- Title
- Investigating the regulatory mechanism of phloem parenchyma transfer cell development in Arabidopsis thaliana
- Creator
- Wei, Xiao-Yang
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Transfer cells (TCs) develop extensive wall ingrowth networks which expand plasma membrane surface area thus facilitating enhanced rates of membrane transport. Consistent with their morphology and predicted functions, TCs occur at anatomical sites that require high rates of nutrient transport at symplasmic/apoplasmic junctions, such as phloem TCs in foliar minor veins, maternal/filial interfaces in seeds, and the gametophyte/sporophyte interface in moss. Because of this link to nutrient flows within the plant, the development of TCs is, in many instances, critical for crop yield, and thus understanding the molecular development of this cell type and its manipulation might provide a contribution to addressing food security. In the model plant Arabidopsis thaliana (Arabidopsis), TCs trans-differentiate from phloem parenchyma (PP) cells abutting the sieve element/companion cell (SE/CC) complex in minor veins of foliar tissues. Based on their anatomy and expression of SUCROSE WILL EVENTUALLY BE EXPORTED TRANSPORTERS (SWEETs) sucrose uniporters, TCs are assumed to play pivotal roles in phloem loading. Previous studies by members of the McCurdy laboratory at the University of Newcastle have developed an efficient modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure to visualize and semi-quantify wall ingrowth deposition using confocal microscopy. Furthermore, Arabidopsis has readily accessible genetic resources. These attributes make Arabidopsis an excellent experimental system to study TC development and the genetic control of wall ingrowth deposition in plants. Therefore, the focus of this thesis is to identify potential regulators of wall ingrowth deposition in PP TCs in Arabidopsis, as well as investigating the developmental regulation of this cell type required to perform its role in active phloem loading. To identify regulatory genes controlling PP TC development, Chapter 2 reports on a forward genetics approach using ethyl methanesulfonate (EMS) mutagenesis to screen for mutants showing disrupted wall ingrowth deposition. As confocal microscopy and mPS-PI staining was not suitable for the rapid processing of a large number of samples, a simpler staining method using ruthenium red was developed to visualize wall ingrowth deposition in PP TCs using DIC (Differential Interference Contrast) microscopy. This method proved to be successful in blind experiments to identify plant lines in which wall ingrowth deposition was known to be reduced. Subsequently, an M2 population of mutagenized seed was established and screening undertaken. However, even though the M2 population yielded numerous mutants deficient in seedling growth, indicating successful mutagenesis of the M2 population, no apparent wall ingrowth mutants were identified after screening over 5000 M2 lines by DIC microscopy. Consequently, due to the time constraints inherent with PhD research, the screen was abandoned. Nonetheless, this work not only developed a reliable and accessible way to visualize wall ingrowth deposition in PP TCs by DIC microscopy, it also provided a pool of successfully mutagenized seed for future mutant screening. Following on from the mutant screen in Chapter 2, cell biology and molecular genetic approaches were then undertaken in Chapters 3 and 4 to further characterise the pattern of wall ingrowth deposition in PP TCs relative to other phloem cell types in minor veins, and to identify signalling factors potentially regulating this process. In Chapter 3, the development and morphology of PP TCs were surveyed in cotyledons and leaves of Col-0 and the transgenic lines pAtSWEET11::AtSWEET11-GFP and pAtSUC2::AtSTP9-GFP. Although temporal differences were present between different tissues, the development of wall ingrowths in foliar tissues generally followed an underlying basipetal pattern whose timing coincided with the onset of phloem loading in source leaves. This developmental pattern suggests a potential association between wall ingrowth deposition and phloem loading activity. To further elucidate the morphology of wall ingrowths and unveil their spatial relationship with other phloem cells, vibratome cross sections of minor veins were surveyed using confocal imaging. PP cells and CCs were identified by their corresponding cell-type-specific fluorescent markers AtSWEET11-GFP and AtSTP9-GFP, respectively. This analysis revealed, unexpectedly, that wall ingrowth deposition was more abundant in PP TCs positioned abaxially in the phloem compared to those positioned adaxially, implying differing contributions to phloem loading across each foliar minor vein. Moreover, wall ingrowths in PP TCs were initiated exclusively at the interface adjacent to SEs, and the deposition of wall ingrowths adjacent to SEs was more abundant than that seen along PP TC/CC interfaces in mature minor veins. Furthermore, only PP cells that were adjacent to SEs had wall ingrowths. Collectively, these results not only demonstrate the dominant influence of SEs on wall ingrowth deposition in PP TCs but also suggest that there are two sub-types of PP cells in Arabidopsis minor veins, one with wall ingrowths (PP TCs) and one without (PP). Moreover, AtSWEET11-GFP levels were significantly higher in PP TCs compared to PP cells, which further supports the role of wall ingrowths in facilitating phloem loading. Chapter 4 investigated the role of sugars in regulating PP TC development in Arabidopsis minor veins. Treatment of cotyledons and juvenile leaves 1 and 2 with exogenously applied sucrose, glucose or fructose demonstrated sucrose-specific repression of wall ingrowth deposition in minor veins. In contrast to exogenously applied sucrose, however, endogenous sucrose promoted wall ingrowth deposition. Thus, elevating endogenous Suc levels by increasing light intensity or extending light period enhanced wall ingrowth formation, whereas lowering endogenous Suc by dark treatment or genetically in the ch-1 mutant resulted in lower levels of deposition. Analysis of pAtSUC2::GFP plants, in which the distribution of AtSUC2-GFP acts as a marker for phloem loading status, illustrated that wall ingrowth deposition was positively correlated with phloem loading activity in PP TCs. Consistent with this observation, expression levels of AtSWEET11 were positively correlated with phloem loading activity and wall ingrowth levels in PP TCs. Analysis of a TREHALOSE 6-PHOSPHATE SYNTHASE over-expression line and the hexokinase-null mutant, gin2-1, suggested that sucrose signaling of wall ingrowth formation is independent of T6P and HXK. The results from this study have been published (Wei et al., 2020, Journal of Experimental Botany 71: 4690-4702). During the Covid pandemic of 2020/2021 when laboratory access was limited, a bioinformatics approach described in Chapter 5 was pursued to identify factors potentially controlling wall ingrowth development. This investigation used published as well as unpublished RNA-seq data sets from the McCurdy laboratory to identify genes commonly up-regulated across developmental stages of wall ingrowth deposition in cotyledons and leaves. These lists were then compared against recently published single cell RNA-seq studies focusing on leaf vascular tissue and 18-day-old leaves, as well as genes co-expressed with AtSWEET11. Collectively, these various pipeline comparisons identified numerous transport-related genes, a confirmatory result given the transport function of PP TCs, in addition to several transcription factors, such as NAC056 previously identified to be associated with wall ingrowth deposition in PP TCs, and other genes with functions predicted to be involved in cell wall synthesis. A role for a sub-set of these genes in PP TC development was then assessed using relevant T-DNA insertional mutants. Due to time constraints, only thirteen T-DNA mutants of nine selected genes were assessed for wall ingrowth phenotypes using confocal microscopy. However, none of the mutants showed statistically different levels of wall ingrowth deposition or morphological differences. This negative result was equivocal to some extent since not all T-DNA insertions analyzed were either homozygous as listed by AraShare or demonstrated knockout lines. Thus, while this time-constrained outcome was disappointing, the approach has identified new candidate genes for future investigations. Chapter 6 provides a concluding discussion of the experimental approaches used in this thesis to investigate genetic control of PP TC development in Arabidopsis, and discusses future directions to be pursued based on findings in this thesis. In summary, this research has clarified that wall ingrowth deposition in PP TCs is tightly associated with adjacent SEs rather than CCs. Moreover, a positive correlation between wall ingrowth deposition and phloem loading activity in PP TCs has been identified. These findings greatly extend the currently limited understanding of interactions among phloem cells and the regulatory mechanism of TC development. This study has provided new insights into the role of sucrose in regulating wall ingrowth deposition in PP TCs and has provided genetic resources for future studies to identify genes involved in TC development.
- Subject
- Arabidopsis; transfer cells; phloem parenchyma; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1504724
- Identifier
- uon:55567
- Rights
- Copyright 2022 Xiao-Yang Wei
- Language
- eng
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