Plant cell walls play a central role in cell growth and morphogenesis. All plant cells have a primary wall. The formation of a secondary cell wall is restricted to particular cell types, such as the xylem cells, highly lignified cells that provide support and transport functions to the plant. The mechanisms regulating secondary cell wall biogenesis remain largely unknown. To identify genes involved in such mechanisms, a genetic screen for mutants with altered xylem development in the primary root of Arabidopsis thaliana has been conducted. Three different classes of mutants were identified. They are characterised by increased number of xylem strands (m"), altered timing of protoxylem differentiation (tpx) and ectopic lignification (eh). Initial characterisation of the mutant phenotypes, establishment of different complementation groups and their map position in the Arabidopsis genome has been determined. Mutations in the EL [I locus have been characterised in further detail. The eli l mutants exhibited ectopic lignification of cells throughout the plant that never normally lignify. Xylem cells in elil were misshapen and failed to differentiate into continuous strands, causing a disorganised xylem. elil mutants also exhibited altered cell expansion resulting in a stunted phenotype. Abnormal distribution of cellulose and lignin was observed in elil cell walls. Ultrastructural analysis of elil cell walls using an anti-lignin antibody has revealed that that the ectopic deposition of lignin-like compounds occurs within an altered secondary wall. Furthermore, other previously described cell expansion mutants, such as lit, rswl (at the conditional temperature) and det3, exhibited lignification patterns reminiscent to that of elil mutants. Analysis of the genetic interactions of elil with the lit mutant revealed that ELlI and LIT genes act in independent pathways to control cell expansion. These results, together with the double mutant analysis of eli l with other cell expansion mutants suggested a link between cell growth and differentiation of secondary thickened walls. Map-based cloning placed the ELJ1 gene in a 140-Kb interval on the top arm of Arabidopsis chromosome V. A candidate gene approach was used that identified a gene encoding a cellulose synthase catalytic subunit (CesA), AthCesA-3 as a candidate. Sequence analysis revealed that the AthCesA-3 gene is mutated in two elil alleles sequenced, both mutations leading to amino acid substitutions. Initial complementation experiments of elil plants with the wild type AthCesA-3 gene appeared to restore the wild type phenotype, suggesting that mutations in the AthCesA-3 gene gave rise to the elil phenotypes. These studies represent an important contribution to our understanding of the molecular mechanism of cellulose deposition during cell expansion and secondary cell wall deposition during plant morphogenesis.

Growth anisotropy, in which cells grow predominantly in one direction, is common in plant cells, and an essential event for plant form and function. The direction and degree of growth anisotropy are governed by the mechanical properties of the primary cell wall. When aligned in a parallel manner, cellulose microfibrils accommodate great resistance in the direction of their alignment to expansion driven by isotropic turgor pressure. Using the Arabidopsis thaliana inflorescence stem as a model system, field emission scanning electron microscopy (FESEM) analysis demonstrated that the establishment of parallel arrangement of microfibrils is closely correlated with anisotropic cell expansion. In the novel anisotropy 1 (any1) mutant allele of the primary cellulose synthase CesA1, growth defects were correlated with random cellulose microfibril patterns in some inflorescence stem tissues. Microtubules have been considered to be the most likely candidates for controlling the orientation of cellulose microfibrils. Recent studies have indeed demonstrated a close association of the plasma membrane-localized cellulose-synthase-complexes (CSCs) that produce cellulose and cortical microtubules. Despite this close association, microtubule disruption did not cause cellulose microfibrils to lose parallel alignment in the radial and inner periclinal walls of cells in the inflorescence stem, suggesting that microtubules influence mechanical properties of cellulose microfibrils other than orientation. X-ray diffraction analysis demonstrated that cellulose crystallinity in wild-type plants declines at the growth-promoting temperature of 29°C, whereas crystallinity fails to adapt and remains high in mor1-1, the temperature-sensitive mutant whose microtubule arrays become disorganized at its restrictive temperature (29°C). This finding suggests that organized microtubules are involved in reducing cellulose crystallinity that normally accompanies increased cell expansion. Live-cell imaging of CSCs by tracking a yellow fluorescent protein (YFP)-tagged CesA6 subunit in hypocotyl cells demonstrated that dynamic and well-organized microtubules affect the velocity, the direction of movement, and the density of CSCs, suggesting that there is a close relationship between microtubules and CSCs. Together with the finding that microtubules also control the distribution of COBRA, a GPI-anchored wall protein that is essential for growth anisotropy, I discuss the variety of roles microtubules play in anisotropic growth.

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Glucans are polysaccharides integral to many materials and biological functions. Under the umbrella of Biomime, the Swedish Center for Biomimetic Fiber Engineering, this work has aimed to improve basic understanding of the biosynthesis of such glucans. This has been achieved through direct investigation of cellulose structure, and by developing the tools to analyze glucan biosynthesis. Notably we have identified a novel chemical effector of glucan synthesis processes and developed a proteomic toolkit useful for analyzing membrane-bound glycosyltransferases, the enzyme group responsible for glucan biosynthesis. During this work, glucan synthesis has been studied using both Gluconacetobacter and Populus cell suspension cultures. Publication I. Gluconacetobacter cellulose (BC) was used as a base to create a novel and well characterized nano-material with improved mechanical properties. This novel composite of BC and hydroxyethylcellulose (HEC) had improved tensile strength compared to pure BC. Through thorough study utilizing dispersion measurements, electron microscopy, nuclear magnetic resonance and X-ray diffraction it was shown that the improved properties derived from a layer of HEC coating each fibril. Publication II. Bacterial cellulose was labeled in specific positions with 13C (C4 and C6). These samples were analyzed by CP/MAS NMR along with cellulose samples from cotton and Halocynthia sp. For each sample spectral fitting was performed and general properties of crystal allomorph composition and fibril widths were determined. Calculations were also made for water accessible surfaces of the fibrils. The results showed that water accessible C4 surface signals are reflective of the allomorph composition of the sample, along with a distorted signal that derives due to fibril imperfections. Water accessible surface signals from the C6 region are instead derived from rotamer conformations of the C6 hydroxymethyl groupsfrom glucose residues. In Publication III, a high-throughput screen was used to identify an inhibitor of Golgi-derived glycosyltransferase activity, termed chemical A. The structural basis for inhibition was determined and in vitro assays of callose synthesis were performed. The in vitro assays revealed chemical A to also be an activator of callose synthesis. To understand this activation kinetic studies were performed, showing that chemical A is a mixed type of activator, which can bind either the free enzyme or the enzyme-substrate complex. Chemical A has uses in chemical genetics for dissecting processes involving callose synthesis, such as stress response and cell-plate formation. In publication IV, we present an in-house developed platform for proteomics with a distributed processing model. This in-house system has been central to many proteomics tasks, including for those presented in publication V, and is being distributed as the Automated Proteomics Pipeline (APP). In publication V, conditions for enrichment of Detergent-Resistant Microdomains (DRM) have been optimized for Populus trichocarpa cell cultures. The proteins enriched in DRM were identified using mass spectrometry based proteomics, and a functional model for DRM was proposed. This model involves proteins specialized in stress response, including callose synthase, and cell signaling. This further strengthens the arguments for DRMs as sites of specific cellular functions and confirms they play a role in glucan synthesis. / <p>QC 20140710</p>

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Morphogenesis in walled organisms represents a highly controlled process by which the variability of shapes arises through changes in the structure and mechanics of the cell wall. Despite taking different evolutionary paths, land plants and some brown algae exhibit great developmental and morphological similarities. In two brown algal model systems: the Sargassum muticum apex and the Fucus serratus embryo, I have used a combination of imaging techniques, growth analyses, surgical and pharmacological treatments, as well as molecular, biochemical and mechanical approaches to characterise the growth patterns and the cell wall contribution to shape change. To understand how the adult algal body is formed, I examined the branching strategy (phyllotaxis) in S. muticum. My results suggest that in S. muticum the spiral phyllotactic pattern and the apical cell division pattern are not linked. The phytohormone auxin and the biochemical changes of the cell wall do not seem to be correlated with the bud outgrowth, contrary to observations in plants. In summary, these results suggest Sargassum convergently developed a distinct growth mechanism with similar shape outcome as observed in plants. This dissertation is one of the first attempts to explore cell wall mechanics in brown algal development and its correlation with underlying cell wall biochemistry utilising the Fucus embryo as a known system. The results suggest a correlation between the wall mechanics and alginate biochemistry with the growing and non-growing regions of the embryo. In addition, altering cell wall deposition or composition has a strong effect on embryo rhizoid elongation and is, in certain cases, accompanied by significant increase in cell wall stiffness and reduction of alginate epitopes. Furthermore, preliminary results exploring transcriptomic changes during development indicate differential expression of particular alginate biosynthesis enzymes (mannuronan C5 epimerases) during development, suggesting alginate conformational modifications might be stage specific. These results contribute to the current knowledge addressing the importance of cell walls in brown algal development using novel tools and approaches. Understanding developmental processes in brown algae will provide a better insight how similar morphogenetic traits are established using different body-building mechanisms.

As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. Unlike tracheophytes that have stomata on anatomically complex leaves and stems, mosses bear stomata exclusively on spore-bearing organs (capsules). However, stomata do not occur in all mosses and, indeed, are absence in the earliest-divergent mosses (Takakia, Andreaea, Andreaeobryum and Sphagnum), suggesting that stomata originated in mosses independently of other plants. The occurrence of structurally unique pseudostomata in Sphagnum further confounds the resolution of homology of moss stomata with those of other plants. The five studies included in this dissertation are aimed at clarifying the structure, development and evolution of moss stomata. The first study focuses on the sporophyte anatomy and stomatal ultrastructure in two structurally and phylogenetically divergent mosses, Oedipodium and Ephemerum. Oedipodium is the sister to peristomate mosses and the first extant moss with true stomata. This monospecific genus has an elaborated capsule with an extended apophysis bearing numerous long-pored stomata. In contrast, Ephemerum nests within the peristomate mosses and has a reduced capsule that lacks an apophysis and has a few round-pored stomata. Ultrastructure of stomata is similar in these two mosses and comparable to that of tracheophytes, except that the stomata of mosses are not as structurally distinct from epidermal cells as are tracheophyte stomata. Anatomical features such as the presence of a cuticle, water-conducting cells, and spongy tissues with large areas for gas exchange are more pronounced in Oedipodium sporophytes and support the role of stomata in gas exchange and water transport during development and maturation. The second study examines changes in pectin composition during development in the model moss Funaria. Stomatal movement in tracheophytes requires guard cell walls to be strong, yet flexible, because they have to undergo reversible deformation to open and close the pore. Pectins are necessary for wall flexibility and proper stomatal functioning in seed plants. In this study of Funaria, immunogold-labeling using five antibodies to pectin epitopes was conducted on guard cell walls during development to relate these features to the limited movement of stomata in moss. Movement of Funaria stomata coincides with capsule expansion when guard cell walls are thin and pectinaceous. Walls dramatically increase in thickness after pore formation and the pectin content significantly decreases in mature guard cell walls, suggesting that a decrease in flexibility is responsible for the inability to open a close previously reported in older moss guard cells. Because this was the first study to demonstrate changes in pectin composition during stomatal development in any plant, a similar study was done on Arabidopsis to identify the main types of pectins in guard cell walls. Localization of pectins in guard cell walls of Arabidopsis is similar to mosses in the stage they can move, with homogeneous walls rich in arabinan pectins that are required for wall flexibility. This study extends knowledge of pectin composition from stomata of the moss Funaria with limited stomatal movement to an angiosperm in which stomatal activity is crucial to the physiological health of the plant. The fourth study describes stomata development and internal changes in sporophyte anatomy that lead to formation of air spaces in the moss Funaria. Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold-labeling was used to investigate the presence of pectin in the newly formed cavities. Stomata in mosses do not develop from a self-generating meristemoid like in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e., there are no stomatal lineage ground cells. This developmental pattern is congruent with the presence of a gene ortholog of FAMA, but not SPCH and MUTE, in Physcomitrella. The final study in this dissertation focuses on the enigmatic Sphagnum. Although true stomata are absent in early-divergent mosses, Sphagnum has specialized epidermal cells, pseudostomata, that partially separate but do not open to the inside. To further understand the structure, function and evolution of pseudostomata, capsule anatomy and ultrastructure of pseudostomata were detailed. As in moss stomata, pseudostomata wall architecture and behavior facilitate capsule dehydration, shape change, and dehiscence, supporting this common function. Unlike other moss stomata, pseudostomata collapse along their ventral walls and they lack a substomatal cavity. Similarities to true stomata include two modified epidermal cells with specialized cell walls that separate by cuticle deposition and respond to drying. Pseudostomata may be interpreted as modified stomata that suppressed substomatal cavity formation, which in turn eliminated pore development. However, clarification of the homology of pseudostomata and moss stomata will require genomic studies integrated with physiological and structural data. The studies described in this dissertation significantly advance our understanding of moss stomatal development and structure, and provide a comparison point to better evaluate the evolution of stomata. Moss capsule anatomy coupled with the exclusive existence of stomata on capsules supports the concept that stomata in moss are involve in gas exchange but also facilitate drying and dispersal of spores. Changes in wall architecture coupled with a decrease in total pectin explain the inability of mature stomata to move. Development and distribution of stomata in Funaria provides evidence of a direct and less elaborated mechanism for stomatal development than described in Arabidopsis. Resolving relationships among early land plants, especially hornworts and mosses, the only bryophyte groups with stomata, is critical to understanding stomata evolution. Evaluated together, the results of this dissertation are consistent with a single origin of stomata in land plants.

Growth anisotropy, in which cells grow predominantly in one direction, is common in plant cells, and an essential event for plant form and function. The direction and degree of growth anisotropy are governed by the mechanical properties of the primary cell wall. When aligned in a parallel manner, cellulose microfibrils accommodate great resistance in the direction of their alignment to expansion driven by isotropic turgor pressure. Using the Arabidopsis thaliana inflorescence stem as a model system, field emission scanning electron microscopy (FESEM) analysis demonstrated that the establishment of parallel arrangement of microfibrils is closely correlated with anisotropic cell expansion. In the novel anisotropy 1 (any1) mutant allele of the primary cellulose synthase CesA1, growth defects were correlated with random cellulose microfibril patterns in some inflorescence stem tissues. Microtubules have been considered to be the most likely candidates for controlling the orientation of cellulose microfibrils. Recent studies have indeed demonstrated a close association of the plasma membrane-localized cellulose-synthase-complexes (CSCs) that produce cellulose and cortical microtubules. Despite this close association, microtubule disruption did not cause cellulose microfibrils to lose parallel alignment in the radial and inner periclinal walls of cells in the inflorescence stem, suggesting that microtubules influence mechanical properties of cellulose microfibrils other than orientation. X-ray diffraction analysis demonstrated that cellulose crystallinity in wild-type plants declines at the growth-promoting temperature of 29°C, whereas crystallinity fails to adapt and remains high in mor1-1, the temperature-sensitive mutant whose microtubule arrays become disorganized at its restrictive temperature (29°C). This finding suggests that organized microtubules are involved in reducing cellulose crystallinity that normally accompanies increased cell expansion. Live-cell imaging of CSCs by tracking a yellow fluorescent protein (YFP)-tagged CesA6 subunit in hypocotyl cells demonstrated that dynamic and well-organized microtubules affect the velocity, the direction of movement, and the density of CSCs, suggesting that there is a close relationship between microtubules and CSCs. Together with the finding that microtubules also control the distribution of COBRA, a GPI-anchored wall protein that is essential for growth anisotropy, I discuss the variety of roles microtubules play in anisotropic growth. / Science, Faculty of / Botany, Department of / Graduate

Cell wall structure was investigated in 20 different red algae. Representatives from all 4- families of the order Ceramiales and one family of the order Gigartinales were investigated. Of these, 3 genera, Polysiphonia, Pterosiphonia and Antithamnion were investigated with regards to both the cellulosic and mucilaginous portions of the cell wall. A new staining technique utilizing a combination of ruthenium red and osmium tetroxide as a postfixation was used in the latter portion of the study. The ultrastructure of pit connections was examined in all algae. The inner cellulosic portion of the cell wall consists of a reticulate pattern of microfibrils which appear densely stained, In Pterosiphonia this cellulosic portion was found to consist of 2 layers; an inner layer of microfibrils which ensheathed individual cells and an outer layer of microfibrils which ensheathed the entire thallus and was in contact with the mucilaginous coat. The microfibrils in the inner layer appear nearly cross-sectioned, while those in the outer layer appear more longitudinally oriented to the plane of sectioning. The outer mucilaginous coat covers the entire thallus. It consists of 4 layers. The first or outermost layer consists of loose bunches of microfibrils extending out from the second layer. The second layer consists of a zone of medium electron density approximately 750 A in thickness. The third layer is wholly contained within the second layer. It is composed of a densely staining band of microfibrils extending from a similarly staining membrane-like structure. The fourth layer is a densely stained membrane-like structure in contact with the cellulosic portion of the cell wall. An additional layer, the D layer, is sometimes found in the cell wall. When present it is found in the outermost portion of the cellulosic wall and obscures the fourth layer of the mucilaginous coat. It consists of a densely staining amorphous material. Investigation of the pit connection showed the occurrence of 2 stages of one basic pit structure. One stage, the single disc stage-pit structure, has been found in all algae investigated. It consists of a solid, lenticular, membrane-bound plug situated within an aperture in the cell wall. The plug consists of a granular material surrounded by a zone of densely staining amorphous material. The other stage, the double disc stage pit structure, is a modification of the single disc stage. It is not found in young cells near the apex of the thallus, but only in cells which have, or are undergoing, rapid elongation and vacuolation. This pit structure has only been observed in axial cells of the family Ceramiaceae in the order Ceramiales. The double disc stage pit structure differs from the single disc stage in that the granular material of the plug is segregated into 2 regions or plates, one on either side of the plug. The central region of the plug at first appears clear but later appears to be partially occupied by a granular to fibrillar material. The differentiation of the double disc stage pit structure from the single disc stage has been described. These observations are thought to support and confirm the earlier work of Jungers (25). However, his observations have been extended through the use of electron microscopy in this study. It has been proposed that the terms used in this study, single disc stage- and double disc stage pit structures, replace the terms Polysiphonia and Griffithsia pits used by Jungers. / Science, Faculty of / Botany, Department of / Graduate