No description available.

Call number: LD2668 .T4 1951 A2 / Master of Science

Fitocistatinas são inibidores competitivos de proteases cisteínicas em plantas que atuam principalmente na inibição de papaínas. Entretanto, há uma demonstração in vitro de uma fitocistatina carboxi-estendida com capacidade de inibir também uma protease do tipo legumaína. Em arroz existem 12 genes de fitocistatinas, mas apenas um deles (OcXII), possui a extensão C-terminal. Desta forma, o objetivo geral deste trabalho é compreender a diversidade e os mecanismos de interação entre as fitocistatinas e suas proteases cisteínicas alvo em arroz, com ênfase nas implicações funcionais da OcXII. Neste trabalho, nós demonstramos que, em arroz, os 12 genes de fitocistatinas têm um perfil de expressão gênica diferenciado na germinação e em respostas ambientais, sendo OcI, OcIII e OcXII os genes mais expressos. O peptídeo recombinante, correspondente à extremidade C-terminal da OcXII possui capacidade inibitória de legumaínas e não afeta a atividade das papaínas. Através do silenciamento transcricional da OcXII, via RNAi, foram obtidas plantas com atividades proteolíticas aumentadas tanto de papaínas quanto legumaínas, em adição a um fenótipo de crescimento inicial acelerado. O fenótipo oposto é observado em plântulas crescendo em condições alcalinas. Plantas expressando o promotor OcXII fusionado ao gene repórter gus demonstraram um perfil de ativação de OcXII durante a germinação, principalmente na região do escutelo das sementes. Esta ativação de OcXII permanece alta quando as sementes são mantidas com níveis elevados de ABA e sob estresse alcalino. Como alvos específicos de OcXII, as legumaínas estão relacionadas com a biossíntese de componentes vacuolares e degradação de proteínas de armazenamento. Em arroz, nós encontramos cinco diferentes loci para as legumaínas. Análises filogenéticas e estudos de expressão gênica demonstraram uma maior associação de OsaLeg2 e OsaLeg3 com tecidos de semente, e OsaLeg1, 4 e 5 com tecidos vegetativos. Também foram observadas formas de splicing alternativo, com potencial de originar diferentes isoformas ativas de legumaínas. Em geral, nossos dados demonstram o envolvimento bifuncional da OcXII nos processos de germinação e defesa interagindo e inibindo a atividade de proteases cisteínicas específicas, onde a região N-term de OcXII inibe papaínas, enquanto a C-term inibe legumaínas. / Phytocystatins are competitive inhibitors of cysteine proteases in plants, principally inhibiting papain-like proteases activity. However, there is one in vitro demonstration that a carboxy-extended phytocystatin can inhibit legumain-like proteases either. In rice there are 12 phytocystatins genes, but only one (OcXII) has the C-terminal extension. Thus, the aim of this study is to understand the diversity and interaction mechanisms among phytocystatins and its cysteine protease targets in rice, focusing on OcXII functional implications. In this work we demonstrated that in rice, the 12 phytocystatins genes have a different gene expression profile during the germination and environmental responses, while OcI OcIII and OcXII were the most expressed genes. A recombinant peptide corresponding to the different C-terminus of OcXII was produced and its inhibitory capacity was confirmed against legumains without affecting the activity of papains. OcXII transcriptional silencing, via RNAi, resulted in plants with increased proteolytic activity for both papain and legumains, in addition to an accelerated initial growth phenotype. The opposite phenotype is observed in seedlings growing under alkaline conditions. Plants expressing the OcXII promoter fused to gus reporter gene demonstrated an OcXII activation profile during germination, especially in the seed scutellum region. This OcXII activation remains high when the seeds are kept at high ABA levels or under alkaline conditions. As specific OcXII targets, legumains are related to the biosynthesis of vacuolar storage components and protein degradation. In rice, we found 5 different loci for legumains. Phylogenetic analyses and gene expression studies demonstrated a greater association of OsaLeg3 and OsaLeg2 with seed tissues, while OsaLeg1, 4 and 5 were more abundant in vegetative tissues. Also alternatively spliced forms were observed, with the potential to produce different isoforms of active legumains. Overall, our data demonstrate the involvement of the bifunctional OcXII in germination processes and defense, interacting and inhibiting the activity of specific cysteine proteases, where its N-term region inhibits papain, while the C-term inhibits legumains.

Abstract not available

No description available.

No description available.

No description available.

No description available.

No description available.

Over the previous quarter century the vine weevil (Otiorhynchus sulcatus) has become a pest of horticultural and agricultural plants. The vine weevil is a polyphagous coleopteran insect and is able to attack over one hundred different plant species. Its spread has been limited by its lack of flight but modern world trade in live container grown plants has spread the insect to new habitats. Damage to plants caused by vine weevil is two fold, with the larvae destroying root balls while the adults attack the, leaves. The larval stage, in particular is difficult to treat with conventional insecticides unless environmentally undesirable soil treatments are used. The current lack of defence against the vine weevil has opened the door for methods of crop protection through the generation of genetically modified plants. The design of an efficient GM approach to control the vine weevil requires a sound knowledge of the insect’s digestive enzymes, which may be used as potential targets for insecticidal proteins. This approach was achieved for the vine weevil through analysis of active digestive proteases in the insects gut and the identification of suitable proteinase inhibitors which would reduce the overall level of protein hydrolysis. Using this method it was discovered that the vine weevil contained both serine and cysteine proteases in addition to a range of other digestive hydrolases. This biochemical data was supported by a molecular approach to isolate cDNA clones associated with the insect's digestive tract. Using a gut specific cDNA library clones encoding a cathepsin B protease, two trypsin proteases, a pectinesterase, a lipase and a cellulase were isolated and characterised. The cellulase isolated from vine weevil has been shown to originate from the insect genome as shown through Southern Blot analysis and sequencing across several intronic regions. Evidence presented herein shows that the vine weevil gut extract hydrolyses both cellulose and cellobiose. Similar results were observed with recombinant protein expressed in the eukaryotic yeast P.pastoris. Furthermore data presented here shows that the vine weevil has the full complement of enzymes needed for the complete digestion of crystalline cellulose, which was until recently believed to be the sole domain of several species of bacteria and yeast. In addition a cDNA clone encoded a vine weevil endogenous chitinase was isolated from the cDNA library. This chitinase cDNA and one encoding the proteinase inhibitor Oryzacystatin-I were used to generate transgenic tobacco plants which have been shown to express the transgene. These transgenic plants are the first step in developing a strategy for plant protection against vine weevil based on genetic modification.