Investigating development of phloem parenchyma transfer cells in Arabidopsis thaliana

Research Doctorate - Doctor of Philosophy (PhD)Recent advances in plant membrane transport research have shown that transport proteins embedded within membranes are key targets for improving plant performance, particularly under stresses such as salinity, pathogens or aluminum toxicity. Many transport processes augment plant performance via defined functions in specialized cell types or tissues. Plant transfer cells (TCs) are an example of such cell types. TCs possess increased densities of membrane transporters due to the increased plasma membrane surface area resulting from deposition of wall ingrowths - the unique structure that defines TC identity. These anatomically specialized cells are located at sites specialized for nutrient transport to facilitate apoplasmic/symplasmic solute exchange. Therefore, optimizing transport performance by manipulating TC development may provide new avenues for improving plant performance and hence crop yield. Despite such potential, mechanisms underlying the process of TC development and wall ingrowth formation in particular are poorly understood. Therefore, the focus of this study was to elucidate the genetic control of TC development. Most TCs are formed via a process known as trans-differentiation in which one differentiated cell type irreversibly switches into another cell type with distinct morphological and functional features. Various differentiated plant cell types including epidermal, vascular, gametophyte cells or root hairs, etc., can trans-differentiate into TCs by developing wall ingrowths on the inner surface of their primary cell wall. Among these diverse TC cell types, adaxial epidermal TCs in cultured Vicia faba cotyledons, endosperm TCs in seeds of maize and barley, and phloem parenchyma (PP) TCs in Arabidopsis thaliana (Arabidopsis), represent the three most extensively studied examples of TC development in plants. Due to the readily accessible genetic resources available in Arabidopsis, studying PP TC development provides an experimental system with unique attributes compared to the other systems. These PP TCs, similar to TCs in many other instances, however, are embedded deep within vascular bundles of leaves and leaf-like organs, and hence have mostly been studied by electron microscopy. To accommodate the use of Arabidopsis as a genetic system to study PP TC development, a rapid way to visualize the extent of wall growth deposition and a simple but efficient method to score this process was imperative. To address this issue, a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure was developed in combination with confocal microscopy to visualize wall ingrowth deposition in PP TCs in Arabidopsis. This approach enabled high-resolution three-dimensional imaging of polarized wall ingrowth deposition in PP TCs. Importantly, simplifying the original staining procedure by using bleach in place of lengthy extractions involving organic solvents, SDS/NaOH and enzymes enabled high-throughput assessment of PP TC development at the whole leaf level using a semi-quantitative scoring system to assess the extent of wall ingrowth deposition. A defoliation experiment was performed as an example of using this scoring system to analyze responses of PP TC development to leaf ablation. This staining technique was also employed successfully to image companion TCs in leaf minor veins of pea, implying its general applicability for imaging wall ingrowths in diverse species. The results from this study have been published in BMC Plant Biology as: Nguyen and McCurdy BMC Plant Biology (2015) 15:109, DOI 10.1186/s12870-015-0483-8. With the robustness of the simplified mPS-PI staining method, the abundance and distribution of PP TC development in shoots of Arabidopsis ecotype Columbia-0 (Col-0) was surveyed. This analysis unexpectedly discovered that wall ingrowth deposition was highly abundant in early-emerged organs such as cotyledons and juvenile leaves, and remarkably less so in later-emerged adult leaves. This survey was extended to other Arabidopsis ecotypes including Landsberg erecta-0 (Ler-0) and Wassilewskja-2 (Ws-2), leading to the conclusion that wall ingrowth deposition in PP TCs represents a novel trait specific to heteroblasty or vegetative phase change (VPC), a phenomenon where plants progress through juvenile and adult stages of vegetative development. Importantly, mPS-PI staining revealed no major differences in xylem development between juvenile or adult leaves, implying that the responses seen in PP TC development across shoot maturation represent a cell wall deposition event specific to VPC. In light of this finding, the potential regulatory role(s) for microRNA miR156 and its targets SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factor genes in controlling wall ingrowth formation in PP TCs was investigated. The abundance of miR156, miR172, SPL3, SPL9, SPL10 and SPL15 all correlated with that of wall ingrowth deposition in PP TCs across shoot maturation from juvenile, transition and adult leaves, and across maturation of individual juvenile and adult leaves. In all cases, levels of miR156 accumulation showed a positive correlation with the extent of wall ingrowth deposition, whereas levels of SPL9, SPL10, SPL15, and to a lesser extent SPL3 and miR172, negatively correlated with wall ingrowth abundance. Additionally, altering the onset and/or progression of VPC by either prolonged leaf ablation, growth of plants under short days, or genetic manipulation of components of the miR156/SPL module, resulted in corresponding changes in levels of wall ingrowth deposition. In particular, overexpression of miR156 caused an increase in PP TC development, whereas reducing its accumulation or activity led to reduced wall ingrowth abundance. The spl9-4/spl15-1 double mutant had increased levels of wall ingrowth abundance compared to Col-0. Analysis of plants carrying miR156-resistant forms of SPLs, including rSPL3, rSPL9, rSPL10 and SPL15-1D lines, showed that wall ingrowth deposition was decreased in SPL9- but not SPL3-group genes, indicating that SPL9-group genes may function as negative regulators of wall ingrowth deposition in PP TCs. These findings represent a significant step towards a better understanding of the genetic pathways required for constructing wall ingrowths in PP TCs. A manuscript describing the novel linkage of PP TC development and VPC and the novel role for the miR156/SPL module in regulating PP TC development has been accepted to Plant Physiology (published on-line 12/01/2017). The novel observation of heteroblastic development of PP TCs also contributes to the knowledge of physiological role(s) for wall ingrowth deposition in plant development. In many anatomical instances, the presence of TCs has been shown to optimize nutrient transport across relevant plasma membranes. Published work on PP TCs in Arabidopsis has also suggested a role for these cells in enhancing export of photoassimilates from collection phloem in leaves. However, the striking differences in wall ingrowth abundance reported here between mature juvenile and mature adult leaves call into question this role in Arabidopsis. Analysis of mutants disrupted in ATSWEET11/12 or SUC2, the two well-characterized classes of proteins functioning in phloem loading in Arabidopsis, did not significantly affect wall ingrowth deposition in PP TCs. These and several other layers of evidence necessitate re-evaluating the general consensus that PP TCs in Arabidopsis, or at least wall ingrowth deposition in these cells, plays an important role in phloem loading. In light of these observations, a potential role for wall ingrowth deposition in PP TCs in acting as a physical barrier to defend access of invading pathogens to sugar-rich sieve elements is also discussed. In summary, the study detailed in this thesis extends the currently limited understanding of the genetic regulation of TC development. The involvement of the miR156/SPL module as a regulator of heteroblastic development of PP TCs in Arabidopsis has been identified. Moreover, the simplified mPS-PI staining procedure used in combination with semi-quantitative assessment of wall ingrowth deposition in PP TCs provides a convenient, accurate and rapid way to analyze PP TC development for future studies of this process. In particular, this approach offers the prospect of using reverse genetics to identify essential genes controlling TC trans-differentiation