The yeast cell wall provides a first barrier to the environment, confers shape and stability to the cells, and serves as a model for fungal cell wall biosynthesis and function in general. During normal growth, during mating and upon cell surface stress, new wall synthesis is induced by a conserved signaling cascade, the cell wall integrity (CWI) pathway. A signal is initiated by plasma membrane-spanning sensors and transduced through a mitogen-activated protein kinase (MAPK) cascade, which ultimately activates a transcriptional activator, Rlm1. The first part of this thesis analyses the role of this MADS-box transcription factor in the milk yeast Kluyveromyces lactis, which has not been investigated, until now. With respect to the distribution of the upstream CWI sensors, evidence for the existence of a special plasma membrane microcompartment, generally referred to as eisosomes, in the milk yeast is provided in the second part of the thesis. Regarding the transcription factor KlRlm1, its impact on the physiology of K. lactis seems to be different from its homolog in Saccharomyces cerevisiae, ScRlm1, although it clearly acts in CWI signaling, too. Thus, in contrast to the Scrlm1 mutant, a Klrlm1 deletion is sensitive, rather than hyper-resistant, towards Congo red and Calcofluor white, typical stress agents used in cell wall research. Data on cross-complementation of the two genes in the respective heterologous yeast indicate that KlRlm1 and ScRlm1 each perform their optimal function only in the native host.To investigate the impact of a Klrlm1 deletion on the transcriptional profile of K. lactis, data from total mRNA sequencing were analyzed in comparison to a wild-type strain. Many of the genes identified did not correspond to known Rlm1 target genes in S. cerevisiae, but many relate to other stress responses (e.g. KlGRE1, KlFMP16, KLLA0C05324g, KLLA0F18766g, KlUGX2) and to chitin synthesis (KlCHS1, KlSKT5 and KlYEA1), both probably connected to cell wall composition. The functions of a large group of KlRlm1 dependent genes identified here are yet uncharacterized or lack homologs in S. cerevisiae. The plasma membrane of fungi is a specialized organelle, which is ordered into several lateral domains, which we define as microcompartments, since each is composed of a special combination of proteins in their lipid environment. Such microcompartments are believed to control a variety of signaling (and transport) processes in all sorts of eukaryotic cells. Microcompartmentalization is also observed in the yeast plasma membrane, e.g. displayed by the CWI sensors in K. lactis, as shown in this thesis. Since distribution of the latter sensors is reminiscent of that of eisosomes, it was also investigated by live-cell fluorescence microscopy, how KlPil1, KlLsp1 and KlSur7 (all homologs of eisosomal proteins in S. cerevisiae) are distributed. Since they form the typical membrane patches, which are not present in deletion mutants of KlPIL1, the major structural component of eisosomes, one can conclude, that eisosomal microcompartments form in K. lactis and are composed similar to their counterparts in S. cerevisiae. The CWI sensors are excluded from these structures and form their separate microcompartments. The exact physiological function of eisosomes in fungi is still a matter of debate and future studies in K. lactis may help to address this role.

Although the cell wall is an essential plant cell structure influencing several important aspects of plant development, little is known about the genes and proteins that regulate its structure and function. In this dissertation, we first examined the regulation of the nuclear proteome of rice (Oryza sativa) in response to cell wall removal. Using labelree comparative proteome analysis we found that, upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 nuclear proteins were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. This study led to a novel discovery that removal of the cell wall results in dynamic changes in the nuclear proteome affecting the regulation of proteins involved in various molecular processes such as chromatin and nucleosome assembly, protein-DNA complex assembly, and DNA packaging. To further study cell wall development, we utilized the unique features of cotton fiber and performed a proteomic study using four stages during cotton fiber cell wall development including 10 days post anthesis (dpa), 15 dpa, 25 dpa, and 35 dpa. In addition, we aimed to improve protein extraction for recalcitrant fiber stages using pressure cycling technology (PCT). To our knowledge, this study identified the largest number of proteins and differentially expressed proteins in the G. hirsutum cotton species including the 35 dpa fiber proteome which has not been examined in prior reports. Additionally, in order to identify key genes regulating cell wall cellulose content, a mutant with a substantial reduction in cellulose was characterized in Arabidopsis. It was found that the mutated gene was VHA-E1. We found that the VHA-E1 protein formed a distinct plate in the boundary of two fusion-destined vacuoles to tether vacuoles together. The eventual vacuole fusion was achieved by pinching off the vacuole-boundary plate producing a stable membrane-bound intravacuolar globoid. These observations demonstrate that VHA-E1 may be involved in a novel cellular process regulating fusion of vacuoles by forming a cellular structure referred to as the vacuole boundary plate. Altogether, these findings suggest plant vacuole fusion and central vacuole biogenesis involve an unprecedented mechanism in Arabidopsis.

The fungal cell wall serves as the interface between the organism and its environment. Complex carbohydrates are a major component of the cell wall, , glucan, mannan and chitin. β-Glucan is a pathogen associated molecular pattern (PAMP) composed of β-(1 → 3,1 → 6)-linked glucopyranosyl repeat units. This PAMP plays a key role in fungal structural integrity and immune recognition. Glycogen is an α-(1 → 4,1 → 6)-linked glucan that is an intracellular energy storage carbohydrate. We observed that glycogen was co-extracted during the isolation of β-glucan from SC5314. We hypothesized that glucan and glycogen may form a macromolecular species that links intracellular glycogen with cell wall β-(1 → 3,1 → 6)-glucan. To test this hypothesis, we examined glucan-glycogen extracts by multi-dimensional NMR to ascertain if glycogen and β-glucan were interconnected. H NMR analyses confirmed the presence of glycogen and β-glucan in the macromolecule. Diffusion Ordered SpectroscopY (DOSY) confirmed that the β-glucan and glycogen co-diffuse, which indicates a linkage between the two polymers. We determined that the linkage is not via peptides and/or small proteins. Our data indicate that glycogen is covalently linked to β-(1 → 3,1 → 6) glucan via the β -(1 → 6)-linked side chain. We also found that the glucan-glycogen complex was present in , and , but was not present in or hyphal glucan. These data demonstrate that glucan and glycogen form a novel macromolecular complex in the cell wall of and other species This new and unique structure expands our understanding of the cell wall in species.

Plants as sessile organisms are susceptible to many environmental stresses such as drought, and salinity. They have therefore evolved mechanisms to acclimate and tolerate environmental stresses. Knowledge of the molecular aspects of abiotic stress gleaned from extensive studies in Arabidopsis has provided much information on the complex processes underlying plant response to abiotic stresses. Nevertheless, there is a need for integration of the knowledge gained and a systematic molecular genetic dissection of the complex responses to abiotic stress. In this study in Arabidopsis, comparative expression profiling analysis of progressive (pDr) and moderate (mDr) drought treatments revealed common drought responses, as well as treatment specific signatures responses to drought stress. Under prolonged moderate drought plants develop different mechanisms for acclimation: induction of cell wall loosening at early stage, and a change in hormonal balance (ABA: JA) at late stage of moderate drought. Taking a reverse genetics approach, a MYB transcription factor (MYB109) has been identified as a regulator of growth under drought and salt stress. Global expression profiling showed possible mechanisms of how MYB109 modulates growth under drought conditions: as a regulator of RNA processing and splicing and as a negative regulator of jasmonic acid biosynthesis and signaling. A forward genetics screen for drought and salt tolerance of transposon activation tag (ATag) lines led to the discovery of novel genes, which shed light on unexplored areas of abiotic stress biology. Utilizing this strategy, a potential role for cell wall modification and MATE transporters in response to drought and salt stress has been discovered, which needs further analysis to integrate this information on the role of these biological processes in plant stress biology. / Ph. D.

<p> The increase in microbes resistant to a wide array of antibiotics has led to the need for the development of novel antimicrobials. However in order to develop new antimicrobials, novel pathways need to be targeted. Teichoic acid is an anionic polymer covalently attached to the cell wall of Gram-positive bacteria. Recent research has demonstrated that teichoic acid genes are indispensable to the viability of Bacillus subtilis. This makes teichoic acid biosynthetic proteins ideal candidates for the development of a new antimicrobial. Of the teichoic acid glycerol phosphate (tag) genes involved in the biosynthesis of teichoic acid in B. subtilis 168, a conditional deletion mutant of tagD, whose protein product encodes the proposed glycerol-3-phosphatecytidylyltransferase, has been previously constructed and was shown to have a lethal phenotype upon depletion of TagD. This was used in a microarray analysis to find genes that were transcriptionally up-regulated upon the depletion of TagD in B. subtilis 168. Ten candidate genes were selected from those up-regulated and used in the design of a novel, real-time, cell-based luminescent reporter system that responds to lesions in wall biosynthesis. Characterization of these reporter systems in tag gene deletion backgrounds and an examination of their response to antibiotics of various mechanism of action led to the identification of our candidate reporter system P ywac, a robust reporter of both lesions in teichoic acid and peptidoglycan synthesis. In a proof-of-principle screen, the use of Pywac as a reporter of lesions in the cell wall was validated. This reporter system is unique in that it combines conventional genetics with a high throughput capacity. It will not only be amenable for screening small molecules to find inhibitors that impinge on teichoic acid biosynthesis, but it can also be used to probe genetic interactions in B. subtilis. </p> / Thesis / Master of Science (MSc)

Corn silage is one of the major components in dairy cattle rations in the United States. Many factors affect the nutritional composition of corn for silage, such as cropping system, including cover crops, and the composition of the corn plant cell wall. The objectives of the first study were to determine the nutritional quality of different winter crops for silage and to determine the impact of the various winter crops on the succeeding productivity of corn and sorghum. Experimental plots were planted with 15 different winter crop treatments: 5 winter annual grasses in monoculture or with one of two winter annual legumes (crimson clover [CC] and hairy vetch [HV]). After harvesting the winter crops, each plot was planted with either corn or forage sorghum. Crimson clover increased DM yield compared to monocultures but HV did not. Adding legumes increased the crude protein concentration, but reduced the fiber and sugar concentrations of the forages. Even though in vitro neutral detergent fiber digestibility was reduced with the addition of legumes, the concentration of highly digestible non-fibrous components is greater in the mixtures than the monocultures, increasing the nutritive value of the silage. The objective of the second study was to determine the cell wall (CW) composition along the corn stalk. Three phytomers of corn plants were examined: center (C) of ear insertion, upper (U) and lower (L) phytomers. Each phytomer was cut into 4 sections: top (T), middle (M), bottom (B), and node (N). The CW, uronic acid (UA), glucose (GLU), and lignin concentrations did not change among phytomers. The concentrations of arabinose (ARA) and xylose (XYL) were greater in the U than in the L phytomers. Concentrations of CW, ARA, and XYL increased from B to T within the phytomer, but UA and GLU concentrations decreased from B to T. Lignin did not change within the phytomer. In mature corn for silage, changes within the corn internode may be more useful in determining how the environment changes the CW. / Ph. D. / Corn silage is one of the major components in dairy cattle rations in the United States. Many factors can affect the nutritional composition and digestibility of corn for silage, including the crops planted before the corn and the maturity of the corn. I first explored the nutritional quality and potential of different winter crops for use as silage and how these various winter crops impacted the succeeding productivity of corn and sorghum. We used 15 different winter crop treatments: 5 winter annual grasses in monoculture or mixed with one of two winter annual legumes (crimson clover and hairy vetch). After harvesting the winter crops, corn and forage sorghum were planted. The addition of legumes increased winter crop yield compared to monocultures. Adding legumes increased the protein concentration, but reduced the reduced the fiber and sugar concentrations of the winter crops. Fiber digestibility was reduced with the addition of legumes. The type of grass and legume did not change the nutrient composition of the corn and sorghum. I then explored how the cell wall (CW) composition changes along the corn stalk. Corn plants are split up into phytomers. Each phytomer contains a leaf, a section of the stalk called an internode, and a node (connects internodes). Phytomers at the top of the corn plant are less mature than ones at the bottom, and maturity increases from bottom to top within a corn internode. Three phytomers of individual corn plants were each cut into 4 sections: top, middle, bottom, and node. In plants, the primary cell wall is deposited first. The primary cell wall contains cellulose, hemicellulose, and pectin. The secondary cell wall is deposited after growth. The secondary cell wall is composed of cellulose, hemicellulose, and lignin. Corn that is harvested for cows to eat is usually at a late stage of maturity. Therefore, in our study we saw very few differences among phytomers as cellulose, hemicellulose, pectin, and lignin had most likely been fully deposited. However, within the corn internode, we did see variability in the corn plant cell wall. The cell wall concentration overall increased with maturity within the corn internode. Further, arabinose and xylose concentrations (sugars from hemicellulose) also incased with maturity. Hemicellulose is important in connecting the rest of the cell wall to lignin later in maturity. The concentration of uronic acids from pectin and glucose from cellulose decreased with maturity because these are typically deposited first within the plat cells and then level off once lignin is deposited. In mature corn for silage, changes within the corn internode may be more useful in determining how the environment changes the CW.

No / Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

Plants colonised land 460 million years ago and charophytes represent the closest living relatives of land plants. The ability to live on land may depend on the presence of certain cell wall polysaccharides such as xyloglucan, a hemicellulose exclusively found in land plants (Popper and Fry, 2003). The cell walls of charophytes are poorly characterised. The aim of this project was to use biochemical techniques to characterise the cell wall polysaccharides of charophytic algae in relation to early land plant phylogeny. Hydrolysis of Coleochaete scutata and Chara vulgaris cell walls in 2 M trifluoroacetic acid yielded predominantly GalA, Gal, Glc and Man residues and also some Ara, Xyl and traces of Fuc and Rha. In addition, hydrolysis of Chara pectin revealed an abundance of an unusual monosaccharide, 3-O-methyl-D-galactose, which was structurally identified by a series of 1-D and 2D NMR spectroscopy by COSY, TOCSY, NOESY and HSQC. 3-O-Methyl-D-galactose is more commonly found in lycophyte cell walls where its presence has been suggested to be related to lycophytes’ evolutionarily isolated position (Popper et al., 2001). The newly discovered presence of 3-O-methyl-D-galactose in charophyte pectin suggests that this polymer may be more complex than previously thought. Coleochaete and Chara hemicellulose extracts were fractionated by anion-exchange chromatography into five classes. A strongly anionic fraction from Chara hemicellulose was found to be rich in Glc, Xyl, Gal and Fuc suggestive of a xyloglucan-like polysaccharide. However, XEG was unable to produce diagnostic xyloglucan oligosaccharides in either Coleochaete or Chara hemicelluloses. Xylanase and mannanase digestion of Coleochaete and Chara hemicelluloses gave xylan- and mannan-oligosaccharides. Furthermore, lichenase digestion of Coleochaete hemicellulose yielded an unusual octasaccharide composed of approximately equimolar xylose and glucose. My work has shown that charophyte cell walls are a source of undiscovered monosaccharides and potentially novel pectic and hemicellulosic domains which may have important functions in enabling the successful colonisation of land by plants.

The present thesis presents new renewable, antimicrobial and biodegradable superabsorbent polymers (SAPs), produced from the cell wall of zygomycetes fungi. The cell wall was characterized and chitosan, being one of the most important ingredients, was extracted, purified, and converted to SAP for use in disposable personal care products designed for absorption of different body fluids. The cell wall of zygomycetes fungi was characterized by subsequent hydrolysis with sulfuric and nitrous acids and analyses of the products. The main ingredients of the cell wall were found to be polyphosphates (4-20%) and copolymers of glucosamine and N-acetyl glucosamine, i.e. chitin and chitosan (45-85%). The proportion of each of these components was significantly affected by the fungal strain and also the cultivation conditions. Moreover, dual functions of dilute sulfuric acid in relation to chitosan, i.e. dissolution at high temperatures and precipitation at lowered temperatures, were discovered and thus used as a basis for development of a new method for extraction and purification of the fungal chitosan. Treatment of the cell wall with dilute sulfuric acid at room temperature resulted in considerable dissolution of the cell wall polyphosphates, while chitosan and chitin remained intact in the cell wall residue. Further treatment of this cell wall residue, with fresh acid at 120°C, resulted in dissolution of chitosan and its separation from the remaining chitin/chitosan of the cell wall skeleton which was not soluble in hot acid. Finally, the purified fungal chitosan (0.34 g/g cell wall) was recovered by precipitation at lowered temperatures and pH 8-10. The purity and the yield of fungal chitosan in the new method were significantly higher than that were obtained in the traditional acetic acid extraction method. As a reference to pure chitosan, SAP from shellfish chitosan, was produced by conversion of this biopolymer into water soluble carboxymethyl chitosan (CMCS), gelation of CMCS with glutaraldehyde in aqueous solutions (1-2%), and drying the resultant gel. Effects of carboxymethylation, gelation and drying conditions on the water binding capacity (WBC) of the final products, were investigated. Finally, choosing the best condition, a biological superabsorbent was produced from zygomycetes chitosan. The CMCS-based SAPs were able to absorb up to 200 g water/g SAP. The WBC of the best SAP in urine and saline solutions was 40 and 32 g/g respectively, which is comparable to the WBC of commercially acceptable SAPs under identical conditions (34-57 and 30-37 g/g respectively). / <p>Disputationen sker fredagen den 1 oktober kl. 10.00 i KA-salen, Kemigården 4, Chalmers, Göteborg</p>

Plant cells are routinely exposed to various pathogens and environmental stresses that cause cell wall perturbations. Little is known of the mechanisms that plant cells use to sense these disturbances and transduce corresponding signals to regulate cellular responses to maintain cell wall integrity. Previous studies in rice have shown that removal of the cell wall leads to substantial chromatin reorganization and histone modification changes concomitant with cell wall re-synthesis. But the genes and proteins that regulate these cellular responses are still largely unknown. Here we present an examination of the nuclear proteome differential expression in response to removal of the cell wall in rice suspension cells using multiple nuclear proteome extraction methods. A total of 382 nuclear proteins were identified with two or more peptides, including 26 transcription factors. Upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. Gene ontology analysis of the differentially expressed proteins indicates that chromatin & nucleosome assembly, protein-DNA complex assembly, and DNA packaging are tightly associated with cell wall removal. Our results indicate that removal of the cell wall imposes a tremendous challenge to the cells. Consequently, plant cells respond to the removal of the cell wall in the nucleus at every level of the regulatory hierarchy.