SPL12 regulates root architecture, nodulation and nitrogen fixation in Medicago sativa by Silencing AGAMOUS-LIKE 6

The root system architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and resistance to biotic and abiotic stresses. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing microRNA156 (miR156) had increased nodulation, improved nitrogen fixation and longer roots. In alfalfa, transcripts of 11 SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL), including SPL12, are targeted 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.
We determined the role SPL12 in root architecture and nodulation by investigating the phenotypic changes associated with altered expression of SPL12 and by determining SPL12 targets. In this study, we used three SPL12-silencing and -overexpression alfalfa plants to investigate the role of SPL12. Furthermore, we conducted transcriptomics analysis of SPL12 RNAi alfalfa roots and identified differentially expressed genes. Phenotypic analysis showed that alfalfa plants with reduced SPL12 level had an increase in nodulation and root regeneration. Illumina next-generation sequencing-based transcriptomics in root tissues of SPL12-silenced genotypes also revealed SPL12 effects on genes involved in nodulation and nitrogen assimilation pathways. In addition, a gene encoding the transcription factor, AGAMOUS-like MADS box protein 6 (AGL6), was also identified as being directly silenced by SPL12 based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays, suggesting that AGL6 may be involved in regulating alfalfa nodulation. The present findings suggest that SPL12/AGL6 module regulates root development and nodulation, as well as nitrogen uptake and assimilation.