Phenotypic characterization of Arabidopsis GSNOR and AKR4C triple mutants in response to iron and nitrosative stress

Nitric oxide (NO) is a signaling molecule regulating plant development and stress responses via reversible post-translational modifications, primarily S-nitrosylation. Intracellular homeostasis of its major mobile reservoir, S-nitrosoglutathione (GSNO), is predominantly regulated by Snitrosoglutathione reductase (GSNOR). However, the aldo-keto reductase 4C (AtAKR4C) subfamily has recently emerged as a potential secondary pathway for modulating nitrosothiol and reactive carbonyl pools under nitro-oxidative stress. This work investigates the roles of GSNOR and the AtAKR4C subfamily in the growth, heavy metal toxicity, and nitrosative stress responses of distinct Arabidopsis thaliana lines. Phenotypic characterization of soil-grown plants revealed that both GSNOR-deficient (hot5-2) and AKR4C triple mutants (akr4c8 akr4c9 akr4c10) exhibit severe growth restrictions, highlighting the essential role of both enzyme classes in baseline metabolic regulation. In stress assays, primary root elongation under severe iron toxicity (200 μM Fe-EDTA) was heavily inhibited in Wild-Type (WT) and hot5-2 lines; remarkably, the akr4c8-11 triple mutant displayed a promotion of root elongation. Conversely, exposure to high exogenous GSNO (1 mM) induced a generalized phenotypic collapse across the WT and the akr4c8-11 triple mutant, masking strain-specific variations. Biochemical assays confirmed that all four recombinant AtAKR4C isoforms catalyze NADPH-dependent GSNO reduction. AtAKR4C8 demonstrated the highest overall catalytic efficiency, while AtAKR4C11 exhibited high specific activity but lower substrate affinity. Collectively, these findings demonstrate that both GSNOR and the AtAKR4C subfamily maintain NO homeostasis, primarily and secondarily respectively, to regulate plant growth and stress tolerance.