A Coupled GSH/GSNOR System Denitrosylates TRXh5 to Allow Activation of SA Signalling by Oxidative Stress

Accumulating evidence shows that reversible protein S -nitrosylation is essential for H 2 O 2 homoeostasis and signalling. However, roles for denitrosylation in such oxidative signalling remain poorly understood. Here, we examined this question using the Arabidopsis catalase-defective mutant, cat2 , in which oxidative stress induces both glutathione accumulation and salicylic acid (SA) pathways. Induction of these pathways was accompanied by enhanced thioredoxin ( TRXH5 ) expression, and oxidative stress-induced activation of the SA pathway was compromised when TRXH5 expression was genetically disabled, whereas TRXH5 overexpression stimulates H 2 O 2 -triggered SA responses. Intriguingly, TRX h5 -reinforced SA responses were antagonised by glutathione (GSH) deficiency when introducing additional pad2 mutation, localised in the GLUTAMATE-CYSTEINE LIGASE gene encoding the first enzyme of glutathione biosynthesis. Further analysis revealed that the two active cysteine residues of recombinant TRX h 5 can be denitrosylated by GSH. Blocking glutathione accumulation increased more TRX h 5-SNO formation in TRXH5-YFP cat2 pad2 trxh5 than in TRXH5-YFP cat2 trxh5 . Furthermore, S -nitrosoglutathione reductase (GSNOR) was capable of physically interacting with TRX h 5, and was also required for GSH-dependent TRX h 5 denitrosylation and TRX h 5-enhanced SA responses during oxidative stress. Collectively, these data suggest that GSH/GSNOR constitutes an active denitrosylating module that works together with the canonical NADPH-dependent TRX-reducing pathway to sustain cytosolic TRX h 5 operation within the oxidative signalling framework.