Master of Science
Traditional breeding involving Medicago sativa (alfalfa), has resulted in minimal yield increases. Moreover, extreme environmental conditions threaten to further limit production. Strategies that make use of molecular tools – such as small non-coding RNA, miR156 – represent an innovative means by which to influence tolerance to abiotic stress. miR156 functions, at least in part, through the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family of transcription factors. In this study, the role of SPL9 in regulating alfalfa development and drought tolerance is evaluated. Examination of alfalfa plants with RNAi-mediated SPL9 (SPL9-RNAi) showed that plant height, stem thickness, and internode length are positively regulated by SPL9, whereas shoot branching is negatively regulated. SPL9-RNAi alfalfa also had enhanced tolerance to drought mediated by elevated anthocyanin content and expression of DIHYDROFLAVONOL 4-REDUCTASE (DFR), an enzyme involved in anthocyanin biosynthesis. Thus, manipulation of SPL9-mediated downregulation of DFR may represent one strategy to improve drought tolerance in alfalfa.
Summary for Lay Audience
Alfalfa is an important forage crop that is used mainly as feed for ruminant animals. While breeding programs have produced winter hardy alfalfa varieties that are capable of growth in harsh Canadian climates, further yield improvements have been limited. Furthermore, climate change is resulting in prime agricultural areas exhibiting extreme weather events such as drought. Drought can limit the growth of alfalfa and cause toxic levels of reactive oxygen species (ROS) to accumulate within the plant. ROS can cause damage to DNA, lipids, and proteins but molecules like anthocyanins, which have stress reducing antioxidant activity, can mitigate ROS accumulation.
Novel molecular tools that can be used to alter alfalfa to promote the induction of desired traits are highly sought after. One such tool, miR156, is a small RNA molecule that influences the expression of a family of proteins – called SQUAMOSA PROMOTER BINDING PROTEIN LIKE or SPL proteins – that are important regulators of development and stress tolerance. In this study, the role of one SPL protein (SPL9) was investigated by comparing alfalfa plants with reduced levels of SPL9 to plants expressing normal levels of SPL9. These studies revealed that SPL9 positively regulates plant height, stem thickness, and internode length and negatively regulates branching. Interestingly, the plants with reduced SPL9 levels were also more tolerant to drought in that they maintained growth, had reduced leaf senescence, and had enhanced relative water content while under water-deficit conditions. Importantly, the plants with reduced SPL9 levels also exhibited increased anthocyanin accumulation and effected DIHYDROFLAVONOL 4-REDUCTASE (DFR) transcript levels, an enzyme involved in anthocyanin biosynthesis. This suggested that the drought tolerance exhibited by plants with reduced levels of SPL9 were at least partly due to the SPL9-mediatd negative regulation of DFR. Taken together, these results indicate that the manipulation of SPL9 can be used as a potential molecular strategy to improve drought tolerance in alfalfa.
Hanly, Alexandria K., "Characterization of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL), and its role in drought stress tolerance in Medicago sativa (alfalfa)." (2020). Electronic Thesis and Dissertation Repository. 7092.
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