Identification and characterization of the aminoadipic semialdehyde dehydrogenase enzyme in plants

Orientador: Paulo ArrudaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O amino ácido lisina é catabolizado em plantas e animais pela via da sacaropina. Nesta via, a lisina é convertida a ?-aminoadipato-?-semialdeído (AASA) pela ação da enzima bifuncional lisina-cetoglutarato redutase/sacaropina desidrogenase (LKR/SDH). O intermediário AASA é então convertido a ?-aminoadipato (AAA) pela enzima ?-aminoadipato-?-semialdeído desidrogenase (AASADH). A LKR/SDH já foi bem caracterizada em plantas e animais, mas a atividade enzimática bem como o possível papel fiiológico da AASADH ainda não foi demonstrada em plantas. A via da sacaropina, além do seu importante papel na regulação dos níveis de lisina, está também envolvida em processos de resposta a estresses. Este trabalho está dividido em dois capítulos. No capítulo I descrevemos a identificação do gene que codifica a AASADH em milho e a caracterização da atividade enzimática da enzima em endosperma imaturo de milho. Mostramos que a AASADH é codificada pelo gene Aldh7b1, um gene muito conservado em eucariotos. A enzima codificada pelo gene Aldh7b1 foi parcialmente purificada de endosperma imaturo de milho e através de eletroforese em condições denaturantes e cromatografia em coluna de gel filtração mostramos que a enzima, na sua forma nativa, apresenta-se como um tetrâmero constituído por quatro subunidades de 55 kDa. A AASADH isolada de endosperma imaturo de milho converte o semi-aldeido AASA em AAA. O produto da reação catalisada pela AASADH foi confirmado por cromatografia em camada delgada. No capítulo II discutimos o papel da via sacaropina no desenvolvimento da semente e na resposta de planas jovens de milho a estresses abióticos. As enzimas LKR/SDH e AASADH são co-expressos nas células das camadas da sub-aleurona do endosperma de milho nas fases intermediarias do desenvolvimento. No entanto, embora a proteína AASADH seja produzida no endosperma e no embrião de sementes imaturas e nos tecidos de plantas jovens, a proteína LKR/SDH é detectada unicamente nas células da sub-aleurona das sementes imaturas. A AASADH mostrou atividade máxima a pH 7,4 e Kms para AASA e NAD+ na ordem de micromolar. Em endosperma imaturo a via da sacaropina é induzida por lisina e reprimida por estresse salino, enquanto prolina e ácido pipecólico são significativamente reprimidos por lisina. Em coleóptiles jovens as enzimas LKR/SDH e AASADH são induzidas transcricionalmente por estresses salino, osmótico e oxidativo, mas enquanto que a proteína AASADH acumula nos tecidos sob estresse, a proteína LKR/SDH não é detectada. Nossos resultados indicam que os genes que codificam as enzimas LKR/SDH e AASADH são co-expressos a nível transcricional, mas não a nível traducional. A ausência da proteína LKR/SDH em plantas jovens sob estresses e os altos níveis do seu transcrito serem detectados mostra um desacoplamento transcrição/tradução que podem ter consequências regulatórias ainda desconhecidasAbstract: Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into ?-aminoadipate-?-semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into ?-aminoadipate (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). LKR/SDH was characterized in higher eukaryotes, but AASADH has not been demonstrated in plants. Furthermore, studies have shown that besides the degradation of lysine, the saccharopine pathway is involved in stress response processes in plants, animals and bacteria. This work was divided into two chapters. Chapter I describes the identification of the gene encoding AASADH and the partial purification and characterization of the enzyme from developing maize endosperm. The enzyme AASADH is encoded by the Aldh7b1 gene, a gene highly conserved among eukaryotes. The enzyme partially purified from developing endosperm and analyzed by SDS-PAGE and gel filtration chromatography behaved, in its native form, as a tetramer constituted by four monomers of 55 kDa. The enzymatic convertion of AASA into AAA was verified by thin layer chromatography. In Chapter II the role of the saccharopine pathway in seed development and stress responses is discussed. LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing seeds. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD+ in the micromolar range. In the developing endosperm the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in stressed tissues, LKR/SDH does not. Our results indicate that the genes encoding the LKR/SDH and AASADH enzymes are co-expressed at the transcriptional level, but not the translational level. The absence of LKR/SDH protein in young plants under stress despite of the high levels of transcripts being detected suggests a decoupling transcription/translation that may have regulatory consequences yet unknownDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecula

Deposition of lignin in four species of Saccharum

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação2014/25994-1; 2008/58035-

Oligomerization, Membrane Association, and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.Fil: Soares, Jose Sergio M.. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes; BrasilFil: Gentile, Agustina. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes; BrasilFil: Scorsato, Valeria. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; BrasilFil: Lima, Aline da C.. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; BrasilFil: Kiyota, Eduardo. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; BrasilFil: Santos, Marcelo Leite Dos . Universidade Federal do Sergipe. Centro de Ciências Exatas e Tecnologia. Núcleo de Química; BrasilFil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Huber, Steven C.. University of Illinois at Urbana-Champaign. Department of Agriculture Agricultural Research Service, and Department of Plant Biology; Estados UnidosFil: Aparicio, Ricardo. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; BrasilFil: Menossi, Marcelo. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes; Brasi

Heterogeneous Parameter Uncertainty and the Timing of Investment During Crisis

We present a model in which investors observe the same macroeconomic data but have varying levels of information about the parameters that determine the distribution of the expected returns on investment. During a crisis that increases macroeconomic uncertainty and reduces asset prices, the threshold required return that triggers investment is lower for an informed investor than for an uninformed investor. Simulation of the model suggests that when macroeconomic uncertainty is high investment may increase, is mostly by informed investors, and as time goes on is progressively more by investors who were initially relatively uninformed about model parameters. For over 10,000 instances of firm-level FDI data for Korea from 1996 to 2001, regression results are consistent with the hypothesis that disproportionably more FDI is made by more informed investors during a period of high macroeconomic uncertainty

Expression, purification and structural characterization of the sugarcane bZIP transcription factors SCF12 and SCF5

Orientadores: Ricardo Aparicio, Marcelo Menossi TeixeiraDissertação (mestrado) - Universidade Esstadual de Campinas, Instituto de QuimicaResumo: Os fatores de transcrição do tipo bZIP estão presentes em organismos eucariotos e estão envolvidos na regulação da expressão gênica e no controle de muitos processos intracelulares. Esses fatores se ligam a seqüências específicas no DNA e são capazes de reconhecer seqüências reguladoras no promotor de um gene. As bZIPs são caracterizadas por uma região conservada rica em resíduos de aminoácidos básicos, e um zíper de leucinas, que possui repetições de uma seqüência de aminoácidos hidrofóbicos onde há uma leucina que ocupa a mesma posição a cada 7 resíduos. Estudos estruturais com bZIPs mostraram que essas proteinas enovelam-se na forma de uma extensa hélice-a e são capazes de formar dímeros através de um arranjo do tipo coiled- coil. Neste trabalho, a parte correspondente à região básica e ao zíper de leucinas de duas bZIPs, SCF5 e SCF12 de cana-de-açúcar, pertencentes a sub-famílias diferentes, foram clonadas, expressas e purificadas para estudos estruturais. O DNA correspondente a SCF12 foi clonado em pET28a e a proteína recombinante foi produzida em E. coli BL21 (DE3) pRil. A SCF12 purificada por cromatografia de afinidade (IMAC) teve sua estrutura secundária caracterizada por dicroísmo circular. A SCF5, clonada em pET3C e expressa em E. coli BL21 (DE3) pLysS foi purificada por cromatografia de troca catiônica. Cristais de um complexo da proteína ligada a uma seqüência de DNA de 24 pares de bases foram obtidos mas não exibiram qualidade suficiente para permitir a determinação da estrutura cristalográfica. Entretanto, foi possível obter um modelo do complexo a partir de experimentos de espalhamento de Raios X a baixos ângulos (SAXS, do inglês Small Angle X-Ray Scattering) em solução, e interpreta-lo à luz de estruturas de homólogas já conhecidas.Abstract: The bZIP transcription factors are present in eukaryotic organisms and are involved in the regulation of gene expression and many intracellular processes. These factors bind specific DNA sequences and are able to recognize regulatory sequences of a gene promoter. The bZIPs are characterized by a conserved region rich in basic amino acid residues as well as by having the leucine zipper region, which possess a sequence of hydrophobic residues where there are leucines every seventh amino acids. Structural studies have shown that bZIP-folding is alpha-helical and these proteins are capable of dimmer formation via coiled-coil arrangement. In this work, the basic region and the leucine zipper of two sugarcane bZIPs, SCF12 and SCF5, belonged to two different bZIP-families were cloned, expressed and purified for structural studies. The corresponding SCF12 DNA was cloned into pET28a expression vector and the protein was produced in E. coli BL21 (DE3) pRil cells. SCF12 protein was purified by affinity chromatography (IMAC) and had its secondary structure characterized by CD. SCF5, cloned into pET3c and expressed in E. coli BL21 (DE3) pLysS was purified by cation exchange chromatography. Crystals of a complex formed by SCF5 protein and a 24-base-pair DNA sequence were obtained but unfortunately with quality insufficient for crystallographic structure determination. However, it was possible to obtain a model of the analyzed complex applying Small Angle X-ray Scattering (SAXS) technique by protein homologous structure comparison.MestradoFísico-QuímicaMestre em Químic

The Saccharopine Pathway In Seed Development And Stress Response Of Maize.

Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into α-aminoadipic semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into aminoadipic acid (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). Here, we show that LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing endosperm. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD(+) in the micromolar range. In the developing endosperm, the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles, the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in the stressed tissues, the LKR/SDH protein is not produced. In the developing seeds, the saccharopine pathway is used for pipecolic acid synthesis although proline may play a major role in abiotic stress response. The results indicate that the saccharopine pathway in maize seed development and stress responses significantly differ from that observed for dicot plants.382450-246

Evolution of phytochemical diversity in Pilocarpus (Rutaceae)

The evolution of phytochemical diversity and biosynthetic pathways in plants can be evaluated from a phylogenetic and environmental perspective. Pilocarpus Vahl (Rutaceae), an economically important medicinal plant in the family Rutaceae, has a great diversity of imidazole alkaloids and coumarins. In this study, we used phylogenetic comparative methods to determine whether there is a phylogenetic signal for chemical traits across the genus Pilocarpus; this included ancestral reconstructions of continuous and discrete chemical traits. Bioclimatic variables found to be associated with the distribution of this genus were used to perform OLS regressions between chemical traits and bioclimatic variables. Next, these regression models were evaluated to test whether bioclimatic traits could significantly predict compound concentrations. Our study found that in terms of compound concentration, variation is most significantly associated with adaptive environmental convergence rather than phylogenetic relationships. The best predictive model of chemical traits was the OLS regression that modeled the relationship between coumarin and precipitation in the coldest quarter. However, we also found one chemical trait was dependent on phylogenetic history and bioclimatic factors. These findings emphasize that consideration of both environmental and phylogenetic factors is essential to tease out the intricate processes in the evolution of chemical diversity in plants. These methods can benefit fields such as conservation management, ecology, and evolutionary biology163132146CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2008/58035-6; 2016/06260-2National Science Foundation Graduate Research FellowshipNational Science Foundation (NSF) [DGE-1650441]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Brasil)National Council for Scientific and Technological Development (CNPq); Fundacao de Amparo Pesquisa do Estado de Sao Paulo (Fapesp)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2008/58035-6]; Cnpq-Brasil; FapespFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2016/06260-2]; Harold E. Moore Jr. and Robert T. Clausen Endowment Fund; BSA; SSB; ASPT; Garden Club of America; Einaudi Center for International Studies; Latin American Studies Program of Cornell; Cornell Graduate Schoo