Orientador: Paulo Mazzafera / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T20:42:22Z (GMT). No. of bitstreams: 1 Cesarino_Igor_M.pdf: 984828 bytes, checksum: 754aac1bc4502a9d5eb3102a955408d0 (MD5) Previous issue date: 2009 / Resumo: Uma grande quantidade de genes que codificam flavina monooxigenases (Flavin containing monooxygenases - FMOs) é encontrada em genomas vegetais, embora poucas funções biológicas tenham sido relacionadas com esse grupo de enzimas em plantas. Um importante papel desempenhado por FMOs é a conversão de triptamina em N-hidroxil niptamina, reação catalisada pelas proteínas YUCCA de Arabidopsis thaliana e que constitui o passo limitante da via de síntese de auxina a partir de triptofano. Proteínas similares às YUCCA foram descobertas e caracterizadas em outras espécies vegetais, como OsYUCCA em arroz. FLOOZY em petúnia, ToFZY em tomate e SPIl em milho, todas comprovadamente envolvidas na produção do hormônio citado. Análises da proteína recombinante CaFM08 de Coffea arabica revelou características similares às YUCCA, sugerindo que esta proteína de café é a primeira YUCCA-like descrita para esta espécie e, inclusive, para a família Rubiaceae. CaFM08 apresenta os mesmos motivos protéicos conservados entre FMOs vegetais, e particularmente entre proteínas YUCCA-like. O padrão de expressão espacial do gene que codifica CaFM08 indica possível relação com o desenvolvimento de raízes, folhas e flores de café. Apesar de grandes semelhanças com as proteínas YUCCA, a atividade de N-hidroxilação da triptamina não foi comprovada para CaFM08 recombinante in vitro. Uma análise minuciosa a respeito da funcionalidade de CaFM08 produzida em E. coli deve ser feita antes de descartar a participação desta proteína na síntese de auxina. / Abstract: A large number of genes coding flavin-containing monooxygenases (FMOs) is found in plant genomes, although only few biological functions have been related with these enzymes in plants. An important role for FMOs is the conversion of tryptamine in N-hydroxyl triptamine, catalyzed by the YUCCA protein family in Arabidopsis thaliana. These proteins perform "the rate-limiting step in tryptophan-dependent auxin biosynthesis. Similar YUCCA proteins were discovered and characterized in other plant species, like OsYUCCA in rice, FLOOZY in petunia, ToFZY in tomato and SPIl in maize. All of them are shown to be involved in auxin synthesis. Analysis of the recombinant CaFM08 from Colfea Arabica showed features similar to YUCCA proteins, suggesting that CaFM08 is the first described YUCCA-like protein from coffee and, indeed, from the entire Rubiaceae family. CaFM08 has the same conserved motifs found in other plant FMOs and particulary conserved in YUCCA like proteins. The spatial expression pattern from the CaFM08 coding gene suggests a probable role in the development of roots, leaves and flowers. Although very similar to YUCCA proteins, the CaFM08-mediated convertion of tryptamine in N-hydroxyl tryptamine has not been confirmed in vitro. A further analysis of CaFM08 functionality should address the relation of CaFM08 to auxin production. / Mestrado / Mestre em Biologia Vegetal

Epilobium hirsutum L. (Onagraceae), a medicinal plant known as hairy willow herb, has been used by people all around the world for treatment or prevention of inflammation, adenoma, rectal bleeding, menstrual disorders, constipates, and prostate. It contains polyphenolics including steroids, tannins such as gallic, ellagic, and p-coumaric acids and flavonoids such as myricetin, isomyricetin, and quercetin. Polyphenols have been known for their multiple biological health benefits, including antioxidant activities. Viscum album L. (Loranthaceae), a species of mistletoe, contains lectins, polypeptides, mucilage, sugar alcohols, flavonoids, lignans, triterpenes, and phenylallyl alcohols. The leaves and twigs of Viscum album L., taken as tea, have been traditionally used for hypertension, stomachache, diarrhea, diabetes, dysuria and also as analgesic and cardiotonic agent in Anatolia, Turkey. In addition, in Europe, sterile extracts of Viscum album L. are among the most common herbal extracts applied in cancer treatment and have been used as prescription drugs, while in US, considered as dietary supplement. Flavin-containing monooxygenases are FAD-containing phase I enzymes responsible for the oxidation of wide-range of nucleophilic nitrogen, sulfur, phosphorus, and selenium heteroatom-containing drugs such as tamoxifen, v methimazole and imipramine, pesticides, neurotoxins, and other chemicals using NADPH as cofactor. The aim of this study was to determine the in vivo effects of Epilobium hirsutum L. and Viscum album L. (subspecies growing on pine trees-subsp. austriacum (Wiesb.) Vollmann) on FMO activity, mRNA and protein expressions in rat liver. The water extracts of Epilobium hirsutum L. (37.5 mg/kg body weight) and Viscum album L. (10 mg/kg body weight) were injected intraperitonally (i.p) into Wistar albino rats for 9 consecutive days. Following the decapitation, the livers were removed and microsomal fractions were prepared by differential centrifugation. Rat liver microsomal FMO activity using methimazole as substrate, mRNA expression by quantitative Real-Time PCR, and protein expression by Western Blot were determined. The results showed that water extract of Epilobium hirsutum L. has no significant effect on FMO activity / however, it decreased significantly (p&lt / 0.05) FMO3 protein and mRNA expression 27.71% and 1.41 fold, respectively, compared as controls. Water extract of Viscum album L. decreased mRNA (2.56 fold), and protein expressions (27.66%) as well as enzyme activity (19%) of FMO with respect to controls. In conclusion, our current data suggest that the metabolism of xenobiotics including drug molecules by FMO-catalyzed reactions may be altered due to the changes in FMO expression and activity by medicinal plants Epilobium hirsutum L. and Viscum album L.

Philosophiae Doctor - PhD / Nitric Oxide (NO) influences a wide range of physiological processes in plants including growth and development, responses to abiotic and biotic stress and pathogen responses. NO binds to the heme group of the mammalian soluble guanylyl cyclase, which activates the enzyme to convert guanosine 5’ triphosphate (GTP) to a second messenger guanosine 3’, 5’ cyclic monophosphate (cGMP). Cyclic GMP further activates other signalling cascades including the regulation of protein kinases, ion gated channels and phosphodiesterases. In plants, a few GCs have been identified and these include AtGC1, AtBRI1, AtWAKL10, and AtPSKR1, however, a GC that contains a heme binding motif that senses NO has yet to be identified. In order to identify such molecules, a search motif based on conserved HNOX domains and the conserved and functionally assigned amino acid residues in the catalytic centres of annotated GCs was designed and used to search the Arabidopsis thaliana proteome. Several candidate molecules were identified including a flavin-containing monooxygenase (FMO)-like protein and the At5g57690 which is currently annotated as a diacylglycerol kinase. FMOs found in bacteria, yeast, and animals are the most important monooxygenases since they are involved in xenobiotic metabolism and variability in drug response. FMOs in plants are implicated in catalysing specific steps in auxin biosynthesis,metabolism of glucosinolates and pathogen defense mechanisms. The human diacylglycerol kinase acts as a lipid kinase that mediates a wide range of biological processes which include cell proliferation, differentiation and turmogenesis. In prokaryotes, the structure of Escherichia coli lipid kinase has been solved however, its function has not yet been demonstrated. So far, the occurrence of the diacylglycerol kinases in plants has not yet been reported, and their structure and function also remain elusive. The domain architecture of the 2 molecules (AtNOGC1 and At5g57690) identified by the HNOX-based search strategy revealed that these molecules contain a GC and a heme-binding motif that is conserved among all known heme-binding proteins.In this study, the role of AtNOGC1, a novel NO binding protein in higher plants was investigated and the results showed that this molecule contains an NO-dependant active GC domain. The sequence was first analysed and the location of the HNOX and the GC motifs highlighted. The protein was then recombinatly expressed as a His-SUMO fusion protein and the purification optimised by a second step of ion exchange chromatography. Electrochemical techniques such as cyclic voltammetry and square wave voltammetry were used to demonstrate the binding of NO and O2 to the AtNOGC1. Electrochemical data revealed that AtNOGC1 has a lower affinity for O2 and a higher affinity for NO, an important signalling molecule in plants.The presence of the GC activity in AtNOGC1 was investigated by conducting GC activity assays in vitro in the presence or absence of NO. The GC activity assays demonstrated that AtNOGC1 can synthesize cGMP from GTP in vitro. It was also noted that NO was required for the maximum activation of AtNOGC1 catalytic activity. NO-activated catalysis resulted in a >2 fold excess of cGMP production compared to an NO-independent GC activity assay. The effect of calcium in regulating the GC activity was also investigated and an increase in cGMP levels was observed however, this was just a preliminary finding that requires further experimentation.3 Homology models for both the FMO-like (AtNOGC1) and the diacylglycerol kinase(At5g57690) were built using Modeller program, and important amino acid residues underlying the heme-binding and GC motifs were identified. Residues corresponding to the motifs, which give signature to AtNOGC1 as an FMO, were also noted. In addition,computational functional prediction also suggested the role of AtNOGC1 in a number of processes which include ion binding and functioning as an FMO.Taken together, these findings suggest that AtNOGC1 is a novel Arabidopsis thaliana hemebinding protein that senses NO with higher affinity than for O2. Though AtNOGC1 is currently annotated as a FMO-like protein, it contains a NO-sensitive GC activity and shares limited sequence similarities with mammalian sGC and the recently identified HNOX domains. Homology modelling strongly suggests that AtNOGC1 and At5g57690 belong to the families of FMOs and diacylglycerol kinases respectively. The domain organisation of AtNOGC1 suggests that more of its functions still remain to be identified. The cloning and characterisation of the At5g57690 gene will provide possible means for further experimentation as well as affording more insights into the exact functions of lipid kinases in plants.