School of BioSciences

Plants possess cell wall, a polysaccharide exoskeleton which encompasses all plant cells. Cell wall gives plant cells mechanical support, defines their shape, enables growth and water transport through a plant. It also has important role in communication with the external environment. Regulation of plant cell wall biosynthesis and cell and organ morphogenesis depends on cell’s ability to detect mechanical signals originating both from the external environment and from internal plant tissues. Thanks to the presence of the cell wall, all living plant cells develop constant internal pressure generated by the active water uptake, known as turgor pressure, which enables them to grow. Thus, actively growing cells in the tissue are exerting mechanical stress to each other. In order to properly coordinate cell growth, tissue morphogenesis and maintain cell-to-cell adhesion, plant cell have to detect these mechanical signals. That is performed by a group of still not well enough characterized plant mechanosensitive proteins. Mechanosensors are proteins capable of detecting changes in mechanical stress patterns and translating them into physiological and developmental outputs. One of plant mechanosensitive proteins, DEFECTIVE KERNEL1 (DEK1) has shown to be a very important in proper plant development. DEK1 bears similarity with animal cysteine proteases of Calpain superfamily. DEK1 is very important for plant development since all null alleles are embryo lethal. During the last 20 years of DEK1 studies, this protein has proven to be a very difficult for different molecular and biochemical manipulations. As a consequence, very little is known about its direct target proteins. Wang and co-workers (2003) and Johnson and co-workers (2008) have given a valuable contribution to biochemical understanding of DEK1 by determining that it functions as Cys-protease in similar way as animal calpains. However, a lot of indirect knowledge was gathered about the effects of disruption and modulation of DEK1 activity. DEK1 is important for proper organ development, epidermal specification, and maintenance. However, some studies have inferred that DEK1 affects expression of different cell wall related genes, and it regulates cell-to-cell adhesion in epidermal cells. This led to two extensive studies (Amanda et al., 2016, 2017) which demonstrated importance of DEK1 in regulation leaf epidermal cell walls in A. thaliana mature leaves and inflorescence stems. These studies demonstrated that DEK1 also influences cell wall thickness and cell-to-cell adhesion and that it could potentially regulate cell growth and expansion. Building up on this research, we decided to try to further characterize molecular and biomechanical aspects of DEK1 mediated cell wall regulation, with special emphasis on regulation of cellulose synthesis. We used two mutant lines, with modulated DEK1 activity, a constitutive overexpressor for DEK1 CALPAIN domain and a point mutant in CALPAIN domain, dek1-4. In Chapter 3 we demonstrated that DEK1 regulates dynamics of Cellulose Synthase Complexes (CSCs). Both lines showed decreased crystalline cellulose contents. This led us to investigate if velocity of CSCs in cotyledons, was affected, since it is known that changes in cellulose contents are often caused by defects in CSC. We found that bothDEK1 modulated lines we used have significantly decreased velocity of CSCs. We have also examined plasma membrane turnover rates of CSCs and found out that after photo-bleaching OE CALPAIN has much faster recovery rates compared to Col-0 wild type, while dek1-4 has lower exocytotic rates of CSCs, and much longer life-time of CSCs inserted into the plasma membrane. These results suggested that DEK1 regulates different aspects of CSC dynamics, possibly through interaction with different regulatory proteins. Decrease in cellulose contents we observed in DEK1 modulated lines, prompted us to investigate how this reflects biomechanics and structural properties of epidermal cotyledon cell walls of DEK1 modulated lines, which is described in Chapter 4. To achieve this, we developed a novel microdissection method for isolation and mechanical and structural characterization of native epidermal cell wall monolayers using atomic force microscopy (AFM). AFM force spectroscopy assays showed that both DEK1 modulated lines had stiffer cell walls compared to Col-0. This was awkward since we initially detected decrease in crystalline cellulose which implied decrease in cell wall stiffness. However, subsequent high-resolution AFM imaging has revealed that DEK1 modulate lines cells walls have their cellulose microfibrils organized in thicker bundles than Col-0. Also, polysaccharide composition analysis has revealed that DEK1 modulated lines have increased abundance of pectins, which could also be responsible for the observed increase in cell wall stiffness. Previous work has shown that different dek1 mutants and modulated lines have defects in cell-to-cell adhesion. This implied that DEK1 may be involved in sensing and/or maintaining cell wall integrity (CWI). We performed several growth assays to determine role of DEK1 in CWI, which is described in Chapter 5. We performed cellulose synthesis perturbation assays with cellulose synthesis inhibitor Isoxaben and obtained very interesting results. While OE CALPAIN plants were hypersensitive to Isoxaben, dek1-4 has shown complete insensitivity. Furthermore, a regular CWI maintenance response, reported in A. thaliana as result of compromised CWI, ectopic lignification in seedlings’ roots was absent in both DEK1 modulated lines we examined. We detected interesting growth response of DEK1 lines to NaCl and mannitol treatments as well. Although these findings are pointing out that DEK1 could be part of CWI signalling pathways, more experiments are necessary to fully elucidate possible role of DEK1 in CWI sensing and/or maintenance pathways, especially to check if DEK1 is interacting with Catharanthus roseus Receptor Like Kinase group of CWI sensors. Studies on 4-month old short day grown DEK1 modulated lines, have shown defects in branching, with development of fasciated stem branches in a DEK1 modulated line overexpressing CALPAIN domain (Amanda et al., 2017). This result pointed out to a possibility that DEK1 may regulate organ morphogenesis and patterning at the level of shoot apical meristem (SAM). Work towards elucidating role of DEK1 in SAM maintenance and organ patterning is detailed in Chapter 6. We determined that OE CALPAIN had significantly larger central zone of SAM as well as larger individual SAM cells in central zone, as well as higher distribution of cell sizes, implying possible cell expansion defects. dek1-4 did not exhibited changes in SAM central zone size or individual stem cell size, but it seemed that it had increased number of stem cells in SAM central zone. Both DEK1 lines had perturbation of phyllotaxis on SAM level, with disturbed divergence angles between floral primordia. Disturbed phyllotaxis was also observed between siliques, in mature plants. In addition to this, OE CALPAIN has exhibited occurrence of multiple (up to four) siliques growing from a single stem node. All this is pointing out that DEK1 might participate in hormone-signalling in the SAM. DEK1 is a highly intriguing protein. However, since it is a unigene, and in addition to that, a regulatory protease, it probably participates in multiple signalling pathways, which makes understanding its function much more complicated.

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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