Investigating molecular regulation of transfer cell development in the model flowering plant Arabidopsis thaliana

- Hou, Jiexi

Title
Investigating molecular regulation of transfer cell development in the model flowering plant Arabidopsis thaliana
Creator
Hou, Jiexi
Relation
University of Newcastle Research Higher Degree Thesis
Resource Type
thesis
Date
2018
Description
Research Doctorate - Doctor of Philosophy (PhD)
Description
Transfer cells (TCs) trans-differentiate from differentiated plant cells by developing highly invaginated wall ingrowths that protrude into the cytoplasm of the cell, thus greatly amplifying plasma membrane surface area. The enlarged plasma membrane surface area consequently enables increased densities of nutrient transporters which facilitate enhanced rates of transport at apoplasmic/symplasmic junctions in plant nutrient transport pathways. Significantly, these processes are primary determinants of plant growth and productivity, and thus TCs can play important roles in nutrient acquisition, distribution and utilization in plants. The study of TCs therefore has the potential to contribute to understanding processes that determine plant productivity and thus crop yield, particularly in seed crops where TCs are commonly present in phloem unloading pathways. This thesis describes a series of experimental approaches where molecular and genetic tools were utilized to gain a deeper understanding of wall ingrowth deposition, in this case in phloem parenchyma (PP) TCs in cotyledons and leaves of the model plant species Arabidopsis thaliana (Arabidopsis). Since PP TCs in Arabidopsis occur in small numbers and are located deep within vascular bundles, Chapter 2 reports attempts to construct a transgenic line(s) enabling live-cell imaging of wall ingrowth development in PP TCs by fluorescence microscopy. Several different combinations of fluorescent reporter protein driven by different promoters were tested to achieve this goal, with some success achieved using a line expressing secreted Red Fluorescent Protein (secRFP) under control of the Root Inducing Loci C (rolC) promoter. In these transgenic plants, secRFP accumulated in wall ingrowths of PP TCs, but specificity was compromised by secretion of this protein into cell wall compartments of other vascular cells, consequently requiring detailed confocal microscopy to resolve accumulation in wall ingrowths of PP TCs. This result limited the usefulness of this transgenic line for live-cell assessment of wall ingrowth deposition, but nonetheless provided a platform to achieve this goal using future modifications. In parallel with the work described in Chapter 2, a new method for confocal imaging of wall ingrowth deposition in PP TCs was developed in the McCurdy laboratory using a modified pseudo-Schiff-propidium iodide procedure for covalent labeling of plant cell walls. This approach was then used to test the role of signalling agents such as methyl jasmonate (MeJA) and hydrogen peroxide (H₂O₂) in regulating wall ingrowth deposition in PP TCs. A liquid culture system was used as an experimental platform for these studies, and initial characterization established that wall ingrowth deposition in PP TCs of cotyledons grown in liquid culture occurred over a 5-d period which correlated with cotyledon expansion. Based on this experimental approach, experiments reported in Chapter 3 demonstrated that altering levels of jasmonic acid (JA) either chemically or genetically, or elevating apoplasmic levels of H₂O₂ had no observable effect on wall ingrowth deposition in PP TCs. However, decreasing apoplasmic H₂O₂ levels by exposure to reactive oxygen species (ROS) scavengers ascorbic acid (AA) and glutathione (GSH) lead to deposition of aberrant wall ingrowths in PP TCs, a wall deposition response that was restricted to PP TCs. Wall ingrowths are cellulose-rich and compositionally-defined as “primary wall-like”, but no molecular analysis of the role of Cellulose Synthase A (CESA) genes has been reported. Chapter 4 therefore provides molecular evidence using CESA mutants that point to the conclusion that the deposition of reticulate wall ingrowths in PP TCs may require both primary and secondary wall CESAs. Altered wall ingrowth deposition was seen in both the primary wall CESA5 mutant cesa5mum3-1 and secondary wall CESA4 mutant cesa4irx5-1. Moreover, live-cell imaging of transgenic plants expressing tdTom-CESA6 under its native promoter showed that the spatial expression of tdTom-CESA6 correlated with wall ingrowth deposition in PP TCs. Chapter 5 provides a general overview of principle findings made in this thesis, and discusses future directions emerging from this research that may provide new understanding of the mechanisms involved in wall ingrowth deposition in PP TCs of Arabidopsis. These findings hopefully will lead to a better understanding more generally of these important and unique cell types in plants.
Subject
transfer cells; plant cells; Arabidopsis thaliana; plant growth; crop yield; wall ingrowth
Identifier
http://hdl.handle.net/1959.13/1387566
Identifier
uon:32631
Rights
Copyright 2018 Jiexi Hou
Language
eng
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