La paroi des cellules végétales subit des modifications afin de s'assouplir ou de se rigidifier selon les besoins de la plante. Cette paroi est une structure complexe, composée de cellulose, d'hémicellulose et de pectines. Les modifications subies par les pectines au cours de l'élongation cellulaire sont encore assez peu caractérisées. Dans ce contexte, le but de ce projet est d'étudier le rôle de deux polygalacturonases (PG) dans le développement racinaire de la plante modèle A. thaliana. Les PG sont des enzymes de dégradation des homogalacturonanes (HG), le composant pectique majoritaire de la paroi primaire. Notrehypothèse est que les PG dégradent partiellement les HG des parois longitudinales des cellules racinaires en élongation. Cette dégradation engendrerait un assouplissement pariétal localisé, permettant la croissance anisotropique des cellules. Nos résultats indiquent que les gènes des deux PG étudiées, nommés PG ROOT APICAL MERISTEM (PG RAM) et PG ROOT (PG R) sontexprimés de façon complémentaire dans la racine, l'un dans le méristème racinaire (PG RAM), et l'autre dans la zone d'élongation et de différenciation (PG R). De plus, la sur-expression de la protéine PG R entraine une augmentation de l’élongation des hypocotyles étiolés, ainsi qu'une augmentation de la densité de racines latérales par rapport au sauvage, démontrant son rôle dans le développement racinaire et dans l'allongement cellulaire. Enfin, nous avons démontré que l'expression des gènes de ces PG était contrôlée de façon différentielle par les facteurs de transcription de la famille PLETHORA (PLT). / Plant cell wall structure is modified to control its stiffness or flexibility according to plant’s requirements. The cell wall is a complex structure, composed of cellulose, hemicelluloses and pectins. Pectin modifications during cellular elongation are not very well characterized. In this context, the aim of this project is to study the roles of two polygalacturonases (PG) in the root development on the model plant A. thaliana. PG are homogalacturonans (HG) degradation enzymes, HG being the major pectic component of the primary cell wall. This degradation would lead to a local parietal relaxation, allowing anisotropic growth of the cells. Our results show that the two studied PG, named PG ROOT APICAL MERISTEM (PG RAM) and PG ROOT (PG R), are expressed in complementary areas of the root, either in the root apical meristem (PG RAM) or in the elongated and differenciated root tissues (PG R). Furthermore, the over-expression of PG R results in longer etiolated hypocotyls and increases root density when compared to wild-type, demonstrating its function in root development and in cell elongation. Finally, we demonstrated that expression of these two PG genes is under the control of PLETHORA (PLT) family transcription factors, by differentially ways

Orientadores: Gonçalo Amarante Guimarães Pereira, Maria Carolina Scatolin do Rio / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T07:03:28Z (GMT). No. of bitstreams: 1 Teixeira_GleidsonSilva_M.pdf: 21908086 bytes, checksum: 84eb6f9c294915ecdd74ee2e9d23a152 (MD5) Previous issue date: 2009 / Resumo: A Vassoura-de-bruxa é uma das principais doenças que acometem o cacaueiro (Theobroma cacao L.). Ela é causada pelo fungo hemibiotrófico Moniliophthora perniciosa. Durante a fase biotrófica, o patógeno coloniza o espaço intercelular e estimula a formação de ramos hipertróficos/ hiperplásicos denominados vassoura verde. No estágio avançado da doença, o fungo passa a colonizar também o interior das células e observa-se a morte do tecido originando a vassoura seca. Homogalacturonano (HG) é um domínio estrutural péctico que constitui a matriz da parede celular primária e define suas propriedades funcionais e estruturais. Durante sua síntese, HG é altamente metilesterificado pela ação de pectinas metiltransferases (PMT) que utilizam S-adenosil-L-metionina (SAM) como fonte de grupos metil. Após ser depositado na parede celular, o padrão de metilesterificações de HG é modelado por pectina metilesterases (PME). Alterações no padrão de metilesterificação de HG são relacionadas à má formação de tecidos, complicações durante o desenvolvimento vegetal e à resistência contra a degradação da parede celular por enzimas microbianas. Neste trabalho, a expressão tecido-específica de genes envolvidos nas reações de síntese de SAM e o grau de metilesterificação do HG foram analisadas em plântulas de T. cacao sadias e infectadas. ESTs (expressed sequence tags) de T. cacao foram utilizadas como molde para a síntese de sondas de RNA específicas para os genes: glicosiltransferase, pectina metiltransferase, pectina metilesterase, S-adenosil-Lhomocisteína hidrolase, adenosina quinase, S-adenosil-L-metionina sintetase (SAMS). Além disso, os anticorpos monoclonais JIM5 e JIM7 foram utilizados na imunolocalização de epítopos de HG com menor ou maior grau de metilesterificação. A detecção de transcritos de SAMS e PMT indicou que esses são diferentemente expressos em plântulas sadias e infectadas. Nas plântulas sadias, o sinal para o gene SAMS foi mais intenso nas amostras coletadas com 14 dias após a inoculação (DAI), comparado com as coletadas 42 DAI. Nas amostras infectadas o sinal não variou, entretanto, foi menos intenso comparado com o de plântulas sadias. A marcação de PMT foi evidente no ápice de plântulas sadias coletadas 42 DAI e ausente nas coletadas com 14 DAI. Interessantemente, a ocorrência foi inversa em plântulas infectadas. Epítopos de HG altamente metilesterificados foram detectados por JIM7 em ambas as amostras sadias e infectadas. Entretanto, com 42 DAI, a ligação de JIM7 foi detectada apenas em plântulas sadias, enquanto resíduos de HG pouco metilesterificados, reconhecidos por JIM5, foram observados nas amostras infectadas. Juntos, os resultados sugerem que 1) com 42 DAI, SAM disponível estaria sendo preferencialmente utilizado como precursor de outros compostos e não para a metilesterificação de pectinas e; 2) a alteração do grau de metilesterificação do HG em plantas infectadas pode ser resultado da menor disponibilidade de SAM, da menor expressão do gene que codifica a enzima PMT e/ou da atividade de PMEs, favorecendo assim, a degradação da parede celular por enzimas do fungo em um período onde a degradação da parede é essencial para progressão da doença. / Abstract: The witches' broom disease is one of the major diseases affecting the cocoa (Theobroma cacao L.). It is caused by the hemibiotrophic Moniliophthora perniciosa fungus. During its biotrophic phase, the pathogen colonizes the intercellular space and stimulates the formation of hypertrophic/hyperplasic branches called green broom. In the advanced stage of the disease, the fungus starts also to colonize the interior of the cells and it is noted the death of tissue originating the dry broom. Homogalacturonan (HG) is a pectic structural domain that constitutes the primary cell wall matrix and defines its functional and structural proprieties. During its synthesis, HG is highly methylesterified by the action of pectin methyltransferases (PMT) which uses S-adenosyl-L-methionine (SAM) as a source of methyl groups. After being deposited in the cell wall, the pattern of HG methylesterifications is modeled by pectin methylesterases (PME). Changes in the pattern of HG methylesterifications are related to poor tissue formation, complications during plant development and resistance to cell wall degradation by microbial enzymes. In this study, the tissue-specific expression of genes involved in the reaction of synthesis of SAM and the degree of HG methylesterification were analyzed in seedlings of healthy and infected T. cacao. ESTs (expressed sequence tags) of T. cacao were used as a template for the synthesis of specific RNA probes for the genes: glycoyltransferase, pectin methyltransferase, pectin methylesterase, S-adenosyl-L-homocysteine hydrolase, adenosine kinase and S-adenosyl-L-methionine synthetase (SAMS). Furthermore, the monoclonal antibodies JIM5 and JIM7 were used in the immunolocalization of HG epitopes with lesser or grater degrees of methylesterification. The detection of transcripts of SAMS and PMT indicated that these are expressed differently in healthy and infected seedlings. In healthy seedlings, the signal of the SAMS gene was more intense than in samples collected 14 days after inoculation (DAI), compared to those collected 42 DAI. In the infected samples the signal did not change, however, it was less intense compared to healthy seedlings. The PMT staining was evident in the shoot apex of the healthy seedlings collected 42 DAI and missing in the ones collected 14 DAI. Interestingly, the occurrence was the inverse in infected seedlings. Highly methylesterified HG epitopes were detected by JIM7 in both healthy and infected samples. However, with 42 DAI, the JIM7 binding was detected only in healthy seedlings, while residues of less methylesterified HG, recognized by JIM5, were observed in the infected samples. Taken together, the results suggest that: 1) with 42 DAI, available SAM would be primarily used as a precursor to other compounds and not to methylesterification of pectins and; 2) the changes in the level of HG methylesterification in infected plants may be the result of the lower availability of SAM, lower expression of PMT and/or the activity of PMEs, thereby favoring the degradation of the cell wall by the fungus enzymes in a time where the degradation of the wall is essential to the progression of the disease. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular