Tsinyk, M. | Search bioRxiv

bioRxiv2026

Stomatal movement in Arabidopsis is driven by guard cell-localized and copper-insensitive CSD1 splice variant

Copper (Cu) is an essential micronutrient whose bioavailability is strongly affected by soil physicochemical properties. During evolution, plants have developed mechanisms to flexibly adjust their metabolism to Cu status. Superoxide dismutases (SODs), including Cu/ZnSOD1 (CSD1) and FeSOD1 (FSD1), are key antioxidant enzymes regulated in Cu dependent manner in Arabidopsis thaliana. Examination of CSD1 cellular distribution and activity revealed that CSD1 is a nuclear and cytosolic SOD whose abundance and activity respond to Cu availability inversely to FSD1. Combined microscopic and biochemical analyses of Cu-dependent dynamics revealed that, unlike FSD1, CSD1 localization in guard cells (GCs) remains independent of Cu availability. CSD1 escapes miR398-mediated regulation in GCs through a cell type-specific splice variant (CSD1.2) that carries an altered miR398-binding site. In silico analyses indicate that this mechanism is also present in crop species. Functionally, the csd1 mutant showed reduced sensitivity to abscisic acid (ABA)-induced stomatal closure, a phenotype rescued by reintroducing CSD1. Biochemical and reactive oxygen species (ROS) level analyses indicate that CSD1.2 most likely acts independently of its canonical enzymatic activity in GCs and functions upstream of the ROS burst in the ABA signaling pathway. Together, we present a novel, cell-type-specific mechanism that safeguards ABA-driven stomatal closure under fluctuating Cu supply.

plant biology↗

bioRxiv2025

Protein and genetic interactions between RACK1A and FSD1 modulate plant development and stress granule-dependent response to salt in Arabidopsis.

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 the FSD1 as an interaction partner of the scaffolding protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). Bimolecular fluorescence complementation analyses revealed that RACK1A interacts with FSD1 predominantly in the cytoplasm. Despite elevated FSD1 activity in rack1a mutants, the abundance of FSD1 protein remained unchanged. Computational predictions of interaction interfaces suggested that RACK1A may interfere with the catalytic site of FSD1. Advanced fluorescence microscopy and genetic studies further confirmed localization and relocation patterns of RACK1A and FSD1 during salt stress responses. 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, fsd1-1 rack1a-1 double mutant displayed reduced salt stress resistance compared to rack1a single mutant, which was substantiated by deregulation of ROS levels. RACK1A and FSD1 accumulated in cycloheximide-sensitive structural condensates in the cytoplasm that colocalized with the stress granule marker protein TSN in roots. However, RACK1A knock-out completely abolished salt-stress-dependent relocation of FSD1 to structural condensates, suggesting that RACK1A recruits FSD1 to these stress granules. Thus, this study uncovers an entirely novel mechanism for the regulation of RACK1/FSD1-dependent antioxidant defense in response to salt stress in Arabidopsis.

plant biology↗