Genome-wide association study, network analysis, and reverse genetics pinpoint novel genes associated with seedling root growth variation of Arabidopsis thaliana under drought

Development of drought-resilient crops requires a precise understanding of molecular signaling in the root, the primary organ encountering drought. This study unraveled novel genetic loci contributing to drought tolerance by exploiting the natural variation in seedling root growth of Arabidopsis thaliana under PEG-induced drought stress. Through a genome-wide association study (GWAS) of 207 worldwide Arabidopsis thaliana ecotypes from regions with varied rainfall, 68 protein-coding genes were identified, associated with the top 50 single-nucleotide polymorphisms ( P < 10 ^{− 3} ), explaining 63% of the observed variation in root length. Subsequent network and functional enrichment analyses of the GWAS-delineated genes demarcated key biological processes crucial for maintaining root growth under drought, including DNA repair, tRNA editing, protein folding and quality control, cell cycle regulation, stress granule assembly, and the pyridoxal 5′-phosphate (PLP) salvage pathway regulating oxidative stress in roots. Expression level polymorphisms, promoter cis -element variations, and amino acid substitutions affecting predicted protein dynamics, with phenotype and climate associations, were identified. Finally, reverse genetic evaluation using T-DNA insertion knockout/knockdown mutants confirmed a direct association of the identified candidate genes, AT1G06690 (PLP pathway), AT4G26990 and RBP45C (stress granule assembly), ACD55.5 (protein folding), PCMP-A4 (RNA modification), SKS6 and ANAC094 (cell wall remodeling), and INCENP (cell cycle regulation), with seedling drought tolerance. Furthermore, the knockdown of AT1G06690 led to higher accumulation of hydrogen peroxide in root tissues, inhibiting growth. Future translation of the current findings into crops will provide new tools for the improvement of drought tolerance by modulating root traits through biotechnology and breeding.