Unveiling the physical interaction between GSNOR1 and TRX-h isoforms

This study aims to investigate any possible interaction between two highly conserved regulators of cellular SNO levels: S-nitrosoglutathione reductase (GSNOR1) and thioredoxin (TRX). These two enzymes control global S-nitrosylation levels and thus impact the cellular redox state during immune responses. A mutant line (Δtrx1trx2sfa1) of Saccharomyces cerevisiae MaV203 strain was generated to explore this possibility rather than using a classical strain. We identified and characterized the physical interaction between GSNOR1 and Arabidopsis cytosolic h isoforms in a yeast two-hybrid screen. Out of the seven TRX isoforms, AtGSNOR1 displayed a high interaction with AtTRXh3, AtTRXh4, and AtTRXh5 (AtTRXh5>AtTRXh4 & AtTRXh3). This interaction was further confirmed through an in vitro pull-down. The interaction between GSNOR1 and TRXh5 remained unaffected by the presence of NO donors, suggesting potential de-/transnitrosylation between these key S-nitrosothiol regulators. However, mutation of either active site cysteine in TRXh5 weakened the interaction, while mutation of both cysteines completely abolished it. This is the first research detailing GSNOR1’s interactions with any TRX isoforms in plants. Without documented evidence of their interaction, we speculate they mutually regulate each other’s activity, aiding SA-mediated responses to enhance plant immunity.