Integrated transcriptomic and metabolomic analyses reveal that MsMYC2-A core transcription factor of the jasmine acid signaling pathway positively regulates NaHCO3-base tolerance in alfalfa (Medicago sativa L.).

Alfalfa is recognized as a high-quality forage crop exhibiting exceptional saline-alkaline tolerance. However, in China's Songnen Plain region, soda saline-alkaline soils dominated by NaHCO3 constitute a critical limiting factor for local ecosystems and alfalfa production. Consequently, enhancing alfalfa adaptation to saline-alkaline stress is considered essential for vegetation restoration in degraded lands and sustainable development of animal husbandry. This study employed physiological, transcriptomic, and metabolomic techniques to investigate the complex response mechanisms of alfalfa cultivated in vermiculite under varying NaHCO3 concentrations (100, 200, and 300 mmol L-1). Preliminary functional characterization was performed for key genes within critical pathways. The results indicated that alfalfa plant growth was progressively inhibited and PSII photochemical activity was significantly reduced with increasing NaHCO3 concentrations. Transcriptomic and metabolomic analyses revealed that differentially expressed genes (DEGs) and metabolites (DMs) in NaHCO3-stressed alfalfa leaves were predominantly enriched in KEGG pathways including starch and sucrose metabolism, proline biosynthesis, and plant hormone signal transduction. Under NaHCO3 stress, substantial accumulations of proline, sucrose, and starch were observed, with concomitant upregulation of genes associated with these metabolic pathways. Genes involved in the biosynthesis and signaling of abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) were activated. Weighted Gene Co-expression Network Analysis (WGCNA) of RNA-Seq data identified the JA signaling pathway, particularly the key transcription factor MsMYC2 (Myelocytomatosis proteins 2), as a pivotal contributor to saline-alkaline tolerance. MsMYC2 was localized to the nucleus and functioned as a transcriptional activator, with its expression significantly induced by NaHCO3 stress. Heterologous expression of MsMYC2 alleviated NaHCO3-mediated growth inhibition in yeast and enhanced saline-alkaline tolerance in transgenic tobacco. Specifically, compared to wild-type (WT) plants under stress, MsMYC2-overexpressing lines exhibited increased accumulations of osmolytes (soluble sugars and proline), mitigated photoinhibition, and reduced oxidative damage. Furthermore, ectopic expression of MsMYC2 activated NbOPR (a key JA biosynthesis gene) while suppressing NbJAZ (a JA signaling repressor). These findings provide novel insights into improving alfalfa resilience to saline-alkaline stress and present promising molecular targets for breeding tolerant cultivars.