Glutathione Deficiency of the Arabidopsis Mutant pad2-1 Affects Oxidative Stress-Related Events, Defense Gene Expression, and the Hypersensitive Response      

Abstract

The Arabidopsis (Arabidopsis thaliana) phytoalexin-deficient mutant pad2-1 displays enhanced susceptibility to a broad range of pathogens and herbivorous insects that correlates with deficiencies in the production of camalexin, indole glucosinolates, and salicylic acid (SA). The pad2-1 mutation is localized in the GLUTAMATE-CYSTEINE LIGASE (GCL) gene encoding the first enzyme of glutathione biosynthesis. While pad2-1 glutathione deficiency is not caused by a decrease in GCL transcripts, analysis of GCL protein level revealed that pad2-1 plants contained only 48% of the wild-type protein amount. In contrast to the wild type, the oxidized form of GCL was dominant in pad2-1, suggesting a distinct redox environment. This finding was corroborated by the expression of GRX1-roGFP2, showing that the cytosolic glutathione redox potential was significantly less negative in pad2-1. Analysis of oxidative stress-related gene expression showed a higher transcript accumulation in pad2-1 of GLUTATHIONE REDUCTASE, GLUTATHIONE-S-TRANSFERASE, and RESPIRATORY BURST OXIDASE HOMOLOG D in response to the oomycete Phytophthora brassicae. Interestingly, oligogalacturonide elicitation in pad2-1 revealed a lower plasma membrane depolarization that was found to act upstream of an impaired hydrogen peroxide production. This impaired hydrogen peroxide production was also observed during pathogen infection and correlated with a reduced hypersensitive response in pad2-1. In addition, a lack of pathogen-triggered expression of the ISOCHORISMATE SYNTHASE1 gene, coding for the SA-biosynthetic enzyme isochorismate synthase, was identified as the cause of the SA deficiency in pad2-1. Together, our results indicate that the pad2-1 mutation is related to a decrease in GCL protein and that the resulting glutathione deficiency negatively affects important processes of disease resistance.

The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis

“…Under the first hypothesis, the oxt6 mutation would be expected to prevent the 2-fold increase in ICS1 expression in the mips1 mutant caused by transfer to LD conditions, and this misregulation could account for cell death suppression. Indeed, impairment of ICS1 induction upon pathogen infection in the pad2-1 mutant has been proposed to account for the impaired SA signaling observed in this mutant (Dubreuil-Maurizi et al, 2011). Based on genome-wide analysis (Thomas et al, 2012) and our assays, ICS1 is unlikely to be a direct target of CPSF30.…”

Role of glutathione in plant signaling under biotic stress

“…However, we showed that pad2-1 contains only 48% of wild-type GCL amount. 16 Based on these results, one hypothesis suggests that pad2-1 mutation could affect GCL protein folding or degradation leading to glutathione deficiency and correlated with cysteine accumulation (Fig. 1.1).…”

“…16 Our hypothesis is that the less reducing E GSH could affect the corresponding gene expression and/or enzyme activity or upstream signaling events. From these results and based on previous studies, their impairment might explain the weak HR establishment observed in pad2-1 during P. brassicae interaction.…”

“…This result was confirmed with the predominant oxidized form of GCL in pad2-1 although the reduced form is observed in wild type. 16 Thus, based on many studies about regulation of gene expression by the redox state, we assume that this altered redox environment could influence gene expression under biotic stress. In that way, marker genes of oxidative stress such as GR1 (Glutathione Reductase1), GSTF6 (Glutathione-STransferase F6) and RbohD (Respiratory Burst Oxidase Homolog D) are upregulated in pad2-1 mutant compared with wild type during P. brassicae infection.…”

“…In that way, marker genes of oxidative stress such as GR1 (Glutathione Reductase1), GSTF6 (Glutathione-STransferase F6) and RbohD (Respiratory Burst Oxidase Homolog D) are upregulated in pad2-1 mutant compared with wild type during P. brassicae infection. 16 Glutathione Deficiency in pad2-1 Affects Defense-related Signaling Events, which Confers a Susceptibility to Pathogens…”