Peroxidases are enzymes that catalyze the reduction of hydrogen peroxide, thus minimizing cell injury and modulating signaling pathways as response to this reactive oxygen species. Using a phylogenetic approach, we previously identified a new peroxidase family composed of a small subset of ascorbate peroxidase (APx) homologs with distinguished features, which we named ascorbate peroxidase-related (APx-R). In this study, we showed that APx-R is an ascorbate-independent heme peroxidase. Despite being annotated as a cytosolic protein in public databases, transient expression of AtAPx-R-YFP in Arabidopsis thaliana protoplasts and stable overexpression in plants showed that the protein is targeted to plastids. To characterize APx-R participation in the antioxidant metabolism, we analyzed loss-of-function mutants and AtAPx-R overexpressing lines. Molecular analysis showed that glutathione peroxidase 7 (GPx07) is specifically induced to compensate the absence of APx-R. APx-R overexpressing lines display faster germination rates, further confirming the involvement of APx-R in seed germination. The constitutive overexpression of AtAPx-R-YFP unraveled the existence of a post-translational mechanism that eliminates APx-R from most tissues, in a process coordinated with photomorphogenesis. Our results show a direct role of APx-R during germinative and post-germinative development associated with etioplasts differentiation.

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterisation of an Arabidopsis thaliana mutant named crk10-A397T in which alanine 397 has been substituted by a threonine in the αC-helix of the kinase domain of CRK10, known to be a crucial regulatory module of mammalian kinase activity. The crk10-A397T mutant is a dwarf which displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type (WT) and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of auto-phosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of WT and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared to WT plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. They are transcriptionally regulated by a wide variety of environmental cues and stresses, however their precise biological roles remain largely unknown. Here we report a novel mutant isolated for the CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) of Arabidopsis thaliana which harbours the substitution of alanine 397 by a threonine in the αC-helix of its kinase domain and which we registered as crk10-A397T in the community database. In situ phosphorylation assays with the His-tagged wild type (WT) and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of auto-phosphorylation with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Phenotypically the mutant is a dwarf and the analysis of thin cross sections with light and transmission electron microscopy revealed that collapsed xylem vessels in roots and hypocotyls are very likely the cause for this reduction in stature. Transcriptomic analysis of WT and mutant hypocotyls revealed that predominantly biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant. Root-infection assays with the vascular pathogen Fusarium oxysporum demonstrated that the crk10-A397T mutant has enhanced resistance to this pathogen compared to WT plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10 and open up new avenues for the investigation of this elusive receptor-like kinase family.