Fumonisin B1-induced cell death in arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways

Abstract

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.

Figure 1.

FB1-Induced PCD in Arabidopsis Protoplasts. (A) Time and dose dependency of FB1-induced cell death. Protoplasts were prepared from wild-type plants and incubated in the light with various concentrations of FB1 (open squares, 0 nM; filled circles, 0.7 nM; filled squares, 70 nM; and filled triangles, 7 μM). Samples were withdrawn at 4, 8, 19, and 25 hr after the addition of FB1, and surviving fractions were determined as described in Methods. For control protoplasts (0 nM FB1), methanol was added instead of the FB1 solution at the final concentration of 0.1%. The methanol did not affect protoplast viability (not shown). The largest standard error in this experiment was 0.05. (B) Requirement of plant cell activities for FB1-induced cell death. Protoplasts isolated from wild-type plants were incubated in the light for 16 hr in the presence of 70 nM FB1 (+FB1) or various inhibitors (100 μM cordycepin [+CORD], 10 μM cycloheximide [+CHX], and 1 μM staurosporine [+ST]), alone or in combination. In experiments 1 to 5, methanol was added instead of FB1 or inhibitor solutions. The final concentrations of methanol were as follows: experiment 1, 0.101%; experiments 2 to 4, 0.001%; and experiment 5, 0.1%. Surviving fractions were determined as described above. Error bars indicate sd.

Figure 2.

Detection of Fragmented DNA in FB1-Treated Protoplasts. Protoplasts were prepared from wild-type plants and incubated for 6 hr in the light in the presence of 0.001% methanol or 70 nM FB1. DNA molecules in the protoplasts then were stained with Hoechst 33342, and free 3′-OH groups in the molecules were labeled by the TUNEL technique as described in Methods. (A) Hoechst 33342 staining of methanol-treated protoplasts. (B) TUNEL staining of methanol-treated protoplasts. (C) Hoechst 33342 staining of FB1-treated protoplasts. (D) TUNEL staining of FB1-treated protoplasts.

Figure 3.

Mutational Analysis of PCD. (A) Effects of various defense-related mutations on FB1-induced cell death. Protoplasts isolated from the indicated plants were incubated for 16 hr in the light, and surviving fractions were determined as described in Methods. FB1 was added at the final concentration of 70 nM (+FB1). To the control experiment (wild type [wt], −FB1), methanol was added instead of FB1 at the final concentration of 0.001%. In the presence of methanol alone, surviving fractions of these mutant protoplasts were similar to that of wild-type protoplasts (not shown). (B) Effects of the acd2-2 and cpr6-1 mutations on protoplast viability. Protoplasts were isolated from the indicated plants and incubated for 16 hr in the light in the presence of 70 nM FB1 (+FB1) or 0.001% methanol (−FB1). Cycloheximide was added at the final concentration of 10 μM (+CHX). Surviving fractions were determined as described in Methods. Error bars indicate sd.

Figure 4.

Lesion Formation by FB1 in Arabidopsis Leaves. Five-week-old plants (wild type [wt], acd2-2, and NahG) were infiltrated with 10 μM FB1 (F) or 0.14% methanol (M) as described by Greenberg et al. (1994) and incubated under a 12-hr light/dark cycle. Leaves were excised 3 or 6 days after infiltration for photographing. (A) After 3 days. (B) After 6 days.

Figure 5.

Inducibility of an SA-Responsive Promoter by FB1. Activity of the Arabidopsis glucosyltransferase promoter was determined in wild-type (wt) and NahG protoplasts by using a transient expression system as described in Methods. The protoplasts were incubated for 16 hr in the light or in the dark in the presence of 70 nM FB1 (+FB1) or 0.001% methanol (−FB1). Promoter activities were normalized to the value obtained with wild-type protoplasts incubated in the light without FB1. Error bars indicate sd.

Figure 6.

Effects of Light on PAL Expression, PAL Activity, and Cell Death. (A) PAL1 promoter activity in wild-type protoplasts analyzed with a transient expression system. Protoplasts transformed with a PAL1 promoter–LUC construct were divided into two identical portions; one was incubated in the light and the other in the dark. LUC activities were assayed 16 hr after transformation, and the activity obtained in the dark was normalized to the activity in the light. See Methods for details of this assay system. (B) PAL enzyme activities in wild-type protoplasts. Protoplasts isolated from wild-type plants were divided into two identical portions; one was incubated in the light and the other in the dark. After 16 hr, samples were taken and the PAL enzyme activity in each sample was determined. (C) Effects of light on FB1-induced cell death. Protoplasts isolated from wild-type (wt) plants were incubated for 16 hr in the light or dark in the presence of 70 nM FB1 (+FB1) or 0.001% methanol (−FB1). Surviving fractions were determined as described in Methods. (D) Effects of light on toxin-independent cell death. Protoplasts were isolated from wild-type (wt) and acd2-2 plants and incubated for 16 hr in the light or dark before determination of the surviving fractions. Error bars indicate sd.

Figure 7.

A Model for Cell Death Signaling in Arabidopsis Protoplasts. Weak and strong SA signals are denoted by smaller and larger letters, respectively. As alternatives to the light-dependent PAL-mediated generation of SA shown in the model, SA might be produced from SA β-glucoside through a pathway independent of PAL and light, and the primary role of light in PCD may be to generate ROS rather than to activate the transcription of PAL genes. See text for details.