- versus from mock-treated wild-type leaves (P < 0.001) as determined by t tests. (B) and (C) Measurements of JA and SA levels in mock-treated (0.02% ethanol; white bars) and AA-treated (black bars) wild-type and aos mutant leaves 3 d after inoculation. Data are means of four independent biological replicates ± se. [See online article for color version of this figure.]

Figure 7.

AA-Treated Tomato Leaves Have Altered JA and SA Metabolite Levels and Display Enhanced Tolerance to Botrytis. (A) and (B) Measurements of JA and SA metabolite levels in tomato leaves treated with 0.02% ethanol (mock) or 100 μM various FAs [LA (18:2), ALA (18:3), EDA (20:2), ETrA (20:3), and AA (20:4)]. (C) Visual symptoms 3 d after spot inoculation with conidia of B. cinerea. Lesion size determined 3 d after inoculation. Data are means of 30 independent biological replicates ± se. Asterisks denote a significant difference from mock treated (P < 0.004) as determined by t tests. [See online article for color version of this figure.]

Figure 8.

AA Elicits Expression of 4xRSRE:LUC and Genes with the RSRE Motif. (A) Image of individual 4xRSRE:LUC transgenic plants before and 90 min after treatment with either 0.02% ethanol (mock) or different FAs (LA, ALA, EDA, EtrA, and AA). Images of empty vector lines, pATM-NOS (Ctrl) before and after AA treatment, are representative of the data obtained from mock experiments as well as exogenous application of other FAs. (B) Total RNA was extracted from AA- (black bars) or mock-treated (white bars) 4-week-old rosette leaves and subjected to real-time quantitative PCR analysis. The transcript levels of CAF1a, ERF#018, and BAP1 genes, which contain RSRE motifs in their promoter sequences, were normalized to At4g34270 and At4g26410 measured in the same samples. Data are means of three biological replicates ± se.