Natural variation in WRKY53 confers cold tolerance at the booting stage between japonica and indica rice.

Natural variation at the WRKY53-C518G SNP drives cold tolerance divergence between rice subspecies.  The japonica-specific WRKY53518C allele, originating from Chinese wild rice and selected in temperate regions, confers cold tolerance by reducing GA repression to protect pollen fertility, and thus provides a valuable target for rice breeding. Cold tolerance at the booting stage (CTB), when inflorescence development begins, is a major factor limiting rice (Oryza sativa L.) productivity and geographical distribution. However, only a few cold-tolerance genes have been identified. We previously showed that WRKY53 regulates CTB by directly associating with the promoters of GA biosynthesis genes and transcriptionally repressing their expression in anthers. In this study, we demonstrate that natural variation in WRKY53 contributes to CTB divergence between japonica and indica subspecies. We identify a functional SNP in the coding region (C518G) that underlies this variation: the japonica-associated WRKY53518C allele confers stronger cold tolerance than the indica-prevalent WRKY53518G allele. Mechanistically, WRKY53518G exhibits higher transcriptional repression activity of GA biosynthesis compared to WRKY53518C, leading to reduced anther GA content and impaired pollen development under low temperature. Population and geographic distribution analyses reveal that the WRKY53518C allele likely originated in Chinese wild rice, was retained in temperate japonica during domestication, and subsequently spread to high-latitude regions, contributing to latitudinal adaptation in rice. Collectively, our findings establish WRKY53 as a key regulator of CTB and highlight the WRKY53518C allele as a valuable genetic resource for enhancing cold tolerance in rice breeding.