Drought response and nodulation are regulated through the miR156/SPL9 gene network in Medicago sativa

Small non-coding RNAs, such as miRNAs, are crucial in regulating gene expression to affect various traits in plants. In particular, miRNA156 (miR156) silences the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family of transcription factors, including SPL9. This study evaluated the role of SPL9 in regulating nodulation and response to drought stress in Medicago sativa (alfalfa). The examination of alfalfa plants with RNAi-silenced SPL9 (SPL9-RNAi) showed that SPL9 positively regulates plant height, stem thickness, and internode length, while negatively regulating shoot branching. SPL9-RNAi alfalfa also had enhanced tolerance to drought stress mediated by elevated anthocyanin content and expression of DIHYDROFLAVONOL 4-REDUCTASE (DFR), an enzyme involved in anthocyanin biosynthesis. Thus, SPL9-mediated downregulation of DFR may represent one strategy to improve drought tolerance in alfalfa. Moreover, phenotypic analyses of SPL9-RNAi plants showed that silencing of SPL9 caused an increase in nodulation. Characterization of phenotypic and molecular parameters revealed that SPL9 modulates nodulation under high nitrate concentration (10 mM KNO3) by regulating the nitrate-responsive genes NR1, NR2, and NRT2.5, as well as the AUTOREGULATION OF NODULATION gene, SUNN. Overexpression of SPL9 upregulated SUNN, NR1, NR2, and NRT2.5, while its silencing downregulated these genes and resulted in a nitrogen-starved phenotype. These results show that SPL9 plays a negative role in regulating nodulation and response to drought stress, which may allow for its use as a molecular tool in the biotechnological improvement of alfalfa and potentially other related crops.