*Corresponding author e-mail: rrmir@skuastkashmir.ac.in; imrouf2006@gmail.com

Wheat is one the most important cereal crops in the world. Cold stress is a major constraint affecting wheat production, particularly in regions with low temperatures. In this study, we undertook a multifaceted approach for cold stress encompassing field screening, membrane stability assessment, biochemical profiling, metabolomic and lipidomic analysis, gene expression studies, proteomics analysis, and a genome-wide association study (GWAS).

The study evaluated 4,560 wheat genotypes over two years, identifying significant genetic diversity for cold stress tolerance. From these, 350 genotypes were selected based on field performance. Membrane stability analysis showed varied results, prompting further selection of 50 genotypes for detailed biochemical profiling. This included assessing reactive oxygen species (ROS) levels, osmoprotectants like proline, and enzymatic antioxidants such as ascorbate peroxidase, superoxide dismutase, guaiacol peroxidase, and catalase.

Correlation analysis of these biochemicals unveiled negative associations between antioxidants and ROS, shedding light on the vital role of antioxidants in alleviating oxidative damage under cold stress conditions. Two contrasting genotypes, SKUA_52 and SKUA_4301, were selected based on their performance in cold stress tolerance. Metabolomic and lipidomic analyses of these genotypes provided valuable insights into the activation of defense mechanisms, highlighting the modulation of key defense hormones such as salicylic acid, jasmonic acid, and abscisic acid in cold-tolerant genotypes.

Additionally, untargeted GC-MS metabolomic analysis revealed upregulation of osmo-protectants and stress-responsive amino acids in the cold-tolerant genotype, while lipidomic analysis underscored the importance of upregulating unsaturated lipids and downregulating saturated lipids to maintain membrane fluidity under cold stress.

Furthermore, gene expression analysis via., qRT-PCR elucidated the coordinated upregulation of specific genes involved in the ICE-CBF-COR pathway, such as COR and CBF genes, in the cold-tolerant genotype. Transcriptomics and proteomics analyses identified highly upregulated genes and proteins associated with antioxidative defense, osmotic adjustment, and signal transduction pathways, providing deeper insights into wheat's cold stress adaptation mechanisms.

Lastly, the GWAS in mini-core set by utilizing 35K Illumina iSelect single nucleotide polymorphism (SNP) array identified 35 marker trait associations and 85 candidate genes associated with cold resistance, offering valuable markers for molecular-assisted breeding efforts aimed at developing cold-tolerant wheat varieties. Overall, this comprehensive study significantly advances our understanding of the complex molecular landscape of cold stress adaptation in wheat, with implications for breeding resilient crop varieties capable of thriving in challenging environments.