A Unique Morpho‐Physiological, Metabolomics, and Transcriptional Shift Under Combined Drought and Salinity Stress in <scp><i>Stevia rebaudiana</i></scp> Bertoni

Stevia rebaudiana, a major source of plant-derived natural low/non-calorie sweeteners (LNCSs), steviol glycosides (SGs), faces severe economic consequences due to environmental stresses like drought (D), particularly when combined with salinity (S). In the present study, integration of 936 million high-quality transcriptional reads with morpho-physiological (eight parameters) and metabolomics (targeted, untargeted) analysis has successfully explicated the combined drought and salinity (CDS) stress responses in S. rebaudiana. Interestingly, non-glandular trichomes recorded a significant growth under CDS, suggesting a potential adaptive response of stevia at 15 days after treatment (15 DAT). Additionally, unique differential gene expression recorded under CDS (9643 DEGs) emphasizes a distinct transcriptional response compared to single (D) and (S) stress in stevia. Moreover, stress-specific specialized metabolism, including sphingolipid, arginine and proline, and biosynthesis of various plant secondary metabolites revealed significant enrichment under CDS. Furthermore, root-specific significant expression of genes involved in stress response (AKT1, HAK12, P5CS), stress perception (WAK5, LRR-RLK, CERK1), and signal transduction (PP2C13, MAPKK, CML38) underscores the severity of CDS. Subsequently, the CDS-specific nucleotide variations were validated with resequencing data of contrasting susceptible and tolerant CDS stevia cultivars, confirming amino acid changes in key genes (AKT1:Pro695Ala, MAPKK5:His317Ile, HAK12:Tyr370Asn, P5CS: Lys495Arg), which suggests wider translational implications. This study represents the first report on the creation of CDS-specific genomic resources with the identification of key genes and regulators that will expedite the breeding of climate-resilient stevia cultivars with optimum accumulations of SGs for commercial utilization.