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Background Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop’s sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. Results To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40–1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40–1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40–1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. Conclusions Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40–1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Heat stress and extreme temperatures negatively affect plant development by disrupting regular cellular and biochemical functions, ultimately leading to reduced crop production. Alfalfa (Medicago sativa) is an important forage crop grown worldwide as forage for livestock feed. Limiting the effects of abiotic stress by developing alfalfa cultivars that are stress tolerant would help mitigate losses to crop production. Members of the microRNA156 (miR156) family regulate the Squamosa Promoter-Binding Protein-Like (SPL) genes that in turn impact plant growth and development by regulating downstream genes in response to various abiotic stresses. In this study, alfalfa with miR156 overexpression and SPL13 RNAi knockdown show increased tolerance to heat stress (40°C). Transgenic plants show high water potential and increased non-enzymatic antioxidant content under heat stress. Moreover, anthocyanin content and chlorophyll abundance were increased under stress. Expression of some important transcription factors and downstream genes involved in abiotic stress response were altered in miR156-overexpressing genotypes under heat. Taken together, our results demonstrate that the miR156/SPL13 network contributes to improving heat stress tolerance in alfalfa.