Serotonin and 24-epibrassinolide enhance physiological performance and saffron quality under water deficit: evidence from two-year field and multivariate analyses

Water deficit constitutes a major constraint on saffron productivity and quality, particularly in arid and semi‑arid regions. Although serotonin (SER) and 24‑epibrassinolide (EBL) have individually been reported to improve plant responses to abiotic stress, their combined effects on saffron under field water deficit remain poorly understood. To address this gap, a two‑year field experiment was conducted to evaluate foliar SER (50 µM) and EBL (6 ppm), individually and in combination, under normal and deficit irrigation regimes. Water deficit reduced flower weight, stigma dry weight, leaf dry weight, relative water content, and total chlorophyll content, while increasing hydrogen peroxide, malondialdehyde, and electrolyte leakage. The application of SER and EBL, particularly in combination, improved physiological and biochemical traits associated with stress adaptation. Under deficit irrigation, the co-application increased flower weight by 51%, stigma dry weight by 52%, relative water content by 32%, and total chlorophyll content by 55% compared with untreated stressed plants. The combined treatment was also associated with higher catalase activity, phenol and flavonoid contents, compatible solute accumulation, and nitric oxide, together with lower oxidative damage indicators. Multivariate analysis revealed distinct physiological and defense‑related dimensions. Principal component analysis explained 88.4% of variation, with PC1 (66%) primarily associated with physiological traits and PC2 (22.4%) linked to antioxidant defense and secondary metabolism. These patterns indicated associations among growth, physiological, biochemical, and quality-related traits. Positive correlations between stigma yield and photosynthetic pigments suggested an association between pigment status and saffron productivity. Additionally, combined application increased crocin, picrocrocin, and safranal contents by 9.2%, 24%, and 55%, respectively, under deficit irrigation versus untreated stressed plants. The combined application of SER and EBL led to greater improvements in all measured traits under water deficit compared to individual treatments, highlighting its potential as a promising agronomic strategy to enhance saffron productivity and quality under water-limited conditions. The complementary effects between these two compounds suggest the feasibility of developing combined biostimulant formulations for high-value crops such as saffron. Although the experiment was conducted over two seasons, multi-location validation is necessary to confirm the generalizability of these findings.