General detoxification and stress responses are mediated by oxidized lipids through TGA transcription factors in Arabidopsis

Abstract

12-oxo-phytodienoic acid and several phytoprostanes are cyclopentenone oxylipins that are formed via the enzymatic jasmonate pathway and a nonenzymatic, free radical-catalyzed pathway, respectively. Both types of cyclopentenone oxylipins induce the expression of genes related to detoxification, stress responses, and secondary metabolism, a profile clearly distinct from that of the cyclopentanone jasmonic acid. Microarray analyses revealed that 60% of the induction by phytoprostanes and 30% of the induction by 12-oxo-phytodienoic acid was dependent on the TGA transcription factors TGA2, TGA5, and TGA6. Moreover, treatment with phytoprostanes and 12-oxo-phytodienoic acid inhibited cell division and root growth, a property also shared by jasmonic acid. Besides being potent signals, cyclopentenones and other lipid peroxidation products are reactive electrophiles that can covalently bind to and damage proteins. To this end, we show that at least two of the induced detoxification enzymes efficiently metabolize cyclopentenones in vitro. Accumulation of two of these metabolites was detectable during Pseudomonas infection. The cyclopentenone oxylipin gene induction profile resembles the defense response induced by a variety of lipophilic xenobiotics. Hence, oxidized lipids may activate chemosensory mechanisms of a general broad-spectrum detoxification network involving TGA transcription factors.

Figure 1.

Regulation of Gene Expression by PPA₁ in Arabidopsis. (A) Classification of genes with at least threefold higher expression in Arabidopsis cell cultures at 4 h after treatment with 75 μM PPA₁ relative to controls. (B) Classification of genes with at least threefold lower expression in Arabidopsis cell cultures at 4 h after treatment with 75 μM PPA₁ relative to controls.

Figure 2.

Regulation of Gene Expression by Different Oxylipins in Arabidopsis. (A) Chemical structures of the different oxylipins used. Phytoprostanes are composed of type I (R′ = C₇H₁₄COOH, R″ = C₂H₅) and type II (R′ = C₂H₅, R″ = C₇H₁₄COOH) stereoisomers. (B) Expression of PPA₁-responsive genes in Arabidopsis cell cultures in response to 75 μM OPDA, JA, and A₁-, B₁-, and dJ₁-phytoprostanes or in cultures not treated (−) or treated with 0.5% methanol in water as control (Con.). Cell cultures were harvested at 4 h after treatments. The experiment was repeated at least three times; a representative RNA gel blot is shown. Eight micrograms of RNA was loaded per lane, and blots were hybridized with the indicated probes. Gel loading was monitored by ethidium bromide staining of the gel (see Supplemental Figure 1 online).

Figure 3.

Venn Diagram Comparing Genes Upregulated by Oxylipins in Wild-Type and tga2-5-6 Plants. Expression was analyzed in wild-type and tga2-5-6 Arabidopsis plants at 4 h after treatment with 75 μM OPDA or PPA₁ in comparison with controls. (A) Number of genes with twofold or greater induction by PPA₁ in wild-type and tga2-5-6 plants. (B) Number of genes with twofold or greater induction by OPDA in wild-type and tga2-5-6 plants.

Figure 4.

Inhibition of Cell Cycle Progression by OPDA and PPA₁ in Tobacco. Synchronized Bright Yellow 2 cells were treated with 15 μM OPDA or PPA_1. The percentage of cells in mitosis was determined after 9 h (means ± sd; n = 3 independent experiments).

Figure 5.

Inhibition of Root Growth by OPDA and PPA₁ in Arabidopsis. Seedlings were germinated on medium containing 25 μM OPDA or PPA₁, and root growth was measured after 8 d (means ± sd; n = 20 seedlings). Three independent experiments were performed with similar results.

Figure 6.

Regulation of OPR1, OPR2, and OPR3 Expression by OPDA and PPA₁. Expression of OPR1 (A), OPR2 (B), and OPR3 (C) in response to OPDA and PPA₁. Arabidopsis cell cultures were treated with 75 μM OPDA, 75 μM PPA₁, or 0.5% methanol/water (Control) and harvested after 4 h. Relative levels of expression were determined by real-time quantitative RT-PCR. Values were normalized to the expression of Actin2/8 (means ± sd; n = 3 independent experiments).

Figure 7.

HPLC-MS/MS Chromatograms of PPA₁ and OPDA Adducts to GSH. Endogenous PPA₁-GSH and OPDA-GSH adducts from Arabidopsis leaves and in vitro–synthesized adducts were analyzed and unequivocally identified by four characteristic MRM transitions (PPA₁-GSH: m/z 616→598, 616→308, 616→273, and 616→179; OPDA-GSH: m/z 600→308, 600→293, 600→275, and 600→179). Chromatograms display the total ion current of the four transitions. Peaks represent different isomers of the conjugate. (A) and (E) Relative levels of PPA₁-GSH (A) and OPDA-GSH (B) at 10 h after mock infiltration of Arabidopsis leaves. (B) and (F) Relative levels of PPA₁-GSH (B) and OPDA-GSH (F) at 10 h after P. syringae infiltration of Arabidopsis leaves. (C) and (G) PPA₁-GSH (C) and OPDA-GSH (G) enzymatically synthesized using recombinant GST6 at pH 7.5. (D) and (H) PPA₁-GSH (D) and OPDA-GSH (H) adducts prepared by nonenzymatic conjugation at pH 10. PPA₁ are a mixture of two racemic regioisomers (in total, 12 stereoisomers), while synthetic OPDA comprises 4 stereoisomers. Stereoisomers are not completely resolved by the HPLC column. Enzymatic conjugation with GSH is expected to result in conjugates ([C] and [G]) with either R or S configuration at the labeled carbon. By nonenzymatic reaction with GSH, R- and S-configured conjugates are formed, which doubles the number of stereoisomers. The peak pattern of endogenously occurring conjugates suggests that they have been formed enzymatically by GST(s).