Evaluation of chickpea (Cicer arietinum L.) varieties under ZnSO4 stress: Insights from growth, physiological, biochemical and metabolomics.

Although plants require an appropriate level of zinc (Zn) for normal growth and development, excessive accumulation, and high concentrations of it can impair plant growth, and disrupt physiological and metabolic processes. In this study, we aimed to identify zinc-tolerant chickpea varieties using multivariate analysis, and to elucidate the contribution of primary metabolites to zinc tolerance. We investigated multiple growth, physiological and biochemical parameters, including plant height, fresh weight and dry weight, Zn uptake, root-to-shoot ratio, relative shoot water content, tolerance index, chlorophyll contents, hydrogen peroxide (H2O2), malondialdehyde (MDA), electrolyte leakage (EL) and enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductases (GR), proline, soluble sugars, and total protein in three chickpea varieties: ICCV89310 (IC8), NC234 (NC2), and ICCV89323-B (IC8-B) under various ZnSO4 concentrations (Ck, 50, 100, and 150 µM) in a hydroponic medium. Metabolite profiling of shoot samples was performed using GC-MS, while Zn accumulation in roots and shoots was quantified with ICP-OES. The results revealed significant differences among the tested varieties. For instance, IC8 and NC2 exhibited robust growth, and physiological and biochmeical performance under elevated ZnSO4 stress, indicating greater Zn tolerance, while IC8-B was the most sensitive variety. Additionally, IC8 and NC2 exhibited greater enzymatic and non-enzymatic antioxidant capacity compared to IC8-B across the Zn treatments. Meanwhile, forty-six responsive metabolites of different categories, including organic acids, amino acids, amines, alcohols, and sugars were quantified in the shoots of three chickpea varieties. Noticeably, the relative tolerance potential of IC8 and NC2 compared to IC8-B was asociated with distinct metabolic adjusments, including changes in histidine, asparagine, tryptophan, allantoin and antioxidant-related metabolites. Overall, this study provides an integrated physiological, biochemical, and metabolomic basis for identifying Zn-tolerant, and Zn-sensitive chickpea germplasm. IC8-B may serve as a sensetive refrence variety for comparative Zn-tolerance studies or to evaluate other chickpeas tolerance capacity, while IC8 and NC2 represent promising germplasm for Zn-contaminated environments; howver, field-scale confirmation remains necessary before practical application.