Electronic Thesis and Dissertation Repository

Thesis Format



Master of Science




Hannoufa, Abdelali

2nd Supervisor

Karagiannis, Jim


The impacts of climate change are expected to increase the demand for crops that are resistant to drought stress. Understanding the molecular mechanisms involved in the response of plants to such stresses is thus crucial for preventing losses in crop yield. In this study, the role of alfalfa SPL4, a target of the non-coding RNA, miR156, was examined in response to drought stress and with respect to the development of trichomes. We found that transgenic alfalfa plants with RNAi-silenced SPL4 exhibited increased trichome density under both control and drought conditions. Furthermore, in response to withholding water for 14 days, SPL4-RNAi plants exhibited increased root length, water content, chlorophyll content, stomatal conductance, and increased water potential in leaves when compared to wild-type plants. RT-qPCR revealed that SPL4-RNAi plants displayed altered expression levels of genes involved in drought tolerance (SPL9, SPL13), antioxidant biosynthesis (CAT), and trichome production (GL1, GL3). This study demonstrates that SPL4 has a role in both trichome development and in the drought stress response, making it a potential target for the improvement of alfalfa and potentially other crops.

Summary for Lay Audience

To maintain food security as the human population increases, more efficient crop production methods must be developed. This need is exacerbated by global climate change, which causes agricultural areas to suffer extreme weather patterns, such as drought. Drought causes the accumulation of reactive oxygen species (ROS), which can interfere with biological processes and limit plant growth. This is of special interest to Canada, as large parts of the prairies are predicted to be subject to even harsher droughts as climate change progresses. Silencing through miRNAs is a promising molecular tool for inducing desirable changes in plant physiology. One example of this is miR156, which through its interaction with proteins named Squamosa Promoter Binding-Like transcription factors (SPLs), acts as a regulator of both development and stress tolerance. In this study, the role of SPL4, a member of the SPL family, was investigated through the comparison of wild-type (WT) alfalfa plants with alfalfa plants exhibiting reduced SPL4 expression. It was found that under drought conditions, plants with reduced SPL4 expression were greener than WT alfalfa, and had increased root length, water content, water potential, chlorophyll content, and stomatal conductance, indicating a greater tolerance for drought conditions. Trichomes, hair-like structures present on most plant leaves and partly responsible for maintaining water content, were found in greater densities on plants with reduced SPL4 expression under both drought and stress-free conditions. The SPL9 and SPL13 genes involved in negatively regulating drought stress tolerance, were found to have lowered expression, while Catalase (CAT), a gene involved in removing ROS, and Glabrous 1 (GL1) and Glabrous 3 (GL3) genes involved in trichome development were found to have increased expression in SPL4 silenced alfalfa. These results suggest that SPL4 is a possible target for molecular manipulation in order to improve drought tolerance in alfalfa.