Integrated transcriptomic and metabolomic analyses reveal that MsSPHK1 - A sphingosine kinase gene negatively regulates drought tolerance in alfalfa (Medicago sativa L.)

Alfalfa is a valuable forage crop but voluntarily affected by drought. Understanding the mechanisms of drought resistance in alfalfa is crucial for improving resilient cultivars. In our study, we used four distinct alfalfa accessions two drought-tolerance (DT) and two drought-sensitive (DS) and identified transcriptional modules and candidate genes associated with the drought tolerance in the DS from transcriptomic analyses. Our metabolic profiling of 520 metabolites revealed significant variations between the DS and DT groups, particularly in the levels of flavonoids and nucleotides and their derivatives. The integrated analysis of transcriptome and metabolome analysis revealed that the glycine, serine, and threonine metabolism and the sphingolipid metabolism are associated with the drought resistance. When drought stress occurs, MsSRR (MsG 0180002649.01) and MsSPHK1 (MsG 0280006618.01) are significantly up-regulated, L-serine and dihydrosphingosine (DHS) significantly down-regulated in DS. By silencing the MsSPHK1 gene we found the drought resistance was significantly improved. This was evidenced by a significant increase in the activity of antioxidant enzymes such as SOD, POD, and CAT, compared to the control group. Additionally, the photosynthetic rate, stomatal conductance, and efficiency of photosystem II measured by Fv/Fm, phi2 and qL, were significantly higher in the silenced plants than in the control group. In conclusion, our results suggest that the increased level of dihydrosphingosine improves alfalfa resistance to drought stress. Moreover, the negative regulatory role of MsSPHK1 in drought tolerance provides a promising target for genetic manipulation to enhance the resilience of alfalfa to drought stress.