Natural variation in the chickpea metabolome under drought stress

Chickpea is the world's fourth largest grown legume crop, which significantly contributes to food security by providing calories and dietary protein globally. However, the increased frequency of drought stress has significantly reduced chickpea production in recent years. Here, we have performed a field experiment with 36 diverse chickpea genotypes to evaluate grain yield, photosynthetic activities and molecular traits related to drought stress. For metabolomics analysis, leaf tissue was collected at three time points representing different pod-filling stages. We identified L-threonic acid, fructose and sugar alcohols involved in chickpea adaptive drought response within the mid-pod-filling stage. A stress susceptibility index for each genotype was calculated to identify tolerance capacity under drought, distributing the 36 genotypes into four categories from best to worst performance. To understand how biochemical mechanisms control different traits for genetic improvement, we performed a differential Jacobian analysis, which unveiled the interplay between various metabolic pathways across three time points, including higher flux towards inositol interconversions, glycolysis for high-performing genotypes, fumarate to malate conversion, and carbon and nitrogen metabolism perturbations. Metabolic GWAS (mGWAS) analysis uncovered gene candidates involved in glycolysis and MEP pathway corroborating with the differential biochemical Jacobian results. Accordingly, this proposed data analysis strategy bridges the gap from pure statistical association to causal biochemical relations by exploiting natural variation. Our study offers new perspectives on the genetic and metabolic understanding of drought tolerance-associated diversity in the chickpea metabolome and led to the identification of metabolic control points that can be also tested in other legume crops.