Introgression of cold tolerance genes from Cicer reticulatum ladiz. to C. arietinum L. and relationships between potential physiological and biochemical selection criteria

Metabolic responses to low temperature and freezing stress play a critical role in determining cold tolerance in plants. However, since most previous studies have focused on cold acclimation, information on metabolic changes occurring under freezing conditions and potential selection criteria for distinguishing tolerant and sensitive genotypes remains highly limited. In this study, the freezing tolerance levels of 163 RILs derived from an interspecific cross between Cicer arietinum L. (CA 2969) and C. reticulatum (AWC 602) were evaluated under freezing conditions. Among these chickpea lines, five freezing-tolerant and five sensitive RILs were selected, and biochemical changes occurring under pre-freezing (4°C), freezing (-12°C), and post-freezing conditions were comprehensively investigated using multivariate statistical approaches and targeted metabolite analyses. The metabolic profiles of tolerant (AWC 602 and tolerant RILs) and sensitive (CA 2969 and sensitive RILs) genotypes were compared based on 11 key metabolites consisting of organic acids [citric acid (CA), succinic acid (SA), fumaric acid (FA), malic acid (MA), and malonic acid (MoA)], fatty acids [palmitic acid (PA), stearic acid (StA), linoleic acid (LA), and linolenic acid (LNA)], and amino acids [aspartic acid (Asp) and tryptophan (Trp)]. PLS-DA analyses demonstrated a clear separation between tolerant and sensitive groups across all treatment conditions. VIP analyses revealed that MoA, MA, and LNA were the most influential variables distinguishing the groups under all conditions. Heatmap analysis showed higher accumulation of LA, MoA, Trp, LNA, MA, and FA in tolerant genotypes pre-freezing; all metabolites except SA during freezing; and CA, MoA, PA, LA, FA, MA, and LNA post-freezing. Metabolite-level evaluation of freezing responses indicated pronounced increases in organic and fatty acids in tolerant genotypes, whereas sensitive genotypes displayed decreases or minimal changes, reflecting a limited and insufficient metabolic adaptation. The marked reduction in metabolite levels during the post-freezing recovery phase in tolerant genotypes suggests their ability to rapidly restore homeostatic metabolic regulation after stress. Overall, the findings indicate that cold-tolerant chickpea genotypes develop a more effective stress adaptation through mechanisms such as maintaining membrane fluidity, enhancing energy production, and reprogramming amino acid metabolism. This study contributes to the identification of metabolic markers associated with cold tolerance and provides valuable biochemical indicators for chickpea breeding programs.