Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1

Abstract

Although ethylene is involved in the complex cross talk of signaling pathways regulating plant defense responses to microbial attack, its functions remain to be elucidated. The lesion mimic mutant vad1-1 (for vascular associated death), which exhibits the light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, is a good model for studying the role of ethylene in programmed cell death and defense. Here, we demonstrate that expression of genes associated with ethylene synthesis and signaling is enhanced in vad1-1 under lesion-promoting conditions and after plant-pathogen interaction. Analyses of the progeny from crosses between vad1-1 plants and either 35SERF1 transgenic plants or ein2-1, ein3-1, ein4-1, ctr1-1, or eto2-1 mutants revealed that the vad1-1 cell death and defense phenotypes are dependent on ethylene biosynthesis and signaling. In contrast, whereas vad1-1-dependent increased resistance was abolished by ein2, ein3, and ein4 mutations, positive regulation of ethylene biosynthesis (eto2-1) or ethylene responses (35SERF1) did not exacerbate this phenotype. In addition, VAD1 expression in response to a hypersensitive response-inducing bacterial pathogen is dependent on ethylene perception and signaling. These results, together with previous data, suggest that VAD1 could act as an integrative node in hormonal signaling, with ethylene acting in concert with salicylic acid as a positive regulator of cell death propagation.

Figure 1.

Ethylene-associated gene expression in wild-type and vad1-1 plants. Quantitative RT-PCR analysis of gene expression in leaves of ACO1, ACO2, EIN2, ERF1, WRKY70, MYC2, PDF1-2, PR4, and PR1 in wild-type (Ws-4) and vad1-1 plants 17 (white bars), 21 (lesion formation in vad1-1; gray bars), and 31 d (black bars) after transplanting under lesion-promoting conditions. Each measurement is an average of two or three replicates, and the experiment was repeated three times with similar results. See “Materials and Methods” for details.

Figure 2.

Ethylene-associated gene expression in wild-type (Ws-4) and vad1-1 plants (26 d posttransplanting plants) after inoculation with the avirulent bacteria Pst DC3000/avrRpm1. Quantitative RT-PCR analysis of gene expression of ACO1, EIN2, ERF1, WRKY70, PDF1-2, and PR1 in wild type (white squares) and vad1-1 (black squares). Each measurement has been performed on 16 to 20 leaves collected from four independent plants, and the experiment was repeated two times with similar results. See “Materials and Methods” for details.

Figure 3.

Effect of ein2-1, ein3-1, and ein4-1 mutations on the cell death and defense phenotypes of vad1-1 plants. A, Six-week-old single- and double-mutant plants 31 d after transplanting (10 d after lesion formation in vad1-1). Arrows indicate lesion formation on plants. B, Transcript levels of the defense-related genes PDF1.2 and PR1 in wild-type, single-, and double-mutant plants evaluated by quantitative RT-PCR. Plants were grown under lesion-promoting conditions and harvested 17 (white bars), 21 (lesion formation in vad1-1; gray bars), and 31 d (black bars) after transplanting. Each measurement is an average of two replicates and experiments were repeated two or three times and similar results were obtained. C, Bacterial populations in wild-type, single-, and double-mutant plants. Inoculations with Pst strain DC3000 and strain DC3000 expressing avrRpm1 were performed on leaves without lesion with a bacterial suspension at 2 × 10⁵ and 5 × 10⁵ cfu mL⁻¹, respectively. Bacterial populations were measured at 0 (white bars) and 3 d (black bars) postinoculation. Mean bacterial densities are shown (three to five replicates with corresponding sds) for one representative experiment from two or three independent experiments. Asterisks denote significantly different values from bacterial number in vad1-1 according to Student's t test (*, P ≤ 0.05; **, P ≤ 0.005). See “Materials and Methods” for details.

Figure 4.

Effect of eto2-1 and ctr1-1 mutations and ERF1 transgene on the cell death and defense phenotypes of vad1-1 plants. A, Single- and double-mutant plants, 17 and 31 d after transplanting. A closeup (3×) of the double mutant vad1-1/ctr1-1 is presented. Arrows indicate lesion formation on plants. B, Transcript levels of defense-related genes PDF1.2 and PR1 in wild-type, single-, and double-mutant plants evaluated by quantitative RT-PCR. Plants were grown under lesion-promoting conditions and harvested 17 (white bars), 21 (lesion formation in vad1-1; gray bars), and 31 d (black bars) after transplanting. Each measurement is an average of two replicates and experiments were repeated two times with similar results. C, Bacterial populations in wild-type, single-, and double-mutant plants. Inoculations with Pst strain DC3000 and strain DC3000 expressing avrRpm1 were performed on leaves without lesion with a bacterial suspension at 2 × 10⁵ and 5 × 10⁵ cfu mL⁻¹, respectively. Bacterial populations were measured at 0 (white bars) and 3 d (black bars) postinoculation. Mean bacterial densities are shown (three to five replicates with corresponding sds) for one representative experiment from two or three independent experiments. Asterisks denote significantly different values from bacterial number in vad1-1 according to Student's t test (*, P ≤ 0.05; **, P ≤ 0.005). See “Materials and Methods” for details.

Figure 5.

VAD1 expression is dependent on ethylene biosynthesis and signaling pathways. Transcript levels were quantified by quantitative RT-PCR. A, VAD1 transcript levels in Col-0 (diamonds), ein2-1 (squares), ein3-1 (triangles), and ein4-1 (circles) plants in response to inoculation with the avirulent pathogen Pst DC3000/avrRpm1. B, VAD1 transcript levels in Col-0 (diamonds), eto2-1 (squares), ctr1-1 (triangles), 35S∷ERF1 (circles) plants, in response to inoculation with the avirulent pathogen Pst DC3000/avrRpm1. C, PDF1.2 transcript levels in Col-0 (diamonds), ein2-1 (squares), ein3-1 (triangles), and ein4-1 (circles) plants in response to inoculation with the avirulent pathogen Pst DC3000/avrRpm1. D, PDF1.2 transcript levels in Col-0 (diamonds), eto2-1 (squares), ctr1-1 (triangles), 35S∷ERF1 (circles) plants, in response to inoculation with the avirulent pathogen Pst DC3000/avrRpm1. Each measurement is an average of two replicates and experiments were repeated two times with similar results. E and F, VAD1 expression in response to ACC treatment. Ten-day-old wild-type (Col-0) plants were treated with water (white squares) or with ACC (black squares) at a final concentration of 100 μm. Mean values with corresponding sds are shown for three independent experiments. G and H, VAD1 and EIN2 transcript levels after treatment of Ws-4 plants (23 d after transplanting) with air (black diamonds), 5 μL L⁻¹ ethylene (black squares), 20 μL L⁻¹ ethylene (black triangles), 80 μL L⁻¹ ethylene (black crosses), and 1 μL L⁻¹ 1-MCP (white circles). Each measurement is an average of two replicates and experiments were repeated two times with similar results. See “Materials and Methods” for details.

Figure 6.

Schematic representation of cell death regulation by VAD1 during plant-pathogen interaction. Black arrows indicate positive regulation, whereas gray arrows indicate the absence of positive regulation. Black end-blocked lines indicate negative regulation, whereas gray end-blocked lines indicate the absence of negative regulation. ET, Ethylene.