Natural variation in OsbHLH150 confers chilling tolerance in rice by increasing ABA biosynthesis

Elucidating the regulatory networks underlying the rice response to chilling stress is critical for breeding resilient varieties, yet the molecular mechanisms governing chilling tolerance remain incompletely understood. Here, through map-based cloning, we identified a chilling tolerance QTL, CSS12b, using chromosome segment substitution lines (CSSLs) derived from a cross between Koshihikari (japonica, chilling-tolerant) and Nona Bokra (indica, chilling-sensitive), and identified OsbHLH150 as its causal gene. Six single-nucleotide polymorphisms in the coding sequence of the two OsbHLH150 alleles underlie their divergent effects on chilling tolerance. OsbHLH150 encodes a basic-helix-loop-helix (bHLH) family transcription factor (TF). Knockout of the Koshihikari OsbHLH150 allele enhances sensitivity to chilling, whereas OsbHLH150 overexpression enhances chilling tolerance. Further mechanistic analyses uncovered a phosphorylation-dependent regulatory axis of OsbHLH150: under chilling stress, OsMAPK3 physically interacts with and phosphorylates OsbHLH150, thereby stabilizing OsbHLH150 and enhancing its ability to activate OsNCED3, which encodes a rate-limiting enzyme in ABA biosynthesis. This cascade elevates ABA biosynthesis and thereby enhances chilling tolerance in rice. This OsMAPK3-OsbHLH150-OsNCED3 module represents a newly identified kinase-TF-metabolic enzyme cascade in rice chilling adaptation. More importantly, the valine residue at position 471 of OsbHLH150 is critical for its transactivation activity, and substitution of this valine with leucine impairs this activity. Moreover, the introduction of OsbHLH150 into indica rice significantly improved chilling tolerance at both the seedling and booting stages. Our findings not only elucidate how allelic variation modulates ABA-mediated chilling resilience but also provide a precise target for breeding chilling-resilient rice varieties.