Multi-omic analysis of maize NILs for chilling tolerance QTLs uncover regulatory and metabolic signatures

Early sowing of maize (Zea mays L.) is increasingly required to mitigate summer drought under climate change, making the acquisition of chilling tolerance a major agronomic challenge. Here, we investigated the molecular and physiological bases of cold tolerance using two maize near-isogenic lines (NILs) differing at two major chilling tolerance quantitative trait loci (QTLs) located on chromosome 4. Plants were exposed to low temperature (14{degrees}C day/10{degrees}C night) for 20 days and analyzed using an integrated multi-omics approach combining transcriptomics, soluble and cell wall proteomics, and metabolomics (primary and specialized metabolites), together with physiological measurements. Univariate and multivariate analyses revealed significant chilling-induced variability across all molecular layers, affecting [~]0.2% of genes, [~]6% of proteins, and a subset of specialized metabolites, while primary metabolites were largely stable. Integrative statistical analyses demonstrated that the soluble and cell wall proteomes contributed most strongly to the genotype effect, highlighting protein-level regulation as a major determinant of chilling tolerance. A restricted 5.15 Mb divergence region on chromosome 4 was sufficient to drive contrasting physiological responses, including differences in photosynthetic charge separation efficiency and leaf development, favoring the chilling-tolerant NIL. Notably, several components of the benzoxazinoid pathway located within the divergence region, including BX1 and associated specialized metabolites (BZX-like glucoside, DIBOA-glucoside-2, HBOA-glucoside-2), were specifically associated with chilling tolerance, suggesting a role in stress signaling and hormonal crosstalk. Overall, this study demonstrates that integrative multi-omics analyses provide a powerful framework to resolve genotype-specific regulatory mechanisms underlying chilling tolerance in maize and to identify candidate molecular targets for breeding. HighlightsO_LIFirst organ-resolved multi-omics dissection of chilling responses in maize NILs. C_LIO_LIA 5.1Mb divergence on chromosome 4 drives major physiological and molecular differences. C_LIO_LIChilling tolerance is linked to more robust photochemical homeostasis and sustained leaf development. C_LIO_LISoluble and cell-wall proteomes dominate the genotype-discriminating -omics signal. C_LIO_LIBenzoxazinoids and defense-related transcriptional modules are differentially activated. C_LIO_LICell wall remodeling enzymes and apoplastic peroxidases emerge as key tolerance players. C_LI