Geoff Wasteneys

The microtubule cytoskeleton is a filamentous network that reinforces cell shape, aids in vesicle transport, and enables cells to divide. In plant cells, the cortical microtubule array lies directly beneath the plasma membrane and is an intermediary between the cell exterior and the cytoplasmic environment. The dynamic flux of microtubules is largely attributed to an assortment of microtubule-associated proteins (MAPs) that bind to the microtubule polymer and promote growth, shrinkage, or rescue of a depolymerizing filament. In Arabidopsis thaliana, the MAP MICROTUBULE ORGANIZATION 1 (MOR1) is a microtubule polymerase essential for plant survival, but how this protein interacts with the microtubule to promote rapid assembly is unclear. Another important MAP is CYTOPLASMIC LINKER ASSOCIATED PROTEIN (CLASP), which prevents microtubule depolymerization and sustains hormone flux by tethering endosomes near the cell surface in root cells. A central question is how CLASP integrates these functions to regulate root meristem growth.In this thesis, I investigated the cellular and molecular roles of MOR1 and CLASP in plant development. By devising an imaging procedure using fluorescence recovery after photobleaching, I visualized fluorescent MOR1 and found that the degree of turnover on the microtubule plus end was correlated to microtubule growth rate. To study the behaviour of CLASP in roots, I used live-cell imaging to observe that cells in the clasp-1 null mutant spent longer in mitosis, and the formation of CLASP-dependent microtubule bundles occurred during G1 and persisted throughout S phase. I found that CLASP was controlled by brassinosteroid hormone signalling, and excess brassinosteroid reduced CLASP expression, protein levels, and changed microtubule organization. This was further demonstrated in a brassinosteroid-insensitive version of CLASP, which showed reduced root meristem growth despite increased CLASP fluorescent protein. Analysis of dark-grown root meristems revealed that a translational checkpoint reduces CLASP protein levels, possibly to inhibit root growth under conditions when hypocotyl expansion is required. Finally, I explored the evolution of the CLASP-SNX1 interaction through a series of BLAST searches and found that this sequence is specific to land plants. This thesis has furthered our understanding of microtubule dynamics and the interplay of MAPs and hormones in root development.

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The plant cortical microtubule array plays a role in the control of directional cell expansion, and the organization and dynamics of the array are subject to control by a variety of microtubule-associated proteins, many of which coordinate organization of the cortical array in response to environmental stimuli. Point mutations affecting MOR1, a microtubule polymerase/depolymerase, result in disruption to the organization and dynamic properties of microtubules under specific conditions: mutations in the N-terminal TOG1 (tubulin-binding) domain have temperature-conditional phenotypes, while the phenotype of a mutation in the C-terminal region is induced by treatment with the microtubule-destabilizing drug propyzamide. In this thesis, I used mor1 mutants with conditional phenotypes to characterize genetic interactions between different domains of the MOR1 protein, microtubules, and components of a microtubule-targeted environmental stress signalling pathway. Analysis of microtubule organization and dynamics in mor1-tubulin double mutants demonstrated that the handedness of helical growth phenotypes does not always correlate with microtubule growth and shrinkage rates, and showed that a mutation in β-tubulin promoted recovery of microtubule dynamics in the temperature-sensitive mor1-1 mutant. I used live-cell imaging to observe interactions between fluorescently tagged MOR1 and microtubules, demonstrating that addition of a fluorescent tag to the MOR1 C-terminus alters MOR1 function and results in phosphorylation of α-tubulin, which is normally a response to environmental stress. Despite this effect, differences in microtubule binding affinity were observed for MOR1 variants with mutations in the TOG1 and C-terminal regions. I determined that mutation of the C-terminal region of MOR1 (mor1-11) results in activation of the tubulin kinase PHS1, though this did not appear to be mediated by MPK18, a previously characterized PHS1-interacting MAP kinase. In order to identify other possible components of this signalling pathway, I carried out a modifier mutant screen in the mor1-11 genetic background, identifying one enhancer and six suppressors of mor1-11.

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Microtubules are dynamic polymers that are important for the growth and development of plant cells. Because of their vital role, the dynamics and organization of microtubules need to be tightly modulated by other proteins. The major focus on this dissertation is to elucidate and characterize the role of specific proteins (ARK1, NEK6, MOR1) that are responsible for orchestrating the dynamics and organization of microtubules in the model plant, Arabidopsis thaliana. The motor protein, ARK1, was previously shown to play an important role in root hair morphogenesis but had an unknown role in modulating microtubule dynamics. Moreover, evidence showed that ARK1 physically interacts with the NEK6 kinase, although for an undetermined reason. Based on my data, I determined that ARK1 functions as a plus-end tracking protein that has a specific role in promoting the depolymerization of microtubules. I also discovered that ARK1 has a secondary microtubule-binding domain in addition to the motor domain, which is the canonical microtubule-binding domain. I noted, however, that this secondary microtubule-binding domain is not essential for ARK1’s ability to induce microtubule depolymerization. While NEK6 and ARK1 both modulate microtubule dynamics, I determined that neither protein requires each other for function or localization, suggesting that they operate independently from each other to control microtubule dynamics and cell elongation. In addition, I provided evidence that shows that ARK1 has a putative yet unknown role in controlling NEK6 gene expression. Finally, the microtubule-associated protein MOR1 was confirmed to be a plus-end tracking protein through live-cell imaging of MOR1 fused to a fluorescent reporter (MOR1-3xYpet). In revealing that MOR1 binds to both growing and shrinking microtubule plus ends, my data corroborated previous studies showing reduced microtubule growth and shrinkage in mor1 mutants, and confirm MOR1’s role as a microtubule polymerase. Comparing MOR1-3xYpet live-cell imaging with previous experiments using fixed samples revealed that the chemical fixation process affects the plus-ends of microtubules, stressing the importance of using non-fixed samples for the most accurate results. My dissertation thus expands our knowledge about how microtubule dynamics and organization is controlled in plant cells by three distinct proteins.

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Shoot and root systems have evolved numerous specialized structures, including stomata and lateral roots. Stomata, which are essential for mediating gas exchange across the shoot epidermis during photosynthesis, consist of two bilaterally symmetrical guard cells arranged around an epidermal pore. The symmetry displayed by mature stomata is essential for stomatal function. Stomata develop via a dedicated pathway defined by two key divisions, an asymmetric division, and a symmetric division which initiates stomatal bilateral symmetry. The symmetric division is followed by pore and guard cell morphogenesis, which maintains the previously established bilateral symmetry. Lateral roots increase the ability of root systems to acquire nutrients and water from the surrounding environment and lateral root development is also dependent on stage specific divisions. Interestingly, several genes regulating symmetric divisions have also been shown to function in lateral root development. The Leucine-Rich Repeat Receptor-Like Kinase, MUSTACHES (MUS), which belongs to a family of four closely related MUS-LIKE kinases (MUSLs), is required to enforce bilateral symmetry post-symmetric division. Mutants in mus display pore and guard cell morphogenesis defects, as well as defects in microtubule array organization and the polarity of microtubule movement. MUS is expressed in both stomatal lineage and root epidermal cells, suggesting that MUS also functions outside stomatal development. Thus, MUS represents an ideal candidate through which mechanisms influencing pore and guard cell morphogenesis, as well as the impact of post-symmetric division stomatal genes on lateral root development, may be explored. Mutant analysis and time-lapse studies demonstrated that MUS enforces bilateral symmetry by ensuring symmetrical positioning of microtubule organizing centres post-symmetric division. Time lapse studies indicated that microtubule organizing centre delocalization occurred before, and is likely responsible for, the subsequently occurring alterations in microtubule polarity and guard cell morphogenesis defects observed in mus. As well, this work revealed that MUS, the cytoplasmic kinase NIMA-RELATED KINASE 6 and the microtubule associated protein CLIP-170 ASSOCIATED PROTEIN are required for stomatal microtubule organizing centre formation. Additionally, a model where pore placement is regulated by opposing MUS and MUSL1 signaling pathways was developed. Finally, a redundant role for MUS and MUSL1 in lateral root development was confirmed.

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Microtubules have long been known to play a vital role in plant growth anddevelopment, which is a complex process and needs to be regulated by bothenvironmental and endogenous hormonal signals. CLASP, an important microtubuleassociatedprotein, has been shown to be involved in both cell division and expansion.The major goal of my thesis was to explore the function of CLASP in new pathways aswell as to identify novel factors that are responsible for its function and distribution.Yeast 2-hybird analysis done independently by two of our collaborators indicated thatCLASP interacts strongly with the endocytic membrane-associated protein SNX1. SNX1had been implicated in the intracellular trafficking of the auxin efflux carrier PIN2. Weproved the direct interaction between CLASP and SNX1 by colocalization and BiFCusing live cell imaging and we found that clasp-1 mutants have an altered distributionpattern of SNX1, reduced abundance of PIN2 and a series of auxin-related phenotypessuch as dwarfism, enhanced lateral branching and aberrant auxin distribution. Druginducedmicrotubule disruption caused clasp-1-like defects on PIN2 stability. This studyillustrated the role of CLASP and microtubules in polar auxin transport and auxinsignalling pathway.Previous studies revealed a cross-talk between auxin and brassinosteroids. I foundthat the two major transcription factors in the brassinosteroid signalling pathway directlytarget the CLASP promoter to repress its transcription. Reduced CLASP expression iscorrelated with a transverse orientation of cortical microtubules in root meristematic cells,switching them from division to differentiation. Also CLASP and microtubules stabilizethe BR receptor BRI1 by preventing its degradation, a similar mechanism as for PIN2.This research highlights the importance of CLASP in BR-modulated meristem cellidentity and the potential role of CLASP as a node between auxin and BR pathways.I carried out an immunoprecipitation experiment to identify putative CLASPinteractors and obtained TRM19. Further analysis failed to confirm a direct interactionbut suggested that TRM19 is a microtubule-associated protein. Its high expression individing cells is consistent with prior report that it functions in PPB formation during thecell cycle.

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Plant cells often display a microtubule reorganization event when encountered with stress. This has been found to be integral for the reaction to stresses such as aluminum toxicity and cold stress. A cDNA microarray was previously conducted that identified MARNERAL SYNTHASE (MRN1), an oxidosqualene cyclase that produces the triterpene marneral, as the most highly upregulated gene when microtubule dynamics are disrupted in Arabidopsis. This work identifies two cytochrome P450s, CYP71A16 and CYP705A12, that are highly coregulated with MRN1 and are located within close proximity to the MRN1 loci. Using GC-FID and GC-MS, MRN1 and CYP71A16 are shown to function together in a single pathway in what is known as a metabolic gene cluster, while further testing shows that they are not in fact regulated by microtubule dynamics. The expression profile of these genes is explored since there is no known function for marneral or its related metabolites. Using a promoter-reporter and real time PCR analysis, it was found that the hormones ABA and methyl jasmonate induce expression of the three genes to different degrees depending on seedling age. Osmotic stressors, including mannitol and NaCl treatments, also induce the expression of these genes. MRN1, in particular, seems to show the highest level of induction suggesting that the pathway is transcriptionally regulated through MRN1. These conditions are shown to not affect the growth response in mutant plants unable to metabolize marneral or plants ectopically expressing different combinations of the three genes. These conditions are intriguing because most triterpenes derived from secondary metabolism are generally thought to play roles in defense, yet these data suggest that the pathway is induced under abiotic stress conditions. The marneral cluster may have evolved to be expressed under osmotic stress conditions in a sense to protect the plants water from pathogens or herbivores. It is also reasonable to speculate that these compounds may play roles in signalling or membrane modification. Further experiments are proposed that could test these hypotheses.

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Plants have developed sophisticated signalling networks that are involved in mediatingdevelopmental transitions and environmental signals. Mitogen-activated protein kinases (MPKs)are a class of signalling proteins that are involved in cellular processes that help plants to detect and initiate appropriate responses to numerous development and environmental inputs. Themicrotubule cytoskeleton plays a pivotal role in plant development and morphogenesis, althoughthe mechanisms that regulate the microtubule-associated proteins and microtubule functions inplant cells are not well understood. I investigated whether perturbations in the microtubule organization triggered by the MICROTUBULE ORGANIZATION] temperature-sensitive mutant(morl-l) could lead to altered transcriptional activity, with a particular interest in the genesencoding signal transduction components. I showed that perturbations in the microtubulecytoskeleton, achieved through the microtubule disruption phenotype of mor1-1, led to changesin the expression of gene transcripts associated with diverse cellular processes, includingchanges in the expression of the PROPYZAMIDE HYPERSENSITIVE 1(PHS1) gene, a memberof MPK-specific signal transduction pathway, which has been previously implicated inmediating cortical microtubule functions in plant cells. Through biochemical, cell biological andgenetic tools, I identified MPK18 as one of the MPKs that interacts with the PHS1 phosphataseand demonstrated through reverse genetics analysis that manipulation of MPK18 results inconditional and subtle defects in the microtubule functionality. In contrast, analysis of MPK12,which was shown to also interact with PHS1, identified no microtubule-specific function. Mylive cell imaging studies revealed that the absence of MPK18 protein appears to have no effecton microtubule plus end growth and shrinkage rates, indicating that MPK18 indirectly influencesmicrotubule functions. Based on the genetic analysis, MOR1 itself does not appear to be a target of the putative MPK18 signalling module. Preliminary attempts to obtain evidence for directimpacts of PHS activity on MOR1 failed to demonstrate that manipulation of PHS1 alteredeither subcellular localization or phosphorylation status of the MOR1 protein. These resultsprovide a platform that should facilitate future investigations aimed at understanding the role ofMPK signalling in the regulation of plant microtubule functions.

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No abstract available.