The role of salicylic acid in the induction of cell death in Arabidopsis acd11

Abstract

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.

Figure 1.

Phenotypes of acd11/eds5, acd11/sid2, and acd11/nahG. A, Phenotypes and trypan blue stainings of leaves at the rosette stage. Trypan blue stainings were performed at day 25. B, Stature of mature acd11/sid2 relative to wild-type Ler.

Figure 2.

Expression of defense and cell death markers in single and double mutants. A, RNA-blot analysis of steady-state levels of FMO, SAG13, and PR1 mRNA in the wild type, in single mutants, and in the acd11/eds5 and acd11/sid2 double mutants. rRNA controls for equal loading are shown at the bottom. In some experiments, weak FMO, SAG13, and PR1 signals were detected in acd11/sid2-2. B, Real-time RT-PCR analysis of mRNA accumulation of ethylene response genes PR3 and ERF1 in single and double mutants. Expression of ubiquitin (UBQ10) was used to normalize cDNA input from the different genetic backgrounds.

Figure 3.

Responses of acd11/eds5 and acd11/sid2 to SA and BTH. The compounds were sprayed onto leaves at concentrations of 2 mm (SA) and 100 μm (BTH). Treatments were done at day 18 and pictures taken at day 25. Trypan blue stainings were also performed at day 25. Nontreated control plants are shown in Figure 1. A, Visible phenotypes and microscopic analysis of trypan blue-stained leaves after treatments. Mock-treated controls behaved as plants shown in Figure 1A. B, Dose-dependent growth inhibition of acd11/sid2 by SA. Plants were allowed to continue growth for 14 d after a single treatment with SA. C, Acceleration of the Acd⁻ phenotype in acd11/eds5 by exogenous SA. Plants were treated at day 12 and photographed 5 d later. D, Accumulation of PR1 mRNA in Ler, acd11/nahG, and acd11/sid2 24 h after treatments with either 2 mm SA or 100 μm BTH. sid2 and nahG PR1 inductions were tested on a separate blot and were similar to the double mutants shown.

Figure 4.

Overview of classes of metabolites required for acd11 phenotypes. A, Elements of biochemical pathways around isochorismate. The ensemble of isochorismate-derived metabolites different from SA is candidate group 1 compounds. The naphthoquinone/anthraquinone intermediate 1,4-dihydroxynaphtoic acid is highlighted due to its structural similarity with NahG substrates. The question mark indicates that there may be other, as yet unknown, biochemical pathways that use isochorismate as a precursor for synthesis of group 1 compounds. B and C, A black box model illustrating contributions to acd11 phenotypes of the two classes of compounds discussed in the text. It is assumed that the two classes of compounds are necessary but not sufficient for cell death and chlorosis and that BTH and INA are able to imitate the actions of both classes of compounds. D, Comparison of the chemical structures of SA, BTH, and INA.