Cold tolerance in Cicer arietinum L. seedlings: multivariate insights into freezing stress resistance.

To enhance the cultivation of chickpeas (Cicer arietinum L.) in regions susceptible to low temperatures, it is imperative to comprehend the physiological and molecular mechanisms that enable plants to tolerate low-temperature stress. This study evaluated eight chickpea genotypes-KAKA and seven cold-resistant lines (MCC194, MCC605, MCC607, MCC613, MCC885, MCC901, and MCC911) from the Mashhad Chickpea Collection (MCC)-under a range of freezing temperatures (0, - 6, -10, - 12, and - 14 °C) using a comprehensive physiological, biochemical, and molecular approach. The plants were cultivated under semi-controlled conditions and underwent a cold acclimation process prior to undergoing freezing treatments. Cold-tolerant genotypes showed higher survival, biomass, and pigment levels under freezing stress, with less electrolyte leakage and more negative osmotic potential. They accumulated more proline, total soluble carbohydrate, phenolic, and antioxidants, especially at - 12 °C. The results of qRT-PCR showed upregulated expression of caCAT, caPOD, and caAPX as antioxidant genes, particularly in genotypes that were tolerant. Chlorophyll fluorescence confirmed better maximum quantum efficiency of photosystem II in these genotypes after cold exposure. Principal component analysis (PCA) and the correlation matrix revealed significant relationships among the measured indices. Partial least squares structural equation modeling (PLS-SEM) indicated that enzymatic activity was the strongest positive predictor of plant dry weight (β = 0.404), while membrane damage had a strong negative effect on it (β = -0.913). Furthermore, comprehensive membership function analysis ranked genotype MCC911 with the highest evaluation value (0.737) as the most tolerant and genotype KAKA with the lowest value (0.222) as the most sensitive. The findings indicate that maintaining membrane integrity, accumulating osmotically compatible compounds, and, particularly, activating the enzymatic antioxidant system are key mechanisms of freezing tolerance in chickpeas. Genotypes MCC911 and MCC901 are identified as promising candidates for use in breeding programs aimed at developing cold-tolerant cultivars for autumn planting. Collectively, these findings provide a detailed physiological and molecular framework for cold stress adaptation in chickpea and identify potential markers for breeding cold-resilient cultivars.