Control of nitric oxide signalling by an interplay between the immune regulators GSNOR1 and TRXh5

Plants are continuously challenged by diverse pathogens and have evolved sophisticated defense networks to counter these threats. Early immune activation is marked by the rapid production of reactive oxygen (ROS) and nitrogen species (RNS), which collectively drive profound alterations in cellular redox balance. Among RNS, nitric oxide (NO) plays a central signalling role through post-translational modifications such as S-nitrosylation, the covalent attachment of NO to the thiol (SH) side chain of reactive cysteine residues, forming S-nitrosothiols (SNOs). S-nitrosylation and its reversal, denitrosylation, are critical regulators of immune signalling. In Arabidopsis thaliana, thioredoxin h5 (TRXh5) directly denitrosylates diverse protein targets, while S-nitrosoglutathione reductase 1 (GSNOR1) regulates S-nitrosylation indirectly by degrading S-nitrosoglutathione (GSNO), a major mobile reservoir of NO. Loss of GSNOR1 function (e.g., gsnor1-3 or par2-1) results in SNO accumulation, impaired salicylic acid (SA)-dependent signalling and enhanced pathogen susceptibility. Post-translational modifications, including stress-induced S-nitrosylation of the cysteine-rich regulator GSNOR1, highlight a dynamic layer of redox control. Here, we investigated the functional relationship between GSNOR1 and TRXh5. Using colocalization, bimolecular fluorescence complementation and co-immunoprecipitation, we demonstrated that the two proteins physically interact in planta, independently of TRXh5 catalytic cysteines. Functional assays revealed that trxh5 mutants retained wild-type levels of pathogen susceptibility and PR genes expression, suggesting compensation by other cytosolic thioredoxins in plant immune response. In contrast, gsnor1 mutants exhibited dramatically increased susceptibility to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and the gsnor1trxh5 double mutant displayed disease susceptibility levels comparable to gsnor1 alone. Consistently, transcription of the immune-responsive genes PR1 and PR2 was robustly induced in wild-type and trxh5 plants but downregulated in gsnor1 and gsnor1trxh5 mutants. Notably, overexpression of GSNOR1 in the trxh5 mutant background did not alter susceptibility but modified PR genes expression, suggesting that excessive GSNO degradation in the absence of TRXh5-mediated denitrosylation perturbs redox balance and defense gene regulation. Finally, we performed a forward genetic screen to identify novel genes potentially involved in GSNO turnover, a critical process in maintaining redox homeostasis. In our forward genetic screen, we identified two classes of par2-1 suppressor mutants, with PSM50 fully restoring the phenotype and the others showing only partial suppression. Together, our findings reveal the possibility of a coordinated interaction between GSNOR1 and TRXh5 in maintaining redox balance during plant immunity and uncover novel regulators that contribute to GSNO turnover. ] These findings extend our understanding of how plants fine-tune S-nitrosylation to balance redox homeostasis and defense.