Abstract: Cold damage during the seedling and reproductive stages has a pronounced impact on rice development and yield. Although significant progress has been achieved in understanding the physiological and molecular mechanisms underlying rice responses to cold stress, the mechanisms of cold stress perception and adaptation in rice remain mostly unclear. Here, we report the functional study of a cold-responsive gene named LENG, which encodes a plasma membrane-localized leucine-rich repeat (LRR) receptor-like pseudokinase. Gene knockout and overexpression analyses indicated that LENG positively regulates chilling tolerance in rice seedlings. LENG interacts with the plasma membrane-localized cytoplasmic protein kinase, cold-responsive protein kinase 1 (OsCRPK1). Knockout of OsCRPK1 in wild-type and leng mutants elevates rice chilling tolerance, indicating that OsCRPK1 plays a negative role and acts downstream of LENG. In vitro kinase assays revealed that OsCRPK1 is an active protein kinase, but it does not phosphorylate LENG, whereas LENG does not have a kinase activity, but it suppresses the kinase activity of OsCRPK1. In addition, LENG interferes with the interaction between OsCRPK1 and the rice 14-3-3 protein OsGF14d (G-box factor 14-3-3 homolog d), which is known to be a positive regulator of chilling tolerance. The polymorphisms in the promoter and coding region of LENG in japonica and indica rice were correlated with the differential gene expression patterns and chilling tolerance in response to chilling treatment. Taken together, these findings suggest that LENG regulates rice chilling tolerance by modulating the kinase activity of OsCRPK1 and eventually the phosphorylation status of OsGF14d protein. The polymorphisms in LENG provide a selection marker for molecular breeding of rice with improved chilling tolerance.

Key words: chilling tolerance, domestication, pseudokinase, receptor‐like kinase, rice