UBC Theses and Dissertations
Investigating the microtubule-associated proteins facilitating transfacial bundle formation and transition zone establishment in arabidopsis thaliana Bali, Breanne
Maintaining balance between division and differentiation in the root apical meristem (RAM) is key to proper root formation. This can be achieved by promoting the proliferation of dividing cells and ensuring their timely transition to differentiation upon entering the transition zone. This thesis aimed to investigate the role that CLASP plays in both of these cellular events. CLASP allows for the proliferation of dividing cells by promoting the formation of transfacial bundles (TFBs). Given that CLASP is not a known microtubule (MT) bundling protein, it likely relies on an additional factor(s) to carry out this role. In yeast and animal models, CLASP orthologues interact with MAP65-1 and MAP65-2 orthologues to facilitate the formation of MT bundles. This, and phenotypic similarities between the clasp-1 null mutant and the map65-1 map65-2 mutant in Arabidopsis support a model whereby CLASP, MAP65-1 and MAP65-2 are functionally interdependent in TFB formation. To investigate this, I attempted to create a map65-1 map65-2 claspCRISPR mutant and characterize its phenotype. I was unable to isolate a homozygous triple mutant and my analysis suggests that CLASP, MAP65-1 and MAP65-2 may all be required for plant viability. A decreased level of auxin (termed the auxin minimum) coincides with the transition zone and instructs cells to stop dividing and begin elongating. The auxin minimum-establishing genes ARR1, SHY2 and GH3.17 were found to be upregulated in clasp-1 which suggests a role for CLASP in downregulating this pathway. Given that CLASP is downregulated by brassinosteroid (BR), we hypothesized that BR downregulation of CLASP is responsible for the upregulation of the auxin-minimum establishing genes. To investigate this, I compared the expression of ARR1, SHY2 and GH3.17 in WT and BR insensitive CLASP mutants (brinCLASP) upon epibrassinolide (eBL) treatment. I found a mild upregulation of ARR1 in WT seedlings that was not observed in brinCLASP, suggesting that BR downregulation of CLASP is required for the upregulation of ARR1 upon eBL treatment. Ultimately, this thesis furthers our understanding of how CLASP plays an important role in both the maintenance of meristematic divisions and suggests a role for BR downregulation of CLASP in establishing the auxin minimum.
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