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Apple scab, the most serious disease of apple is caused by fungal pathogen Venturia inaequalis. Knowledge about the apple response to apple scab attack on the cellular and tissue level is insufficient. For studies of Malus-Venturia interaction on the cellular and tissue level, the establishment of methods for cell structures visualization in apple leaves is necessary. In this work, the experimental plant material grown in vitro and ex vitro was successfully established and the method of apple infection by conidia of V. inaequalis was optimized. Various methods of cell components visualization such as vital staining, in situ immunolocalization, transformation, environmental scanning electron microscopy and confocal microscopy, were tested. Cell structures, such as the cytoskeleton, the cell wall and the cuticle were visualized in apple leaves. Preliminary experiments following specific the changes of cell wall structures induced by V. inaequalis attack were performed. Further, changes of cuticle structure, the first barrier for penetration of pathogen to plant tissues during infection, were observed during the leaf ontogenesis. Powered by TCPDF (www.tcpdf.org)

Doctor of Philosophy / Department of Biochemistry / Subbaratnam Muthukrishnan / In order to grow and develop, insects must undergo a process of molting, wherein the old cuticle is replaced with a new one. A thin envelope layer has been predicted to act as a physical barrier between molting fluid chitinases and the site of new chitin synthesis ensuring selective protection of newly synthesized chitin. The factors that help the new exoskeleton withstand the deleterious effects of chitinolytic enzymes remain poorly understood. In the current study a mechanistic role for two proteins, Knickkopf (Knk) and Retroactive (Rtv), was explored in organization and protection of the newly synthesized procuticular chitin. Our study demonstrated colocalization of molting fluid chitinases (chitinase-5) with chitin in T. castaneum pharate adult elytral cuticle. Presence of chitinases in the new cuticle, disproved the old theory of the envelope being a protective barrier against chitinases. Confocal and transmission electron microscopic imaging of T. castaneum pharate adult elytral cuticle suggested that Knk protein selectively colocalizes with chitin in the new procuticle, organizes chitin into laminae and protects it from the activity of molting fluid chitinases. Down-regulation of Knk expression resulted in reduction of procuticular chitin, disruption of the laminar architecture of the procuticle and severe molting defects that are ultimately lethal at all stages of insect growth. The presence and activity of Rtv protein ensures the trafficking of Knk into the procuticle. Down regulation of Rtv transcripts showed molting defects and a significant decrease in chitin content similar to those following Knk dsRNA treatment. Confocal microscopic analysis revealed an essential role for Rtv in proper trafficking of Knk from epithelial cells to within the newly synthesized procuticule. Once released into the procuticle, Knk organizes and protects chitin from chitinases. The conservation of Knk and Rtv in all insect species suggests a critical role for these proteins in maintenance and protection of chitin in the insect exoskeleton.

Doctor of Philosophy / Department of Biochemistry / Subbarat Muthukrishnan / The extracellular matrices of cuticle and peritrophic matrix of insects are composed mainly of chitin complexed with proteins, some of which contain chitin-binding domains. This study is focused on the identification and functional characterization of genes encoding proteins that possess one or more copies of the six-cysteine-containing ChtBD2 domain (Peritrophin A motif =CBM_14 =Pfam 01607) in the red flour beetle, Tribolium castaneum. A bioinformatics search of T. castaneum genome yielded previously characterized chitin metabolic enzymes and several additional proteins. Using phylogenetic analyses, the exon-intron organization of the corresponding genes, domain organization of proteins, and temporal and tissue-specificity of expression patterns, these proteins were classified into three large families. The first family includes 11 proteins essentially made up of 1 to 14 repeats of the peritrophin A domain. Transcripts for these proteins are expressed only in the midgut and only during feeding stages of development. We therefore denote these proteins as “Peritrophic Matrix Proteins” or PMPs. The genes of the second and third families are expressed in cuticle-forming tissues throughout all stages of development but not in the midgut. These two families have been denoted as “Cuticular Proteins Analogous to Peritrophins 3” or CPAP3s and “Cuticular Proteins Analogous to Peritophins 1” or CPAP1s based on the number of ChtBD2 domains that they contain. Unlike other cuticular proteins studied so far, TcCPAP1-C protein is localized predominantly in the exocuticle and could contribute to the unique properties of this cuticular layer. RNA interference (RNAi), which down-regulates transcripts for any targeted gene, results in lethal and/or abnormal phenotypes for some, but not all, of these genes. Phenotypes are often unique and are manifested at different developmental stages, including embryonic, pupal and/or adult stages. The experiments presented in this dissertation reveal that while the vast majority of the CPAP3 genes serve distinct and essential functions affecting survival, molting or normal cuticle development. However, a minority of the CPAP1 and PMP family genes are indispensable for survival under laboratory conditions. Some of the non-essential genes may have functional redundancy or may be needed only under special circumstances such as exposure to stress or pathogens.

Le développement de la graine est une étape cruciale du cycle de vie des Angiospermes. La graine est composée de trois compartiments : (1) Le tégument, assurant un rôle protecteur, (2) l’albumen, qui a un rôle principalement nourricier pour (3) l’embryon, qui donnera la future plante. Ainsi, ces trois tissus se développent de concert pour former une graine viable. Une telle coordination présuppose que les différents compartiments communiquent entre eux. Dans ce contexte, j’ai étudié les gènes impliqués dans la formation de la cuticule embryonnaire, une structure hydrophobe recouvrant la plante et essentielle pour limiter les pertes d’eau, assurant ainsi sa survie. Au début de ma thèse, différents gènes étaient déjà connus, certains spécifiques de l’embryon, et d’autres spécifiques de l’albumen, renforçant l’idée de l’existence d’une communication moléculaire entre les deux tissus. Côté albumen, le facteur de transcription ZOU contrôle l’expression d’ALE1, une protéase. Côté embryon, deux récepteurs, GSO1 et GSO2, sont impliqués. L’étude de l’interaction génétique de ces différents gènes a permis de prouver leur appartenance à la même voie de signalisation. L’identité de ces gènes nous a amené à supposer l’existence d’un ou plusieurs peptides agissant comme messagers entre l’embryon et l’albumen. Ainsi, mes travaux de thèse ont permis de caractériser de nouveaux gènes impliqués dans ce processus, ainsi que certaines propriétés de la cuticule. Le principal est CERBERUS, dont l’expression est contrôlée par ZOU, un peptide sécrété par l’albumen qui est nécessaire pour la mise en place d’une cuticule fonctionnelle et la mise en place d’une structure non encore décrite à ce jour, la gaine embryonnaire. Un nouveau rôle pour GSO1 et GSO2 a aussi été démontré. Des résultats préliminaires suggèrent que TPST, une enzyme impliquées dans la sulfation des peptides, est impliquée dans la voie de signalisation étudiée. Enfin, mes travaux ont identifiés un autre gène, FRIABLE1, qui est aussi essentiel à la mise en place de la cuticule et joue dans la même voie de signalisation. Les découvertes associées à mes travaux de thèse ont permis de compléter et d’approfondir les connaissances sur les gènes impliqués dans la formation de la surface de l’embryon chez Arabidopsis. / Seed development is a crucial step in Angiosperms life cycle. The seed is composed of three distinct compartments: (1) The testa, ensuring a protective function, (2) the endosperm, which plays a key nutritive role supporting (3) the embryo, the fate of which is to become the future plant. These three tissues develop concomitantly to form a viable seed. Such developmental coordination necessitates the involvement of communication between the compartments. In this context, I have studied genes involved in the establishment of the embryonic cuticle, a hydrophobic structure that surrounds the embryo, plays an essential post-germination function in regulating water loss and is thus critical for plant survival. At the beginning of my PhD, several proteins were known to be involved in the process of cuticle establishment, some of which were expressed in the endosperm and others in the embryo, hinting at the existence of molecular communication between the two tissues. On the endosperm side, the transcription factor ZOU controls the expression of ALE1, a subtilisin-like serine protease. On the embryo side, two receptors, GSO1 and GSO2, are involved. Genetic interaction between the genes encoding these proteins had confirmed their involvement the same signalling pathway. The molecular identities of these proteins led us to propose the existence of one or more unidentified peptides acting as messengers between the embryo and the endosperm. My research has allowed the characterization of novel proteins involved in the process of embryonic surface formation. The principal subject of my research has been CERBERUS, a peptide produced in the endosperm, the expression of which is controlled by ZOU, and which is necessary both for the formation of an intact embryonic cuticle and the production of a previously uncharacterised structure, the embryo sheath. I have demonstrated novel roles for GSO1 and GSO2 in embryo sheath deposition. Furthermore, I have generated preliminary data suggesting that a protein involved in peptide sulfation, TPST, is involved in the GSO1 GSO2 signalling pathway. Finally, I have shown that another protein involved in posttranslational protein modification, FRIABLE1 is involved in this same pathway. My results have advanced knowledge of the molecular mechanisms controlling embryonic surface formation in Arabidopsis.

The main function of cuticular lipids in insects is to limit water loss through the surface. However they are also involved in various types of chemical communication between species and reduced penetration insecticides chemicals, and toxins as well as provide protection against the attack of microorganisms, parasites and predators. Numerous species of insect vectors of highly virulent diseases of plants, play a critical role as agricultural pests as in the case of Rhynchophorus palmarum which is vector in nematode transmitter red ring disease is responsible for causing damage to the agricultural economy of palm trees, in particular coconut in various regions of the country. However, the susceptibility or resistance of various insect species, infection can result from several factors, including differences in structure and composition of the exoskeleton, and the presence of antimicrobial compounds in the cuticle. The role of the host surface lipids in the pathogenesis of insects is still poorly understood. The determination of cuticular lipids profile is therefore of great importance to understanding insect susceptibility or resistance to infection. This study aimed to characterize the cuticular profile adult insect R. Palmarum male and female, and evaluate the presence of antimicrobial activity. The analyzes were performed by gas chromatography-mass spectrometry of the hexane fractions (extract I) and dichloromethane (extract II and III). the main cuticular components were identified as hydrocarbons, alcohols, ketone and aldehyde, both in male and in female, including the presence of alkanes (C25; C27; C43) trimethyl-alkane (C40) identified in the extract II female insect unsaturated hydrocarbons (C20:5) in the extract I female and male, and methyl branched alkene (C24:6) only in female extract II. They identified 10 fatty acid esters of C14 to C23, with variation between the sexes in C22:0, C18:1, C21:0 and C20:2. Showed relevant amounts of C16 and C18, the common cuticular lipids extract (II) and internal (III). The extracted chitin cuticle, chitosan and derivative thereof, in the study were characterized by IR and X-ray diffraction. The extracts of cuticle were satisfactory inhibition against Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Candida albicans and Candida tropicalis. / Fundação de Amparo a Pesquisa do Estado de Alagoas / A principal função dos lipídios cuticulares em insetos é limitar a perda de água através da superfície. Porém também estão envolvidos em vários tipos de comunicação química entre espécies e redução da penetração de inseticidas, produtos químicos e toxinas além de fornecer proteção contra o ataque de microorganismos, parasitas e predadores. Numerosas espécies de insetos transmissores de doenças altamente virulentas de plantas, desempenham um papel crítico como pragas agrícolas, como é o caso do Rhynchophorus palmarum que é vetor no nematoide transmissor da doença Anel vermelho responsável por causar danos à economia agrícola de palmeiras, em especial o coqueiro, em diversas regiões do país. No entanto, a susceptibilidade ou resistência de várias espécies de insetos a infecção pode resultar de vários fatores, incluindo as diferenças de estrutura e composição do exoesqueleto, assim como a presença de compostos antimicrobianos na cutícula. O papel dos lipídios de superfície de acolhimento na patogênese de insetos ainda é pouco compreendida. A determinação do perfil de lipídeos cuticulares é, portanto, de grande importância para a compreensão da suscetibilidade do inseto ou a resistência à infecção. Este estudo teve como objetivo caracterizar o perfil cuticular do inseto adulto R. palmarum macho e fêmea, e avaliar a presença de atividade antimicrobiana. As análises foram executadas por cromatografia gasosa acoplada a espectrometria de massa das frações cuticulares em hexano (extrato I) e diclorometano (extrato II e III). Foram identificados os principais constituintes cuticulares, como hidrocarbonetos, álcoois, cetona e aldeído, tanto em macho quanto em fêmea, incluindo a presença de alcanos (C25; C27; C43), trimetil-alcano (C40), identificado no extrato II do inseto fêmea, hidrocarboneto insaturado (C20:5) no extrato I fêmea e macho, e alceno metil- ramificado (C24:6) apenas no extrato II da fêmea. Foram identificados 10 ésteres de ácidos graxos de C14 a C23, com variação entre os sexos em C22:0, C18:1, C21:0 e C20:2. Apresentou quantidade relevante de C16 e C18, comum no extrato de lipídeos cuticulares (II) e internos (III). A quitina extraída da cutícula, e o seu derivado quitosana, foram caracterizados em estudo por infravermelho e por difração de raio- X. Os extratos da cutícula apresentaram inibição satisfatória contra Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Candida albicans e Candida tropicalis.

Doctor of Philosophy / Department of Biochemistry / Karl J. Kramer / Subbarat Muthukrishnan / Chitin, a linear homopolymer of beta-1,4-linked N-acetylglucosamine, is the second most abundant biopolymer next to cellulose. It is the major structural polysaccharide in the insect’s exoskeleton and gut lining. An extensive study of two of the major genes encoding enzymes involved in chitin metabolism, chitin synthases (CHSs) and beta-N-acetylglucosaminidases (NAGs), was undertaken. CHS genes from the tobacco hornworm, Manduca sexta, and NAG genes from the red flour beetle, Tribolium castaneum, were identified and characterized. In general, chitin deposition occurs in two major extracellular structures of insects, the cuticle that overlays the epidermis, and the peritrophic membrane (PM) that lines the midgut. Only two CHS genes were identified in M. sexta using Southern blot analysis. Extensive expression studies of both M. sexta CHS genes, MsCHS1 and MsCHS2, suggest a strict functional specialization of these two genes for the synthesis of epidermal and PM-associated chitin, respectively. Furthermore, two alternatively spliced transcripts of MsCHS1, MsCHS1a and MsCHS1b, were identified. Analysis of the levels of these transcripts in different tissues and stages of development indicated that the MsCHS1a transcript predominates in the integument during the feeding and pupal stages, whereas the MsCHS1b transcript is more abundantly present in the tracheae, foregut, and hindgut during all developmental stages tested. Four genes encoding putative NAGs (TcNAG1, TcNAG2, TcNAG3, and TcNAG4) were identified by searching the Tribolium genomic database. The full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organizations were determined. Studies on developmental expression patterns of each gene indicated that they are expressed during most developmental stages with TcNAG1 being the predominant one. The function of each NAG was assessed by down regulating the level of each transcript at various developmental stages using RNA interference. Selective knock down of each transcript, without significant reduction in the expression levels of the other NAG transcripts, was verified and the resulting phenotypes were documented. Knockdown of TcNAG1 interrupted larval-larval, larval-pupal, and pupal-adult molting, and the insects were unable to completely shed their old cuticles.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-27T12:03:27Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_SilviaPereira..pdf: 1700069 bytes, checksum: de95e5af421e528465694aadb5cdee58 (MD5) / Made available in DSpace on 2017-07-27T12:03:27Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_SilviaPereira..pdf: 1700069 bytes, checksum: de95e5af421e528465694aadb5cdee58 (MD5) Previous issue date: 2016-02-23 / CAPES / Adaptações e mecanismos de tolerância à seca são fatores fundamentais para a sobrevivência das espécies vegetais de clima árido ou semiárido. É devido a adaptações como área foliar, profundidade de raízes, manutenção do status hídrico, espessura da cutícula e densidade estomática que tais espécies obtêm sucesso neste ambiente. Contudo, diante das mudanças climáticas há a necessidade de avaliação da eficiência desses mecanismos diante de uma condição ainda mais limitante. Dessa forma, nosso objetivo foi analisar os principais mecanismos fisiológicos de tolerância a seca de Cynophalla flexuosa (L.) J.Presl., uma espécie lenhosa e sempre verde de uma floresta tropical sazonalmente seca, avaliando de que forma a cera epicuticular (CE) das folhas dessa espécie são eficientes diante das condições do seminário em diferentes épocas do ano. O trabalho foi realizado em três períodos (julho de 2014, dezembro de 2014 e fevereiro de 2015), ao longo de um dia em uma área de Caatinga, utilizando folhas intactas e com parte da CE removida mecanicamente. Foi calculado o balanço hídrico do solo e mensurado o potencial hídrico foliar, trocas gasosas, fluorescência da clorofila a, bioquímica e anatomia foliar. Foi observado que C. flexuosa reduz seu potencial hídrico foliar nos horários mais quentes, sendo uma espécie anisohídrica, capaz de manter sua condutância estomática mesmo diante de um balanço hídrico negativo do solo em todos os períodos de avaliação. A concentração da CE foi maior no mês de fevereiro e seu principal componente foram as cadeias de n-alcanos, compostos muito eficientes em manter a impermeabilidade da cutícula e proteger as folhas. O desempenho fotossintético não mostrou alterações ao longo do dia em folhas que tiveram a CE removida, além da manutenção do metabolismo bioquímico, com poucas variações nos diferentes períodos avaliados. Diante disso, C. flexuosa possui eficientes mecanismos de adaptação à seca, podendo suportar ambientes ainda mais limitantes, como o previsto para as próximas décadas devido às mudanças climáticas. / Adaptations and drought tolerance mechanisms are key factors for the survival of plant species in arid or semi-arid climate. It is due to adaptations such as leaf area, depth of roots, maintenance of water status, cuticle thickness and stomatal density these species succeed in this environment. However, given the climate change there is a need of efficiency evaluation of these mechanisms before a further limiting condition. Therefore, our objective was to analyze the main physiological mechanisms of drought tolerance of Cynophalla flexuosa (L.) J.Presl., a woody and evergreen specie in a seasonally dry tropical forest, assessing how the leaves epicuticular wax (EW) of this species are efficient before the semiarid conditions in different seasons. The study was conducted in three periods (July 2014, December 2014 and February 2015), throughout the one day in an area of Caatinga, using intact leaves and the EW removed mechanically. We calculated the the hydric balance of the soil and measured leaf water potential, gas exchange, chlorophyll fluorescence, biochemistry and leaf anatomy. It was observed that C. flexuosa reduces its leaf water potential at the hottest times, one anisohydric species, able to maintain its stomatal conductance in the face of a negative soil water balance in all evaluation periods, which reduced its xylem potential. The concentration of EW was higher in the month of February and its main component was n-alkane chains, very efficient compounds in maintaining a waterproof cuticle and protect the leaves. The photosynthetic performance showed no change throughout the day in leaves that had the EW removed, as well as maintenance the biochemical metabolism, with few variations in different periods. Therefore, C. flexuosa has efficient mechanisms of adaptation to drought and can withstand even more limiting environments, as predicted in the coming decades due to climate change.

1.Adult specimens of Ascaris lumbricoides of similar weights were incubated under nitrogen for 24 hours in a synthetic medium with 1 mM 2,2′-dipyridyl.2.Under these conditions, the viability of the parasites was not affected as evidenced by the amount of ATP in the whole sample and the mobility after mechanical stimulus.3.Incorporation of [14C]proline into non-collagenous proteins in the body wall and cuticle was reproducibly higher in 2,2′-dipyridyl-treated specimens than in untreated worms. Synthesis of collagenous hydroxyproline was inhibited in the cuticle and, to a greater extent, in the muscle layer.4.After transferring the specimens into a fresh medium enriched with 0·1 mM ferrous ions and incubated under 70% oxygen, the muscle collagen remained underhydroxylated. The synthesis of hydroxyproline, however, was almost completely normalized in the cuticle collagen.5.We interpret the data as further evidence of the existence of at least two different enzymes hydroxylating collagenous proline, one located in the subcuticle and the other in the muscle layer of Ascaris lumbricoides.

No description available.