Structural Characterization Of Plant Cell Walls Using Synchrotron X Ray

Download or read book Structural Characterization of Plant Cell Walls Using Synchrotron X-ray written by Sintu Rongpipi and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Plant cell walls are complex, dynamic biological assemblies consisting of several biopolymers and some structural proteins. They form an extracellular matrix that helps in growth, morphogenesis, mechanics, intercellular communication, and defense against pathogens. Besides having important structural and functional roles in plants, cell walls form the primary source for the most abundant biopolymers on earth which include cellulose, hemicellulose, pectin, and lignin. They form raw materials for several important commercial products and hold promise as renewable and sustainable source of energy and materials. Although the chemical composition of cell walls has been known for several decades now, detailed knowledge of the structure, interactions between the cell wall components, and their roles in the cell wall remain unknown. X-ray diffraction and scattering have been invaluable towards understanding the structure and organization of plant cell walls. Synchrotron-based X-ray techniques provide us with an opportunity to characterize the structure of plant cell walls over a broad length scale with high spatial resolution. The higher flux and collimation help obtaining good quality data in shorter measurement times from plant samples which are, in general, weakly scattering. In this dissertation, we explored the applicability of several synchrotron-based X-ray techniques to structural characterization of plant cell walls. We developed experimental designs and analytical approaches for successful application of techniques such as transmission X-ray scattering, grazing incidence X-ray scattering, X-ray absorption spectroscopy, and resonant X-ray scattering on plant cell walls. We begin by introducing GIWAXS as a technique to probe preferred orientation of cellulose crystals (texture) with respect to cell wall plane, a previously unexamined structural parameter of cell walls. Using GIWAXS, we revealed that scattering from cellulose crystals and cuticular wax crystals can be decoupled. We also established a method to measure degree of preferred orientation of cellulose in cell walls through pole figures. Altogether, our GIWAXS results contradict the predominant notion of twisting of cellulose microfibrils in primary cell walls. We then applied GIWAXS to investigate how defects in biosynthesis of cellulose, pectin, and xyloglucan affect cellulose preferred orientation in primary cell walls of etiolated hypocotyls of a model plant, Arabidopsis thaliana wild type, cellulose mutants, pectin mutants, and xyloglucan mutants. With the newly developed ability of GIWAXS to measure cellulose preferred orientation in plant cell walls, we found that the preferential orientation of cellulose is disrupted in cellulose and pectin deficient mutants, but not in xyloglucan deficient mutants. This suggests that pectin might act as a cementing substance in the cell wall matrix that maintains the preferred orientation of cellulose crystals. We also found that degree of cellulose preferred orientation is correlated with the rate of hypocotyl elongation, leading us to hypothesize that the degree of cellulose preferred orientation could be a measure of tension, cells experience during elongation. We then used transmission small angle X-ray scattering (SAXS) to evaluate the effects of defects in biosynthesis of cellulose, pectin, and xyloglucan on anisotropy of mesoscale cellulose organization. We focused on the anisotropic organization of cellulose microfibrils in primary cell walls of etiolated hypocotyls of Arabidopsis thaliana. We introduced a parameter called anisotropy ratio to describe relative alignment of microfibrils in direction parallel versus perpendicular to cell growth axis and found that xyloglucan mutants have higher anisotropy ratio when compared to wild type. Our results suggest that global anisotropic organization of cellulose microfibrils might be an essential factor for growth and mechanical anisotropy of plant cells. We also used near-edge X-ray absorption fine-structure spectroscopy (NEXAFS) to quantify calcium content in plant samples. We tried to eliminate the effects of sample thickness in (a) transmission NEXAFS near calcium L-edge by normalizing NEXAFS spectra by edge jump at carbon K-edge and in (b) fluorescence yield NEXAFS near calcium K-edge by normalizing NEXAFS spectra by pre-edge region. Using a calibration curve for calcium concentration between calcium NEXAFS signal and calcium concentration from ICP-MS for different onion scales, we could estimate calcium concentration in hypocotyls of Arabidopsis thaliana, which have similar chemical composition of cell walls. Finally, we combined SAXS and NEXAFS to carry out a chemically sensitive characterization of primary cell walls in onion epidermis. This technique called resonant X-ray scattering has been used previously with soft X-rays (near Ca L-edge) to extract interfibrillar spacings between cellulose in primary cell walls. In the current work, we explored the possibilities of obtaining higher resolution structural information using tender X-rays near calcium K-edge (~ 4.05 keV). While the works presented in this chapter are in progress, we indeed see a structural feature corresponding to a length scale of ~ 2 nm in primary cell walls of onion epidermis which we have demonstrated to be originating from distribution of calcium in the cell wall. We hope that the results established throughout this work will help in further understanding of the structure of plant cell walls, particularly in relating nanoscale structure with macroscale properties of cell walls. This work demonstrated that the unique opportunities offered by synchrotron-based X-ray techniques which are routinely applied to other soft materials can be used for furthering our understanding of the plant cell walls, either by looking at an unexamined structural aspect or by developing a new analytical approach towards an already known structural parameter. In several sections of this dissertation, we have tried to answer fundamental questions relating to plant growth by using methods we developed. We believe that this dissertation has just scratched the surface of plant cell wall characterization using advanced X-ray techniques, and we hope that the techniques and protocols established here will enable answering several questions surrounding fundamental structure-function relationships in plant morphogenesis, growth, and mechanics through new directions.