Morindae Officinalis Radix (MOR), Bajitian in Chinese, is the dried root of Morinda officinalis F.C.How. (Rubiaceae). It is one of the most popular herbal medicines used in the southeast region of China. Various types of chemical constituents have been experimentally shown to be bioactive components of MOR, among which secondary metabolites and saccharides predominate. Pharmacological studies revealed MOR shows kidney tonifying, anti- osteoporosis, antidepressant, anti-inflammatory and antioxidant effect. Since 2002, MOR has been approved as a food supplement for daily healthcare, hence increasing consumption and demand for better quality of MOR. However, selection of MOR with superior quality is largely based on traditional experience which lacks scientific basis. For example, 3-4-year-old MOR is usually used without xylem; and processed MOR are believed to show different bioactivities. Therefore, to promote the rational utilization and ensure efficacy of MOR, overall qualitative and quantitative characterization of MOR in different traditional usage is needed. Anthraquinones, iridoid glycosides and oligosaccharides are the common reference compounds for chemical characterization of MOR. However, they are usually selectively characterized, which is not comprehensive enough in herbal quality evaluation. To deal with this, metabolomics targeting secondary metabolome and glycomics targeting glycome can be applied. And the integration of metabolomics and glycomics could be a promising approach to investigate overall chemical variations in MOR according to its traditional usage. Therefore, in this study, chromatographic methods for metabololmics and glycomics were firstly developed to study the traditional usage of MOR. In Chapter 2, they were applied for studying chemical variation and differences in growth year and plant tissue of MOR. In Chapter 3, chemical differences in processed products of MOR were also studied using the established metabololmics and glycomics methods. Further bioactivity differences of them were studied by cell metabolomics with HEK 293 cells under high glucose microenvironment. Besides that, in Chapter 4, consumption method of not only MOR, but other herbal medicines were studied. Conventional boiling water extraction (BWE) and ultrasound-assisted extraction (UAE) were compared to understand their effects on polysaccharides. For the study of growth year and tissues of MOR, the results showed that various types of bioactive components reached a maximum between 3-4 years of growth; and that xylem contained more potentially toxic constituents, but less bioactive components, than cortex. For the study of processing products, the results showed that secondary metabolome and glycome of raw MOR and other processing products was found qualitatively and quantitatively different. Contents of secondary metabolites were generally increased in processed products, while saccharides were decreased instead. Also, steamed MOR (F) seemed to show preventive effect of diabetic nephropathy and different MOR processing products had induced different metabolic changes on high glucose induced HEK 293 cells. In the study of extraction methods, the results showed that the polysaccharides from the herbal medicines by UAE were quantitatively and qualitatively different with those by BWE. The powerful extraction ability and polysaccharide degradation caused by ultrasound collectively contributed to these differences. It was revealed that not only the UAE conditions but also the polysaccharide structures could affect the extraction ability and polysaccharide degradation To conclude, metabolomics and glycomics were integrated in this study to investigate the variations in secondary metabolome and glycome in MOR. We had successfully applied these methods to study and provide scientific basis for traditional practice of MOR. We had proved that 3rd to 4th years of growth are the key period for the development of the biochemical signature of MOR. Xylem and cortex of MOR were qualitatively and quantitatively different and removing xylem could help to remove potentially toxic components. This study also provided scientific evidences for the justification of MOR and its processed products, as well as their metabolic effects on high glucose induced DN in HEK 293 cells. Besides, this study revealed both UAE parameters and structural properties of polysaccharides affects extraction recovery of polysaccharides in herbal medicines. Hence, we suggest UAE should be carefully considered before employing it in relevant chemical and pharmacological analysis.

Abstract not available

Collection of the author's previous publications / Includes bibliographies / 1 v. (various pagings) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D. Sc.)--University of Adelaide, 1989

Offprint of the author's journal article in pocket / Bibliography: leaves 162-193 / x, 193 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1986

Orientador: Sonia Nair Bao / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T17:33:24Z (GMT). No. of bitstreams: 1 Fank-de-Carvalho_SuzaneMargaret_D.pdf: 16381065 bytes, checksum: 37dffed971377c79c257dea1b955e34c (MD5) Previous issue date: 2011 / Resumo: A família Amaranthaceae no sensu lato, incluindo Chenopodiaceae, é formada por cerca de 2.360 espécies, 145 delas encontradas no Brasil; 94 espécies subsistem em diversas fitofisionomias do Bioma Cerrado, 71 são endêmicas de diferentes regiões e biomas brasileiros e 27 aparecem em listas regionais de espécies ameaçadas de extinção. Visando contribuir para o conhecimento das espécies dessa família, foram estudados aspectos ecológicos, micromorfológicos, anatômicos e ultraestruturais de folhas de Amaranthaceae nativas do Brasil, com ênfase nos gêneros Alternanthera, Gomphrena, Froelichiella, Hebanthe e Pfaffia. As espécies nativas de regiões abertas de cerrado apresentam adaptações que favorecem a sobrevivência em condições adversas (seca e fogo), tais como raízes tuberosas ou lenhosas e xilopódios, hábito herbáceo e/ou subarbustivo, pilosidade densa nas porções aéreas, senescência de ramos aéreos durante as fases mais secas, dependência de fogo ou chuva para rebrotação e/ou floração, frutificação rápida seguida de dispersão anemocórica, epidermes com cutículas bem desenvolvidas e metabolismo fotossintético C4. O comportamento pirofítico das espécies favorece o estabelecimento pioneiro das mesmas, principalmente nas áreas abertas do Cerrado. As superfícies foliares de algumas espécies do gênero Gomphrena apresentam cristais de cera epicuticular do tipo plaquetas, orientadas paralelamente, aspecto anteriormente descrito apenas em espécies de Chenopodiaceae. Em duas espécies foram encontrados fungos Ascomyceto colonizando folhas, cujos aspectos ultraestruturais foram descritos. A anatomia Kranz foi caracterizada em seis espécies do gênero Gomphrena, que também possuem cloroplastos dimórficos, demonstrando estrutura foliar compatível com o metabolismo fotossintético C4. Na análise das duas espécies do gênero Alternanthera (uma C3 e outra intermediária C3-C4) verificou-se que a posição das organelas nas células da bainha pode ser um elemento chave na determinação do tipo metabólico. As espécies de Froelichiella, Hebanthe e Pfaffia possuem anatomia e ultraestrutura compatíveis com o metabolismo C3. A anatomia foliar e a ultraestrutura das espécies estudadas apresentam um padrão já descrito para outras espécies da família Amaranthaceae, exceto para os gêneros Hebanthe e Froelichiella, cuja descrição anatômica e ultraestrutural foi realizada pela primeira vez. A evolução do metabolismo C4 pode estar relacionada, pelo menos em parte, ao desenvolvimento da anfiestomia associada à maior espessura do limbo foliar em espécies herbáceas. Plastoglóbulos bem desenvolvido foram encontrados em cloroplastos de algumas das espécies do Cerrado e parecem associados aos mecanismos de defesa, além do metabolismo de lipídios. A família Amaranthaceae pode ser um bom marcador da biodiversidade de dicotiledôneas de pequeno porte e da capacidade de regeneração das áreas de campos rupestres, campos úmidos e outros tipos de vegetação aberta dos cerrados. A riqueza de informações obtidas durante o estudo de espécies dessa família ilustram a importância da ampliação das pesquisas básicas e aplicadas em suas espécies, especialmente as que ocorrem naturalmente no Cerrado. A ampliação do conhecimento relativo à anatomia e à especialização das organelas de diferentes gêneros para o metabolismo fotossintético pode contribuir para o entendimento da evolução da via C4 e do ambiente onde as plantas se especializaram. O estudo do Bioma Cerrado pretende ampliar as justificativas para esforços de preservação de sua biodiversidade. / Abstract: The Amaranthaceae family sensu lato, including Chenopodiaceae, is comprised of approximately 2,360 species, 145 of them found in Brazil. 94 species exist in various vegetation types of the Cerrado, 71 are endemic to different regions and Brazilian biomes, and 27 appear in regional lists of endangered species. To contribute to the knowledge of this family species the ecological aspects, micromorphology, anatomy and ultrastructure of leaves of brazilian Amaranthaceae were studied, with emphasis on the genera Alternanthera, Gomphrena, Froelichiella, Hebanthe and Pfaffia. The native species of open areas of Cerrado (a savannah-like vegetation) exhibit adaptations that promote survival in adverse conditions (drought and fire), such as tuberous or woody roots, xylopodium, herbaceous or subshrub habit, dense pubescence in aerial portions, senescence of shoots and leaves during the driest season, dependence on rain or fire to resprout and/or flowering, fruiting followed by rapid wind dispersion, tick cuticle on epidermis and C4 photosynthetic metabolism. The species' fire behavior favors their establishment as pioneers, especially in open areas of the Cerrado. Leaf surfaces of some species of the genus Gomphrena present epicuticular wax crystals in platelet form, oriented in parallel, an aspect previously described only in Chenopodiaceae species. In two of the species studied Ascomycete fungi were found colonizing its leaves, and ultrastructural aspects were described. Kranz anatomy was found in six species of the genus Gomphrena, which also have dimorphic chloroplasts, showing leaf structure compatible with the C4 photosynthetic metabolism. In the analysis of two species of Alternanthera (a C3 and a C3-C4 intermediate) showed that besides the Kranz anatomy, the position of organelles in bundle sheath cells can be a key element in determining the metabolic type. The Froelichiella, Hebanthe and Pfaffia species have leaf anatomy and ultrastructure consistent with C3 metabolism. The leaf anatomy and ultrastructure have a pattern already described for other species of the Amaranthaceae family, except for the genera Hebanthe and Froelichiella, whose anatomical and ultrastructural aspects were described for the first time. The evolution of C4 metabolism may be related, at least in part, to the development of amphystomy associated with increased leaf thickness in herbaceous species. Large plastoglobuli were found in chloroplasts of some Cerrado species and appear associated with defense mechanisms, and lipid metabolism. The Amaranthaceae family can be a good marker of the biodiversity of nonwoody Eudicotyledons with ability to regenerate areas of rocky fields, wet grasslands and other open vegetation of the cerrado. These results illustrate the importance of expansion of basic and applied research in Amaranthaceae species, especially those that occur naturally in Brazilian cerrados. The expansion of knowledge concerning the anatomy and the specialization of organelles of different genera to perform its photosynthetic metabolism may contribute to understanding of the evolution of C4 pathway and the environment where plants specialize. The study of Cerrado Biome aims to broaden the justification for efforts to preserve its biodiversity. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

M.Sc. / Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.

Success in evaluating effects of a 75% oxygen atmosphere, containing an ambient concentration of carbon dioxide, on levels of protein and oxidative enzymes in cotton, Gossypium herbaceum L. C.B. 1697, leaf tissue was dependent upon generation of genetically uniform and physiologically similar leaves. Sufficient quantities of 2-4 week old leaves for experimental procedures were obtained from plants grown in a 1:1 perlite:vermiculite potting medium supplied with 20% Hoagland's nutrient and maintained in a controlled environment growth chamber under the following conditions: 16 hr day/8 hr night cycle, irradiances of 200-300 µE m⁻² sec⁻¹, 70% humidity, and 30°. Maximum yields of soluble protein (10-12 mg/g tissue) and active enzymes were obtained when freshly harvested leaves were crushed in liquid nitrogen and then homogenized in 0.1 M Tris-Cl, pH 6.9, containing 0.01 M isoascorbate and polyvinylpyrrolidone [2% (w/v) PVP-10 and 0.5 g dry Polyclar AT/g tissue]. Routinely 90% of the solubilized protein was obtained following centrifugation and chromatography on Sephadex G-50. Introduction of cotton plants into vinyl chambers of 95% humidity resulted in foliar symptoms of physiological stress, including chlorosis, cellular edema, leaf curling, and loss of turgidity. Of the oxidative enzymes analyzed, catalase activity decreased 30%, glycolate oxidase activity decreased 42%, and peroxidase. increased 89% while soluble protein decreased 27% in plants maintained in the high humidity for 5 days. Exposure of plants to 75% oxygen, 350 ppm carbon dioxide for 48 hr under these conditions resulted in a 70% increase in glutathione reductase and a 25% increase in malate dehydrogenase. Other enzymes, superoxide dismutase, catalase, peroxidase, glyoxylate reductase, and acid phosphatase, appeared to be independent of the oxygen treatment. When humidity was carefully controlled at 70%, treatment with 75% oxygen, 350 ppm carbon dioxide resulted in a 180% increase in glutathione reductase, an 85% increase in acid phosphatase, and a 22% increase in peroxidase. In the same experiment, catalase decreased by 32% and total soluble protein decreased by 15%. Activities of glyoxylate reductase and both cyanide-sensitive and insensitive superoxide dismutases appeared to be unaffected by the 75% oxygen treatment after 48 hr. Obviously the impact of humidity as a component of the regulation of metabolism in leaf tissue must be considered. It is concluded that oxidoreductase activities located in the chloroplast, peroxisome, and cytosol are not equally sensitive to high oxygen tensions, and effects of environmental oxygen are not restricted to oxidative enzymes. / Ph. D.

The NRT2 (high affinity nitrate transporter 2) family is a part of the iHATS (inducible high affinity system) that studies have shown is responsible for the influx of nitrate into the plant cell after provision of nitrate. The ZmNRT2.1 from Zea mays was constitutively expressed in Nicotiana tabacum. To assess how over-expression of this foreign NRT2.1 affects nitrate influx by plants, nitrate content in leaf and root tissue, gene expression, and vegetal growth were analyzed in media with deficient or high nitrate concentrations (0.1, 1, or 10 mM). Compared to wild type plants: the transgenic lines had a significantly larger fresh weight in all nitrate conditions; primary root length was significantly longer in the 0.1 and 1 mM nitrate conditions; both the fresh weight and the primary root length were significantly higher when 50 mM NaCl was applied as a stress factor to medias containing 0.1 and 10 mM nitrate. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

[Truncated abstract] The mitochondrion is the energy powerhouse that provide energy to many metabolic functions in the form of ATP. Mitochondria in plants are also known to carry out a variety of other important biochemical processes within the cell, including the anaplerotic function of tricarboxylic acid (TCA) cycle, one-carbon metabolism and portions of photorespiration. Dynamics of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles under different developmental and environmental conditions. A quantitative comparative proteomic approach was carried out to analyze mitochondria isolated from non-photosynthetic models, cell culture and root, and compared them to mitochondria isolated from photosynthetic shoots. The glycinedependent respiration rate and the protein abundance of the photorespiratory apparatus was found to be higher in shoot than cell culture and root mitochondria. Also, there were major differences in the abundance and/or activities of enzymes in the TCA cycle between the three systems examined. The metabolic pathways that relied on the supply of intermediates from TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation, and branched-chain amino acids degradation. To further provide insight into the extent of mitochondrial heterogeneity in plants, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, stem and silique were analyzed. Of the 251 protein spots on a 2D-gel of the mitochondrial soluble/matrix fraction, the abundance of 213 spots were significantly varied between different samples. Identification of these spots revealed a non-redundant set of 79 proteins which were differentially expressed between organ/cell types. ... Importantly, posttranslational modifications played a significant role in the dynamics of the leaf mitochondrial proteome during the diurnal cycle. Overall, these findings indicated that the mitochondrial proteome is dynamic in order to fulfil different functional and physiological requirements in response to organspecific growth and changes in the external environments. These results also indicated that the majority of the changes in the mitochondrial proteome occurred in the matrix and suggested differences in substrate choice/availability in various plant organs and during the diurnal cycle. Further, these analyses demonstrate that, while mitochondrial proteins are regulated transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the activation state and/or regulation of proteins in key biochemical pathways in plant mitochondria. The integration of proteomics data with respiratory measurements, enzyme assays and transcript datasets will allow the identification of organ-enhanced and/or light/darkresponsive metabolic pathways as well as providing potential targets for reverse genetic approaches for further functional analysis of plant mitochondria.

CAPES / Durante a dormência, as plantas de clima temperado cessam temporariamente o crescimento visível. No caso da macieira sabe-se que a temperatura é o fator ambiental que mais influencia a dormência. A planta necessita de baixas temperaturas para induzir a entrada e posteriormente a saída da dormência, para então retomar o crescimento vegetativo. Sabe-se que quando não ocorre acúmulo suficiente de frio, as plantas apresentam crescimento errático, com brotação ocorrendo predominantemente na porção distal dos ramos, comprometendo a brotação satisfatória. No entanto, há pouca informação sobre o metabolismo destes carboidratos e sua relação com a dormência de plantas em condições de inverno ameno. Contudo o objetivo deste trabalho foi avaliar as diferenças biológicas e bioquímicas entre as porções distal e proximal de ramos de macieira que influenciam a acrotonia. De abril a outubro dos anos de 2012, 2013 e 2014, foram coletados ramos e esporões de três cultivares de macieira (Eva, Galaxy e Fuji Suprema) cultivadas em um pomar comercial no município de Plamas-PR. Para avaliar a intensidade da dormência em gemas vegetativas foi realizado o teste de estacas de uma só gema, e o teste de Tabuenca para as gemas florais. Variáveis bioquímicas também foram avaliadas como o teor de proteínas solúveis; atividade de alfa-amilase; açúcares (glicose, frutose, sacarose e sorbitol); amido e umidade. De forma geral as cultivares apresentaram tempo médio de brotação (TMB) menores do que em clima temperado. A porcentagem de brotação manteve-se alta na maioria das coletas para as três cultivares. O teste de Tabuenca confirmou a precocidade da cultivar Eva e mostrou comportamento muito semelhante entre a ‘Galaxy’ e a ‘Fuji Suprema’. Além disso foi observado um intervalo muito curto entre a saída da endodormência em condições de forçagem e em condição de campo. A umidade ponderal variou conforme o padrão da temperatura em cada ano, sendo que em 2013, ano de maior acúmulo de frio a umidade ponderal apresentou os menores valores para todas as cultivares. Em 2013 a tendência de aumento da atividade da alfa-amilase e no teor de proteína coincidiram com o menor valor de TMB. A glicose e frutose apresentaram comportamento muito semelhantes entre si durante as observações. O teor de sacarose variou de ano para ano, não apresentando um padrão fixo. O sorbitol, açúcar mais abundante no xilema, tende a ser mais constante durante a dormência com leve tendência de redução próximo à brotação. O teor de amido tende a diminuir com os primeiros acúmulos de horas frio, no entanto frequentemente sofre ressíntese, devido às ondas de calor comuns no inverno. Existe diferenças entre as porções dital e proximal dos ramos para todas as variáveis analisadas, com teor de carboidrato, atividade enzimática, proteínas e umidade predominantemente maiores na porção distal dos ramos, com algumas exceções pontuais. Isso pode estar associado à ocorrência de acrotonia em macieiras. As plantas avaliadas parecem não entrar em dormência profunda em condição de inverno ameno, tendo seu metabolismo muito sujeito às oscilações de temperatura. O metabolismo das plantas tende a mudar de ano para ano de acordo com o padrão do inverno em cada ano. / During dormancy, temperate plants temporarily stops visible growth. In apple trees, it is known that temperature is the environmental factor that most influences dormancy. The plant requires low temperatures to induce input and then to output endodormancy, thus resumption vegetative grow. It is known that when there isn’t sufficient chilling accumulation, plants present erratic growth, with budbreak occurring predominantly in the distal portion of the branches, compromising the satisfactory budbreak. However, there is little information on the metabolism of these carbohydrates and their relationship to plant dormancy in subtropical humid climates. This work aimed to evaluate the biological and biochemical differences between the distal and proximal portions of apple tree branches that influence acrotonia. From April to October of 2012, 2013 and 2014, branches and spurs of three apple cultivars (Eva, Galaxy and Fuji Suprema) were harvested in a commercial orchard in Palmas-PR. In order to evaluate the dormancy intensity in vegetative and floral buds, single bud cuttings test and Tabuenca test were carried out, respectively. Biochemical variables were also evaluated such as soluble protein content; Alpha-amylase activity; Soluble sugars (glucose, fructose, sucrose and sorbitol); Starch and water content. In general, the cultivars showed the Mean Time to Budbrst (MTB) lower than in temperate climate. The budburst percentage remained high in most of the observation dates for the three cultivars. The Tabuenca test confirmed the precocity of the cultivar Eva and showed very similar behavior between 'Galaxy' and 'Fuji Suprema'. In addition, a very short interval between the endodormancy output under forcing conditions and in field condition was observed. The water content varied according to the temperature pattern in each year, and in 2013, the year of greatest cold accumulation, the lowest values for all cultivars were observed. In 2013, the trend of increased alpha-amylase activity and protein content coincided with the lower value of MTB. Glucose and fructose presented very similar behavior among themselves during the observations. The sucrose content varied from year to year and did not present a fixed pattern. Sorbitol, sugar most abundant in the xylem, tends to be more constant during dormancy with a slight reduction tendency close to budding. The starch content tends to decrease with the first chilling accumulations, however it often undergoes resynthesis due to the heat waves common in winter. There are differences between the distal and proximal portions of the branches for all variables analyzed, with carbohydrate content, enzymatic activity, proteins and humidity predominantly larger in the distal portion of the branches, with some occasional exceptions. This may be associated with the occurrence of acrotonia in apple trees. The evaluated apple trees do not appear to fall into deep endodormancy in a mild winter condition, their metabolism seems to be very subject to temperature oscillations. Plant metabolism tends to change from year to year according to the winter pattern in each year.