The plant cell wall is a living and dynamic compartment of the plant cell. Its many diverse functions range from cell expansion and differentiation to defence and signalling. Furthermore, there is currently a growing body of evidence which suggests that the cell wall/apoplast also plays an important role in cell water relations. The aim of this study was to highlight the importance of apoplastic solutes in plant cell water relations, particularly in turgor regulation. The water relations parameters of two members of the family Chenopodiaceae, Suaeda maritima L. Dum. and Beta vulgaris L., were studied at single cell resolution using the cell pressure probe, single cell sampling and analysis techniques, and the xylem pressure probe. These species share a common peculiarity, in that certain cell types, namely the leaf epidermal cells in Suaeda maritima and the taproot storage parenchyma cells in Beta vulgaris, maintain cell turgor pressure (Pau) at a level which is dramatically lower than the respective cell osmotic pressures (III�). This phenomenon is attributed to the properties of the cell wall/apoplast. The hydrostatic component of the apoplast (P, uau) accounts for only a small fraction of the difference between P. u and H.,, in these species. In light of this the discrepancy between P. u and H can only be due to the presence of osmotically active solutes in the adjacent apoplast Suaeda maritima leaf epidermal cells accumulate NaCl in response to an increase in external NaCl concentration. This accumulation of solutes leads to an increase in leaf epidermal osmotic pressure, which exactly mirrors the increase in the osmotic pressure of the external medium (ITI). Leaf epidermal turgor pressure (P, -. u), however, is maintained at a constant level over a range of external salinities. In the short term the leaf epidermal cells are shielded from abrupt changes in flea by the properties of the root system, and a root reflection coefficient which is close to 0. In the longer term, as NaCl accumulates in the protoplast, Pcen is apparently maintained by the parallel adjustment of solutes in the protoplast and apoplast. Page III Changes in Suaeda maritima leaf epidermal turgor pressure (P. u), induced by modulating the solute content of the apoplast (11. u) in excised leaves, initiated a mechanism which regulated P., u back to in vivo levels within 40 minutes. Turgor regulation was not accompanied by equivalent changes in cell osmotic pressure (H n), suggesting that osmotic adjustment leading to turgor regulation is apoplastic rather than protoplastic in nature. This apoplastic osmotic adjustment mechanism was dependent on the permeant nature of the apoplastic solutes and on the volume of the apoplast. A comparable upward turgor regulation mechanism was observed in excised Beta vulgaris taproot tissue, within 40 - 80 minutes. The presence of apoplastic KK apparently facilitated the turgor regulation mechanism in this case. Proton efflux studies on Beta vulgaris taproot tissue revealed that the driving force behind this osmotic adjustment mechanism is likely to be turgor/external osmotic pressure (P,. u/H, ) dependent modulation of plasma membrane proton ATPase activity. It was concluded that the apoplast should be regarded as a true osmotic compartment in higher plants.

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Plant cell walls determine cellular shape and provide structural support for the entire plant. Polysaccharides, comprising the major components of the wall, are actively remodelled throughout development. Xyloglucan endotransglucosylase (XET)/hydrolase (XTH, EC 2.4.1.207) cleaves xyloglucan (XyG), the donor substrate, and attaches a portion to another XyG chain, the acceptor substrate. Recently, a novel transglucosylase called mixed-linkage β-glucan (MLG) : XyG endotransglucosylase (MXE) was discovered in horsetails (Equisetum spp.) that could attach a portion of MLG to XyG, resulting in a hetero-polymer product. My aims were to further investigate the nature of this activity, biochemically characterize the enzyme, and explore its physiological role. MXE activity was attributable to an enzyme unlike Equisetum XTHs. MXE had a p1 of 4.1 (XTHs were 6.6-9), a pH optimum of 6.3 (XTHs preferred 5.5), and had higher activity using smaller oligosaccharide acceptor substrates like XXXGol (XTHs were more active using XLLGol). Importantly, the MXE protein was shown to utilize both MLG and XyG as donor substrates, and therefore have both MXE and XET activity. Also, the enzyme was capable of using various glucan oligosaccharides (O) as substrates, including MLGO, XyGO, and cello-O, but not laminari-O. By using a novel ex vivo approach, the proportion of extractable MXE product to XET product was found to increase in older tissues. Transglucosylase products were localized in sclerenchyma and structural parenchyma by in situ assays, implying a strenghening function for MXE. Surprisingly, another novel activity was discovered that could covalently attach cellulose to XyG, and termed cellulose : xyloglucan endotransglucosylase (CXE). This activity was attributed to the MXE enzyme, implying that the protein is a promiscuous endotransglucosylase. The presence of CXE in other plants has not yet been tested. Besides being a novel discovery in plant cell biology, the modification of cellulose has applications in a number of industries.

This study provides information to support the use of plant cell wall, from sugarcane bagasse, to produce cellulosic ethanol. Therewith, cell wall proteins from sugarcane cells cultures, leaves and culms were identified. To do so, different protocols were used. Using two-month-old leaves and culms, the extractions were performed using a destructive method, based on griding the tissues, submitting them to a growing gradient of succrose and centrifugation, being the cell wall extract later isolated by washing on a nylon net. After that, the cell wall proteins were extracted using two salts, 0,2 M CaCl2 and 2 M LiCl. Using cultured cells, a similar protocol was used, but it had a previous step of separation of the cell wall through grinding and precipitation in glycerol 15%. Using culms of the same age, a nondestructive protocol was tested based on vacuum infiltration of the tissues in the same salts already described, 0,2 M CaCl2 and 2 M LiCl, and posterior centrifugation. Two replicates were used from two-month-old plants and three in the case of suspension cells. The complex samples were digested, fractionated and sequenced through mass spectrometry, using SYNAPT G2HDMS coupled to nanoACQUITY, both from Waters. Peptides were processed using ProteinLynx 2.5 Global Server against sugarcane translated-EST database. Using bioinformatic programs, such as Blast2GO, it was possible to find the annotation and classification of similar proteins. Only proteins equally found in all repetitions were considered in the main analysis. SignalP, WolfPSORT, TargetP, TMHMM and Predotar were used to predict the subcellular location, both from ESTs and blasted proteins, and only the proteins predicted to be secreted in two or more programs were considered as cell wall proteins. Altogether, 157 different SAS related to sugarcane cell wall were found. Among these, 101 different cell wall proteins were characterized from eight functional classes. The method based on vacuum infiltration seems to be the most efficient one, since it had almost half, 48,84% of the proteins predicted to be secreted, which is a good percentage when comparing to other studies. From secreted proteins most of them were related to lipid metabolism, as lipid-transfer proteins, oxido-reductases, such as peroxidases, cell wall modifying enzymes, like glycoside-hydrolases, proteases, proteins with interacting domains, signaling proteins and several others. Results are in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and signaling phenomena. Therefore, this work provided valuable information about sugarcane cell wall that can lead to future studies to enhance cellulosic ethanol production. / Este estudo fornece informação para auxiliar o uso da parede celular vegetal, a partir do bagaço de cana, para a produção de etanol celulósico. Com isso, as proteínas da parede celular de folhas, colmos e células em suspensão foram identificadas. Para isso, foram utilizados diferentes protocolos. Utilizando folhas e colmos de cana-de-açúcar de dois meses de idade, as extracções foram realizadas por meio de método destrutivo, com base na trituração dos tecidos, submetendo-os a gradiente crescente de sacarose e centrifugação, sendo a parede da célula extraída e depois isolada por lavagem sobre uma rede de nylon. Depois disso, as proteínas de parede celular foram extraídas utilizando dois sais, 0,2 M de CaCl2 e 2 M de LiCl. Para células em suspensão, um protocolo semelhante foi utilizado, contendo, no entanto, um passo anterior de separação da parede celular por meio de maceração e precipitação em glicerol 15%. Usando colmos da mesma idade, dois meses, um protocolo não destrutivo foi testado com base na infiltração a vácuo dos tecidos nos mesmos sais já descritos, 0,2 M de CaCl2 e 2 M de LiCl, e posterior centrifugação. Duas repetições foram usadas nos experimentos com plantas de dois meses de idade, e três, no caso de células em suspensão. As amostras complexas foram digeridas, fracionadas e seqüenciadas por espectrometria de massas, utilizando o equipamento SYNAPT G2HDMS acoplado ao cromatógrafo nanoACQUITY, ambos da Waters. Os peptídeos foram processadas utilizando ProteinLynx 2,5 comparando com a base de dados de ESTs traduzidos da cana. Utilizando programas de bioinformática, como Blast2GO, foi possível encontrar a anotação e classificação de proteínas semelhantes. Apenas proteínas igualmente encontradas em todas as repetições foram consideradas na análise principal. SignalP, WolfPSORT, TargetP, TMHMM e Predotar foram softwares utilizados para prever a localização subcelular, tanto para ESTs como proteínas, e apenas as proteínas preditas para serem secretadas por dois ou mais programas foram consideradas como proteínas de parede celular. Ao todo, 157 SAS diferentes relacionados à parede celular da cana foram encontrados. Dentre eles, 101 diferentes proteínas de parede foram caracterizadas em oito classes funcionais. O método baseado na infiltração a vácuo mostrou-se o mais eficiente, uma vez que apresentou quase metade, 48,84%, das proteínas preditas para serem secretadas, o que é um bom valor quando comparado com outros estudos. A maioria das proteínas secretadas estava relacionada com o metabolismo lipídico, como proteínas de transporte de lípidos, oxido-redutases, tais como peroxidases, enzimas modificadoras da parede, como as glicosil-hidrolases, proteases, proteínas com domínios de interação, proteínas sinalizadoras, entre outras. Os resultados estão de acordo com o papel que se espera da matriz extracelular no metabolismo de polissacarídeos e fenômenos de sinalização. Portanto, este trabalho forneceu informações valiosas sobre a parede celular da cana, tornando possível a utilização desses dados em futuros estudos para otimizar a produção de etanol celulósico.

Orientador: Marcos Silveira Buckeridge / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T07:05:13Z (GMT). No. of bitstreams: 1 Cavalari_AlineAndreia_D.pdf: 2066715 bytes, checksum: 503395a4dfdc3832fc20cbf4193ea790 (MD5) Previous issue date: 2009 / Resumo: A parede celular é um componente particular dos tecidos vegetais e conhecer a composição dos polissacarídeos que a constituem e suas interações é essencial para compreender a textura dos alimentos e suas alterações pós-colheita, em especial em frutos climatérios, como é caso do mamão. A parede celular esta dividida por três domínios: o primeiro é formado por celulose e hemiceluloses, o segundo domínio é formado por pectinas e o terceiro um domínio composto por proteínas. As modificações dos polímeros e suas proporções nestes respectivos domínios são resultados de ações enzimáticas, que no caso dos frutos carnosos, leva ao amaciamento da polpa. Portanto, estudar as modificações nesses polímeros através da análise dos OXG obtidos por hidrolise com celulase, é um caminho importante para entender as alterações neste polissacarídeo ao longo do desenvolvimento de frutos. O presente trabalho teve como objetivo compreender as modificações da parede celular durante o desenvolvimento do fruto do mamoeiro. Foram utilizados frutos de Carica papaya L. cv. Sunrise solo, coletados diretamente do produtor (Caliman S/A- Unhares- ES). As amostras de frutos foram colhidas em intervalos de 30 dias, sendo os estádios analisados de 30 a 150 após a antese (dpa). Os resultados demonstram queda acentuada na proporção de parede celular em relação a outros compostos, como açúcares, por exemplo, o que é possivelmente uma indicação do processo de expansão celular e conseqüentemente uma alteração de textura da parede celular durante o desenvolvimento. Observou-se que o principal açúcar solúvel é a sacarose, sendo esta provavelmente a principal fonte energética para o desenvolvimento do fruto de mamão, uma vez que este não sintetiza amido. De maneira geral, a proporção de oligossacarídeos de xiloglucano menos ramificados diminuiu aos 120 dpa, enquanto que os de maior peso molecular e ou grau de ramificação (com fucose) aumentaram proporcionalmente. Estes resultados sugerem que xiloglucanos (ou parte das moléculas destes) pobremente ramificados com fucose, são retirados da parede celular, consequente enriquecimento de oligosasacarídeos fucosilados. Como estes últimos tornam o xiloglucano mais interativo com ele próprio e com a celulose, é possivel que estes sejam os principais efeitos que as transformações na parede promovam no fruto. As alterações na parede foram acompanhadas pelo aumento concomitante nas ativades de beta­galacosidase e beta-glucosidase, duas das principais hidrolases de xiloglucano. Concumitantemente, observou-se uma diminuição acentuada na proporção de celulose na parede. Com base nestas observações, sugere-se que as paredes celulares sofrem transformações importantes nos frutos do mamoeiro até os 120 dpa I sendo que a partir deste estádio a parede se torna mais acessível à hidrólise e denotando a preparação do fruto para o amadurecimento. / Abstract: The plant cell wall is a unique component of plant tissues and its polysaccharide composition essential to understand food texture and its changes during post-harvestingl especially of climateric fruits, as is the case of papaya. The plant cell wall is composed of three domains: the first is formed by cellulose and hemicelluloses, the second of pectins and the third of proteins. The changes in polymers and their proportions in these domains are a result of enzyme action, which in the case of fleshy fruits lead to the softening of the pulp. Hydrolysis of hemicelluloses such as xyloglucan can play important functions in cell expansion, cell growth and cell wall degradation. Therefore, studying the modifications in xyloglucan by looking at is fine structure (Le. oligosaccharide (OXG) pattern obtained after cellulase action) may be an important way to understand polysaccharide change during fruit development. The present work aimed at understanding the modifications in cell wall during the development of the papaya fruit. Fruits of Carica papaya L. Cv.Sunrise solol were collected directly by the producer (Calimanl SAI Unharesl Espirito Santol Brazil). Samples of fruits were harvested at intervals between 30 and 150 days after anthesis (daa). Our results showed that there were drastic changes in the cell wall of the mesocarp in relation to other compoundsl such as soluble sugars. This is probably an indication that cell expansion process is at least part of the cause of the changes in texture during development. We observed that the main soluble sugar found in fruits is sucrose, this being probably the principal source of energy for development of the organ, as no starch is synthesised in this fruit. In general, the proportion of less branched xyloglucan oligosaccharides decreased at 120 daa, whereas the OXG branched with fucose increased constantly during development up to the same stage. These results suggest that xyloglucans (or part of their molecules) that are poorely brached with fucose are retrieved from the cell wall. This seems to lead to enrichment of fucosylation of xyloglucan. As these OXG turn xyloglucan more interactive with itself and with cellulose, it is possible that these would be the principal effects that the cell walls provoke in the fruit. The changes in the wall were followed by a concomitant increase in activities of beta-galactosidase and betaglucosidase, both thought to be related to xyloglucan hydrolysis in vivo. At the same time, we observed a decrease in the proportion of cellulose in the walls during development. On the basis of these results, we suggest that the cell walls of papaya fruits undertake structural changes untill 120daa after which the wall becomes more accessible to hydrolases denoting the preparation of the papaya fruit for ripening. / Doutorado / Doutor em Biologia Vegetal