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In strongly acidic soils (pH <4.5) aluminium (Al) becomes soluble in quantities that can lead to Al phytotoxicity. It is estimated that approximately 30 % of the worlds’ potentially arable lands are acidic, with Al toxicity the most limiting factor for plant growth on acid soils. With increasing use of marginal land in cropping systems, this area could reach 70 %. Cell wall pectin provides up to 70 % of the root cation exchange capacity. Pectin is suggested to control a number of physiological properties of the plant cell wall such as porosity, charge density, microfibril spacing and pH. The ability of pectin to bind cations is not only important for the uptake of nutrients but is implicated in metal toxicity, in particular Al. Despite over a century of research, the mechanisms of Al toxicity are yet to be fully elucidated or agreed upon. Gluconacetobacter xylinus is a gram-negative, soil dwelling bacterium which produces extracellular cellulose. It is an established archetype for the study of cellulose biogenesis. In the presence of pectin in the growth medium, the bacterium can form cellulose-pectin composites. Recently, the bacterium has been used to form composites as model cell walls to understand plant cell wall deposition. Additionally, bacterial cellulose composites in their natural hydrated state mimic the hydration state of primary plant cell walls. The aim of this project was to attempt to incorporate this novel cell wall analog into laboratory investigations into metal interactions with plant cell walls. Preliminary work was undertaken to optimise the bacterial culture medium, growth conditions, analysis of the composites and developing an overall general methodology. The medium buffering system was altered, growth under non-optimal pH conditions was evaluated and Al was successfully incorporated into the composites. Appropriate sample preparation for scanning electron microscopy (SEM) of the composites was determined. This work resulted in the successful production of bacterial cellulose-pectin composites with 30 % w/w pectin incorporation. The effect of Al on the tensile properties of the composites was examined. Aluminium had no effect on the stress/strain profiles, confirming the hypothesis that pectin is not the main load bearing component of the cell wall. The composites were used to investigate the effects of Al and other trace metals (copper (Cu), gadolinium (Gd), lanthanum (La), ruthenium (Ru) and scandium (Sc)) on the hydraulic conductivity of the composites. Hydraulic conductivity was reduced to ≈ 30 % of the initial flow rate by 39 μM Al and 0.6 Cu μM, ≈ 40 % by 4.6 μM La, 3 μM Sc and 4.4 μM Ru, and ≈ 55 % by 3.4 μM Gd. These metal concentrations were selected based on the concentrations causing a 50 % reduction in root elongation in cowpea (Vigna unguiculata L.). This study demonstrated that all the trace metals caused a similar decrease of hydraulic conductivity, despite the different concentrations of the metals used. Scanning electron microscopy showed changes in pectin porosity with metal binding which may account for the decreases in hydraulic conductivity observed. As the composites could not be used as a model material in all investigations, pectin-only systems were employed in a rheological study to investigate the effect of increasing concentrations of Al, Ca, Cu or La at pH 4 on pectin (degree of esterification 30 %, 1 % w/v) gel physical strength. Comparing similar saturation levels, La formed the weakest gel, followed by Ca, which was similar to Al, while the strength of Cu gels was almost an order of magnitude stronger than the other cations. This study was the first to investigate Al and La pectate gel strength. The swelling of the gels also varied, with Ca gels being the most swollen. Pectin was also used to determine the exchange selectivity of Al, Cu, Gd, La, Ru and Sc toward Ca pectate. The order of selectivity was found to be Sc>Gd>La>Cu>Ru>Al. There were some parallels between this sequence and the rhizotoxicity data of the metals, suggesting that the strength with which metals bind to pectin is an indication of their rhizotoxicity. Through the use of synthetic pectate gel systems new information was discovered about the strength of pectin gels and the selectivity of trace metals towards pectin. These findings were in keeping with those of a number of related studies, as well as with studies of plant root tissue. Overall, the novel bacterial cellulose-pectin cell wall analog was successfully integrated into research into Al and other metal toxicity in plants, and offers a useful system that can overcome some of the difficulties encountered when using plant cell wall tissue. Further research may be warranted on manipulating the growth system to produce composites in the presence of the metal (ie. metal added to the growth medium), as opposed to post-formation treatments. Moreover, the production of a three way composite of cellulose, hemicellulose and pectin would likely be another useful analog for plant cell wall material.

Pinus radiata is by far the dominant species grown in New Zealand plantations as a renewable source of wood. Several wood quality issues have been identified in the material produced, including the high incidence of compression wood, which is undesirable for end users. At present our understanding of the complex array of developmental processes involved in wood formation (which has a direct bearing on wood quality) is limited. Hence, the forest industry is interested in attaining a better understanding of the processes involved. Towards this goal, and for reasons of biological curiosity, the experiments described in this thesis were carried out to investigate several aspects of xylem cell development. In an in arbor study, changes in the orientation of cortical microtubules and cellulose microfibrils were observed in developing tracheids. Results obtained provide evidence that cortical microtubules act to guide cellulose synthase complexes during secondary wall formation in tracheids. The mechanisms involved in controlling cell wall deposition in wood cells are poorly understood, and are difficult to study, especially in arbor. A major part of this thesis involved the development of an in vitro method for culturing radiata pine wood in which hormone levels, nutrients, sugars and other factors, could be controlled without confounding influences from other parts of the tree. The method developed was used in subsequent parts of this thesis to study compression wood development, and the influence of the hormone gibberellin on cellulose microfibril organisation in the cell wall. Results from the in vitro compression wood experiments suggested that: 1. when a tree is growing at a lean, the developing cell wall was able to perceive compressive forces generated by the weight of the rest of the tree, rather than perceive the lean per se. 2. ethylene, rather than auxin, was involved in the induction of compression wood. Culture of stem explants with gibberellin resulted in wider cells, with steeper cortical microtubules, and correspondingly steeper cellulose microfibrils in the S2 layer of developing wood cells. This observation provides further evidence that the orientation of microtubules guides the orientation of cellulose microfibrils. Overall, the work described in this thesis furthers our knowledge in the field of xylem cell development. The stem culture protocol developed will undoubtedly provide a valuable tool for future studies to be carried out.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 115-123).

Abstract Two regions of the genome of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H were characterized, representing 14 % of the phage genome. The first region of 2497 bp contained genes encoding phage structural proteins and the second region of 2498 bp genes involved in lytic functions. The nucleotide sequences of the major capsid protein gene g34, a putative capsid morphogenesis gene (ORF178A), the gene mur encoding phage cell wall hydrolase (lysin), the gene hol (ORF107) encoding the cell membrane permeabilizing phage holin, and six other genes with unknown function were found. Identification of these genes was performed by amino acid sequencing of their encoded proteins (genes g34 and mur), by their physiological effect on E. coli (genes hol and mur), by sequence comparison (genes mur, hol, ORF178A), and by biochemical analysis of their encoded purified protein (gene mur). A promoter for the capsid protein encoding gene cluster was determined by primer extension method. A purification method suitable for large scale processing (cation exchange chromatography by expanded bed adsorption method) was developed for the phage LL-H lysin protein Mur. Purified Mur was biochemically determined as a N-acetylmuramidase, which was effective on cell walls of Lb. delbrueckii, Lb. helveticus, Lb. acidophilus and Pediococcus damnosus. Some biotechnological applications for the lysis genes hol and mur or the purified protein Mur are suggested. Mur digests E. coli cell walls inefficiently, but could still be used for lysis of E. coli. Coexpression of the phage LL-H lysin and holin genes yielded to lysis of the E. coli host only at low culture densities. Therefore, some chemicals were tested for their ability to trigger lysis of E. coli cells overexpressing the phage LL-H gene mur. Thymol was found to mimic the physiological effects of the phage holin in a bacterial growth state independent manner. An efficient lysis method utilizing intracellular production of Mur and triggering the lysis with thymol was developed.

The bonding of fibres in paper is influenced by environmental changes (e.g. moisture) that may cause unstable fibres to move. These movements include cell-wall swelling, fibre lifting and/or puffing that break inter-fibre bonds and lead to reduced strength and surface roughness. Fibre puffing is defined as the expansion of the lumen area as result of changes in the environment. Puffing was investigated through image analysis of scanning electron micrographs. Detailed images were obtained with samples that were embedded in resin and then etched. Puffing of fibres was then quantified by calculating the ratio of lumen area to fibre area. Stability of softwood and hardwood fibres was studied in this way, and to simulate printing, handsheets were calendered and rewetted. This method was later validated against commercial sheets. Compared to softwood, hardwood fibres were more stable and most of the handsheet properties were retained after rewetting. Mannanase and/or endoglucanase treatments resulted in improved fibre stability by increasing fibre bonding, fibrillation or fibre collapse. Mannanase improved handsheet smoothness and strength as well as fibre stability, but endoglucanase was less effective. The effect of the enzymes was more difficult to observe on hardwood fibres, because even untreated fibres were more stable under moist conditions. Thin-walled fibres such as earlywood were less stable than latewood fibres, but it responded better to mannanase treatment. Thick-walled fibres (latewood), on the other hand, were more difficult to improve with enzymes. The potential of enzymes to improve fibre stability of commercial pulp was tested on chemi-thermo-mechanical pulp (CTMP) and bleached CTMP. Enzyme treatment improved fibrillation and reduced beating energy of bleached CTMP. Mannanase again resulted in the most improved fibre stability. On rejects, a lack of response to enzymes was overcome by pre-treating the pulp with alkaline peroxide. This study provided new insights into the stability of fibres with different morphology. It was also demonstrated that fibre stability can be improved with enzyme treatment and it is expected that this knowledge could have significant commercial value. / Dissertation (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Uma alternativa para aumentar a produção de bioetanol por área de cana plantada no Brasil seria utilizar os resíduos de sua biomassa para conversão em etanol. O conhecimento de como processos de degradação da parede celular se dão em plantas usadas para a produção de bioenergia e a compreensão de como eles funcionam pode ser de grande utilidade para esta tecnologia. Na investigação da anatomia de cana encontramos evidências da formação de um aerênquima lisígeno na raiz de cana, espaços gasosos no córtex da raiz decorrentes da morte celular e degradação da parede. Assim, decidiu-se aprofundar os estudos neste sistema através de técnicas de bioquímica de parede celular, microscopia de luz e transmissão e imunolocalização. A formação do aerênquima nas raízes de cana-de-açúcar tem início com a morte celular programada e a degradação de &beta;-glucano e pectinas, principalmente daquelas associadas às lamelas médias, resultando na separação das células. As hemiceluloses arabinoxilano e xiloglucano mostram apenas modificações em suas estruturas finas, mas permanecendo nas paredes. Além disto, foram observados em microscopia de transmissão alguns pontos onde houve a degradação completa de parede celular, porém a presença de diversas paredes celulares colapsadas nas lamelas entre o aerênquima e ao seu redor parece ser mais importante para a formação do aerênquima. As modificações dos polissacarídeos estão possivelmente associadas com a alteração de características físicas das paredes, tornando-as mais suscetíveis a dobras e colapsos, gerando os espaços de gás e lamelas resistentes, que sustentam estes espaços. Mais do que a \"degradação da parede celular\", como é tratado em definições de aerênquima, pudemos observar que este fenômeno é resultado de uma sequência de eventos que permitem modificações da parede celular, e não necessariamente a sua completa degradação, resultando na abertura dos espaços gasosos / An alternative to increase bioethanol production per area of sugarcane plantation in Brazil would be to use its biomass residue for conversion into ethanol. The knowledge of how cell wall degradation processes occur in plants used for bioenergy production and understanding how they work can be of great use for this technology. Studying the sugarcane anatomy, we found evidences for the formation of a lysigenous aerenchyma in the roots, gas spaces in the root cortex originated from cell death and cell wall degradation. Thus, we decided to deepen the studies in this system using cell wall biochemistry, light and transmission microscopy and immunolabeling. The aerenchyma formation in sugarcane roots starts with programmed cell death and degradation of &beta;-glucan and pectins, especially those from middle lamellae, resulting in cell separation. The hemicelluloses arabinoxylan and xyloglucan only show modifications in fine structure, but they remain in the cell wall. Besides, complete cell wall degradation was observed in a few spots through transmission electron microscopy, although the collapsing of cell walls seems to be more important for aerenchyma formation. Modifications in the polysaccharides are possibly associated with changes in cell wall physical properties, making them more susceptible to folding and collapsing, generating gas spaces and resistant lamellae that support these spaces. Described as \"cell wall degradation\" in aerenchyma definition in literature, we observed that this phenomenon is the result of a series of events that allow cell wall modifications, and not necessarily its complete degradation, resulting in the formation of gas spaces

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第23395号 / エネ博第422号 / 新制||エネ||80(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻 / (主査)教授 河本 晴雄, 教授 亀田 貴之, 教授 杉山 淳司 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

This study is interested in properties of fungal transglycosylases, specifically Phr1, Phr2 and Crh2. These enzymes are involved in the remodelling of yeast cell walls due to their cleavage of structural donor polysaccharides and transfer of their fragments to the other acceptor (poly)saccharide molecules. The mammalian cells do not contain cell walls, nor cell wall transglycosylases, that´s why these enzymes are possible targets for antifungal agents. In this diploma thesis the effect of 67 commercially available inhibitors on Phr1 and Phr2 enzymes was studied by rapid screening. In the case of the Phr1 enzyme, two inhibitors showed a potential effect which was subsequently tested by size exclusion chromatography column incorporated into HPLC device. None of the inhibitors were found to have an inhibitory effect on Phr1 or Phr2 enzymes in contrast to DMSO in which all inhibitors were dissolved. The mode of action of Phr enzymes was also studied by thin layer chromatography and high performance liquid chromatography. The first method allowed to monitor the formation of products only in the later stages of the reaction, but more sensitive size exclusion chromatography showed the product formation at the beginning of the reaction. Phr1 cleaved the donor substrate near the non-reducing end and forms small fragments that are transfered to labeled acceptors during the whole reaction. Phr2 utilized random action pattern, thus creating products with higher molecular weight from the beginning of reaction. The effect of the polymerization degree of acceptor on it´s affinity with the Crh2 was also studied. The Michaelis-Menten constants showed no effect of acceptor lenght on the affinity between enzyme and substrate.

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