Date of Award
8-12-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Plant cell walls and extracellular matrices (ECMs) are important to cellular activities in plant/animal organs. The organization of plant cell wall/ECM components (e.g., local composition, arrangement, and ultrastructure of cell wall/ECM polymers) plays a role in determining their local mechanical properties. As a result, the natural processes of cell and tissue growth, development, and function rely on the control of cell wall/ECM composition, arrangement, and ultrastructure to manipulate local mechanical properties. However, directly correlating cell wall/ECM composition, organization, and mechanics has been challenging. To overcome this barrier, one goal of this thesis is to develop probing methodologies at a sub-cellular scale to investigate these properties within the plant cell wall/ECM. Study 1 developed high-resolution atomic force microscope infrared spectroscopy (AFM-IR) imaging methodologies and investigated the correlations between cell wall composition and local mechanical properties using Arabidopsis thaliana (A. thaliana) as a model plant. This study enables quantification of chemical composition from IR spectral signatures and visualization of chemical heterogeneity at nanoscale. Cross-correlation analysis of the spatial distribution of chemical components and mechanical properties suggests that increased carbohydrate composition of cell wall junctions correlates with increased local stiffness. In Study 2, AFM-IR was applied to quantify the compositional and mechanical properties in the primary cell walls of cellulose-deficient procuste1 (prc1) and xyloglucan (XG)-absent xxt1xtx2 mutants at a sub-cellular level. Correlation analysis results indicated that the increase in carbohydrate contents led to an increase in the elastic stiffness of the prc1 epidermal cell walls. On the other hand, with the absence of XG, the xxt1xxt2 mutant, expectedly, had a lower elastic stiffness in epidermal cell walls than the wildtype. In the xxt1xxt2 cell walls closer to the outer environment, the increase in carbohydrate composition resulted in reduced wall stiffness, possibly due to cell wall disruption. The mechanochemical changes revealed in this study demonstrated how primary cell wall components play a key role in the wall mechanics. Study 3 used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to investigate the types and spatial distributions of the cell wall components across stem cross-sections of both wildtype and helical growth mutant strains (spr1-3, spr2-2, and tua4) of A. thaliana. Linear discriminant analysis (LDA) indicated the epidermal cells in both right-handed mutants spr1-3 and spr2-2 had higher cellulose composition than the cortical cells, opposite to what was found for the left-handed mutant tua4. For the right-handed mutants, spr1-3 had higher XG composition in the cortex while spr2-2 had lower XG composition as compared to the wildtype. The left-handed tua4 mutant had higher XG composition in the epidermis as compared to the wildtype based on the LDA results. As cellulose is the major load-bearing component of cell walls, such compositional changes might underlie the handed twisting of cell files in Arabidopsis mutants. The second part of the thesis focuses on spatial distribution analysis of glycosaminoglycans (GAGs) in electrospun gelatin-based fibers (Study 4) and the mechanochemical correlations in ECMs (Study 5). In Study 4, to leverage the interactions with ECM components and mimic ECM composition, solutions of gelatin and gelatin doped with chondroitin sulfate-C (CSC) were electrospun into fibrous scaffolds. A K-means clustering approach was used to quantify the surface aggregation of CSC and showed a heterogeneous spatial distribution on the surface CSC-gelation fibers. ToF-SIMS depth profiling revealed that while the extent of CSC spatial heterogeneity remains similar across different depths, however, the relative CSC concentration is higher at the fiber surface. Study 5 used AFM-IR to quantify the composition and mechanical properties of the ECMs in neonatal and adult rat extrahepatic bile ducts (EHBDs) at a sub-cellular level. The reduced Young’s moduli of the ECMs measured in neonatal as compared to adult EHBDs make neonatal EHBDs more susceptible to external mechanical stress and possible injury, which supports the finding that ECM stiffness is closely related to fibrotic contracture and fibrotic diseases. A sub-cellular analysis of the ECM stiffness revealed that decreased Young’s moduli was observed in the near-edge ECMs closer to the outer environment of neonatal EHBDs. Using a random forest regression model, the ECM stiffness was found to correlate with residual carbonyl groups and amide bonds in collagen near the edges and amide III vibrations in ECM proteins near the lumen. The mechanochemical changes in neonatal EHBDs in Study 5 facilitated the understanding of how ECM components play a key role in the stiffening process for inducing biliary atresia in liver tissue.
Language
English (en)
Chair
Marcus Foston