Systemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the salicylic acid induction deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a KD of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 (PAD4) is required for a slight NHP-mediated increase in NPR1 protein levels and for NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signalling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic.

Glutaredoxins (GRXs) are small proteins that interact with the atypical tripeptide glutathione (GSH), a redox active metabolite that forms a disulfide (GSSG) upon oxidation. GRXs encoding variants of a CPYC motif at a conserved active site act as GSH-dependent thiol-disulfide oxidoreductases (class I). GRXs with a CGFS site (class II) bind GSH in a way that is non-permissive for thiol-disulfide oxidoreductase reactions and favours transient iron-sulfur cluster binding. The biochemical functions of CCxC/S-type (class III) GRXs, which are found only in land plants, have remained largely unexplored. In this study, we characterized the in vitro properties of one of the Arabidopsis thaliana class III GRXs, namely ROXY9. In contrast to class I GRX AtGRXC2, ROXY9 was inactive as a reductase on the small substrate bis(2-hydroxyethyl)disulfide (HEDS) and as an oxidase on the redox-sensitive fluorescent protein roGFP2. Redox titrations with different GSH/GSSG ratios revealed formation of a disulfide bond (S2) between the first and the last cysteine of the CCLC motif at -220 to -230 mV. Glutathionylation (SG) at the first reactive cysteine was not observed. In contrast, AtGRXC2 and another class I GRX, HsGRX1, were oxidized to roughly equal amounts of GRX-SG and GRX-S2 over a wide range of GSH/GSSG ratios. The different reactivity of ROXY9 towards GSSG as compared to class I GRXs is most likely due to subtle differences in the GSH binding mode and explains why ROXY9 does not function as a GSH-dependent oxidoreductase on standard substrates. This feature might have evolved to avoid overlapping functions with class I GRXs in planta.

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