School of BioSciences - Theses
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ItemUnderstanding the role of companion of cellulose synthase1 (CC1) in maintaining cellulose synthesis under salt stress( 2022)Salt stress is one of the most detrimental abiotic stresses for plants, and substantially impacts plant biomass and agricultural productivity. In the last decades, revealing how plants cope with stress conditions and maintain growth under salt stress has been an important focus in plant research and agricultural development. The plant cell wall, which encases plant cells and functions as a cellular exoskeleton, is an important structure to cope with such stresses. One of the main components of the cell wall is cellulose, which is synthesized by cellulose synthase (CESA) complexes (CSC) at the plasma membrane by moving along underlying cortical microtubules. COMPANION OF CELLULOSE SYNTHASE (CC) 1 and 2 are components of the CSCs and links the CSCs to the microtubules. CC1 and CC2 function in maintaining cellulose synthesis under salt stress by supporting microtubules and CESA behaviors. However, the exact regulatory mechanisms of the CC1 and CC2 proteins remain largely unknown. In this thesis, the regulatory mechanisms of CC1 are investigated in more details. In Chapter 2, a comprehensive phylogenetic analysis shows that the CC protein family contains up to seven members in land plants, with six CCs present in the Arabidopsis thaliana (A. thaliana) genome. The chimeric constructs swapping the N-terminus of CC1 with different homologs, i.e., CC2-CC6, were generated and transformed into cc1cc2 double mutants. The phenotypic analyses showed that CC1 to CC4 and CC6 behaved similar to CC1 in supporting plant growth under salt stress, while CC5 did not. These inabilities of CC5 were due to its defects in microtubule-binding, microtubule bundling, and maintenance of microtubules stability under salt stress, as well as other changes in functionalities of the N-terminal part of the protein. CC5 was found to have a dominant negative effect on plant root growth. Furthermore, CC5 was specifically expressed in pollen and inhibited pollen germination under salt stress. These findings reveal functional differences among CC protein family members and improve our understanding of the four microtubule-binding motifs of CC1. In Chapter 3, an immunoprecipitation-mass spectrometry (IP-MS) analysis was performed to identify potential interactors of CC1 under normal conditions and salt stress. Among the candidates, FERONIA (FER) was selected for further study. It was found that FER interacted and phosphorylated CC1, and the phosphorylation of CC1 by FER negatively affected its in vitro microtubule-binding and microtubule-bundling abilities. In Chapter 4, in planta phospho-proteomic analysis was performed and potential phosphorylation sites of CC1 were identified. Here, BRASSINOSTEROID INSENSITIVE 2 (BIN2) was found to phosphorylate CC1, and the relevant phospho-null and phospho-mimetic mutants were generated and briefly characterized. These findings provide further insights into the regulatory mechanisms of CC1, which are important to better understand how plants maintain cellulose synthesis and growth under salt stress.
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ItemThe LEUNIG regulatory complex: How does it control shoot apical meristem formation and its post-embryonic activity?( 2021)Patterning along the apical-basal (A-B) axis is a crucial step during the early stages of plant embryogenesis and leads to the establishment of two poles of which each will develop their own stem cell niches. The activity of these meristems is responsible for post-embryonic growth, with the shoot apical meristem (SAM) generating the above-ground organs and the root apical meristem (RAM) producing the subterranean structures of the plant. While several transcriptional regulators governing A-B patterning have been identified, precisely how their regulatory function is orchestrated remains elusive. This study focuses on transcriptional co-regulators LEUNIG (LUG) and closely related LEUNIG_HOMOLOG (LUH) and their role in the formation of A-B patterning during embryogenesis as well as their post-embryonic maintenance. A link between the LUG regulatory complex and SAM formation and maintenance comes from the observation that lug mutants heterozygous for the luh allele (lug luh+/-) often have enlarged SAMs resulting from misregulated cell divisions. A more severe phenotype is observed in lug luh double mutants which are embryonically lethal. In this study, a detailed characterisation of lug luh embryo phenotype reveals that these mutants display aberrant cell divisions along the A-B axis, which correlates with defects in auxin distribution, complete loss of apical identity, and altered expression of transcription factors determining basal fate. Like other co-regulators, LUG and LUH lack intrinsic DNA-binding domains and instead must interact with DNA-binding cofactors to ensure recruitment to regulatory elements of target genes. This either involves direct contact between the co-regulators and transcription factors (TFs) or the formation of higher-order complexes with adaptor proteins such as SEUSS (SEU) or related SEUSS-LIKEs (SLKs), which facilitate binding to specific TFs. Results presented in this study provide insight into the molecular framework for the LUG regulatory complex activity during embryogenesis. Both yeast and in planta assays showed that LUG/LUH and SEU/SLKs physically associate with a variety of WUSCHEL-RELATED HOMEOBOX (WOX) TFs including members of the WOX2-module. Furthermore, genetic interactions between members of the WOX2-module and the LUG regulatory complex, support their mutual action during embryogenesis. Based on the reduced activity of HOMEODOMAIN LEUCINE-ZIPPER CLASS III (HD-ZIPIII) promoters in lug luh embryos, a model is proposed in which the LUG regulatory complex functions together with WOX2-module to promote apical identity and subsequent SAM initiation through regulation of the HD-ZIPIIIs. The activity of the LUG complex in promoting basal embryo identity through positive regulation of microRNA165/166 suggests that this complex also has functions that are independent of the WOX2-module. Preliminary work reported in this study further uncovered the role of the LUG regulatory complex in post-embryonic development. While the fasciated inflorescence meristems of lug luh+/- plants displayed defects in auxin transport and altered activity of stem cell markers, embryonically rescued lug luh mutants formed flat and differentiated SAMs. In addition, rescued lug luh mutants exhibited severely disorganised RAM and defects in quiescent center (QC) specification, supporting the involvement of the LUG complex in post-embryonic RAM maintenance.
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ItemFrom nucleotide sugars to polysaccharides: How do plants control the delivery of substrates for cell wall biosynthesis and protein glycosylation?( 2021)Plant cell walls constitute one of the most abundant raw biomaterials on Earth. The synthesis of long-chain olysaccharides, the main components of plant cell walls starts ab ovo in the cytoplasm where most of the building blocks for polysaccharide synthesis, so-called nucleotide sugars, are produced. The monosaccharide moieties of nucleotide sugars are incorporated into growing polysaccharide chains either directly at the plasma membrane by lycosyltransferases (GTs) that form cellulose synthase complexes or by those residing in the Golgi apparatus. In the latter case, nucleotide sugars have to pass the Golgi membranes with the help of nucleotide sugar transporters (NSTs). Once inside, they are used by Golgi GTs which assemble polysaccharide chains and decorate proteins and lipids with sugar residues. Recent evidence suggests that in plants nucleotide sugars can be guided to specific polysaccharides and/or glycan decorations, yet the molecular mechanisms of these processes are not fully understood. This PhD research attempts to explore the phenomenon of the targeted substrate delivery by investigating two possible hypotheses: the spatiotemporal distribution of proteins within the Golgi apparatus and the occurrence of direct interactions between NSTs and GTs. The author of this dissertation has tested both of those hypotheses by investigating protein-protein interactions, localising the individual components to their specific sub-compartments within the cell and tracking changes in mutant plants where these processes are modulated. The bifunctional UDP-RHAMNOSE/UDP-GALACTOSE TRANSPORTER (URGT) family from the model plant Arabidopsis thaliana was selected for this study. While this family has six members which in vitro are capable of transporting the same substrates, plant mutant studies indicate substrate preference and targeted delivery to specific cell wall components in vivo. This thesis presents the first evidence that members of the URGT family localise to specific sub-Golgi compartments. The colocalisation studies undertaken as part of this thesis place URGT1 and URGT5 in the cis-Golgi, URGT2, URGT4 and URGT6 – in the medial Golgi, while URGT3 seems to localise to trans-Golgi stacks. Protein-protein interactions studies have identified multiple interaction partners for the six URGTs. Notably, many of those are known galactosyltransferases, which aligns with the transport function of the URGTs. It is therefore highly likely that the identified candidates are true interactors, which use the proximity of the transporter to increase the process efficiency by substrate channelling. This finding is further supported by the fact that observed interactions between URGT family members and GTs often localise to the same sub-Golgi compartment. The study identified new potential galactosyl- and rhamnosyltransferases, including two putative arabinogalactan protein galactosyltransferases. The data obtained during this project suggest, that URGTs may determine the flow of substrate through both spatial separation within the sub-Golgi stacks and direct interactions with GTs. The study presents new insight into the sugar substrate delivery processes in plants, suggests that similar processes may take place in other NST families and that their specificity may be similarly tuned by localisation and interactions.
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ItemRegulation of mucilage production in the Arabidopsis seed coat through MADS-box TF family members( 2018)During Arabidopsis seed development, the epidermal cells of the seed coat produce large quantities of pectinaceous mucilage polysaccharides. Following imbition, mucilage is released from these cells and forms a thick protective layer around the germinating seed that can be visualised with Ruthenium Red staining. The seed coat epidermis is therefore an attractive system to investigate the production of cell wall polysaccharides, particularly pectin. Previously, mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) have been shown to be associated with a mucilage extrusion defect that is caused by reduced expression of the β-galactosidase MUCILAGE MODIFIED2 (MUM2). To better understand the role of LUH in regulating MUM2, RNA-Seq analysis was performed on whole seeds and identified genes that were differentially expressed in luh mutants. This transcriptomic analysis not only revealed elevated expression of transcription factors with a known role in epidermal cell differentiation, but also several MADS-box transcription factors that perform roles in floral development and silique shattering such SHATTERPROOF1 (SHP1) and SHP2. This thesis presents evidence that SHP1, SHP2 and SEPALLATA3 (SEP3) are a new class of regulators involved in mucilage polysaccharides modification, as loss of their activity result in mucilage extrusion defects. These mucilage defects are enhanced in shp1 shp2 double mutants and correlate with reduced MUM2 expression. While carbohydrate analysis data failed to show an expected increase in galactose residues attached to pectin in shp1 shp2 and sep3 mutants, the decrease of MUM2 in these mutants is shown to contribute to their mucilage defects as partial rescue of mucilage defects in shp1 shp2 and sep3 was achieved with rescue of MUM2 expression. This is further supported by the use of SRDX repressor motif fusions to SHP2 that not only produced mucilage extrusion defects when introduced into Col-0, but also were correlated with the level of reduced MUM2 levels. Finally, the positive regulation of MUM2 by MADS-box TFs was established through a combination of CArG-box motif mutations in the regulatory sequences of MUM2, demonstrating the requirement of MADS-box TFs for proper MUM2 seed coat expression. This was further supported by luciferase-based transactivation assays, which produced increased MUM2 promoter activity on the addition of SHP1, SHP2 and SEP3. In addition, this positive regulation of MUM2 by SHP1, SHP2 and SEP3 was shown to occur in a complex with LUH, mediated by the co-regulator SEUSS (SEU), in protein-protein interaction assays such as yeast 2-hybrid (Y2H), Y3H, and bimolecular fluorescence complementation (BiFC). Overall, this study provides novel, clear evidence that MADS-box TFs can directly bind to MUM2 and mediate its activation with LUH. Future work includes expanding on and integrating MADS-box TFs into a working model that describes the regulatory pathways controlling mucilage polysaccharides production in the Arabidopsis seed coat.
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ItemThe role of LEUNIG_HOMOLOG in regulating mucilage release from the Arabidopsis testa( 2016)Upon hydration, Arabidopsis seeds release pectinaceous mucilage from the seed coat, which is primarily composed of rhamnogalacturonan I (RG-I). Mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) result in mucilage extrusion defects that are associated with increased galactose (Gal) residues present on the RG-I backbone. This structural modification is correlated with reduced expression of MUCILAGE MODIFIED 2 (MUM2), a gene encoding a β-galactosidase belonging to the Glycoside Hydrolase Family 35, suggesting that BGAL6/MUM2 is positively regulated by LUH. In considering how LUH promotes BGAL6/MUM2 expression in the seed coat, two contrasting models of regulation have been proposed. According to the ‘direct’ model, LUH and associated proteins, bind to BGAL6/MUM2 regulatory sequences and promote transcription of the gene. In the ‘indirect’ model, the LUH complex regulates activity of a transcription factor (TF) that then targets BGAL6/MUM2. In one version of this model, the intermediary TF is an activator and thus LUH promotes BGAL6/MUM2 expression indirectly by activating this TF. However, as LUH shares significant sequence similarity and functional redundancy with the transcriptional co-repressor LEUNIG (LUG), it has been proposed that LUH functions as a transcriptional repressor. According to this possibility, LUH promotes BGAL6/MUM2 indirectly by limiting the activity of a TF(s) that represses BGAL6/MUM2. In order to resolve the molecular mechanism by which LUH is regulating BGAL6/MUM2, various molecular approaches were undertaken in this study. Firstly, RNA sequencing (RNA-seq) was performed on developing wildtype and luh seed, which together with extensive bioinformatic analysis, led to a number of seed coat-expressed genes that are responsive to LUH being identified. Within the group of genes that were elevated in luh mutant seeds were TFs that are known to play a role in epidermal differentiation, raising the possibility that mis-expression of one or more of these genes interferes with expression of BGAL6/MUM2. Another interesting finding was that MADS-box TFs are elevated in the seed coat of luh mutant seeds, suggesting that these transcriptional regulators may play a role in the mucilage synthesis/modification process. To help identify the TF(s) involved in BGAL6/MUM2 regulation, regulatory elements in the MUM2 gene were characterised. Initial experiments showed that robust expression of BGAL6/MUM2 in the seed coat requires the presence of both the BGAL6/MUM2 promoter as well as the large first intron. Use of phylogenetic shadowing coupled with deletion analysis of the intron identified a conserved block of sequence in the 5’ region of the intron that contains both enhancer and repressor elements. Binding sites for MADS-box TFs were subsequently found in this region and, based on deletion experiments, categorized as enhancers that ensure robust seed coat expression. Finally, this study used a combination of techniques to determine whether LUH functions as an activator or repressor in the seed coat, and whether LUH activity in the seed coat is mediated through interactions with the co-regulators SEUSS and/or SEUSS-LIKE; proteins that are required for LUG activity. None of these results proved conclusive, but were consistent with LUH being a repressor that probably forms a complex with these LUG-associated factors. Overall this study provides clear evidence that LUH is a global regulator of genes active in the developing seed and that BGAL6/MUM2 regulation occurs indirectly via an as yet unidentified repressor.