SPL13 regulates shoot branching and flowering time in Medicago sativa.

KEY MESSAGE: Our results show SPL13 plays a crucial role in regulating vegetative and reproductive development in Medicago sativa L. (alfalfa), and that MYB112 is targeted and downregulated by SPL13 in alfalfa. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing microRNA156 (miR156) show a bushy phenotype, reduced internodal length, delayed flowering time, and enhanced biomass yield. In alfalfa, transcripts of seven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL13, are targeted for cleavage by miR156. Thus, association of each target SPL gene to a trait or set of traits is essential for developing molecular markers for alfalfa breeding. In this study, we investigated SPL13 function using SPL13 overexpression and silenced alfalfa plants. Severe growth retardation, distorted branches and up-curled leaves were observed in miR156-impervious 35S::SPL13m over-expression plants. In contrast, more lateral branches and delayed flowering time were observed in SPL13 silenced plants. SPL13 transcripts were predominantly present in the plant meristems, indicating that SPL13 is involved in regulating shoot branch development. Accordingly, the shoot branching-related CAROTENOID CLEAVAGE DIOXYGENASE 8 gene was found to be significantly downregulated in SPL13 RNAi silencing plants. A R2R3-MYB gene MYB112 was also identified as being directly silenced by SPL13 based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays, suggesting that MYB112 may be involved in regulating alfalfa vegetative growth.