- Title
- Spatiotemporal biosynthesis and organization of the polysaccharide composition of the transfer cell wall labyrinth
- Creator
- Wheeler, Simon
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Transfer cells (TCs) are an anatomically specialized plant cell type found at sites of apo /symplasmic nutrient exchange. Morphologically, TCs are defined by the presence of an architecturally distinct invaginated wall labyrinth (WL), in many cases polarized to the direction of nutrient transport. The WL provides a platform to increase the surface area of the plasma membrane that is enriched in nutrient transporters. The plasma membrane/WL complex facilitates an increased maximal rate of membrane transport at potential bottlenecks for nutrient transport. As photoassimilate uptake by sink tissues and nutrient flux within/from source tissues influence photosynthetic rates, and subsequently biomass accumulation, TCs are important players in determining crop yield. Based on appearance, the WL can be considered to have either a flange or reticulate type morphology. The latter is the most common, occurring in all plant taxa and across a variety of cell types. The deposition of the reticulate WL comprises two distinct wall building processes. Initially, a thin, foundational uniform wall layer (UWL) is deposited on top of the original wall (OW) of the cell, polarized toward the direction of increased nutrient transport. This is followed by the deposition of a network of wall ingrowths (WIs) at distinct loci on the inner surface of the UWL. Their appearance at maturity can vary from small, sparse projections that rarely branch or fuse, to a dense and bulky labyrinth that penetrates the cytoplasm to occupy a substantial proportion of the cell volume. The cell wall is a polysaccharide-rich extracellular matrix ubiquitous to all plant cells. The polysaccharide composition of the cell wall can vary widely based on cell type, developmental stage and species and in most instances is the key determinant of the mechanical properties and structure of the cell wall. Previous investigations into the TC WL have indicated that its polysaccharide composition is largely similar to that of the underlying primary cell wall. These investigations have predominantly been restricted to immunocytochemical approaches and have not considered the separate contributions of the UWL and WI to the WL, nor the spatiotemporal regulation of polysaccharide deposition during WI development. The paucity of published information is largely due to the technical challenges posed in examining TCs, specifically their low abundance and typical location deep within the vascular and reproductive tissues of plants. The cotyledons of the legume Vicia faba (Faba bean), provide a novel experimental system to circumvent these technical challenges, as the epidermal cells on the adaxial surface trans differentiate to a TC morphology when exposed to culture conditions. An optimized protocol for the chemical fixation of epidermal cells for transmission electron microscopy was developed. When leveraged in conjunction with immunocytochemistry at the light and electron microscopic levels, together with the parallel linkage analysis of a de-starched, homogenous population of trans-differentiating epidermal TCs, this protocol identified previously unidentified aspects of the polysaccharide composition of the OW, UWL and WIs. The polysaccharide phase of the OW was found to consist primarily of de-branched arabinans and cellulose. Cellulose constituted a higher proportion of the UWL than the OW, commensurate with an increase in xyloglucan, while the composition of the WIs was regulated with regard to their stage of development. Nascent WIs were comprised of mostly cellulose, xyloglucan, branched arabinans and highly-branched rhamnogalacturonan-I. The debranching of arabinans and rhamnogalacturonan-I, which likely serve to further mechanically reinforce the cellulose microfibril scaffold, were associated with WI maturation, defined by the formation of a fenestrated layer. The variation in cell wall composition between the OW, UWL, nascent WI papillae and WIs in the process of branching, fusion and consolidation established that strict, but highly dynamic, spatial and temporal control of fine polysaccharide structure, characterizes WL development in trans-differentiating Faba bean epidermal TCs. To determine how these compositional dynamics may be interrelated with the transcriptional drivers of UWL and WI deposition, an RNAseq dataset generated to examine transcript dynamics during UWL and WI deposition was mapped to a Faba bean transcriptome chosen from those publicly available based on several metrics of ‘completeness’. A pipeline to identify the cohort of carbohydrate active enzymes (CAZymes) in the transcriptome was employed. Expression of those CAZymes putatively implicated in cell wall polysaccharide biosynthesis and post-secretory modification of their structure was identified and investigated, with a particular focus on those differentially expressed during UWL and/or WI deposition. Distinct cohorts of CAZymes were associated with UWL and WI deposition that complemented their predicted composition. The transcriptional upregulation of genes associated with cellulose, xyloglucan and homogalacturonan deposition and modification were identified as likely key contributors to the UWL, cumulatively responsible for the cessation of cell expansion and reorganization of the cellulose microfibril network described previously as critical to its formation. The transcript dynamics of cell wall biosynthetic genes during WI deposition were consistent with a shared polysaccharide composition between the OW and WIs. The differential expression of genes associated with the modification of polysaccharides, particularly xyloglucan, mannan and rhamnogalacturonan-I, in conjunction with the regulation of cellulose microfibril organisation, were identified as strong candidates that may be key determinants of the unique morphology of the reticulate WI network. Additionally, several genes associated with WL formation during pathogen infection were expressed in similar patterns during TC trans-differentiation in Faba bean, which were previously undocumented parallels between these systems. In summary, this work extended the body of knowledge pertaining to the polysaccharide composition of the TC UWL and WIs. The phasic modulation of polysaccharide deposition, and post-secretory modification of their structures in accordance with the specific UWL and WI developmental stages, were identified. The TC WL is delineated from other examples of cell walls deposited as a secondary developmental event in that its composition closely follows that of the underlying wall material, with morphology largely driven instead by putative changes in polysaccharide structure and polysaccharide: polysaccharide interactions. Insights into the transcriptomic drivers of these processes identified a cohort of candidates that, based on their function in other examples of primary cell wall morphogenesis, have the capacity to be determinants of, and critical to, polysaccharide deposition and structure within the WL, and thus TC function.
- Subject
- transfer cell; cell wall; polysaccharide; RNA-seq; transmission electron microscopy; confocal microscopy
- Identifier
- http://hdl.handle.net/1959.13/1412418
- Identifier
- uon:36482
- Rights
- Copyright 2020 Simon Wheeler
- Language
- eng
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