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

Elucidating the regulatory networks underlying rice response to chilling stress is critical for breeding resilient varieties, yet the molecular mechanisms governing chilling tolerance remain elusive. Here, through map-based cloning, we successfully cloned 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), identifying OsbHLH150 as its causal gene. Six SNPs in the coding sequences of the two OsbHLH150 alleles account for their allelic divergence in chilling tolerance. OsbHLH150 encodes a bHLH family transcription factor (TF). Knockout lines of OsbHLH150 from Koshihikari allele exhibit enhanced sensitivity to cold, while its overexpression lines enhance chilling tolerance in rice. Further investigations mechanistically uncovered a phosphorylation-dependent regulatory axis of OsbHLH150: under chilling stress, OsMAPK3 phosphorylates and stabilizes OsbHLH150 through physical interaction, which in turn directly promotes the OsbHLH150-driven activation of ABA biosynthetic rate-limiting enzyme OsNCED3, thereby enhancing chilling tolerance in rice through elevating ABA biosynthesis. This OsMAPK3-OsbHLH150-OsNCED3 module represents the novel reported kinase-TF-metabolic enzyme cascade in rice chilling adaptation. More importantly, the valine residue at 471 of OsbHLH150 protein is critical for its transactivation activity, and the activity is impaired by substitution of valine with leucine. Moreover, 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 precision target for chilling-resilient rice breeding.