Exploring metabolite profiles and phenylpropanoids biosynthesis gene expression in Pimpinella anisum L. under water deficit stress.

Water deficit stress (WDS) is a major environmental constraint limiting crop productivity by affecting physiological and biochemical processes. This study aimed to investigate the physiological responses, secondary metabolite accumulation, antioxidant activity, and gene expression associated with drought tolerance in two anise (Pimpinella anisum L.) genotypes, Kerman (tolerant) and Gilan (sensitive). Under WDS, Kerman exhibited significantly higher osmotic potential (-2.80 MPa) and relative water content (80.6%) compared to Gilan (-6.19 MPa and 38.2%, respectively), indicating superior water retention capacity. Electrolyte leakage was significantly lower in Kerman (25.9%) than in Gilan (53.2%) after 12 days of stress, suggesting enhanced membrane stability. Drought stress markedly increased total phenolic content (TPC), total flavonoid content (TFC), and anthocyanin accumulation, with peak values in Kerman reaching 241.45 mg GAE g- 1 DW, 27.33 mg QE g- 1 DW, and 133.25 µmol g- 1 FW, respectively. Antioxidant capacity, assessed by DPPH scavenging and reducing power assays, was significantly higher in Kerman, showing up to a 70.3% increase in DPPH activity and a 2.11-fold rise in reducing power. HPLC profiling revealed elevated levels of rutin (7.08 mg g- 1 DW), caffeic acid (4.5-fold increase), and apigenin (2.87-fold increase) in Kerman under WDS. Significant positive correlations were observed between TPC, TFC, and antioxidant parameters (r ≥ 0.92**), confirming the functional role of polyphenols in oxidative stress mitigation. Gene expression analysis demonstrated coordinated upregulation of key phenylpropanoid pathway genes (PAL, 4CL, F3H, CHI, and 3GT), particularly in Kerman, with PAL and F3H showing 3.51- and 7.1-fold increases, respectively, after 9 days of stress. These findings suggest that enhanced osmotic adjustment, polyphenolic biosynthesis, and gene co-regulation contribute to the superior drought tolerance of the Kerman genotype.