Técnicas y métodos de laboratorio para el estudio en Fisiología Vegetal

Trabajo de Suficiencia ProfesionalEl presente informe contiene la experiencia profesional adquirida en la empresa Novoliz S. A. la que con un gran sentido de altruismo permitió mi iniciación en el campo de la investigación al financiar mi capacitación en la Universidad Federal de Viçosa (UFV). Los trabajos de investigación fueron realizados en el Laboratorio de Biología Molecular de Plantas y en la Unidad de Crecimiento de Plantas de la UFV-Brasil. En el presente informe se describen algunas técnicas utilizados en investigación en el la Fisiología Vegetal, dando énfasis a las técnicas de Biología Molecular como métodos de clonación génica, análisis de PCR (Reacción en cadena de la polimerasa), análisis de la expresión génica y transformación de plantas, todas estas técnicas direccionadas para caracterizar un gen en particular. Adicionalmente se describen algunas técnicas de análisis bioquímico para determinar como la alteración de un gen en una planta transformada, ya sea por super-expresión o por silenciamiento, repercute en su desarrollo especialmente en el metabolismo donde se analiza el contenido de clorofilas, aminoácidos y proteínas y dentro de los compuestos con carbón, almidón y ácidos orgánicos. Dentro de los análisis fisiológicos, se describe algunas técnicas para evaluar la tasa fotosintética, conductancia estomática, concentración interna de CO2 y la tasa de transpiración, adicionalmente se describe el análisis de la fluorescencia de la clorofila para determinar la disipación fotoquímica, tasa de transporte de electrones y la disipación no fotoquímica. Los métodos de análisis molecular, bioquímico y fisiológico tienen un gran potencial en el campo de la Agronomía, al permitir analizar el desempeño de diferentes especies cultivadas y dentro de cada especie, analizar variedades en condiciones óptimas y en condiciones estresantes en nuestra región

Functional analysis of the NIK-mediated antiviral signaling in tomato

A proteína NSP de begomovírus facilita o transporte do DNA viral do núcleo para o citoplasma e coopera com a proteína de movimento MP para promover o transporte do DNA viral às células adjacentes não infectadas através dos plasmodesmas. A proteína NSP interage com membros da família LRR-RLK ( leucine- rich repeat receptor like kinase ), designados NIK ( NSP-Interacting Kinase ). A ligação de NSP na alça de ativação de NIK inibe a atividade quinase, e conseqüentemente, a proteína viral inibe a atividade de autofosforilação desses receptores e sua atividade de defesa antiviral. Estudos de mutagênese na alça de ativação de NIK demonstraram que o resíduo Treonina 474 é fosforilado in vitro e exerce uma função crucial para atividade de quinase que é requerida para sinalização antiviral. Mutação no resíduo de Thr-474 para aspartato resulta no mutante T474D que exibe ativação constitutiva, atividade de fosforilaçao do substrato aumentada e menor efeito inibidor de NSP. Este trabalho teve como objetivo caracterizar o domínio quinase de NIK na resposta de defesa antiviral em tomateiros. Tomateiros foram transformados com a construção que codifica para NIK super ativa (35S-AtNIK-T474D). Os transformantes primários foram selecionados por PCR e a expressão do transgene em linhagens independentes foi confirmada por RT-PCR normal e em tempo real. A super expressão da NIK1 e NIK- T474D super ativa em tomateiros promoveu um alongamento de entrenós, mas afetou negativamente o desenvolvimento do sistema radicular, demonstrando uma possível comunicação cruzada entre a via de sinalização antiviral mediada por NIK e vias de sinalização de desenvolvimento. Experimentos de infectividade foram conduzidos em linhagens transgênicas superexpressando AtNIK ou AtNIK-T474D, utilizando o vírus ToYSV-[MG-Bi2]. Super expressão de NIK super ativa alterou a taxa de infecção por ToYSV e interferiu no desenvolvimento dos sintomas. Comparado com as plantas não transformadas e a linhagem transgênica 35S-AtNIK1-6 superexpressando NIK normal, a taxa de infecção foi inferior e os sintomas mais atenuados em linhagens transgênicas independentes superexpressando AtNIK-T474D. Estes resultados confirmam in planta o papel essencial da fosforilação do resíduo de Treonina 474 de NIK e indicam a possibilidade de se desenvolverem estratégias de tolerância a geminivirus mais eficientes.The begomovirus NSP (nuclear shuttle protein) facilitates the transport of viral DNA from the nucleus to the cytoplasm and cooperates with the movement protein MP to promote the translocation of viral DNA to the adjacent, uninfected cells through plasmodesmata. NSP interacts with members of the LRR-RLK ( leucine-rich repeat receptor like kinase ) family, designated NIKs ( NSP-Interacting Kinase ). Binding of NSP to the activation loop of NIK inhibits kinase activity and hence the viral protein suppresses receptor autophosphorylation and defense responses. Mutagenesis assays in the activation loop of NIK have demonstrated that the threonine 474 residue is phosphorylated in vitro and plays a crucial role in the kinase activity that is required for signaling. Replacement of Thr-474 with aspartate produces the T474 mutant, which exhibits constitutive activation, enhanced substrate phosphorylation activity and less inhibitory effect by NSP binding. The goal of this investigation was to analyse the NIK kinase domain in defense responses against begomovirus in tomato. The NIK mutant T474D cDNA was placed under the control of 35S promoter into a binary vector for plant transformation (35S-AtNIK-T474D). Primary transformants were selected by PCR and the expression of the transgene was confirmed by normal and quantitative RT- PCR in independently transformed lines. NIK and NIK-T474D overexpression in tomato plants affected the overall developmental performance of transgenic lines, which display elongated stems and a root system less developed. These phenotypes were consistent with a cross-communication between the NIK-mediated antiviral signaling and developmental signaling pathways. Infectivity assays were carried out in AtNIK- and AtNIK-T474D-overexpressing lines, with the virus ToYSV-[MG-Bi2]. Overexpression of super active AtNIK-T474D altered the infection rate by ToYSV, and interfered in symptom development. As compared to untransformed plants and NIK- overexpressing 35S-AtNIK1-6 transgenic lines, independent transgenic AtNIK-T474D lines displayed lower infection rate and attenuated symptoms. These results confirmed in planta the essential role for phosphorylation of the Thr-474 residue for NIK function and underlined the possibility for the development of more efficient tolerance strategies against geminiviruses

Análise fisiológica e metabólica de Arabidopsis thaliana com baixa expressão das subunidades da 2-oxoglutarato desidrogenase

One of the roles of the tricarboxylic acid (TCA) cycle in plants is the production of 2-oxoglutarate (2-OG) required for nitrogen assimilation. In this cycle, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase (2- OGDH) is involved in synthesis and consumption of 2-OG, respectively. 2- OGDH complex is composed of three subunits responsible for decarboxylation of 2-OG to succinyl CoA with the consequent reduction of NAD + . Notably the 2-OGDH plays an essential role in overall metabolic activity in plants, being limited for respiration and playing important role in the carbon-nitrogen interactions. In Arabidopsis thaliana, E 1 and E 2 subunits are encoded each by two genes. Here, I used two T-DNA insertion mutant lines in each gene encoding E 1 and E 2 subunits of 2-OGDH. For both subunits of 2-OGDH mutant plants exhibited substantial reduction in respiration. The photosynthesis was also altered in plants with low expression of E 1 subunit. Several changes were observed for primary metabolites, with decreased levels of the main nitrogen containing metabolites and increase in metabolites related to carbon metabolism, culminated in alterations of plant growth and seed production. Although both E 1 and E 2 subunits each one are encoded by two genes, they display partial redundant roles in metabolism and plant growth.Uma função do ciclo dos ácidos tricarboxilicos (TCA) em plantas é a produção de 2-oxoglutarato (2-OG) necessário para a assimilação do nitrogênio. No ciclo TCA, o isocitrato desidrogenase e a 2-oxoglutarato desidrogenase (2-OGDH) estão envolvidos na síntese e consumo do 2-OG respectivamente. O complexo 2-OGDH é formado por três subunidades responsáveis pela descarboxilação do 2-OG a succinil CoA com a consequente redução do NAD + . Notavelmente a 2-OGDH tem uma função essencial na atividade metabólica geral em plantas, limitante na respiração e importante na interação carbono-nitrogênio. Em Arabidopsis thaliana, as subunidades E 1 e E 2 são codificadas cada uma por dois genes. Neste trabalho foram utilizados duas linhagens mutantes caracterizadas pela inserção do T-DNA para cada gene que codifica a subunidade E 1 e E 2 da 2-OGDH. As linhagens mutantes para as duas subunidades da 2-OGDH apresentaram uma diminuição substancial na respiração. A fotossíntese também foi alterada nas plantas com baixa expressão da subunidade E 1 . Muitas mudanças foram observadas para os metabólitos primários, diminuição dos níveis dos principais metabólitos que contem nitrogênio e aumento dos metabólitos relacionados com o metabolismo do carbono, culminando em alterações no crescimento vegetal e na produção de sementes. Embora as duas subunidades E 1 e E 2 são codificadas cada uma por dois genes, estes genes apresentam funções parcialmente redundantes no metabolismo e crescimento vegetal.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Maize heat shock proteins—prospection, validation, categorization and in silico analysis of the different ZmHSP families

Abstract Among the plant molecular mechanisms capable of effectively mitigating the effects of adverse weather conditions, the heat shock proteins (HSPs), a group of chaperones with multiple functions, stand out. At a time of full progress on the omic sciences, they look very promising in the genetic engineering field, especially in order to conceive superior genotypes, potentially tolerant to abiotic stresses (AbSts). Recently, some works concerning certain families of maize HSPs (ZmHSPs) were published. However, there was still a lack of a study that, with a high degree of criteria, would fully conglomerate them. Using distinct but complementary strategies, we have prospected as many ZmHSPs candidates as possible, gathering more than a thousand accessions. After detailed data mining, we accounted for 182 validated ones, belonging to seven families, which were subcategorized into classes with potential for functional parity. In them, we identified dozens of motifs with some degree of similarity with proteins from different kingdoms, which may help explain some of their still poorly understood means of action. Through in silico and in vitro approaches, we compared their expression levels after controlled exposure to several AbSts' sources, applied at diverse tissues, on varied phenological stages. Based on gene ontology concepts, we still analyzed them from different perspectives of term enrichment. We have also searched, in model plants and close species, for potentially orthologous genes. With all these new insights, which culminated in a plentiful supplementary material, rich in tables, we aim to constitute a fertile consultation source for those maize researchers attracted by these interesting stress proteins

Using Principal Component Analysis and RNA-Seq to Identify Candidate Genes That Control Salt Tolerance in Garlic (Allium sativum L.)

To examine physiological responses of garlic to salinity, 17-day-old seedlings of eight soft-neck accessions were treated with 200 mM NaCl for seven days in a hydroponic system. Several morphological and physiological traits were measured at the end of the treatment, including shoot height, shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, photosynthesis rate, and concentrations of Na+ and K+ in leaves. The principal component analysis showed that shoot dry weight and K+/Na+ ratio contribute the most to salt tolerance among the garlic accessions. As a result, salt-tolerant and sensitive accessions were grouped based on these two parameters. Furthermore, to investigate the molecular mechanisms in garlic in response to salinity, the transcriptomes of leaves and roots between salt-tolerant and salt-sensitive garlic accessions were compared. Approximately 1.5 billion read pairs were obtained from 24 libraries generated from the leaves and roots of the salt-tolerant and salt-sensitive garlic accessions. A total of 47,509 genes were identified by mapping the cleaned reads to the garlic reference genome. Statistical analysis indicated that 1282 and 1068 genes were upregulated solely in the tolerant leaves and roots, whereas 1505 and 1203 genes were downregulated exclusively in the tolerant leaves and roots after NaCl treatment, respectively. Functional categorization of these genes revealed their involvement in a variety of biological processes. Several genes important for carotenoid biosynthesis, auxin signaling, and K+ transport were strongly altered in roots by NaCl treatment and could be candidate genes for garlic salt tolerance improvement