Unravelling transfer cell development: wall labyrinth construction and reaction oxygen species

Xia, Xue

Title: Unravelling transfer cell development: wall labyrinth construction and reaction oxygen species
Creator: Xia, Xue
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2018
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: Transfer cells are plant cells specialized to sustain high rates of nutrient transport across plasma membranes. The defining feature of transfer cells is their wall ingrowth papillae with adjacent high densities of transporter proteins in their enclosing plasma membranes. The wall ingrowth papillae invaginate into the cytosol forming a wall labyrinth that dramatically amplifies the plasma membrane surface area and hence accelerates nutrient transport rates by up to 20-fold. The positioning of transfer cells at “bottlenecks” of nutrient transport in plants, with their amplified nutrient transport rates, overcomes these nutrient transport constraints to enhance plant growth and crop yield. Upon transfer of Vicia faba (V. faba) cotyledons to culture, their adaxial epidermal cells spontaneously initiate a trans-differentiation process leading to the development of a reticulate cell wall labyrinth and transport function comparable to their abaxial epidermal transfer cell counterparts formed in planta. We have used this experimental system to understand processes, and their regulation, that result in the formation of the transfer cell wall labyrinth. The knowledge gained has the potential to open up technological opportunities to manipulate nutrient transport and hence crop yields. The wall labyrinth of transfer cells is the product of two separate wall-building processes. The first process involves construction of a uniform wall layer formed evenly across the entire outer periclinal wall of the trans-differentiating epidermal cells. This is followed by deposition of wall ingrowth papillae upon the uniform wall layer at discrete loci. The important finding, from a literature survey, was that the uniform wall layer is an evolutionary conserved feature of reticulate wall labyrinths of transfer cells. This finding inspired an investigation of how the less-well studied uniform wall layer was deposited and the role it plays in the subsequent assembly of wall ingrowth papillae. Uniform wall layer formation commences once adaxial epidermal cell expansion has ceased. The deposition follows a unique pattern from the junction of the anticlinal and outer periclinal walls radially inward to the centre of the outer periclinal wall. Unlike the densely packed lattice of cellulose microfibrils in the original primary wall, cellulose deposition in the uniform wall layer formed a sparsely-dispersed parallel array of linear cellulose microfibrils. Interestingly their deposition patterns were found to be microtubule independent. Despite their presence, these cellulose microfibrils did not form an essential scaffold for formation of the uniform wall layer. Rather a sub-set of these cellulose microfibrils, characterized by a 3-fold greater diameter, converge to form a core scaffold essential for constructing wall ingrowth papillae. Formation of these thickened cellulose microfibrils correlated with the up-regulated expression of a transfer cell-specific cellulose synthase isoform, VfCesA3B. A sequential epidermal-specific signalling pathway has been shown to be responsible for regulating wall labyrinth assembly. A core hub of this signalling pathway is comprised of an extracellular reactive oxygen species (ROS) signal and plumes of elevated cytosolic calcium. These signals co-localize to, and are essential for, the assembly of the wall labyrinth. The present study focused on elucidating the identity and sub-cellular organization of the cohort of ROS-related enzymes responsible for generating the polarized ROS signal and on gaining some insights into how their ROS metabolic activity is regulated. In the absence of a transformation system for V. faba, the identities and roles of the ROS-related biosynthetic/catabolic enzymes generating the polarized ROS signature were investigated using a pharmacological approach combined with a RNAseq analysis of their expression profiles. Among the forms of ROS generated, hydrogen peroxide (H₂O₂) was confirmed to be the signalling molecule essential for assembly of both the uniform wall layer and wall ingrowth papillae. The extracellular H₂O₂ signal was shown to be polarized to the outer periclinal wall throughout the trans-differentiation process. It was concluded that the observed temporally-displaced two-peak burst in H₂O₂ biosynthesis likely was a wound response that also carried information essential for wall labyrinth formation. Appearance of the first peak correlated with the electron-dense band deposition that heralds uniform wall layer formation. Biosynthesis of this H₂O₂ signature was catalyzed by two groups of Class III cell wall peroxidases (Prxs). The catalytic activity of one group was dependent on an interaction with a complex formed between polyamine oxidases (PAOs) and a respiratory burst oxidase (rboh). The other Prx group was PAO/rboh independent. The former Prx group was likely located in the cell wall while the latter was located on the plasma membranes lining the developing uniform wall layer. A cohort of plasma membrane-localized PAO/rboh-independent Prxs were joined by copper-containing diamine oxidase (CAO) to generate the H₂O₂ localized to the tips of wall ingrowth papillae associated with the second peak in extracellular H₂O₂ production. A peak in catabolism of extracellular H₂O₂ coincided with the trough in H₂O₂ biosynthesis and was probably catalyzed by the extracellular-located V. faba ascorbate peroxidase 1 (VfAPX1). Significantly, H₂O₂ catabolism was shown to be essential for sustaining polarization of the extracellular H₂O₂ signal. Modelling suggested that the catabolic enzymes were likely localized to the junction of the outer periclinal and anticlinal walls of the trans-differentiating epidermal cells. The temporal regulation of H₂O₂ biosynthesis was mediated at a post-translational level for the two H₂O₂ peaks but a component of the intervening trough was controlled at the transcriptional levels. The latter control was exercised on a sub-set of the PAO/rboh-independent Prxs. Ethylene and extracellular calcium (Ca²⁺) were found to regulate H₂O₂ biosynthesis while extracellular Ca²⁺ influenced H₂O₂ catabolism. Ethylene was shown to exert its regulatory influence through affecting extracellular Ca²⁺. Of the H₂O₂ biosynthetic enzymes, extracellular Ca²⁺ was found to regulate the activity of the PAO/rboh-dependent Prxs contributing to the first burst in extracellular H₂O₂ levels and the Prxs participating across the trough and second burst in H₂O₂ production. A component of the Ca²⁺ regulatory influence on Prx activity appeared to be mediated by formation of a pectin-Ca²⁺-Prx complex. Other mechanisms contributing to the extracellular H₂O₂ signature were vesicle trafficking during the first H₂O₂ peak and trough while microdomains, known to be polarized to the outer periclinal wall plasma membrane, played no role. Polarization of the H₂O₂ signature to the outer periclinal wall was shown to result from the fine-tuned balance between H₂O₂ biosynthesis and catabolism regulated by ethylene, extracellular Ca²⁺ and vesicle trafficking. In summary, this work has extended our understanding of the never before understood development of the uniform wall layer and the role it plays in subsequent wall ingrowth papillae construction. In addition, the study has provided valuable insights into the generation of the polarized extracellular H₂O₂ signal essential for directing wall labyrinth formation in terms of establishing the identities, relative roles and spatial organization of the H₂O₂ biosynthetic and catabolic enzymes along with some insights into how their activities are regulated.
Subject: seed; transfer cell; respiratory burst oxidase; uniform wall layer; cell wall; thesis by publication; wall ingrowth labyrinth; cellulose microfibril; cellulose synthase; cortical microtubule array; reactive oxygen species; cell wall peroxidase; hydrogen peroxide; superoxide
Identifier: http://hdl.handle.net/1959.13/1391421
Identifier: uon:33230
Language: eng
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