葉綠體是一重要的植物細胞器。除在光合作用上扮演重要角色外，葉綠體同時也與其他生理過程有關，例如油脂，氨基酸及植物賀爾蒙的生成等等。葉綠體的發育及作用由一系列的調控因子所調控。如有任何一個調控因子無法正常運作，都可能導致葉綠體什至整棵植物無法正常生長及發育。本人在碩士論文研究期間利用T-DNA激活標籤法進行了一項的bzr1-1D抑制子的突變體篩選實驗。在篩選過程中，意外發現一個幼苗階段致死的隱性白化突變體，命名為bt20。bzr1-1D是一個對油菜素内酯（brassinosteroids, BRs）高度敏感的單點突變體，而該單點突變發生於BR信號傳導中的轉錄因子BZR1基因上。我在該碩士論文研究中取得的主要結果如下：白化突變體的T-DNA被確定插入於擬南芥的第二染色體上，而該插入點與白化的表現形相連鎖。本人曾嘗試用多種方法克隆有關基因，並已發現若干候選基因。生理學研究顯示該白化突變體的葉綠體無法正常發育，而其光合作用也無法正常進行。該些實驗數據顯示BT20極有可能在葉綠體的生物发生及發育當中扮演重要角色。綜上所述，本人在該碩士論文的研究中發現一全新的、可能受BR調節的葉綠體發育調控因子，對於將來研究BR與葉綠體發育的關係具重要科學價值。 / Chloroplasts are a type of organelles in plants that not only capture light for photosynthesis but are also involved in other important biological processes such as the synthesis of lipids, amino acids and phytohormones. The development and functioning of a chloroplast are coordinated by multiple regulators. Loss of function of any of the regulators may result in abnormal development of chloroplasts and even the whole plant. In my thesis project, I characterized a recessive seedling-lethal albino mutant, named bt20, which was isolated from a genetic mutant screen for bzr1-1D suppressor mutants screen using a T-DNA activation tagging approach in Arabidopsis thaliana. bzr1-1D is a brassinosteroid (BR) hypersensitive mutant that was caused by a dominant mutation in the transcription factor BZR1 in the BR signaling pathway. I confirmed that the T-DNA insertion in the bt20 mutant is located in the chromosome 2 of Arabidopsis and is linked with the albino phenotype. Attempts for the mutant gene cloning have identified several candidate genes but the exact responsible gene(s) remain to be determined. Physiological studies indicated impairment of chloroplasts and photosynthesis within the mutant, suggesting that BT20 gene plays an important role in regulating chloroplast development. We anticipate that our study may lead to the identification of a novel regulator involved in the biogenesis and development of chloroplasts and may establish a molecular link between BR and chloroplast development. / Detailed summary in vernacular field only. / Wong, King Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 87-95). / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Part 1 --- Introduction --- p.1 / Chapter 1.1 --- Brassinosteroids --- p.1 / Chapter 1.1.1 --- Brassinosteroids and its discovery --- p.1 / Chapter 1.1.2 --- Brassinosteroid signal transduction --- p.2 / Chapter 1.1.3 --- The transcription factor BZR1 --- p.5 / Chapter 1.1.4 --- bzr1-1D suppressor mutant screen and the identification of an albino mutant --- p.6 / Chapter 1.2 --- Chloroplast --- p.7 / Chapter 1.2.1 --- Chloroplasts in plants and their origin --- p.7 / Chapter 1.2.2 --- Structure of chloroplasts in plants --- p.8 / Chapter 1.2.3 --- Chloroplast development in cotyledons --- p.11 / Chapter 1.2.4 --- Nuclear gene transcription in chloroplast development --- p.14 / Chapter 1.2.5 --- Protein import for chloroplast development --- p.15 / Chapter 1.2.6 --- Chloroplast gene transcription --- p.16 / Chapter 1.2.7 --- Chloroplast RNA processing and protein translation --- p.18 / Chapter 1.2.8 --- Chloroplast protein processing --- p.19 / Chapter 1.2.9 --- Defects in regulators involving chloroplast development and biogenesis --- p.20 / Chapter 1.2.10 --- Potential relationship between BR and chloroplast development --- p.21 / Chapter Part 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Plant materials and growing conditions --- p.22 / Chapter 2.2 --- bzr1-1D suppressor mutant screen --- p.23 / Chapter 2.3 --- Genotypic PCR for bzr1-1D background and T-DNA insertion --- p.24 / Chapter 2.4 --- Fresh weight measurement of Arabidopsis plants --- p.26 / Chapter 2.5 --- Genetic crossing of Arabidopsis plants --- p.27 / Chapter 2.6 --- T-DNA insertion site identification --- p.28 / Chapter 2.7 --- Gene Cloning --- p.33 / Chapter 2.8 --- Reverse Transcription PCR (RT-PCR) --- p.36 / Chapter 2.9 --- Gene Cloning and vector constructions --- p.39 / Chapter 2.10 --- Gene transformation into Agrobacteria and Arabidopsis --- p.42 / Chapter 2.11 --- Transgenic plant screen --- p.44 / Chapter 2.12 --- Chlorophyll extraction and measurement --- p.45 / Chapter 2.13 --- Chlorophyll fluorescence assay --- p.46 / Chapter 2.14 --- Electronic microscopy (EM) --- p.48 / Chapter 2.15 --- Brassinolide (BL) and brassinozole (brz) treatments --- p.49 / Chapter Part 3 --- Results --- p.50 / Chapter 3.1 --- Summary of the bzr1-1D suppressor mutant screen --- p.50 / Chapter 3.2 --- Isolation of the albino bt20 mutant from the suppressor mutant screen --- p.52 / Chapter 3.3 --- T-DNA insertion site of bt20 is located in the chromosome 2 of Arabidopsis and is linked to the phenotype --- p.57 / Chapter 3.4 --- Genes flanking the insertion site are over-expressed --- p.59 / Chapter 3.5 --- T-DNA insertion does not cause base pair change in genes flanking the insertion site --- p.60 / Chapter 3.6 --- Over-expression of the selected candidate genes do not reproduce the albino phenotype --- p.62 / Chapter 3.7 --- bt20 seedlings have very low chlorophyll content --- p.64 / Chapter 3.8 --- bt20 has defect in chloroplast development --- p.65 / Chapter 3.9 --- bt20 has very low photosynthesis efficiency --- p.67 / Chapter 3.10 --- The expression of genes encoding plastid proteins in the bt20 mutant --- p.71 / Chapter 3.11 --- bt20 can respond to BL and brz treatment --- p.73 / Chapter 3.12 --- bt20 mutation does not affect the expression of BR biosynthetic gene --- p.75 / Chapter Part 4 --- Discussion --- p.77 / Chapter 4.1 --- bzr1-1D suppressor mutant screen and identification of the bt20 albino mutant --- p.77 / Chapter 4.2 --- Characterization of the bt20 albino mutant and cloning of the BT20 gene --- p.79 / Chapter 4.3 --- The possible role of BT20 role in plant growth and chloroplast development --- p.82 / Chapter 4.4 --- BR can improve the growth of the albino bt20 mutant --- p.85 / Chapter Part 5 --- Conclusion --- p.86 / Chapter Part 6 --- Reference --- p.87

Jiang, Tiantian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 61-75). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.v / Acknowledgements --- p.vi / List of Figures and Tables --- p.vii / Chapter Part 1 --- Introduction --- p.-0- / Chapter 1.1 --- Brassinosteriods (BRs) and BR signaling --- p.-0- / Chapter 1.1.1 --- Discovery of BRs --- p.-2- / Chapter 1.1.2 --- Functions of BRs --- p.-4- / Chapter 1.1.3 --- BR signaling pathway --- p.-6- / Chapter 1.2 --- Nitrogen (N) and N responses --- p.-10- / Chapter 1.2.1 --- Hormones involved in plant N responses --- p.-11- / Chapter 1.3 --- Anthocyanin and anthocyanin synthesis --- p.-13- / Chapter 1.3.1 --- Anthocyanin structures --- p.-13- / Chapter 1.3.2 --- Functions of anthocyanins --- p.-14- / Chapter 1.3.3 --- Biosynthesis of anthocyanins --- p.-14- / Chapter 1.3.4 --- Regulations of anthocyanin biosynthesis --- p.-15- / Chapter 1.4 --- Hormones and plant nutrient stresses --- p.-19- / Chapter Part 2 --- Materials and Methods --- p.-20- / Chapter 2.1 --- Plant materials and growth conditions --- p.-20- / Chapter 2.2 --- Measurement of anthocyanin content --- p.-21- / Chapter 2.3 --- Yeast two-hybrid (Y2H) assay --- p.-22- / Chapter 2.4 --- Bimolecular fluorescence complementation (BiFC) assays --- p.-23- / Chapter 2.5 --- Quantitative real-time PCR --- p.-25- / Chapter 2.6 --- Electrophoretic mobility shift assay (EMSA) and competition assay --- p.-26- / Chapter 2.7 --- Histochemical staining of GUS activity --- p.-28- / Chapter Part 3 --- Results --- p.-29- / Chapter 3.1 --- 24-epibrassinolide (24-eBR) increases plant tolerance to N-starvation in Arabidopsis - --- p.-29- / Chapter 3.2 --- BR treatment enhances anthocyanin accumulation under N deprivation conditions --- p.-31- / Chapter 3.3 --- BZR1 interacts with PAP1 in vitro and in vivo --- p.-35- / Chapter 3.4 --- BR and BZR1 promote the expression of the 'late' anthocyanin biosynthetic genes during N deprivation - --- p.-39- / Chapter 3.5 --- BZR1 binds to the promoter of DFR --- p.-43- / Chapter 3.6 --- BR-enhanced anthocyanin accumulation is specific to N-deprivation --- p.-46- / Chapter 3.7 --- BZR1 differently regulates PAP1 and PAP2 --- p.-48- / Chapter 3.8 --- Endogenous GL3 is required for BR-enhanced anthocyanin biosynthesis --- p.-52- / Chapter 3.9 --- N status affects the expression of BR biosynthetic gene CPD --- p.-52- / Chapter Part 4 --- Discussion --- p.-54- / Chapter 4.1 --- BRs confer plant tolerance to low-N stress and the tolerance is mediated by BR enhancement of low-N-induced anthocyanin biosynthesis --- p.-54- / Chapter 4.2 --- BRs enhance anthocyanin accumulation under N starvation through BZR1-PAP1 interaction or direct control of the expression of anthocyanin biosynthetic genes --- p.-55- / Chapter 4.3 --- BRs are specifically involved in low-N induced anthocyanin production --- p.-56- / Chapter 4.4 --- Transcription factors that specifically control BR-regulated anthocyanin biosynthesis --- p.-57- / Chapter 4.5 --- DFR is an important target of BR-regulation of anthocyanin biosynthesis --- p.-58- / Chapter Part 5: --- Conculsions --- p.-59- / Chapter Part 6: --- References --- p.-61-

As sementes do gênero Brachiaria apresentam desuniformidade de maturação, degrana natural e dormência, causando problemas para a colheita e estabelecimento de estande uniforme de plantas em campo; a desuniformidade de maturação acarreta a produção de sementes com grau de dormência diferenciado. Por outro lado, o condicionamento fisiológico é uma opção interessante para uniformizar o processo germinativo. O objetivo desta pesquisa foi verificar os efeitos do condicionamento fisiológico com bioestimulantes, em sementes de Brachiaria humidicola colhidas de diferentes maneiras, armazenadas sob diferentes condições de temperatura e umidade relativa do ar, procurando, também, identificar a possível superação da dormência e verificar a atividade enzimática das sementes durante esse período. As sementes foram obtidas mediante dois procedimentos de colheita: a) sementes colhidas das inflorescências inteiras e b) sementes caídas ao solo. Após a colheita foi realizada a escarificação com H2SO4, obtendo sementes sem escarificação e escarificadas de cada procedimento de colheita. Foram realizados cinco tratamentos nas sementes escarificadas ou não, para os dois procedimentos de colheita: 1- sem condicionamento e papel umedecido com água; 2- sem condicionamento e papel umedecido com KNO3; 3- biocondicionamento com 24-EpiBL, na concentração de 10-8 M; 4- biocondicionamento com GA3, na concentração de 10-4 M e 5- biocondicionamento com 24-EpiBL + GA3, nas concentrações de 10-8 e 10-4 M, respectivamente. Após os tratamentos as sementes foram armazenadas em câmara fria e seca e ambiente natural e realizadas avaliações em quatro épocas (zero, três, seis e nove meses de armazenamento), mediante os testes de germinação e vigor (primeira contagem, velocidade de germinação e, análise computadorizada de imagens de plântulas - SVIS®) e emergência de plântulas, além da determinação da atividade das enzimas α-amilase e polifenoloxidase no início e aos nove meses de armazenamento. Os resultados indicam que o armazenamento das sementes de Brachiaria humidicola, em câmara fria e seca reduz a porcentagem de sementes dormentes; o biocondicionamento com ácido giberélico e 24-EpiBL + ácido giberélico, favorece a velocidade de germinação e o crescimento de plântulas; o condicionamento fisiológico com ácido giberélico isolado ou combinado com 24-epibrassinolídeo, é vantajoso para sementes de Brachiaria humidicola, armazenadas em câmara fria e seca ou ambiente natural e, a atividade da enzima α-amilase aumenta e da polifenoloxidase diminui à medida que a dormência das sementes de Brachiaria é superada durante o armazenamento. / Seeds from the genus Brachiaria present uneven maturation, natural threshing and dormancy, leading to issues during harvest and in the establishment of a uniform plant stand; the uneven maturation entails to the production of seeds with different degrees of dormancy. On the other hand, physiological priming is an interesting option to uniform the germination process. The aim of this research was to verify the effects of physiological priming with biostimulants, on seeds of Brachiaria humidicola harvested by different methods, stored under different conditions of temperature and air relative humidity, also looking to identify the possibility of dormancy breakage and verify seed enzymatic activity during this period. Seeds were obtained through two harvest procedures: a) seeds harvested from whole inflorescences and b) dropped seeds on the ground. After harvest, scarification was performed with H2SO4, obtaining seeds with or without scarification for each harvest procedure. Five treatments were performed on seeds scarified or not, for the two harvest procedures: 1- without priming and paper moistened with water; 2- without priming and paper moistened with KNO3; 3- Bio-priming with 24-EpiBL, in the concentration of 10-8 M; 4- Bio-priming with GA3, in the concentration of 10-4 M and bio-priming with 24-EpiBL + GA3, in the concentrations of 10-8 and 10-4 M, respectively. After treatment seeds were stored in a dry cold chamber and natural environment and evaluations were performed on four periods (zero, three, six and nine months of storage), through germination and vigor tests (first count of germination, speed of germination, computerized image analysis of seedlings - SVIS® and seedling emergence), besides the determination of the activity of the α-amylase and polyphenol oxidase enzymes at the beginning and at the ninth month of storage. The results indicate that the storage of Brachiaria humidicola seeds, in a dry cold chamber reduces the percentage of dormant seeds; bio-priming with 24-EpiBL + gibberellic acid accelerates germination and favors seedling growth; the physiological priming with gibberellic acid alone or combined with 24-epibrassinolide is advantageous for Brachiaria humidicola seeds, stored in dry cold chambers or natural environments and, the activity of the α-amylase enzyme increases and of the polyphenol oxidase decreases as the dormancy of Brachiaria seeds is broken during storage.

Background: The EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described. Results: The exo loss of function mutant showed diminished leaf and root growth and reduced biomass production. Light and scanning electron microscopy analyses revealed that impaired leaf growth is due to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were smaller in comparison to the wild-type. The exo mutant showed reduced brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were significantly more sensitive to the inhibitory effect of synthetic brassinolide. Apart from reduced growth, exo did not show severe morphological abnormalities. Gene expression analyses of leaf material identified genes that showed robust EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and genes involved in primary and secondary metabolism showed weaker expression in exo than in wild-type plants. However, the vast majority of BR-regulated genes were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to the apoplast. Conclusion: The EXO gene is essential for cell expansion in leaves. Gene expression patterns and growth assays suggest that EXO mediates BR-induced leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the shoot. EXO presumably is involved in a signalling process which coordinates BR-responses with environmental or developmental signals. The hypersensitivity of exo roots to BR suggests that EXO plays a diverse role in the control of BR responses in the root.

<p>In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised.</p>

<p>In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised.</p>

Orientador: Claudia R. B. Haddad / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T21:05:30Z (GMT). No. of bitstreams: 1 Dalio_RonaldoJoseDurigan_M.pdf: 923106 bytes, checksum: 4cfd1d62015260abdbf750e8baf62aa6 (MD5) Previous issue date: 2007 / Resumo: Plantas de Cajanus cajan receberam aplicações de brassinosteróide ou de clotrimazol (inibidor de síntese de brassinosteróides) e foram submetidas à salinidade. O objetivo deste trabalho foi verificar o efeito de duas concentrações de brassinosteróide (1 x 10-7 e 0,5 x 10-9 M) e uma de clotrimazol (1 x 10-4 M), no crescimento, composição bioquímica e transporte de aminoácidos em plantas de C. cajan, submetidas à duas concentrações de NaCl (200 e 400 mM). A salinidade afetou os parâmetros de crescimento (massas frescas e secas, comprimento da parte aérea e da maior raiz, número de folhas, área e suculência foliar), os parâmetros bioquímicos (teores de nitrato, prolina, aminoácidos livres, proteínas totais, açúcares solúveis, sacarose, clorofilas e carotenóides) e o padrão dos aminoácidos transportados. A proporção da maioria dos aminoácidos transportados no xilema diminuiu sob salinidade, com exceção de alanina e serina. A concentração de NaCl a 400 mM provocou as maiores alterações. A aplicação de clotrimazol mostrou-se eficiente na inibição dos efeitos provocados por brassinosteróide na grande maioria dos parâmetros estudados, sob salinidade. Não houve diferença em relação à eficiência das duas concentrações de brassinosteróide utilizadas na maioria dos parâmetros avaliados. A aplicação de brassinosteróide amenizou o efeito do estresse salino na maioria dos parâmetros de crescimento, dos parâmetros bioquímicos e no padrão de aminoácidos transportados em plantas de C. cajan, submetidas à salinidade / Abstract: Plants of Cajanus cajan received applications of brassinosteroid or clotrimazol (brassinosteroids synthesis inhibitor), and were subjected to salt stress. The goal of this study was to verify the effect of two concentrations of brassinosteroid (1.0 x 10-7and 0.5 x 10-9M), and one concentration of clotrimazol (1.0 x 10-4 M) on growth, biochemical composition and transport of amino acids in plants of C. Cajan, cultivated under two concentrations of NaCl (200 and 400 mM). The salt affected growth (fresh and dry mass, shoot length and main root length, number of leaves, leaf area and succulence of leaves) and biochemical parameters (nitrate, proline, free aminoacids, total protein, soluble sugars, sucrose, chlorophylls and carotenoids concentrations), and changed the pattern of amino acids transported. The proportion of most of the amino acids transported in the xylem was reduced by salinity. Alanine and serine were exceptions. The largest alterations were caused by 400 mM of NaCl. Clotrimazol was effective in inhibiting the effects caused by brassinosteroid for the great majority of studied parameters. There was no difference between the two concentrations of brassinosteroid for most parameters evaluated, under salinity. The application of brassinosteroid was effective diminishing the effect of salt on most of the growth and biochemical parameters, and in the patterns of amino acids transported in plants of C. Cajan subjected to salt stress / Mestrado / Mestre em Biologia Vegetal

Philosophiae Doctor - PhD / In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised. / South Africa

Orientador: Claudia Regina Baptista Haddad / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T21:10:07Z (GMT). No. of bitstreams: 1 Chone_RosanaMarySartor_M.pdf: 46520912 bytes, checksum: 140ac9102b951f96df903dee18f16c8a (MD5) Previous issue date: 2015 / Resumo: O cultivo do cafeeiro é de grande importância econômica mundial. O consumo crescente de café arábica tem levado os pesquisadores a buscar variedades que produzam bebidas de melhor qualidade e com baixos teores de cafeína. Os programas de melhoramento genético concentram esforços na busca destas características com destaque para cultivares da espécie Coffea arabica, a mais cultivada no mundo. A embriogênese somática é importante para manter características selecionadas em programas de melhoramento genético e produzir os indivíduos selecionados em grande escala, além de contribuir para estudos fisiológicos e moleculares. A embriogênese somática direta apresenta vantagens pela redução de insumos e mão de obra, devido à eliminação da fase calogênica, porém, apresenta baixa eficiência, quando comparada com a via indireta, para a espécie C. arabica. Apesar da importância da embriogênese somática direta, nada se sabe sobre os fatores que controlam a sua ocorrência em genótipos de C. arabica e tampouco há estudos com utilização de brassinosteróides na fase de indução desse tipo de embriogênese in vitro. O brassinosteróide, 24-epibrassinolídeo, foi utilizado isoladamente e em conjunto com uma citocinina, N6-(2-isopentenyl) adenina (2-iP), para avaliar seus efeitos sobre a embriogênese somática direta e verificar possíveis alterações endógenas no metabolismo desses hormônios durante a indução da embriogênese em explantes foliares da cultivar Mundo Novo de Coffea arabica. Os explantes foram cultivados em meio de cultura MS modificado, adicionado de 2-iP a 10 µM, acrescido ou não de 24-epibrassinolídeo. Foram utilizadas as seguintes concentrações do brassinosteróide: 0,01 µM, 0,10 µM e 1 µM, em dois experimentos realizados em diferentes épocas do ano. Análises de microscopia de luz e microscopia eletrônica de varredura foram realizadas para compreensão dos eventos anatômicos e morfológicos da via de embriogênese relacionada às diferentes combinações hormonais praticadas. Avaliações de variáveis qualitativas e quantitativas dos explantes foliares dos dois experimentos foram realizadas in vitro durante 130 e 240 dias. Na tentativa de compreender melhor a participação dos brassinosteróides na via de embriogênese somática, explantes foliares induzidos apenas com a citocinina, 2-iP, foram submetidos a análises de Ressonância Magnética Nuclear (RMN). Os embriões somáticos obtidos tanto no tratamento com uso exclusivo de citocinina, como no tratamento com citocinina associada ao 24-epibrassinolídeo foram cultivados em meio de cultura para germinação e desenvolveram-se em plântulas. A aplicação do brassinosteróide levou à iniciação do processo de embriogênese somática direta, promovendo a formação de estruturas embriogênicas. Sua aplicação conjunta com citocinina aumentou o número de embriões, o número de explantes com embriões, levou à formação de embriões em condição na qual apenas a aplicação de citocinina foi insuficiente e acelerou a via embriogênica. Promoveu o desenvolvimento de embriões mais bem definidos, com características meristemáticas típicas. O aumento de picos na região do espectro de RMN, característico de moléculas esteroidais, coincidente com o período em que ocorre a embriogênese somática direta, pode ser indicativo da participação de brassinosteróides endógenos durante esse processo. Este trabalho abre perspectivas para a utilização de brassinosteróides em embriogênese somática direta em processos produtivos de C. arabica / Abstract: Coffee cultivation is of worldwide economic importance. The increasing consumption of coffee has led to search for varieties that produce better quality beverages with lower caffeine contents. Genetic improvement have concentrated their efforts on the search for these characteristics with emphasis on cultivars of the species Coffea arabica, the most cultivated in the world. Somatic embryogenesis is important to maintain selected characteristics in genetic improvement and produce selected individuals on a large scale, as well as contributing to physiological and molecular studies. Direct somatic embryogenesis shows the advantages of reduced consumables and less manual labor, due to elimination of the callus phase, but is inefficient with the species C. arabica when compared with the indirect pathway. Despite the importance of direct somatic embryogenesis, nothing is known about factors controlling its occurrence in C. arabica genotypes, nor are there any studies on use of brassinosteroids in the in vitro induction of this type of embryogenesis. The brassinosteroid 24-epibrassinolide was used alone and together with cytokinin N6-(2-isopentenyl) adenine (2-iP) to evaluate its effects on direct somatic embryogenesis and verify endogenous alterations in metabolism of these hormones during induction of embryogenesis in foliar explants of cultivar Mundo Novo of species Coffea Arabica. The explants were cultivated in modified MS culture medium with the addition of 10 µM 2-iP, with or without 24-epibrassinolide. The following concentrations of brassinosteroid were used: 0.01 µM, 0.10 µM and 1 µM, in two experiments carried out at different times of the year. Light microscopy and scanning electronic microscopy analyses were carried out in order to understand anatomic and morphologic events of embryogenic pathway as related to different hormone combinations applied. The qualitative and quantitative variables of the foliar explants were evaluated in the two experiments at points in time from 130 and 240 days. In an attempt to better understand the participation of the brassinosteroids in the somatic embryogenesis pathway, foliar explants induced only by the cytokinin 2-iP were submitted to nuclear magnetic resonance (RMN) analyses. The somatic embryos obtained both using the treatment with the exclusive use of cytokinin and in treatment with cytokinin associated with 24-epibrassinolide were cultivated in a culture medium for germination and developed into plant embryos. The application of brassinosteroid led to initiation of direct somatic embryogenesis process, promoting the formation of embryogenic structures. Its application together with cytokinin increased number of embryos, the number of explants with embryos, led to formation of embryos in a condition in which only application of cytokinin was insufficient and accelerated embryogenic pathway. It also promoted development of much better defined embryos with typically meristematic characteristics. The increase in peaks in the region of the RMN spectrum, characteristic of steroidal molecules, coinciding with the period in which direct somatic embryogenesis occurred, could be an indication of participation of endogenous brassinosteroids during the process. This work widens the perspectives for the use of brassinosteroids in direct somatic embryogenesis for productive processes with C. arabica / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

Les brassinostéroïdes (BR) sont des hormones stéroïdes végétales qui jouent un rôle dans la croissance et le développement des plantes. Ils sont perçus à la surface cellulaire par le récepteur kinase BRI1 (BR insensitive 1) situé au niveau de la membrane plasmique. La voie de signalisation des BRs implique la cis et trans-phosphorylation de BRI1 et de son corécepteur BAK1 (BRI1 Associated Receptor Kinase 1) et aboutit à la déphosphorylation des facteurs de transcription BZR1 (Brassinazole Resistant 1) et BES1 (bri1-EMS-Supressor 1) et l’activation des réponses génomiques aux BRs. Les mécanismes permettant la dé-activation du complexe de perception des BRs sont encore peu connus mais peuvent impliquer la déphosphorylation de BRI1, la fixation de régulateur négatif comme BKI1, et l’endocytose de BRI1. Mon travail de doctorat a consisté à étudier les mécanismes d’endocytoses et de dégradation de BRI1 et leurs régulations par les conditions environnementales.L’ubiquitination est une modification post-traductionnelle impliquant l’attachement d’un polypeptide d’ubiquitine (Ub) sur une ou plusieurs lysines (K) d’une protéine cible. L’ubiquitine elle-même peut être sujette à l’ubiquitination, créant ainsi des chaînes de poly-ubiquitine (polyUb) qui peuvent adopter des topologies différentes selon le résidu K de l’ubiquitine impliqué dans la formation de la chaîne. Parmi ces chaînes de polyUb, la chaîne impliquant la lysine-63 (K63) est connue pour être impliquée dans la dégradation des protéines par endocytose chez les levures et les mammifères. Néanmoins, peu de choses sont connues sur l’endocytose dépendante de l’ubiquitine chez les plantes. Durant la première partie de ma thèse, j’ai démontré que BRI1 est modifié in vivo par des chaînes de polyUb K63 et j’ai pu identifier des sites putatifs d’ubiquitination dans BRI1. En utilisant une forme artificiellement ubiquitinée de BRI1 ainsi qu’une forme non ubiquitinable de BRI1, j’ai montré que l’ubiquitination joue un rôle sur l’internalisation de BRI1 à la surface cellulaire et est essentielle pour son adressage à la vacuole. Par ailleurs, j’ai établi que la dynamique de la protéine BRI1 médiée par son ubiquitination joue un rôle important dans le contrôle des réponses des BR.La deuxième partie de ma thèse m’a permis de découvrir une connexion entre l’endocytose dépendante de l’ubiquitine de BRI1 et la réponse des plantes à une élévation de température. J’ai notamment montré que l’accumulation de la protéine BRI1 est réduite dans les racines lorsque les plantes sont cultivées à une température plus élevée (i.e. 26°C). Cependant, la forme non ubiquitinable de BRI1 ne répond pas à cette élévation de la température suggérant une implication de l’endocytose dépendant de l’ubiquitine dans de telles conditions. Cette déstabilisation de BRI1 observée à température plus élevée se traduit au niveau moléculaire par une inhibition de la voie de signalisation des BRs et une hypersensibilité à des traitements BRs exogènes. Les plantes répondent à une montée subite de la température par une augmentation de la longueur de l’hypocotyle, des pétioles et de la racine principale ; processus largement sous le contrôle de l’auxine. J’ai accumulé au cours de ma thèse des évidences génétiques et génomiques montrant que la déstabilisation du BRI1 et l’inhibition de la signalisation des BR à plus hautes températures contrôle l’élongation des racines indépendamment de l’auxine. En conclusion, les résultats obtenus indiquent que BRI1 intègre les informations de température et de signalisation des BRs pour ajuster la croissance des racines lors de variations des conditions environnementales. / Brassinosteroids (BRs) are plant steroid hormones that play important roles on plant growth and development, and that are perceived at the plasma membrane (PM) by the receptor kinase BRI1 (BR insensitive 1). The perception of BRs by BRI1 triggers the cis and trans-phosphorylation of BRI1 and its co-receptor BAK1 (BRI1 Associated Receptor Kinase 1) initiating this way the BR signaling cascade that culminates with the de-phosphorylation of the transcription factors BZR1 (Brassinazole Resistant 1) and BES1 (bri1-EMS-Supressor 1), allowing these transcription factors to activate the transcription of thousands of BR-responsive genes. The mechanisms allowing the deactivation of the BR receptor complex are still elusive but may involve dephosphorylation, binding to negative regulators such as BKI1 and endocytosis. My PhD work focused on the endocytosis and the degradation of BRI1, and its regulation by environmental conditions.Ubiquitination is a post-translational modification that consists of the attachment of ubiquitin (Ub) polypeptides to one or several lysine (K) of a target protein. Ubiquitin itself can undergo self-ubiquitination thereby creating polyubiquitin (polyUb) chains that can harbor different topologies depending on the lysine residue from ubiquitin involved in the chain formation. Among these, polyUb chains involving lysine-63 (K63) are known to be involved in the degradation of proteins by endocytosis in yeast and mammals, while very little is known in plants. On a first part of my thesis, I demonstrated that BRI1 is post-translationally modified by K63 poly-ubiquitin chains and I identified putative ubiquitination sites in BRI1. Using both artificial ubiquitination of BRI1 and the generation of an ubiquitination-defective BRI1, I showed that ubiquitination plays a role on the internalization of BRI1 from the cell-surface, and is essential for its vacuolar targeting. Furthermore, ubiquitin-mediated BRI1 protein dynamics was also shown to play an important role in the control of BR responses by stabilizing BRI1 at the PM.The second part of the PhD uncovered a connection between BRI1 ubiquitin-mediated endocytosis and plant responses to elevated ambient temperature. I showed that BRI1 protein accumulation is lower at elevated temperature (i.e. 26°C) in roots, while the ubiquitin-defective form of BRI1 failed to respond, indicating a role of the BRI1 ubiquitin-mediated endocytosis in temperature responses. This temperature-mediated destabilization of BRI1 correlates with a downregulation of BR signaling and BR responses. Plants respond sudden rise in temperature by increasing their primary root length, a process that is under the control of auxin. Importantly, I accumulated genetic and genomic evidence that BRI1 destabilization and downregulation of BR signaling controls root elongation upon elevated ambient growth temperature, with little or no contribution of auxin. Altogether, our results establish that BRI1 integrates temperature and BR signaling to regulate root growth upon long-term changes in environmental conditions.