The generation of reactive oxygen species (ROS) and their regulation by antioxidant enzymes, such as IRON SUPEROXIDE DISMUTASE 1 (FSD1), are critical for managing plant responses to salt stress. However, the protein networks modulating ROS levels during salt stress remain incompletely understood. Our co-immunoprecipitation analysis identified FSD1 as an interaction partner of the scaffolding protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). Bimolecular fluorescence complementation analysis revealed that RACK1A interacts with FSD1 predominantly in the cytoplasm. Despite elevated FSD1 activity in rack1a mutants, the abundance of FSD1 protein remained unchanged. Additionally, we found that the RACK1A-FSD1 module was involved in root hair tip growth, highlighting the developmental significance of this interaction. While rack1a mutants exhibited salt resilience, the fsd1-1 rack1a-1 double mutant displayed reduced salt stress resistance, which was substantiated by reduced ROS levels. Upon salt stress, a distinct pool of RACK1A and FSD1 proteins accumulated in cycloheximide-sensitive structural condensates in the cytoplasm that colocalized with the stress granule (SG) marker protein RNA-BINDING PROTEIN 47. FSD1 activity was lower in SGs compared to the soluble extract. RACK1A also interacted with TUDOR STAPHYLOCOCCAL NUCLEASE 2, which participates in SG formation. However, RACK1A knock-out completely abolished salt-stress-dependent accumulation of FSD1 in structural condensates, suggesting that RACK1A likely mediates the recruitment of FSD1 to SGs. Thus, this study uncovers a mechanism for the regulation of RACK1/FSD1-dependent antioxidant defense in response to salt stress in Arabidopsis thaliana.

Department of Biotechnology Faculty of Science Palacký University Olomouc Olomouc Czech Republic

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