uORF-mediated translational regulation of drought stress response gene MASP1 in Arabidopsis thaliana
Human-induced climate variability can subject global regions to water scarcity, prompting widespread crop failure. This calls for innovations that preserve global food security that do not further contribute to climate change. Plants have existing drought responses in which energy is allocated to translate genes that specifically promote plant survival. This process can compromise translation of growth-related genes, ultimately affecting agricultural yield. Releasing growth-related genes from translational suppression could enhance the plant response to better withstand drought-related conditions. Microtubule-Associated-Stress-Protein-1 (MASP1) is a gene associated with plant growth. MASP1 protein levels have been seen to increase during the drought response in the model plant, Arabidopsis thaliana (At). Ribosome profiling experiments in the Macknight lab found that MASP1’s main open reading frame (mORF) is host to an upstream open reading frame (uORF). uORFs are short protein-coding sequences known to differentially regulate translation of downstream mORFs by interacting with the initiating ribosome. Our research aimed to investigate MASP1-uORF involvement in regulation of the MASP1-mORF in stressed and non-stressed conditions. We hypothesised that the MASP1-uORF regulates the MASP1-mORF, and that ribosomal recognition of the initiation codon is vital to the mechanism of regulation.To test whether the MASP1-uORF differentially responds to drought stress, At plants containing gene fusions of the MASP1-uORF and PAP1, a reporter gene that promotes anthocyanin biosynthesis, were grown in non-stressed and drought stress-mimicking conditions. Anthocyanin expression increased in MASP1-uORF-PAP1 plants subject to stress conditions, with no change seen in non-stressed conditions, suggesting the MASP1-uORFs involvement in differentially regulating PAP1 per condition. Another reporter-gene fusion technique was used to confirm the regulatory potential of the MASP1-uORF. Constructs containing mutated versions of the MASP1 uORF were generated using site-directed mutagenesis and fused to firefly luciferase (F-LUC) in vectors containing renilla luciferase (R-LUC). WT and mutated MASP1-uORF dual-luciferase vectors were transiently expressed in N. benthamiana, respectively, where light emission of F-LUC was measured and normalised to R-LUC expression. Disrupting the uORF coding sequence increased F-LUC expression, further associating the role of the MASP1-uORF in regulating a downstream gene.Where uORF-mediated regulation had already been implicated in developmental and metabolic contexts, these findings demonstrate that uORF’s may also differentially regulate translation in response to stress. They also establish that manipulation of the MASP1 uORF manipulates translation of the downstream mORF, which could be harnessed by growth-promoting technologies for future crop improvement.
Advisor: Macknight, Richard
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Department of Biochemistry
Publisher: University of Otago
Keywords: New Zealand; uORF; translational; regulation; drought; stress; response; gene; MASP1; Arabidopsis; thaliana; post-transcriptional; molecular; climate; smart; crops; translation; plant; growth; agriculture; upstream; open; reading; frame
Research Type: Thesis