Characterization of fasciclin-like arabinogalactan proteins in Arabidopsis thaliana

© 2018 Dr Edgar LiuAll plant cells are surrounded by an elastic primary cell wall and, in some specialised cell types/tissues such as the vasculature, the cell deposits a secondary wall that is capable of withstanding enormous compressive forces important for water conducting tissues and plant support (Doblin et al., 2010). The primary wall largely consists of polysaccharides such as cellulose, hemicelluloses and pectins and a minor portion of protein components. An abundant superfamily of plant-specific wall proteins are the hydroxyproline-rich glycoproteins (HRGPs), which includes the arabinogalactan-proteins (AGPs). AGPs are characterised by a protein backbone rich in non-contiguous proline (P) residues that direct O-linked glycosylation with type II arabino-(3,6)-galactans (AGs) (Schultz et al., 2002; Johnson et al., 2003; Basu et al., 2015a). AG glycans have been proposed to be important in secretion and trafficking of proteins, and in modulating their biological function (Ellis et al., 2010; Kovall and Blacklow, 2010). Within the AGP family, chimeric AGPs, with recognised PFAM domains other than the AGP regions, include an important class known as the fasciclin-like arabinogalactan-proteins (FLAs) (Schultz et al., 2002; Johnson et al., 2003). In FLAs, AGP regions are found adjacent to fasciclin (FAS) domains (Johnson et al., 2003). FAS domains are known to be conserved in proteins from a broad range of living organisms playing important biological roles related to adhesion (Elkins et al., 1990a; Johnson et al., 2003; Hamilton, 2008; Moody and Williamson, 2013). The combination of O-linked glycans and FAS domains is proposed to confer unique biological roles to FLAs. Bioinformatic studies have shown that FLAs belong to multigene families in all plant species, for example, 21 FLAs have been identified in the Arabidopsis genome (Johnson et al., 2003). The function of some FLAs has been inferred through investigation of their tissue-specific expression profiles and phenotypes of the respective fla mutants. Based on these studies, FLAs are proposed to play roles in cell expansion, plant growth and development (Shi et al., 2003; Seifert and Roberts, 2007; Li et al., 2010; MacMillan et al., 2010). In particular, a subset of FLAs specifically expressed in stems have been shown to influence stem biomechanical properties. MacMillan et al. (2010) demonstrated that loss of FLA11 and FLA12 resulted in changes in cell wall composition and the microfibril angle of cellulose in Arabidopsis stems. No obvious growth phenotypes were observed in fla11 fla12 mutants suggesting further redundancy with other FLA members. FLA16 was also identified as having stem-specific expression yet its role in stem biomechanics was not explored. In this study, multi-disciplinary approaches, including molecular biology, microscopy, biochemistry, biomechanics and genetics, have been employed to investigate the biological function(s) and genetic relationships of FLA11 (AT5G03170), FLA12 (AT5G60490) and FLA16 (AT2G35860). We developed molecular resources to further explore the role of Arabidopsis FLA11 and FLA12 during stem development and characterise FLA16 as a novel regulator of stem development. Chapter 1 provides an overview of AGP/FLA structures, classification and known biological roles. Chapter 2 provides a detailed description of the experimental materials and research methods used in this thesis. This is followed by Chapter 3, an investigation of FLA11, FLA12 and FLA16 at the protein level. Fluorescent protein fusions of FLA11, FLA12 and FLA16, driven by either their endogenous promoters or the 35S promoter were generated and transformed into either Arabidopsis or tobacco (Nicotiana tabacum), respectively. The tissue/cellular distributions of the FLA proteins in different plant organs as well as their sub-cellular location was determined. In addition, several enrichment methods, including: chelation, immunoprecipitation (IP) and hydrophobic interaction chromatography (HIC), were explored in preparation for structural analyses and identification of potential interacting partners of the FLAs (see Chapter 3, Appendices A3.1). FLA11, FLA12 and FLA16 were shown to be predominantly expressed in inflorescence stems, siliques and branches with lower abundance in cotyledons and roots. The location of FLAs was largely confined to cells with secondary walls, such as interfascicular fibres and xylem cells in stems/branches, endocarp b and replum cells in the siliques, guard cells in the leaves and vasculature in the main roots. Membrane fractionation studies suggest the FLAs are present at the plasma membrane (PM) and in the wall and are glycosylated. Enrichment of detergent-solubilised FLAs showed IP and chelation are the most effective methods, however, improved methods for enriching FLAs from Arabidopsis are needed given the difficulty of extracting proteins from stems due to the abundance of secondary walls. These data suggest that FLA11, FLA12 and FLA16 are likely to function at the PM and/or wall of cells with secondary walls in inflorescence stems. In Chapter 4, the function of FLA16 in stem development is explored through characterisation of a fla16 mutant at different growth stages and tissues. Phenotypic analyses of the fla16 mutant shows a delay in root growth during early seedling development, early bolting and reduced stem length and thickness. Morphological analysis of stems using light microscopy shows a reduced number of pith cells with no obvious changes in fibre and xylem cells. Cell wall compositional analyses of the stem showed that fla16 has reduced cellulose compared to wild-type (wt). Similar to FLA11 and FLA12, loss of FLA16 altered biomechanical properties of the stems, with increased tensile stiffness in the middle stems and decreased flexure strength in the basal stems. Quantitative RT-PCR (Q-PCR) was used to study the expression of primary and secondary wall-specific cellulose synthases (CesAs) in stems and showed changes of the secondary-wall active CesA7 occurs in fla16. Our results suggest FLA16 plays a role in maintaining biomechanical properties in the stem, likely via regulating cellulose in the secondary walls. In Chapter 5, a series of comparative and functional analyses are undertaken for FLA11, FLA12 and FLA16, based on analysis of the double fla11 fla12, fla11 fla16, fla12 fla16, and triple fla11 fla12 fla16 mutants compared to the respective single fla11, fla12 and fla16 mutants and wt. Through comparative analyses of growth parameters such as the length, thickness and cellulose content of the stems, we found all fla mutants show similar phenotypes, suggesting they act in the same genetic pathway. Q-PCR analyses of the expression of CesAs active in either primary or secondary wall cellulose synthesis show CesA7 expression is consistently increased in fla16, fla11 fla12 and fla11 fla12 fla16 mutant stems. Our results suggest FLA11, FLA12 and FLA16 likely act in similar pathways to regulate stem growth and cellulose biosynthesis during secondary wall formation, and the genetic relationship of these FLAs seems to be epistatic. In Chapter 6 we summarise the implications of these data on plant growth and development and speculate on the roles and properties of FLAs. We pose questions as to whether they function as wall biosynthesis regulators, architectural components, or both. This research provides valuable insights into the biology of FLA11, FLA12 and FLA16, and provides future directions in the study of these cell wall glycoproteins. Greater knowledge of how FLAs influence cellulose deposition and biomechanics will underpin future applications in the forest industry and the agri-food sector