Characterization of the Role of miR156-SPL12 Regulatory Module in Root Architecture and Stress Response in Medicago sativa (alfalfa)
Abstract
The highly conserved plant microRNA156 (miR156) regulates various aspects of plant development and stress response by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. The Hannoufa lab previously showed that transgenic alfalfa (Medicago sativa L.) plants overexpressing miR156 display increased nodulation, nitrogen fixation, and root regenerative capacity during vegetative propagation. In alfalfa, transcripts of 11 SPLs, including SPL12, are targeted by miR156. Our understanding of the functions of SPLs and their mode of action in alfalfa’s nodulation and nitrogen fixation is still elusive, and thus this study was aimed at filling this gap in knowledge.
Here, I carried out a functional characterization of SPL12 by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and ‑overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 and AGL21 that encode respective AGAMOUS-like MADS box transcription factors were identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Phenotypic and molecular analysis showed that silencing AGL6 also increased nodulation in alfalfa.
The role of SPL12 and AGL6 in nodulation was also investigated under osmotic stress using SPL12-RNAi and AGL6-RNAi plants, where the SPL12/AGL6 module appears to have a negative role in maintaining nodulation. Additionally, examination of the role of SPL12 in nodulation under nitrate treatment, suggested that SPL12 may regulate nodulation under nitrate treatment in alfalfa by targeting AGL21. Moreover, I also investigated the role of the alfalfa SPL12 homolog, LjSPL12, in the model legume Lotus japonicus for nodulation and found that LjSPL12 negatively affects the nodulation in spl12 mutant plants. Taken together, these results suggest that SPL12, AGL6 and AGL21 form a genetic module that regulates root development and nodulation in alfalfa.
Considering the important role already shown for another SPL, SPL13, in vegetative state transition and abiotic stress tolerance in alfalfa, I also successfully applied the CRISPR/Cas9 technique to edit the SPL13 gene in alfalfa, however, the overall efficiency was low.