School of BioSciences - Theses
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ItemThe influence of circadian clock variation on local adaptation in Arabidopsis and agronomic traits in wheat( 2023-11)Plants have evolved diverse mechanisms to cope with changes in their environment. Among the most important of these, the plant circadian clock adjusts physiology and development in response to daily and seasonal environmental rhythms. The cues perceived by plant circadian clocks are non-uniform across the biogeographical environment, and variation of circadian function is required between and within species. The overarching aim of this thesis was to identify how this functional clock variation arises in plants. Extant phenotypic variation in circadian rhythms across a naturally occurring species, Arabidopsis thaliana, and a cultivated species, bread wheat (Triticum aestivum), was quantified and compared. The respective contributions of this variation to local adaptation in Arabidopsis and agronomic traits in wheat were rigorously assessed. In Chapter 2, a transient luciferase imaging assay was used to measure circadian rhythms of 287 natural Arabidopsis accessions. Through genome-wide association mapping, three SNPs were identified in the evening-expressed clock gene EARLY FLOWERING 3 (ELF3) that were highly associated with variation in circadian period. Accessions harbouring these SNPs primarily occupy continental climates of Eastern Europe and Central Asia, and through physiological and population genetic analyses, evidence is provided that ELF3 has aided local adaptation to highly seasonal climates. The circadian rhythms of elite Australian wheat cultivars were measured using delayed leaf fluorescence in Chapter 3, and a large range in circadian period was detected. By leveraging existing and novel clock gene markers, specific combinations of clock gene alleles (chronotypes) were defined that are associated with circadian period. To test the importance of circadian rhythm variation to agricultural traits, the timing of leaf senescence and grain nutrition traits were measured across the same cultivars, and strong associations with circadian period were observed. A specific effect on timing of senescence and grain protein content was found for a widespread deletion in TaELF3-D1 using pairs of near-isogenic lines (NILs). To define the global transcriptional response of circadian rhythms to senescence, in Chapter 4 48-hour ‘circadian transcriptomes’ were generated in both mature and senescent flag leaves. This analysis revealed that the output of the clock expands and diversifies at senescence, and this response is associated with increasing rhythmicity of WRKY transcription factor expression. The average circadian period of transcripts shortens by 0.5 h in senescent tissue, akin to previous studies of circadian rhythms during ageing. Interestingly, the pace of circadian oscillator genes is largely unchanged. Instead, clock genes are enriched amongst transcripts that exhibit significant advancement of phase, which is perhaps a driver of the changing period of global gene expression. These findings demonstrate abundant phenotypic variation in the circadian clocks of naturally occurring and domesticated plant species. This variation is not only consequential for traits related to seasonal development (e.g. flowering or senescence); it can also have pleiotropic effects on traits like response to high temperature and nutrient use efficiency. Clock gene variation has been co-opted by the forces of natural and artificial selection and thus holds promise for the finetuning of agricultural traits in future changing environmental conditions.
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ItemThe role of lipids in the formation of beneficial interactions between plant roots and soil microbiota under heat stress( 2022-11)Climate change, which is characterized by the rise of global atmospheric temperatures known as global warming, has serious detrimental effects on crop production because of the direct influence of elevated temperature on plant development. One novel strategy to increase crop productivity while mitigating heat stress is the use of soil microbes, which is slowly gaining popularity because of its low-cost approach, availability, sustainability, and quick turnover. Specific soil microbes can form symbiotic relationships with the roots, whose beneficial effects on plant growth and development, as well as on plant responses to biotic and abiotic stresses, lead to improved plant performance. The plant-microbe interaction is complex and involves below-ground communication, followed by modifications of molecular, biochemical, and morphological processes in the plant. Plant roots display extreme plasticity in adapting to a range of environmental stimuli and are therefore important indicators of plant-level responses to microbial colonization, via changes in architecture and metabolic processes. Lipids, which are essential constituents of the plasma membrane with diverse functions in cellular processes and homeostasis, have been proposed to play significant roles in the rhizosphere. Because heat stresses have a profound effect on membrane stability and lipid composition, rising global temperatures are likely to impact the formation of plant-microbe symbiosis. This study aimed to characterize and quantify the bacteria-induced growth promotion and heat tolerance in plants, and to investigate how plant root lipid profiles are altered under both bacteria and high-temperature conditions. For that, advanced phenotyping and lipidomics technology were employed to monitor plant responses to developmental and environmental changes. By using the high-resolution, high-throughput phenotyping platform GrowScreen-Agar II, an open-top plant-bacteria co-cultivation system was optimized utilizing the model plant Arabidopsis thaliana and the plant-growth-promoting rhizobacteria (PGPR) Paraburkholderia phytofirmans PsJN. This allowed for in-depth, tissue- and time-specific root-and-shoot morphological trait characterization, which elucidated the dynamics of bacterial promotion on plant growth. We have quantified the magnitude of bacterial-induced plant stimulation between ambient and elevated temperatures, confirming the excellent benefit of the PGPR in ameliorating the adverse effects of heat stress. These morphological traits were also associated with the root lipid profile using state-of-the-art lipidomics technology, which revealed specific lipid species and their functions in this tripartite interaction. Knowledge gained from this study, besides being fundamental in the understanding of plant-microbe interactions, can also inform research agenda of future directions for microbial studies as potential agricultural and biotechnological solutions in the endeavor to address global food security under climate change.
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ItemBuilding a wall: Developing small molecule biosensors to visualize cell wall biosynthesis and untangling mechanisms underlying nucleotide sugar transport( 2022)The cell wall is one of the main energy sinks in plants constituted of many polysaccharides and glycoproteins. The synthesis of most polysaccharides and the proteins glycosylation occur in the Golgi apparatus. The nucleotide sugars are the precursors of the cell wall building blocks. These molecules are biosynthesized in the cytosol from sugars of the primary metabolism and are transported from the cytosol to the Golgi by the nucleotide sugar transporters (NSTs). The glycosyltransferases (GTs) then consume these nucleotide sugars to produce polysaccharides and the glycoproteins. Although we have a generalized overview, our knowledge of the exact roles of NSTs and nucleotide sugars in regulating the cell wall synthesis is still sparse. The recent determination of the crystal structures of two NSTs, the GDP-D-mannose transporter VRG4 from yeast and the orthologue of CMP-sialic acid transporter from maize indicate that conformational changes occur during the transport process. Therefore, NSTs are excellent candidates to develop fluorescent proteins-based sensors to study the flow of nucleotide sugars. In chapter II, our aim was to generate fluorescent protein- biosensors based on the UDP-XYLOSE TRANSPORTER 1 (UXT1) to follow the import of UDP-D-xylose into the Golgi in vivo as a proxy for xylan and xyloglucan biosynthesis. We show that the UXT1-based FRET sensors maintain the physiological localization of UXT1 in planta, while transporting UDP-D-Xyl in vitro. We also designed ratiometric sensors based on the sfGOMatryoshka. However, these sensors were found to disrupt the Golgi localization of UXT1. In chapter III, we aimed to probe the existence of protein complexes involved in arabinosylation and study the regulation of the arabinosylation pathway. Investigating higher order mutants of the UDP-ARABINOFURANOSE TRANSPORTERs (UAfTs), allowed us to decipher the relative contribution of each of the UAfTs to arabinosylation. These newly generated mutants in combination with other mutants of the arabinosylation pathway led us to propose a mechanistic model to explain a glucose hypersensitivity phenotype in the dark. Phenotypic assessment of nucleotide sugar levels and hypocotyl elongation allowed us to make progress towards dissecting the role of cytosolic UDP-GLUCOSE EPIMERASE 1 and 3 in regulating nucleotide sugar metabolism. Finally, using affinity purification, split-ubiquitin assays and in silico co-expression approaches, we unravel putative NST-GT complexes and suggest that these complexes also involve nucleotide sugar interconverting enzymes.
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ItemRibosome Heterogeneity and Specialization during Temperature Acclimation in Plants( 2022)Ribosomes decode mRNA to synthesize proteins. Ribosomes, once considered static, executing machines, are now viewed as dynamic modulators of translation. Increasingly detailed analyses of structural ribosome heterogeneity led to a paradigm shift toward ribosome specialization for selective translation. As sessile organisms, plants cannot escape harmful environments and evolved strategies to withstand. Plant cytosolic ribosomes are in some respects more diverse than those of other metazoans. This diversity may contribute to plant stress acclimation. The goal of this thesis was to determine whether plants use ribosome heterogeneity to regulate protein synthesis through specialized translation. I focused on temperature acclimation, specifically on shifts to low temperatures. During cold acclimation, Arabidopsis ceases growth for seven days while establishing the responses required to resume growth. Earlier results indicate that ribosome biogenesis is essential for cold acclimation. REIL mutants (reil-dkos) lacking a 60S maturation factor do not acclimate successfully and do not resume growth. Using these genotypes, I ascribed cold-induced defects of ribosome biogenesis to the assembly of the polypeptide exit tunnel (PET) by performing spatial statistics of rProtein changes mapped onto the plant 80S structure. I discovered that growth cessation and PET remodeling also occurs in barley, suggesting a general cold response in plants. Cold triggered PET remodeling is consistent with the function of Rei-1, a REIL homolog of yeast, which performs PET quality control. Using seminal data of ribosome specialization, I show that yeast remodels the tRNA entry site of ribosomes upon change of carbon sources and demonstrate that spatially constrained remodeling of ribosomes in metazoans may modulate protein synthesis. I argue that regional remodeling may be a form of ribosome specialization and show that heterogeneous cytosolic polysomes accumulate after cold acclimation, leading to shifts in the translational output that differs between wild-type and reil-dkos. I found that heterogeneous complexes consist of newly synthesized and reused proteins. I propose that tailored ribosome complexes enable free 60S subunits to select specific 48S initiation complexes for translation. Cold acclimated ribosomes through ribosome remodeling synthesize a novel proteome consistent with known mechanisms of cold acclimation. The main hypothesis arising from my thesis is that heterogeneous/ specialized ribosomes alter translation preferences, adjust the proteome and thereby activate plant programs for successful cold acclimation.
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ItemRole of phosphorylation in regulating secondary cell wall cellulose synthesis in Arabidopsis( 2022)Plant secondary cell walls (SCWs) are important for plant growth and development as the vascular tissues and fibers support plants with water and mineral transport. Cellulose is the major component of SCWs, and its synthesis is a highly complex process regulated by transcription factors as well as post-translational modifications. Cellulose synthase (CESA) 4, 7 and 8 are essential enzymes that catalyze the synthesis of SCW cellulose and form a cellulose synthase complex (CSC) that is active at the plasma membrane. The CSCs move at the plasma membrane; a process driven by the catalytic activity of the CESAs. The behaviour of the CSC is an important character of cellulose synthesis and SCW patterning. Protein phosphorylation is arguably the most common post-translational modification in many cells and affects CESA behaviour during primary wall synthesis. However, how SCW CESA phosphorylation contributes to secondary wall production is not understood well. Chapter 1 provides a brief overview about plant cell wall cellulose synthesis, especially secondary cell wall biosynthesis. There are five main aspects discussed, including secondary cell wall patterns, transcriptional regulation during SCW formation, CESA structures and the function of each domain, the effects of phosphorylation on cellulose synthesis, and environmental effects on SCW production. In Chapter 2, proteomic and phospho-proteomic changes were characterized during the transition from primary to secondary wall synthesis using the VASCULAR-RELATED NAC-DOMAIN7 (VND7)-inducible system. A vast number of phosphorylation sites, especially in SCW-related proteins, were detected. The phosphorylation changes of putative and selected phosphorylation sites in primary and secondary cell wall CESAs were analyzed in detail. This phospho-proteomic dataset provides more insights into phospho-protein changes during the process of SCW biosynthesis. In Chapter 3, phosphorylation sites in each SCW CESA were analyzed and mutated to examine if and how phosphorylation regulates SCW biosynthesis. Most of the selected phospho-mutants, either phospho-null or phospho-mimic versions, restored the phenotype of SCW cesa mutants, and did not show significant differences from wild type control. However, one conserved phosphorylation sites in CESA4, S374, did affect SCW biosynthesis, as single-site phospho-null mutant (CESA4S374A) showed dwarf phenotype with deformed xylem vessels, similar to cesa4 mutant. Sequencing and qRT-PCR confirmed the correct amino acid substitutions and gene expression, respectively. Further, both bioinformatic analysis of protein structure and sequence alignments indicated that S374 in CESA4 was likely to be externally exposed and phosphorylated. Thus, phosphorylation in the position of S374 in CESA4 potentially works to positively regulate SCW cellulose biosynthesis. In Chapter 4, an immunoprecipitation approach of a YFP tagged CESAS7 in the VND7-inducible system was used to pull out potential proteins interacting with SCW CESAs, focusing on protein kinases. Thirteen highly enriched kinases were in this way found to potentially associate with the CESAs. One interesting but unknown receptor-like kinase, AT1G09440, may potentially play a role in SCW formation. Subcellular localization analysis further showed that this protein kinase was secreted from the Golgi to the plasma membrane where it is likely to have its main function. In Chapter 5, the conclusion for this research and some future work directions are proposed.
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ItemCircadian-regulated dynamics of translation in Arabidopsis thaliana( 2021)Circadian clocks are an endogenous timing mechanism that allows an organism to fine tune diverse cellular processes in anticipation of external stimuli. To date, most plant circadian studies have focussed on transcriptional control, so our knowledge at the level of mRNA translation is very limited. Thus, the overall goal of this thesis was to close this gap in knowledge by monitoring translational dynamics in Arabidopsis thaliana (Arabidopsis) over a diel and circadian cycle in order to uncover translational regulation imposed by the circadian clock. To achieve these goals, the ribosome profiling method was first optimized for 2-week old Arabidopsis seedlings. Quality control assessment revealed that this optimized method generates translatomic data that exceeds the sequencing depth of currently published plant Ribo-seq studies. This protocol was therefore applied to generate a diel and circadian translatome with 2 h temporal resolution. Data integration with complementary transcriptomes revealed widespread translational regulation. Most notably, many genes were identified that are not rhythmic at the level of transcript abundance, but maintained rhythms at the level of translation output. These translational unique-cyclers are phased at select times of the day, revealing that select transcripts involved in coordinated biological processes are preferentially translated by ribosomes. Such translational preference is observed at subjective dawn for genes involved in the general translation machinery, and at subjective dusk for genes involved in protein catabolism. Thus, it is reported here that the translational machinery itself appears to be under translational control by the plant circadian clock. Finally, an Agrobacterium-mediated seedling transformation approach was tailored for circadian studies as an independent method for defining the molecular mechanisms of regulatory elements that were identified from the ribosome profiling data. Using luciferase reporters, the promoter activity of core clocks genes following this “transient” transformation system was reported to be highly comparable to equivalent stable transgenic approaches. Furthermore, transient transformation of reporter constructs into clock mutants reproduced published circadian defects. Together, this verified the robustness and reliability of this approach. Subsequently, the transient transformation system was used to assess the functionality of candidate upstream open reading frames (uORFs) found in the leader sequence of core clock genes. The results suggest that CCA1, GI and PRR7 contain uORFs that may contribute to rhythm phase. In summary, this thesis describes widespread translational regulation in a diel and circadian context, and implicates uORFs as potential translational regulators of the core circadian oscillator.
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ItemDevising computational tools to quantify the actin cytoskeleton and pavement cell shape using network-based approaches( 2020)Recent advances in microscopy have led to an improved visualization of different cell processes. Yet, this also leads to a higher demand of tools which can process images in an automated and quantitative fashion. Here, we present two applications that were developed to quantify different processes in eukaryotic cells which rely on the organization and dynamics of the cytoskeleton. In plant cells, microtubules and actin filaments form the backbone of the cytoskeleton. These structures support cytoplasmic streaming, cell wall organization and tracking of cellular material to and from the plasma membrane. To better understand the underlying mechanisms of cytoskeletal organization, dynamics and coordination, frameworks for the quantification are needed. While this is fairly well established for the microtubules, the actin cytoskeleton has remained difficult to study due to its highly dynamic behaviour. One aim of this thesis was therefore to provide an automated framework to quantify and describe actin organization and dynamics. We used the framework to represent actin structures as networks and examined the transport efficiency in Arabidopsis thaliana hypocotyl cells. Furthermore, we applied the framework to determine the growth mode of cotton fibers and compared the actin organization in wild-type and mutant cells of rice. Finally, we developed a graphical user interface for easy usage. Microtubules and the actin cytoskeleton also play a major role in the morphogenesis of epidermal leaf pavement cells. These cells have highly complex and interdigitated shapes which are hard to describe in a quantitative way. While the relationship between microtubules, the actin cytoskeleton and shape formation is the object of many studies, it is still not clear how and if the cytoskeletal components predefine indentations and protrusions in pavement cell shapes. To understand the underlying cell processes which coordinate cell morphogenesis, a quantitative shape descriptor is needed. Therefore, the second aim of this thesis was the development of a network-based shape descriptor which captures global and local shape features, facilitates shape comparison and can be used to evaluate shape complexity. We demonstrated that our framework can be used to describe and compare shapes from various domains. In addition, we showed that the framework accurately detects local shape features of pavement cells and outperform contending approaches. In the third part of the thesis, we extended the shape description framework to describe pavement cell shape features on tissue-level by proposing different network representations of the underlying imaging data.
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ItemThe effect of sub-optimal temperature on the cellular metabolism of wheat and Arabidopsis thaliana( 2020)Low or suboptimal temperature stress is one of the primary abiotic conditions limiting the growth and productivity of economic crops in many regions of the world. Wheat is one of the major crops in Australia, it is grown during winter to avoid hot summers and they flower in early spring. The sensitive flowering stage of wheat is therefore frequently exposed to spring frost. In Australia, the frequency of spring frosts during the flowering stage has increased significantly since 1960, and the reoccurrence of frost events led to an estimated $360 million of losses in the Australian wheat industry per annum. It is therefore important for breeders to minimize the loss via the development of more chilling/frost-tolerant wheat varieties, especially during their reproductive stages. Two approaches could be employed to achieve this goal. The first one is by employing metabolomics approaches to understand the underlying molecular mechanisms involved in cold responses of wheat upon cold stress. The second approach is via bioengineering of cold responsive genes into wheat to create chilling/frost-tolerant varieties. With this in mind, my PhD study was carried out with three main objectives. The first objective was to investigate and understand metabolic traits involved in the cold acclimation of two Australian wheat varieties with contrasting cold tolerance using targeted metabolomics and lipidomics approaches. The cold-sensitive variety used in this study was Wyalkatchem and the cold tolerant variety used was Young. The second objective of this study was to identify potential metabolite and lipid responsible for chilling tolerance in the two studied wheat varieties. The third objective was to evaluate the potential of REIL (Required for isotropic bud growth1 – like) protein as cold acclimation factor in Arabidopsis thaliana for potentially enhancing wheat cold tolerance. Chapter 1 consists of a review of the recent literature covering cold stress responses (physiologically and metabolically) of plants and how plants adopt to cold stress. It describes how metabolomics and lipidomics can be used as promising tools to decipher cold stress responses in wheat and discuss the role of cold-induced genes to increase cold tolerance in plants. The targeted protein in this study, REIL, as a new potential cold acclimation factor in Arabidopsis thaliana and wheat is also reviewed. To achieve the first and second objectives of this study, work described in Chapter 2 was conducted to investigate the cold acclimation of two Australian wheat varieties with contrasting cold tolerance using targeted metabolomics and lipidomics approaches. The selected cold-sensitive spring wheat variety used in this study was Wyalkatchem and the selected cold-tolerant spring wheat variety was Young. Samples of flag leaves and spikes at the young microspore stage were collected and analysed in this study. The results obtained provide us with a better understanding of the cold responses of wheat, and pointed out the potential of several sugars, amino acids, amines and glycerolipids to confer cold-tolerance to the Young variety. The outcomes gained from this study have been published in Cheong et al., (2019) for the study on flag leaves, and in Cheong et al., (2020) for the study on spikes. The outcomes also pointed out the profound potential of lipid species as biomarkers that can be explored to distinguish the two varieties. This further motivated us to expand the lipidomics study on the underground part of wheat, the roots (Chapter 3). There are limited cold stress studies on the lipidome of whole roots and to the best of our knowledge, no data are available on responses of specific root developmental zones. In Chapter 3, the lipid profiles of the spatial root zones derived from young seedlings of Wyalkatchem and Young grown at optimal, chilling and freezing temperatures were investigated. The outcomes indicate the involvement of not only glycerolipids in discriminating Young from Wyalkatchem, but sphingolipids are also involved in conferring cold-tolerance of Young. Next, to fulfil the third objective of this study, REIL, a protein that has been postulated to act as a potential cold acclimation factor in the mature leaves of Arabidopsis thaliana, was evaluated in roots in Chapter 4, followed by the evaluation of its potential in wheat in Chapter 5. REIL proteins have been postulated to be involved in late ribosomal biogenesis and affect the accumulation of 60S large subunits in the mature leaves of A. thaliana upon cold stress. To validate these roles in A. thaliana, a systematic analysis of roots grown at optimized and cold temperatures was conducted in Chapter 4. The outcomes substantiate the role of REIL proteins as a cold acclimation factor in Arabidopsis by being involved in ribosomal biogenesis during cold acclimation. In Chapter 5, three REIL homologs are found to be expressed in wheat. Evaluation of the REIL expressions in wheat subjected to cold stress through the re-analyses of published transcriptomics datasets show the potential cold and heat responsiveness of REILs. A real-time PCR analysis was then performed to evaluate the REIL expressions in Wyalkatchem and Young under cold stress, but no significant changes of expressions were observed in both varieties upon cold stress. It is then yet-to-be-known whether the cold acclimation function of REIL is conserved among dicots (A. thaliana) and monocots (wheat). Therefore, more in-depth investigation such as overexpression or silencing of the REIL expression in Australian spring wheat varieties is needed. The last chapter of this thesis (Chapter 6) summarizes the key results from each research chapter (Chapter 2 to 5) and also discusses the future directions and perspectives.