Year of Publication

2016

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Plant and Soil Sciences

First Advisor

Dr. Seth Debolt

Abstract

The primary cell wall is a highly organized multi-layered matrix of polysaccharides (cellulose, hemi-cellulose, and pectin). The ability of the rigid cell wall to sufficiently loosen to allow growth is a complex process that differs considerably between grasses monocots and dicots. Cellulose is the major structural component required for anisotropic cell expansion and is synthesized by CELLULOSE SYNTHASE A (CesA) proteins. Here, our objectives were two-fold: 1) dissect cell walls and cellulose biosynthesis in dicots and grasses using chemical biology and reverse genetic approaches 2) characterize and classify the inhibitory mechanisms of cellulose biosynthesis inhibitors (CBIs). A reverse genetics TILLING experiment was conducted to study CesAs in the model grass Brachypodium (Bd). New mutant alleles of BdCesA1 and BdCesA3 were identified and characterized. On average, Bdcesa1S830N and Bdcesa3P986S mutants had 15% and 8% less cellulose than wild type plants, respectively. No obvious vegetative growth phenotypes were detected in mutants. However, at reproduction, inflorescence stems of cesa1S830N were 62% shorter than that of the wild type while cesa3P986S mutants were 20% longer. To classify CBIs, time-lapse confocal microscopy data were used to categorize CBIs based on how they disrupted the normal tracking and localization of fluorescently labeled CesAs. Furthermore, biochemical and confocal microscopy data were used to characterize the putative CBI, indaziflam. Three different inhibitory mechanisms were discovered within the CBI mode of action. Next, CBIs were used as molecular probes to study grass cell walls. However, grasses were found to be inherently tolerant to isoxaben and other CesA targeting CBIs. Isoxaben-tolerance was investigated but could not be explained by target and non-target site mechanisms. Thus, it was hypothesized mixed linkage glucans (MLGs), a unique grass cell wall polysaccharide, have cell wall strengthening characteristic and may partially compensate for reduced cellulose content. Bdcslf6 mutants deficient in MLGs were 2.1 times more susceptible to isoxaben than wild type plants indicating MLGs do have a structural role in expanding cells, but likely cannot explain tolerance. These data, collectively, support a conclusion that the non-cellulosic fraction of grass primary cell walls has more load-bearing capacity than dicot cell walls.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2016.453

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