School of BioSciences - Theses

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    The role of lipids in the formation of beneficial interactions between plant roots and soil microbiota under heat stress
    Macabuhay, Allene Andaya ( 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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    Building a wall: Developing small molecule biosensors to visualize cell wall biosynthesis and untangling mechanisms underlying nucleotide sugar transport
    Mariette, Alban Philippe Yves Marie ( 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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    Ribosome Heterogeneity and Specialization during Temperature Acclimation in Plants
    Martínez Seidel, Federico ( 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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    Role of phosphorylation in regulating secondary cell wall cellulose synthesis in Arabidopsis
    XU, HUIZHEN ( 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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    Circadian-regulated dynamics of translation in Arabidopsis thaliana
    Ting, Michael Kien Yin ( 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.