The objective of this study was to identify functional genes regulating root branching and drought resistance that could be used in developing drought-tolerant alfalfa (Medicago sativa L.) cultivars. Heterologously expression of an Arabidopsis orphan gene QQS was found to enhance root branching and drought tolerance in alfalfa. By yeast two-hybid (Y2H) and luciferase complementation imaging (LCI) assay, a transcription factor MsNF-YC4 was confirmed as an endogenous interactor of QQS. We speculated it could play a positive role in regulating alfalfa root branching and drought tolerance. Transgenic alfalfa plants overexpressing MsNF-YC4 were generated by Agrobacterium-mediated transformation. Through RNA sequencing (RNA-seq), we noticed that the abscisic acid and auxin signaling pathways were affected in the MsNF-YC4 overexpressing plants. Consistently, exogenous application of indoleacetic acid also enhanced root branching and drought tolerance of alfalfa seedlings. Further, MsNF-YC4 was found to activate the transcription of MsARF8 by yeast one-hybrid (Y1H) and dual-luciferase (LUC) reporter Assay. Silencing of MsARF8 by antisense oligonucleotide (as-ODN) method suppressed both root branching and drought tolerance of alfalfa seedlings. Collectively, we prove that an endogenous transcription factor MsNF-YC4 interacts with foreign protein QQS, and by activating MsARF8 to regulate alfalfa root branching and drought resilience. Our study also provides a regulatory pathway for drought tolerant alfalfa breeding.

Alfalfa (Medicago sativa L.) is the most widely cultivated perennial leguminous forage crop woldwide. MicroRNA156 (miR156) precursor genes from dicotyledonous species are reportedly useful for improving alfalfa plant architecture and abiotic stress resistance. However, there has been no report on whether a miR156 precursor gene from a monocotyledonous species functions in alfalfa. We introduced two tandem precursor genes of miR156, rice Osa-MIR156b and Osa-MIR156c (Osa-MIR156bc), into alfalfa. The expression of miR156 in the transgenic (TG) alfalfa was significantly elevated. Compared to wild-type plants, the TG plants overexpressing miR156 had more branches and leaves and showed improved salt and drought tolerance. Overexpression of miR156 slightly reduced plant height, but the biomass yield of TG plants grown in flowerpots was still increased. Forage quality of TG plants was markedly improved by reduction of acid detergent lignin (ADL) content and increase in crude protein content. The expression of the putative miR156 target genes MsSPL6, MsSPL12, and MsSPL13 in TG plants was repressed by miR156 overexpression, and that of all tested MsSPLs would be sharply increased under drought or salt stress. RNA sequencing revealed that overexpression of miR156 affected the expression of genes associated with abiotic stress resistance and plant development in multiple pathways. This first report of overexpression of monocot miR156 precursors in alfalfa sheds light on the function of miRNA156 precursors from the monocot species rice that could be used for genetic improvement of the dicot forage crop alfalfa.