MsNF-YC4 positively regulates root branching and drought resistance in alfalfa by activating MsARF8

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.