Characterization of the Role ofSPL9in Drought Stress Tolerance inMedicago sativa

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality.Medicago sativa(alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silencedMsSPL9(SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. InSPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected.SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating thatSPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly,SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting thatMsSPL9may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targetingMsSPL9is a suitable means for improving alfalfa resilience towards drought conditions.