A major gene for chilling tolerance variation in Indica rice codes for a kinase OsCTK1 that phosphorylates multiple substrates under cold

Summary Rice is susceptible to chilling stress. Identifying chilling tolerance genes and their mechanisms are key to improve rice performance. Here, we performed a genome‐wide association study to identify regulatory genes for chilling tolerance in rice. One major gene for chilling tolerance variation in Indica rice was identified as a casein kinase gene Os CTK1 . Its function and natural variation are investigated at the physiological and molecular level by its mutants and transgenic plants. Potential substrates of OsCTK1 were identified by phosphoproteomic analysis, protein–protein interaction assay, in vitro kinase assay, and mutant characterization. OsCTK1 positively regulates rice chilling tolerance. Three of its putative substrates, acidic ribosomal protein OsP3B, cyclic nucleotide‐gated ion channel OsCNGC9, and dual‐specific mitogen‐activated protein kinase phosphatase OsMKP1, are each involved in chilling tolerance. In addition, a natural Os CTK1 chilling‐tolerant (CT) variant exhibited a higher kinase activity and conferred greater chilling tolerance compared with a chilling‐sensitive (CS) variant. The CT variant is more prevalent in CT accessions and is distributed more frequently in higher latitude compared with the CS variant. This study thus enables a better understanding of chilling tolerance mechanisms and provides gene variants for genetic improvement of chilling tolerance in rice.