Characterizing the Function of CSLD Proteins During Plant Cell Wall Deposition in Arabidopsis

Abstract

As one of the most significant features of plant cells, the cell wall not only defines plant cell shape but also provides strength and rigidity to the plant. During plant development, changes in cell shape are primarily driven by cell expansion, which is controlled by cell wall deposition and modification. The two major mechanisms that control these changes are called diffuse growth and tip growth. During diffuse expansion, cell wall materials are synthesized and integrated in a polarized fashion along the entire expanding face of the cells. In contrast, during tip growth new cell wall deposition is restricted to a limited plasma membrane domain, leading to the highly polarized cell expansion associated with this directed cell wall construction. As the major load-bearing component in plant cell walls, cellulose is also the most abundant biopolymer on earth. Unlike other cell wall polysaccharides, cellulose is synthesized in the plasma membranes by large integral membrane protein complexes called cellulose synthase complexes (CSCs). The catalytic subunits of the CSCs are encoded by members of the Cellulose Synthase (CESA) family. Previous research showed that CESA1, CESA3, and CESA6 are required for the formation of active CSCs involved in the synthesis of cellulose in the primary cell wall of cells undergoing diffuse growth in Arabidopsis. Interestingly, our laboratory previously demonstrated that CSCs containing CESA3 and CESA6 did not appear to be required for new cellulose synthesis at the apical plasma membranes of root hair cells undergoing tip growth. Instead, members of a related family of Cellulose Synthase-Like D (CSLD) proteins showed tip-specific localization in these membranes and provided cell wall synthase activity required for maintenance of structural integrity of the cell wall in these tip-growing root hairs. However, while these CSLD cell wall synthases are essential, the nature of the polysaccharides generated by CSLD proteins has remained elusive. Here, I use genetic and biochemical approaches to characterize the catalytic activity of one member of the CSLD family, CSLD3. Genetic complementation of a cesa6 mutant with a chimeric CESA6 protein containing a CSLD3 catalytic domain demonstrated that the CSLD catalytic domains can successfully generate β-1,4-glucan polymers for cellulose synthesis. Time-lapse fluorescence microscopy demonstrated that these CESA6-CSLD3 chimeric proteins assembled into CSC complexes with similar mobility as CESA6-labeled complexes in hypocotyl cells. Proteoliposomes containing purified, detergent-solubilized CSLD3 and CESA6 proteins could specifically utilize UDP-glucose as an enzymatic substrate and synthesize products that are only sensitive to endo-β-1,4-glucanase. Taken together, these data strongly support the conclusion that CSLD3 represents a UDP-glucose-dependent β-1,4-glucan synthase. However, whether CSLD proteins require the formation of higher-order complexes to perform β-1,4 glucan synthase activities remained unclear. Here, I used genetic methods to demonstrate that CSLD2 and CSLD3 proteins are functionally interchangeable with each other during root hair elongation and cell plate formation. CSLD5 could partially rescue the root hair elongation defects in csld3 mutants. However, it plays a unique and essential function during cell plate formation. Proteoliposomes containing CSLD2 and CSLD5 displayed conserved β-1,4 glucan synthases activities similar to those described for CSLD3. Taken together, these results indicate that while all three vegetatively expressed CSLD proteins possess conserved β-1,4 glucan synthases activities, CSLD5 has a more complicated and specialized role during cell plate formation. To sum up, my dissertation research further supports that CSLD proteins represent a distinct family of cellulose synthase in Arabidopsis.