Background: Cold stress is a major environmental constraint limiting wheat productivity worldwide. Although numerous cold-responsive pathways have been identified, the molecular basis of differential cold tolerance among genetically related wheat lines remains poorly understood. In this study, two wheat sibling lines derived from a single progeny plant of the same parental cross, Luyan951 (cold-tolerant) and Luyan955 (cold-sensitive), were employed to investigate the regulatory mechanisms of cold adaptation through integrated physiological, transcriptomic, and metabolomic analyses. Results: Physiological assays revealed that Luyan951 exhibited markedly enhanced cold tolerance, with a survival rate of 52.67% following cold treatment compared with 20.67% in Luyan955. This enhanced tolerance was accompanied by 1.90–2.41-fold greater increases in antioxidant enzyme activities (SOD, CAT, and POD) and 1.84–4.50-fold greater accumulation of proline and soluble sugars relative to Luyan955, along with substantially lower MDA accumulation. Transcriptomic and metabolomic analyses identified phenylpropanoid biosynthesis and jasmonic acid (JA) signaling as key pathways associated with cold adaptation. Compared with Luyan955, cultivar Luyan951 exhibited stronger activation of these pathways under cold stress. Key genes involved in phenylpropanoid biosynthesis ( CAD , and 4CL ) and JA signaling ( JAZ , MYC2 ) were significantly upregulated in Luyan951, as confirmed by qRT-PCR. Bioinformatic analyses further suggested that AP2/ERF transcription factors may act as upstream regulators of these pathways. Furthermore, subcellular localization and transcriptional activation experiments confirmed the nuclear localization and transactivation function of three AP2/ERF genes ( TraesCS5D02G318400 , TraesCS6A02G381000 , TraesCS6D02G366100 ). Conclusions: Our findings indicate that the phenylpropanoid biosynthesis pathway plays a significant role in the cold tolerance of wheat, and together with the jasmonic acid signaling pathway, it forms a crucial regulatory network. This network promotes the scavenging of reactive oxygen species, maintains osmotic homeostasis, and stabilizes metabolism under low-temperature stress. Integrated analyses further suggest that this network may be coordinated by upstream ERF transcription factors. These findings provide comprehensive insights into the molecular mechanisms of wheat cold adaptation and offer valuable candidate genes and pathways for the genetic improvement of cold tolerance in wheat.

Introduction: The CCT (CONSTANS, CO-LIKE, and TIMING OF CAB EXPRESSION 1) gene family regulates flowering time, circadian rhythm maintenance, and abiotic stress responses. However, previous investigations in wheat have relied on single-reference genomes, limiting the understanding of inter-varietal structural variation. Methods: Here, we performed a pan-genomic analysis of the CCT gene family across 16 wheat accessions. Results and Discussion: Copy numbers varied from 27 in diploid Triticum urartu to 69–103 in modern common wheat cultivars, with subgenomic contributions displaying an A > D > B gradient. Orthogroup classification based on the 12 T. aestivum cultivars identified 22 Core, 9 Softcore, 2 Dispensable, and 2 predicted Private orthogroups, all under strong purifying selection; the Private orthogroups require assembly-level validation because most cultivars used projected gene models. Protein-protein interaction network analysis identified a small set of highly connected nodes, with enrichment in circadian rhythm and photoperiodic flowering pathways, though this is largely expected given the query sequences; DeepGOPlus annotation further supported their roles as transcriptional regulators. Hexaploid wheat possessed longer mRNA lengths than tetraploids and diploids, but this may partly reflect annotation gaps. Promoter cis -elements were conserved across subgenomes, while paired analysis of homoeologous triads revealed significant expression divergence among subgenome copies. Tissue-specific profiling in Chinese Spring identified grain-preferential genes enriched for seed-specific elements, and stress treatments revealed candidate cis-elements, with WUN-motif and CGTCA-motif remaining significant after FDR correction. These findings provide a comprehensive view of CCT gene family evolution and regulatory diversification, offering candidate genes for stress tolerance and yield improvement in wheat.

Pumilio (Pum)-family proteins are evolutionarily conserved RNA-binding proteins that regulate post-transcriptional processes; however, their specific functions in staple crops, for example, wheat (Triticum aestivum), remain poorly understood. We identified 56 TaPum genes and classified them into three phylogenetic subgroups (I–III). The larger TaPum family size in allohexaploid wheat was associated with retention of A-, B-, and D-subgenome homoeologs, segmental duplication, and tandem duplication. After separating homoeologous pairs, 17 segmental duplication pairs and five tandem duplication pairs were identified, and ratios of nonsynonymous to synonymous substitutions (Ka/Ks) below 1 were consistent with purifying selection. Subgroup-specific motifs and the uneven distribution of cis-acting elements, particularly abscisic acid (ABA)- and methyl jasmonate (MeJA)-responsive elements, suggested potential divergence in promoter regulatory architecture. Transcriptomic profiling and independent quantitative reverse transcription-polymerase chain reaction analysis showed more consistent response of TaPum genes to salt stress than to cold or drought stress. Salt stress strongly promoted TaPum-III-5b expression, and its encoded protein exhibited nuclear and cytoplasmic localization. Heterologous overexpression of TaPum-III-5b in Arabidopsis thaliana increased salt tolerance, as indicated by enhanced survival; augmented peroxidase, superoxide dismutase, and catalase activities; and diminished accumulation of reactive oxygen species and malondialdehyde. Electrophoretic mobility shift assay (EMSA) demonstrated sequence-specific binding of TaDREB2B to a dehydration-responsive element (DRE)-containing sequence in the TaPum-III-5b promoter, whereas dual-luciferase (Dual-LUC) analysis showed activation of the wild-type promoter. Together, these results support a proposed regulatory framework in which TaPum-III-5b may contribute to salt-stress responses through as-yet-unidentified downstream RNA targets. This study characterizes the Pum gene family in wheat and identifies the TaDREB2B-TaPum-III-5b module as a candidate regulatory pathway associated with salt-stress responses. Its potential application in wheat improvement requires further validation in wheat.