Identification of CaOSB2, a CabHLH-79-interacting protein that enhances cold stress tolerance by maintaining cellular homeostasis in Capsicum annuum

Cold stress is a major limiting factor for pepper ( Capsicum annuum L.) production worldwide, yet the molecular regulators governing cold stress tolerance remain incompletely understood. Here, we identified CaOSB2 ( Capsicum annuum Organellar Single-stranded DNA Binding Protein; LOC107863843), a cold-inducible chloroplast-localized protein, as a novel interacting partner of the bHLH transcription factor CabHLH-79, through yeast two-hybrid screening of a cold-stress cDNA library. Bioinformatic analysis revealed that CaOSB2 spans 4975 bp, contains a conserved OB-fold DNA-binding domain, and clusters within the Solanaceae clade in phylogenetic analyses. The CaOSB2–CabHLH-79 interaction was validated by BiFC, split-luciferase complementation, and subcellular co-localization assays, with BiFC signals confirming that the CaOSB2 precursor interacts with CabHLH-79 in the nucleus prior to chloroplast import. β-glucuronidase (GUS) transactivation assays demonstrated that CaOSB2 promotes transcriptional activity of the CabHLH-79 promoter in a sequence-specific manner, establishing a feedforward transcriptional regulatory module. CaOSB2 transcript levels increased progressively ∼10-fold under cold treatment (4°C, 48–72 h). VIGS and transient overexpression in pepper, combined with heterologous overexpression in Arabidopsis thaliana , demonstrated that CaOSB2 positively regulates cold tolerance by enhancing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) antioxidant enzyme activities, reducing reactive oxygen species (ROS) accumulation, alleviating membrane damage, preserving chlorophyll content and photosystem II efficiency (Fv/Fm↑), maintaining stomatal integrity, and promoting proline accumulation. Our findings reveal a novel regulatory mechanism linking organellar stress sensing with nuclear transcriptional responses, and identify CaOSB2 as a promising target for genetic improvement of cold tolerance in pepper and other crops.