Natural Variations in the Promoter of CHILLING TOLERANCE DIVERGENCE 8 Contribute to the Functional Divergence in the Chilling Tolerance of Rice.

Improvement of the chilling tolerance is one of the key points in rice breeding since it is highly sensitive to chilling stress, which often limits rice production. Here, we identified a quantitative trait locus for chilling tolerance, qCTS8, from a recombinant inbred line (RIL) population derived from crossing Dongxiang wild rice (DXWR) with Guangluai 4 (GLA4). Map-based cloning revealed CHILLING TOLERANCE DIVERGENCE 8 (COLD8) as the underlying major gene. RT-qPCR showed that the COLD8^jap from japonica exhibited a significantly higher expression than the COLD8^ind from indica. Transgenic lines demonstrated that COLD8 negatively regulated chilling tolerance at the seedling stage. Transactivation assays, yeast one-hybrid and EMSA suggested that the transcription factor OsbZIP23 directly bound to the ABA-responsive element (ABRE) of the COLD8 promoter, accounting for the higher expression of COLD8 in NIP than in GLA4. Evolutionary analysis indicated that the japonica allele COLD8^jap originated from Chinese Oryza rufipogon and was selected during japonica domestication. COLD8 encodes a triacylglycerol lipase localized in chloroplast membrane. Loss-of-function mutants of COLD8 exhibited enhanced chilling tolerance associated with increased accumulation of unsaturated fatty acids 16:1 and 18:3. Together, these findings uncover a molecular link between lipid metabolism and chilling adaptation and establish COLD8 as a promising target for breeding chilling-tolerant rice.