Difficulties in controlling and forecasting avocado fruit ripening and the highly perishable nature of the crop once harvested, are the major causes of concern for avocado traders. In particular, the simultaneous presence of many suppliers may account for increased fruit variability during ripening. Avocado is a climacteric fruit with consistent ethylene production after harvest which is also related to high perishability. However, the mechanisms regulating ethylene biosynthesis and mesocarp softening are not completely understood. In order to study such effects, avocado fruit from different growing areas and harvested at various maturity stages, were investigated and the biochemical and physiological changes during ripening at both 18 and 23°C were studied. Mesocarp softening and fatty acid content discriminated fruit maturity and growing area, respectively, whereas C7 sugars (D-mannoheptulose and perseitol) discriminated length of fruit shelf life. For the first time, oleic acid content presents in the oil mesocarp was found to depend on fruit sources making of this a suitable indicator of avocado fruit growing area. In contrast, sugar content declined along fruit maturity and ripening. In particular the mannoheptulose presents in avocado mesocarp might be use to estimate avocado fruit shelf life. Indeed, fruit harvested late in season were found to have a lower C7 content than earlier harvest fruit and a faster softening, regardless fruit source. However, sugars content changed between growing area, thus a general C7 threshold defining fruit storability seems to be not definable. Furthermore, other possible indicators of fruit maturity and/or ripening stage have been searched in the cell wall constituents of avocado mesocarp. Thus, the structural carbohydrates profile of avocado mesocarp investigated with a new immunological method changed during ripening and harvest time (early and late season), suggesting a possible effect of cell wall composition on fruit ripening regulation. Also, the possible use of ethylene application in reducing the high heterogeneity noted on imported fruit from South Africa was also evaluated through different consignments. Results showed ethylene efficacy changed depending on harvest time and fruit dimension with less efficacy of the treatment on fruit harvested at the end of the season and characterised by smaller size.One of the most commercialized avocado cultivars, Hass, is peculiar in that its skin colour changes from green to deep purple as ripening progresses. The most common ripening indicator of avocado fruit is the mesocarp firmness and the destructive nature of this evaluation increases losses in the avocado industry. The availability of a non-destructive indicator of fruit ripening represents an important advantage for avocado consumers and importers. Thus, the possible relationship between mesocarp softening, skin colour were objectively evaluated (C*, L*, and H°), and the main pigment, cyanidin 3-O-glucoside, was investigated. Cyanidin 3-Oglucoside was confirmed to be the main anthocyanin present in avocado cv. Hass peel, regardless of preharvest factors. However, differences in its content were noted between shelf life temperatures. A higher relationship between hue angle and firmness was detected in late harvest fruit, whereas no correlation was found between anthocyanin content and firmness. Avocado skin is also involved in defence mechanisms due to the presence of antifungal and phenolic compounds. These phenolic compounds represent a natural protection against pathogenic infections and seem to be down regulated during ripening. The main phenolics were identified and quantified, using a new analytical method which was validated and optimised. Epicatechin, chlorogenic acid and procyanidin B2 were found to be present in the skin tissue and quantified using this assay and found to vary during shelf life and seasons. Although phenolics were present in minor amounts, in avocado pulp they are involved in mesocarp discoloration incidence, and therefore with fruit postharvest quality. Due to a lack of information, a new straightforward method for the identification and quantification of the main phenolics present in avocado mesocarp was developed. Finally, a commercial trial was undertaken to ensure that the results obtained in the laboratory can be reproduced in the market place. In conclusion, postharvest markers can define avocado fruit maturity and growing area and give guidelines in the control of avocado shelf life. Moreover, new methods for the investigation of the phenolic profiles (peel and mesocarp) and the characterisation of cell wall structures can be further tools in the management of avocado fruit postharvest quality.

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.

No description available.

The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis) fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Diploma thesis Author: Dana Kettnerová Supervisor: PharmDr. Jan Martin, Ph.D. Title of diploma thesis: Isolation of plant organelles and study of transport mechanisms Key words: isolation, chloroplast, protoplast, vacuole, cell wall Isolation of plant organelles and other cellular components is essential for the study of physiological and pathological processes within the plant cell. It is possible to analyze cell structures, detect accumulation of certain metabolites, ions, enzymes and other substances thanks to the isolation. The goal of this diploma thesis was to provide an overview of isolation methods used for the isolation of cell wall, protoplasts, chloroplasts and vacuoles of plant cells. Isolation processes used for individual types of cell structures, the pros and cons of the various isolation methods, components of used media and their functions, as well as the structure and function of individual plant structures were described.

Plant primary cell walls are hydrated extracellular complexes composed largely of polysaccharides: cellulose, hemicellulose and pectin. Cell wall constituents and composition vary in cell-, environment-, and species-dependent manners. For example, within land plant hemicelluloses xyloglucan is ubiquitous while mixedlinkage (1→3),(1→4)-β-D-glucan (MLG) is found only in the Poales and Equisetum. Glycosyl hydrolase 16 (GH16) enzyme family members include numerous enzymes with pertinence to the understanding of the ‘lives’ of cell wall hemicelluloses. However, despite this, the details of the interactions between GH16 enzymes and their substrates have often not been elucidated. Likewise, the true preferences of many of these enzymes and the range of substrates which they can utilise remain to be fully explored. By providing a greater wealth of information for the correlation of enzyme structure with reaction catalysed, such an understanding would enable better predictions of the activities of novel enzymes. Crucially, this would also allow better identification of roles performed by these enzymes in planta as well as of the potential applications of these enzymes. This work sought to further our understanding of the interactions between GH16 enzymes and their substrates by the study of five activities exhibited by GH16 enzymes – xyloglucan endotransglucosylase (XET), xyloglucan endoglucanase/hydrolase (XEG/XEH), mixed-linkage glucan : xyloglucan endotransglucosylase (MXE), lichenase and cellulose : xyloglucan endotransglucosylase (CXE). All of the analysed activities act on xyloglucan and/or MLG. Of particular focus is the novel enzyme MXE from the evolutionarily isolated genus Equisetum (horsetail), which acts on both. Notable findings include: identification of MXE/CXE gene; determination of the substrate specificity of MXE; defining of the sites of attack of lichenase, XEG, XET and MXE; discovery of novel xyloglucan structures and discrepancies between the xyloglucan present in different barley organs.

A parede celular vegetal é composta primariamente de celulose, hemicelulose e lignina. A lignina é o segundo biopolímero mais abundante na biosfera, atrás apenas de celulose, e é formado principalmente a partir da ligação entre três monômeros chamados monolignóis. A formação desses monolignóis é catalisada por pelo menos 10 enzimas membros das famílias gênicas AMP_binding (gene 4CL), p450 (C3H, C4H e F5H), ADH_N (CAD), Epimerase (CCR), Methyltransf_3 (CCoAOMT), Methyltransf_2 (COMT), Transferase (HCT) e Lyase_aromatic (PAL), que compõem a via de biossíntese dos monolignóis. Até o momento, cerca de 25 sequencias da via de biossíntese de lignina já foram identificados em cana-de-açúcar (Saccharum spp). Ainda, o sequenciamento do genoma desta espécie se mostra uma difícil tarefa devido ao tamanho do genoma e ploidia nuclear. Em virtude da disponibilidade de transcriptomas oriundos de diferentes órgãos de cana-de-açúcar e a importância na identificação correta das sequencias ortólogas, o presente trabalho visa à identificação, anotação e análise filogenética dos genes das famílias gênicas envolvidas na biossíntese de lignina em cana-de-açúcar. Para isso, genes destas famílias gênicas da planta modelo de eudicotiledôneas Arabidopsis thaliana e gramíneas tais como Oryza sativa (arroz), Brachypodium distachyon, Zea mays (milho) e Sorghum bicolor (sorgo) foram identificados, anotados e filogeneticamente categorizados. Em seguida, as sequências de cana-de-açúcar foram identificadas em cinco transcriptomas distintos, anotados e avaliados quanto à identidade e cobertura em relação às proteínas de sorgo. As análises filogenômicas entre cana-de-açúcar e as demais espécies estudadas revelaram 23 sequências candidatas envolvidos na biossíntese de lignina em cana-de-açúcar. / Plant cell wall is composed of cellulose, hemicellulose, and lignin. Lignin is the second most abundant biopolymer on biosphere, after only cellulose, and is formed primarily from three monomers, called monolignols. Monolignol biosyntesis is catalysed by at least 10 enzymes members of AMP_binding (4CL enzyme), p450 (C3H, C4H e F5H), ADH_N (CAD), Epimerase (CCR), Methyltransf_3 (CCoAOMT), Methyltransf_2 (COMT), Transferase (HCT) e Lyase_aromatic (PAL) gene families involved in the biosynthetic pathway of monolignols. So far, there is 25 sugarcane (Saccharum spp.) genes identified involved in the lignin biosynthesis. Sequencing sugarcane\'s genome is still a difficult task due to large genome and ploidy level. Due to the availability of transcriptome data from distinct organs of sugarcane and the importance in the accuracy on the identification of the orthologous sequences, this work aim the identification, annotation and phylogenetic analysis of proteins members of distinct gene family involved in monolignol biosynthesis in sugarcane. To conduct it, we identified and annotated all genes from the model plant eudicots Arabidopsis thaliana and grasses such as Oryza sativa (rice), Brachypodium distachyon, Zea mays (mayze) and Sorghum bicolor (sorghum) and analysed the phylogenic relationship among them. The sugarcane sequences were retrieved and annotated from five distinct transcriptome data and analysed by the identity and coverage against the best sorghum orthologous genes. The phylogenomics analysis revealed a total of 23 sugarcane sequences putatively involved in the biosynthesis of lignin.

Plant cell wall is critical for the regulation of cell shape, cell growth, and responses to abiotic stress and pathogen infection. The plant cell wall is composed of several monosaccharides including glucose, galactose, mannose, xylose, fucose, rhamnose, and arabinose. Arabinose-containing polymers account for ~20 % of the total cell wall saccharides in rice and Arabidopsis. Arabinose is a plant-specific monosaccharide that is required for the decoration of several cell wall polysaccharides, including rhamnogalacturonan I (RGI)-arabinan, arabinoxylan, and rhamnogalacturonan II (RGII). Arabinose is also involved in the modification of some cell wall glycoproteins, including arabinogalactan-proteins (AGPs), extensins, and leucine-rich repeat extensin (LRX) proteins. In addition, arabinose is conjugated to signaling peptides like CLAVATA3 and some cytoplasmic arabinosylated flavonols, such as quercetin 3-O-l-arabinoside and myricetin. The only known enzyme in the final step of the arabinose de novo biosynthesis pathway is the Golgi-localized UDP-D-xylose 4-epimerase (MUR4), which converts UDP-xylose to UDP-arabinose. There is a 50% reduction of cell wall arabinose in mur4 mutant, indicating that other enzymes may also be involved in the de novo biosynthesis pathway. Under salt stress, mur4 mutant plants exhibit reduced root elongation and abnormal cell-cell adhesion. The roles of three MUR4 paralogs, MURL, DUR, and MEE25, in arabinose biosynthesis and salt stress tolerance are described. Data are also shown regarding the importance of AGPs in salt tolerance. Analysis of higher order mutants of mur4 with its three paralogs reveals that the three proteins also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. The salt-hypersensitivity of the mur4 mutant is rescued by exogenous arabinose or gum Arabic (a commercial AGP product). Taken together, my work reveals the importance of arabinose metabolism in salt stress tolerance and provides new insights into the enzymes involved in UDP-Ara biosynthesis in plants. Plants have evolved cell-wall integrity sensing and signaling pathways to maintain cell-wall homeostasis in response to stress conditions, but the cellular components involved in the perception and transduction of cell-wall signals are largely unknown. I found that the cell wall-localized leucine-rich repeat extensins (LRX) 3/4/5 interact with RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23 to transduce cell wall signals. Mature RALF22/23 peptides convey signals to the plasma membrane-localized FERONIA (FER) to induce intracellular stress responses. The lrx345 and fer mutants and RALF22/23 overexpressing transgenic plants display similar phenotypes, including retarded growth and increased sensitivity to salt stress. These results suggest that LRX3/4/5, RALF22/23, and FER function as a module to regulate plant growth and salt stress tolerance. Further analyses show that the LRXs-RALF-FER module negatively regulates the accumulation of the phytohormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA), and the simultaneous over-accumulation of these stress hormones can be detrimental to plants. Based on genetic and biochemical data, we propose that salt-induced perturbations of the cell wall may be sensed by the LRXs, triggering the release of RALF peptides in the extracellular space; these peptides are then perceived by FER, inducing its internalization and down-regulating its function as part of a homeostatic mechanism to halt growth and to acclimate to salt stress through the activation of ABA, JA and SA signaling. Taken together, my work offers valuable insights into how salt stress is sensed in the apoplast by the LRXs-RALFs-FER signaling module, which subsequently modulates hormone signaling to establish a homeostatic mechanism coordinating growth and stress responses. In brief, my study contributes to the understanding of the role of MUR4 family of enzymes in plant arabinose biosynthesis and the role of arabinose-containing macromolecules in salt stress sensing and adaptation.<br>

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

A parede celular vegetal é composta primariamente de celulose, hemicelulose e lignina. A lignina é o segundo biopolímero mais abundante na biosfera, atrás apenas de celulose, e é formado principalmente a partir da ligação entre três monômeros chamados monolignóis. A formação desses monolignóis é catalisada por pelo menos 10 enzimas membros das famílias gênicas AMP_binding (gene 4CL), p450 (C3H, C4H e F5H), ADH_N (CAD), Epimerase (CCR), Methyltransf_3 (CCoAOMT), Methyltransf_2 (COMT), Transferase (HCT) e Lyase_aromatic (PAL), que compõem a via de biossíntese dos monolignóis. Até o momento, cerca de 25 sequencias da via de biossíntese de lignina já foram identificados em cana-de-açúcar (Saccharum spp). Ainda, o sequenciamento do genoma desta espécie se mostra uma difícil tarefa devido ao tamanho do genoma e ploidia nuclear. Em virtude da disponibilidade de transcriptomas oriundos de diferentes órgãos de cana-de-açúcar e a importância na identificação correta das sequencias ortólogas, o presente trabalho visa à identificação, anotação e análise filogenética dos genes das famílias gênicas envolvidas na biossíntese de lignina em cana-de-açúcar. Para isso, genes destas famílias gênicas da planta modelo de eudicotiledôneas Arabidopsis thaliana e gramíneas tais como Oryza sativa (arroz), Brachypodium distachyon, Zea mays (milho) e Sorghum bicolor (sorgo) foram identificados, anotados e filogeneticamente categorizados. Em seguida, as sequências de cana-de-açúcar foram identificadas em cinco transcriptomas distintos, anotados e avaliados quanto à identidade e cobertura em relação às proteínas de sorgo. As análises filogenômicas entre cana-de-açúcar e as demais espécies estudadas revelaram 23 sequências candidatas envolvidos na biossíntese de lignina em cana-de-açúcar. / Plant cell wall is composed of cellulose, hemicellulose, and lignin. Lignin is the second most abundant biopolymer on biosphere, after only cellulose, and is formed primarily from three monomers, called monolignols. Monolignol biosyntesis is catalysed by at least 10 enzymes members of AMP_binding (4CL enzyme), p450 (C3H, C4H e F5H), ADH_N (CAD), Epimerase (CCR), Methyltransf_3 (CCoAOMT), Methyltransf_2 (COMT), Transferase (HCT) e Lyase_aromatic (PAL) gene families involved in the biosynthetic pathway of monolignols. So far, there is 25 sugarcane (Saccharum spp.) genes identified involved in the lignin biosynthesis. Sequencing sugarcane\'s genome is still a difficult task due to large genome and ploidy level. Due to the availability of transcriptome data from distinct organs of sugarcane and the importance in the accuracy on the identification of the orthologous sequences, this work aim the identification, annotation and phylogenetic analysis of proteins members of distinct gene family involved in monolignol biosynthesis in sugarcane. To conduct it, we identified and annotated all genes from the model plant eudicots Arabidopsis thaliana and grasses such as Oryza sativa (rice), Brachypodium distachyon, Zea mays (mayze) and Sorghum bicolor (sorghum) and analysed the phylogenic relationship among them. The sugarcane sequences were retrieved and annotated from five distinct transcriptome data and analysed by the identity and coverage against the best sorghum orthologous genes. The phylogenomics analysis revealed a total of 23 sugarcane sequences putatively involved in the biosynthesis of lignin.