School of BioSciences - Theses
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ItemRole of phosphorylation in regulating secondary cell wall cellulose synthesis in Arabidopsis( 2022)Plant secondary cell walls (SCWs) are important for plant growth and development as the vascular tissues and fibers support plants with water and mineral transport. Cellulose is the major component of SCWs, and its synthesis is a highly complex process regulated by transcription factors as well as post-translational modifications. Cellulose synthase (CESA) 4, 7 and 8 are essential enzymes that catalyze the synthesis of SCW cellulose and form a cellulose synthase complex (CSC) that is active at the plasma membrane. The CSCs move at the plasma membrane; a process driven by the catalytic activity of the CESAs. The behaviour of the CSC is an important character of cellulose synthesis and SCW patterning. Protein phosphorylation is arguably the most common post-translational modification in many cells and affects CESA behaviour during primary wall synthesis. However, how SCW CESA phosphorylation contributes to secondary wall production is not understood well. Chapter 1 provides a brief overview about plant cell wall cellulose synthesis, especially secondary cell wall biosynthesis. There are five main aspects discussed, including secondary cell wall patterns, transcriptional regulation during SCW formation, CESA structures and the function of each domain, the effects of phosphorylation on cellulose synthesis, and environmental effects on SCW production. In Chapter 2, proteomic and phospho-proteomic changes were characterized during the transition from primary to secondary wall synthesis using the VASCULAR-RELATED NAC-DOMAIN7 (VND7)-inducible system. A vast number of phosphorylation sites, especially in SCW-related proteins, were detected. The phosphorylation changes of putative and selected phosphorylation sites in primary and secondary cell wall CESAs were analyzed in detail. This phospho-proteomic dataset provides more insights into phospho-protein changes during the process of SCW biosynthesis. In Chapter 3, phosphorylation sites in each SCW CESA were analyzed and mutated to examine if and how phosphorylation regulates SCW biosynthesis. Most of the selected phospho-mutants, either phospho-null or phospho-mimic versions, restored the phenotype of SCW cesa mutants, and did not show significant differences from wild type control. However, one conserved phosphorylation sites in CESA4, S374, did affect SCW biosynthesis, as single-site phospho-null mutant (CESA4S374A) showed dwarf phenotype with deformed xylem vessels, similar to cesa4 mutant. Sequencing and qRT-PCR confirmed the correct amino acid substitutions and gene expression, respectively. Further, both bioinformatic analysis of protein structure and sequence alignments indicated that S374 in CESA4 was likely to be externally exposed and phosphorylated. Thus, phosphorylation in the position of S374 in CESA4 potentially works to positively regulate SCW cellulose biosynthesis. In Chapter 4, an immunoprecipitation approach of a YFP tagged CESAS7 in the VND7-inducible system was used to pull out potential proteins interacting with SCW CESAs, focusing on protein kinases. Thirteen highly enriched kinases were in this way found to potentially associate with the CESAs. One interesting but unknown receptor-like kinase, AT1G09440, may potentially play a role in SCW formation. Subcellular localization analysis further showed that this protein kinase was secreted from the Golgi to the plasma membrane where it is likely to have its main function. In Chapter 5, the conclusion for this research and some future work directions are proposed.