Electronic Thesis and Dissertation Repository

Thesis Format



Doctor of Philosophy




Hannoufa, Abdelali

2nd Supervisor

Kohalmi, Susanne



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.

Summary for Lay Audience

With an increasing global population that is projected to reach nine billion people by 2050, demand for more resource-intensive foods is predicted to rise even faster than it currently is. In addition, agricultural production is predicted to be severely affected by climate change, resulting in major challenges for crop production and food security. The availability of major nutrients in the plant rhizosphere is critical for sustainable crop production, including nitrogen a major limiting factor in crop growth and productivity. Leguminous plants, including alfalfa (Medicago sativa L.), can withstand nitrogen scarcity to a certain extent due to their ability to host nitrogen-fixing bacteria in root nodules. At the molecular level, the highly conserved plant microRNA156 (miR156) affects plant growth and development, and is involved in regulating response to various stress conditions, including nutritional scarcity, drought and diseases, by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. It is thus critical to determine if the miR156-SPL regulatory network plays a role in modulating alfalfa’s root-related traits.

In the current study, the role of the transcription factor protein, SPL12, as well as downstream genes that are regulated by SPL12 were investigated to understand their potential roles in root-related traits, including root development, nodule formation and nitrogen assimilation. This study involved the phenotypic and molecular genetic characterization of alfalfa plants with increased and decreased levels of SPL12 and other downstream genes. The analyses showed that SPL12 plays a negative role in root regeneration, nodulation and nitrogen fixation by regulating downstream target genes, such as AGL6 and AGL21. Phenotypic and molecular analyses further showed that silencing AGL6 also increased nodulation in alfalfa. Analysis of plant-wide changes in gene expression revealed that at least 169 genes were affected by SPL12 silencing in alfalfa. Alfalfa plants with reduced SPL12 levels maintained nodulation under osmotic stress by partially regulating sulfate transportation.

Understanding the molecular function of miR156-targeted SPL12 and its targets in alfalfa root architecture and nodulation will provide an important molecular tool that can be used in marker-assisted improvements not only for alfalfa, but also potentially for other legume crops.