Genome-wide identification of the P5CS gene family in Psammochloa villosa and functional characterization under drought stress

Abstract Psammochloa villosa is a perennial herbaceous plant belonging to tribe Stipeae (Poaceae). It is mainly distributed in the arid and sandy Inner Mongolia Plateau and adjacent areas, demonstrating remarkable resilience to arid, windswept sandy environments. Proline is shown to be involved in plant development under normal conditions and contributes to resistance against both biotic and abiotic stresses. Δ1-pyrroline-5-carboxylate synthetase (P5CS), one of the two main enzymes in the proline biosynthesis pathway of the glutamate precursor, has been demonstrated to play a significant role in proline accumulation in plants under water stress. In this study, we identified nine PviP5CS1-PviP5CS9 genes in the P. villosa genome, which exhibit the closest phylogenetic relationship to same gene family in the closely related Neotrinia splendens, a dominant plant species of temperate degraded grassland in Inner Mongolia. The promoter region of PviP5CS harbors different cis-regulatory elements involved in light and hormonal regulation, as well as stress responses. Collinearity analysis revealed that the expansion of the PviP5CS gene family occurred via segmental duplication and underwent purifying selection during species evolution. In P. villosa, P5CS genes exhibited distinct expression patterns across different tissues and showed differential regulation in response to drought stress, suggesting potential tissue-specific roles in proline biosynthesis and adaptive stress tolerance mechanisms. Most importantly, functional characterization in the model organism Arabidopsis thaliana demonstrated that overexpression of PviP5CS4 significantly enhances drought tolerance, which is attributed to elevated proline accumulation and increased antioxidant enzyme activity. In addition, we performed a yeast one-hybrid (Y1H) analysis to investigate the potential interactions between P. villosa PviP5CS4 and several candidate transcription factors, including DREB3, GBF3, serine/threonine protein kinase, bZIP, and pyruvate phosphate dikinase. These findings provide novel scientific insights into the molecular mechanisms underlying the proline biosynthesis pathway in P. villosa and reveal its functional role under drought stress, offering valuable targets for improving drought tolerance in P. villosa and related species through genetic engineering or breeding strategies.