ANÁLISIS in silico Y EXPRESIÓN GÉNICA DEL FACTOR DE TRANSCRIPCIÓN TgNACO1 IMPLICADO EN XILOGÉNESIS Y ESTRÉS ABIÓTICO EN Tectona grandis / In silico Analysis and Gene Expression of TgNACO1 Transcription Factor Involved in Xylogenesis and Abiotic Stress in Tectona grandis

RESUMEN El xilema secundario es el componente más abundante de la biomasa vegetal. Por tanto, conocer los genes que regulan su formación ayudaría a diseñar estrategias para el mejoramiento genético de la madera. Así, el objetivo de este trabajo fue realizar el análisis computacional de la estructura primaria y secundaria del factor de transcripción (FT) TgNACO1 de Tectona grandis, además de evaluar su historia evolutiva, dominios conservados y expresión génica en tejidos lignificados de árboles de 12 y 60 años. Para ello, se realizó una evaluación del potencial de interacción ion-electrón (PIIE), mediante el método del espectro de la información (MEI) utilizando la librería SFAPS de R-Project, seguido del modelamiento estructural utilizando el software MODELLER y visualizado mediante PyMol. Además, el análisis de alineamiento de secuencia múltiple y filogenia fue mediante el software Bioedit y MrBayes respectivamente. También se evaluó los niveles de síntesis del FT TgNACO1 mediante qRT-PCR. Como resultados, se evidenció que el FT mantiene una estructura (3-hoja antiparalela retorcida, que se compacta contra una a-hélice en la región N-terminal, teniendo así tres dominios a hélice y siete dominios (3 plegada. Asimismo, mediante el MEI se demostró que tiene alrededor de cinco funciones biológicas y mutaciones sobre los aminoácidos con mayor PIIE, lo que conlleva a evoluciones sobre las redes de regulación genética. Finalmente, el FT TgNACO1 podría presentar un papel fundamental en la organización y desarrollo de las partes que componen la albura, como las células radiales de la zona cambial, los vasos, fibras y los anillos de crecimiento.

ABSTRACT Secondary xylem is the most abundant component of plant biomass. Therefore, knowing the genes that regulate its formation would help to design strategies for wood genetic improvement. Thus, the objective of this work was to perform computational analysis of the primary and secondary structure of the TgNACO1 transcription factor (FT) of Tectona grandis, and to evaluate its evolutionary history, conserved domains and gene expression in lignified tissues of trees with 12 and 60 years old. For this, an ion-electron interaction potential (IEP) was evaluated using the information-spectrum method (IEM) using the R-Project and SFAPS library, followed by structural modeling using the MODELLER software and visualized by PyMol program. In addition, the analysis of multiple sequence alignment and phylogeny was performed using Bioedit and MrBayes software, respectively. We also evaluated the qRT-PCR levels of TgNACO1. As results, it was found that TgNACO1 maintains a twisted antiparallel 3-sheet structure, which is compacted against an a-helix in the N-terminal region, having three a-helix domains and seven folded ((-domains. Also, through the IEM, it was demonstrated that it has about five biological functions, and mutations on amino acids with higher IEP, which leads to evolutions on genetic regulation networks. Finally, the FT TgNACO1 could play an esential role in the organization and development of the parts that make up the sapwood, such as the radial cells of the cambial zone, the vessels, fibers and the growth rings.

Biotecnologia na agricultura / Biotechnology in agriculture

A expectativa de o crescimento populacional atingir 9 bilhões de habitantes em 2050 em adição às questões da sustentabilidade e do aquecimento global nos desafiam a aumentar a oferta de alimentos. Uma metodologia alternativa que contribua para a redução do impacto desse cenário envolve a biotecnologia, que, nas últimas décadas, trouxe marcantes oportunidades tecnológicas na agricultura, resultando em relevante desenvolvimento na obtenção de novas variedades de plantas, na melhoria da qualidade de diversos alimentos e atualmente também na bioenergia. As técnicas biotecnológicas envolvendo os marcadores moleculares, a genômica e a transformação genética estão transformando a agricultura e são discutidas neste artigo.

The expected population growth to reach 9 billion by 2050 in addition to issues of sustainability and global warming challenges us to increase the supply of food. An alternative approach to help reducing the impact of this scenario involves biotechnology which in recent decades has brought remarkable technological opportunities in the agriculture that resulted in relevant development in obtaining new plant varieties, improved quality of different foods, and now also in bioenergy. The biotechnology techniques involving molecular markers, genomics and genetic transformation are transforming agriculture and will be discussed in this article.

Signaling pathways in a Citrus EST database

Citrus spp. are economically important crops, which in Brazil are grown mainly in the State of São Paulo. Citrus cultures are attacked by several pathogens, causing severe yield losses. In order to better understand this culture, the Millenium Project (IAC Cordeirópolis) was launched in order to sequence Citrus ESTs (expressed sequence tags) from different tissues, including leaf, bark, fruit, root and flower. Plants were submitted to biotic and abiotic stresses and investigated under different development stages (adult vs. juvenile). Several cDNA libraries were constructed and the sequences obtained formed the Citrus ESTs database with almost 200,000 sequences. Searches were performed in the Citrus database to investigate the presence of different signaling pathway components. Several of the genes involved in the signaling of sugar, calcium, cytokinin, plant hormones, inositol phosphate, MAPKinase and COP9 were found in the citrus genome and are discussed in this paper. The results obtained may indicate that similar mechanisms described in other plants, such as Arabidopsis, occur in citrus. Further experimental studies must be conducted in order to understand the different signaling pathways present.

Identification of protein kinase SNF1 in CitEST

SnRKs (Sucrose non-fermenting-1 related kinases) is a family of protein kinases found in many crops, such as Arabidopsis, rice, sugarcane, tomato and several other plant species. This family of proteins is also present in other organisms like Saccharomyces cerevisiae (sucrose non-fermenting-1 - Snf1) and in mammals (AMP-activated protein kinases - AMPKs). There is evidence that SnRKs play an important role in plant responses to nutritional and environmental stresses and that SnRKs also play a major role in controlling key enzymes in the biosynthetic pathways of plants. In this work, we identified 18 contigs and two singletons encoding putative SnRKs in the CitEST database. All of them present highly conserved N-terminal catalytic domain, which is found in the SnRKs families of several plant species. Through comparison with known SnRKs, we were able to classify them into three subfamilies.

Putative resistance genes in the CitEST database

Disease resistance in plants is usually associated with the activation of a wide variety of defense responses to prevent pathogen replication and/or movement. The ability of the host plant to recognize the pathogen and to activate defense responses is regulated by direct or indirect interaction between the products of plant resistance (R) and pathogen avirulence (Avr) genes. Attempted infection of plants by avirulent pathogens elicits a battery of defenses often followed by the collapse of the challenged host cells. Localized host cell death may help to prevent the pathogen from spreading to uninfected tissues, known as hypersensitive response (HR). When either the plant or the pathogen lacks its cognate gene, activation of the plant’s defense responses fails to occur or is delayed and does not prevent pathogen colonization. In the CitEST database, we identified 1,300 reads related to R genes in Citrus which have been reported in other plant species. These reads were translated in silico, and alignments of their amino acid sequences revealed the presence of characteristic domains and motifs that are specific to R gene classes. The description of the reads identified suggests that they function as resistance genes in citrus.

Genes associated with hypersensitive response (HR) in the citrus EST database (CitEST)

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.br sebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.

Identification of Astyanax altiparanae (Teleostei, Characidae) in the Iguaçu River, Brazil, based on mitochondrial DNA and RAPD markers

Astyanax fishes are among the most important food-web components of South America rivers. In the Iguaçu River basin, the Astyanax genus is represented mainly by endemic species. For millions of years, that hydrographic basin has been geographically isolated from the Paraná River basin by the Iguaçu Falls. Recently, a species from the Upper Paraná River basin identified as Astyanax bimaculatus was revised and described as a new species named Astyanax altiparanae Garutti e Britski, 2000. Fauna endemism and geographic isolation triggered interest in investigations to evaluate the identification and genetic relatedness among two A. altiparanae populations from the Upper Paraná River basin and the population identified as A. bimaculatus in the Iguaçu River, upstream from the Iguaçu Falls. Mitochondrial DNA sequences and RAPD markers revealed high genetic diversity within each population, as well as low genetic distance, high gene flow, and high mitochondrial DNA similarity among all three populations. In conjunction with morphological similarities, these results demonstrated that the population presently known as Astyanax bimaculatus in the Iguaçu River should actually be stated as Astyanax altiparanae. Furthermore, it could be inferred that the A. altiparanae population is not endemic and most likely it was recently introduced in the Iguaçu River basin, maintaining the ancestral genetic identity

In silico characterization and expression analyses of sugarcane putative sucrose non-fermenting-1 (SNF1) related kinases

Quinases de proteínas relacionadas a SNF1 (SnRK) podem desempenhar um papel importante na regulaçäo da expressäo gênica em células vegetais. Essa família de proteínas regulatórias é representada pela quinase de proteínas SNF1 (sucrose non-fermenting-1) em Saccharomyces cerevisiae, AMPKs (quinase de proteínas ativadas por AMP) em células de mamíferos e SnRKs (quinase de proteínas relacionadas a SNF1) em células vegetaìs. A família de SnRKs foi reorganizada em três subfamílias de acordo com suas relações filogenéticas com base nas seqüências de aminoácidos das proteínas. Membros das subfamílias de SnRKs foram identificados em diversas plantas. Existem evidências mostrando que essa família de proteínas está envolvida em resposta a estresses (nutricional e ambiental), apesar de seu papel näo ser totalmente compreendido. Nesse trabalho nós identificamos 22 contíguos de ESTs (expressed sequence tags) de cana-de-açúcar codificando SnRKs putativas. O alinhamento das seqüências de aminoácidos das SnRKs putativas de cana-de-açúcar com seqüências de aminoácidos de SnRKs identificadas em outras plantas revelaram um domínio catalítico N-terminal altamente conservado. Além disso, nossos resultados indicaram que em cana-de-açúcar há pelo menos um membro de cada subfamília de SnRKs. Análìse do padräo de expressäo dos contíguos de EST codificando para SnRK putativas nas 26 bibliotecas selecionadas do banco de dados do Sucest, indicou que membros dessa família de quinases säo expressos por toda planta. Membros de cada subfamília näo apresentaram padrões de expressäo específicos, sugerindo que suas funções näo estäo correlacionadas com sua relaçäo filogenética, com base nas seqüências de aminoácidos da regiäo N-terminal.