QUERYING PLANT CELL WALL MATRIX ORGANIZATION WITH CLICK CEHMISTRY
Open Access
- Author:
- Mc Closky, Daniel Dennis
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 25, 2019
- Committee Members:
- Charles T Anderson, Dissertation Advisor/Co-Advisor
Charles T Anderson, Committee Chair/Co-Chair
Daniel J Cosgrove, Committee Member
Gabriele Brigitte Monshausen, Committee Member
William O Hancock, Outside Member - Keywords:
- Arabidopsis
metabolic labeling
plant cell wall
cellulose
pectin
click labeling
bioorthogonal chemistry - Abstract:
- Plant cells accomplish growth through the controlled deformation of their walls in response to internal turgor. This control is exerted only indirectly by the protoplast: it synthesizes an extracellular matrix, and it is the biomechanical properties of the resulting wall that directly determine the shape, size, and strength of the resulting cell. The ordered synthesis of paracrystalline cellulose microfibrils, major load-bearing components of the wall, occurs at the cell surface, but the remainder of the polysaccharides in the wall are synthesized intracellularly and must be delivered to the wall. Recent research highlights underappreciated roles for both pectins and hemicelluloses in determining wall mechanical properties, through interactions with cellulose microfibrils; the ordered delivery of these matrix polysaccharides thus may have important consequences for emergent physical properties of the wall, and by extension, overall plant form. To directly observe and study matrix polysaccharide delivery to the growing wall, we have employed a metabolic labeling technique. This has revealed (1) subcellular heterogeneity in the deposition patterning of different matrix polysaccharides, and (2) clues as to the drivers of this patterning. The roles of matrix polysaccharides in establishing cell wall mechanical properties are becoming more well-supported in recent years, but how they are delivered and organized into the growing wall remains unclear. Expanding on a metabolic labeling approach, we observed that in some cell types of the model plant, Arabidopsis thaliana, newly-synthesized matrix assumes a non-random organization in developing primary walls. In our approach, this resulted in fluorescent features on the μm scale.In primary root epidermal cells, subcellular regions destined to transition to tip-growing root hairs differentially accumulated a glucose analog, showing that particular chemical reporters can highlight regions of differential accumulation of special matrix polysaccharides, in this case, callose. Experiments with a different chemical reporter modeled on fucose that is incorporated with striated patterns of deposition into the walls of differentiating root epidermal cells, revealed that it is the cellulose microfibril array that drives the organization of recently-deposited matrix, not underlying cortical microtubules, which exert a more indirect effect on matrix patterning. However, microtubules do appear to play some role in orchestrating matrix deposition, since metabolic labeling revealed defects in matrix polysaccharide deposition in a series of mutants in a common genetic pathway involving the kinesin, Fragile Fiber1(FRA1).In summary, I demonstrate an expansion of the metabolic labeling toolkit in Arabidopsis, and an extension of the method to address fundamental questions of wall matrix organization and delivery, including a dissection of the differential influences of the cytoskeleton, the pre-existing cellulose microfibril array, and a kinesin. This work reveals new aspects of the assembly of plant cell walls, which can serve as renewable sources of fuel, food, and fiber.