Plant immunity and beyond: Signals from proteins & peptides

Plants are the primary and most important source of food for human consumption, besides their ecological importance, since they define the diverse ecosystems worldwide. Because of their position in the ecological chain as primary supplier of biomass and food for other organisms, plants are also fundamental for animals, fungi and microorganisms, and some of them are also beneficial to other plants. Humans rely mostly on plant products for food, and many plants provide important non-food products, including wood, textiles, medicines, cosmetics, soaps, rubber, plastics, paints and other industrial chemicals. Moreover, plants are also fundamental for animal feeding, including not only mammals (e.g. cattle, sheep and goats), but also poultry and aquaculture (e.g., fish and shrimp farming)

GENOSOJA - The Brazilian Soybean Genome Consortium: high throughput omics and beyond.

Plant genomes are among the most complex and large ones of our planet, with high levels of redundancy when compared to other eukaryotic groups, leading to intricate processes for gene regulation and evolution. Such a complexity demands interdisciplinary and multidimensional approaches in order to allow a better understanding of the processes able to exploit the whole potential of the existing genes in different species, including crop plants. Among cultivated plants, soybean [Glycine max (L.) Merr.] occupies an outstanding position due to its importance as source of protein and oil that may also be converted into biodiesel. The seeds are rarely consumed in natura, but many traditional food products have been consumed, as soymilk, and tofu, as well as fermented products as soy sauce, and soy paste among others, besides its wide use for animal feed. Soybean cultivation has been highly concentrated geographically, with only four countries (USA, Brazil, Argentina and China) accounting for almost 90% of world output. Asia (excluding China) and Africa, the two regions where most of the food insecure countries are located, account for only 5% of production. Among countries classified as 'undernourished', only India and Bolivia are significant producers of soybeans (FAO, 2009). Available evidences indicate that the cultivated soybean was domesticated from its wild relative Glycine soja (Sieb. and Zucc.) in China about 5,000 years ago (Carter et al., 2004). Since then, soybean has been grown primarily in temperate regions for thousands of years, first in Northern Asia and in more recent years in North America and countries of the Southern Cone of Latin America (FAO, 2009). The remarkable success of this crop in temperate zones is well known, but the crop presents also an important role in cropping systems of the tropics and subtropics, also in low fertile regions, as the Brazilian cerrado savannah (Spehar, 1995). The actual area cultivated worldwide with soybean is estimated to cover 103.5 millions of hectares, from which 24.2 only in Brazil, with considerable increases in the production achieved without significant increase in the cultivated area (Embrapa Soybean, 2011). As a legume, soybean is able to develop symbiotic interactions with rhizobia, allowing the fixation and assimilation of atmospheric N2, bearing quite specific mechanisms to coordinate this complex interaction (Oldroyd and Downie, 2008), absent in most angiosperm groups. Besides this peculiarity, soybean presents 2n = 40 chromosomes and was early characterized as an ancient polyploid (paleopolyploid) through genetic mapping studies that identified homeologous chromosome regions based upon duplicate RFLP markers (Shoemaker et al., 1996; Lee et al., 1999; 2001). Due to its allopolyploid nature, the first approaches regarded the generation of expressed sequences from different library tissues and conditions, including mainly ESTs (Expressed Sequence Tags; Nelson et al., 2005) partially in annotated databases, including ca. 40.000 full length cDNA clones available (Umezawa et al., 2008, see also RIKEN Soybean Full-Length cDNA Database), besides analyses regarding RNAseq under different tissues and development stages, as well as under different stressing situations (e.g. Libault et al., 2010; Severin et al., 2010). Also a complete shotgun genome sequence of the soybean cultivar Williams 82 was released, comprising 1.1-gigabase genome size allowing the integration of physical and high-density genetic maps, including 46,430 predicted protein-coding genes (Schmutz et al., 2010). The total amount of data publicly available at GenBank (NCBI) includes more than 120,000 nucleotide sequences (mainly mRNA), ~1,460,000 ESTs, ~368,000 genome sequences, ~80,000 proteins, 118 deposited structures and more than 6,2 million SNPs. Such numbers show that working with soybean is a very challenging task. By the other hand, despite of the wide data availability, most data regard cultivars from temperate regions (as Williams 82), not adapted for cultivation under tropical conditions, as it is the case of central Brazil and many other tropical countries that are subjected to distinct environmental stresses. The proposition of creating the GENOSOJA consortium was submitted in 2007 to the National Council for Scientific and Technological Development (CNPq), an agency linked to the Brazilian Ministry of Science and Technology (MCT), starting its activity in March 2008 with the participation of nine Brazilian institutions from different regions (Figure 1). The proposal aimed to study the soybean genome from its organization into the structural level, seeking to characterize and sequence important genomic regions and their products, thus contributing to the identification of genes using transcriptional and proteomic methods, especially considering the plant response to different biotic and abiotic stresses that affect the culture in the Southern hemisphere. Still, the GENOSOJA network aimed to approach not only whether a gene is induced or suppressed under a given condition, but also to determine the levels at which it is expressed, the interactions with other genes, their physical location and products, allowing the identification of important genes and metabolic pathways, vital for the development and study of plants tolerant to challenging situations. The GENOSOJA project is still in course and is structured into six Project Components (Figure 2), including management and addressing of different aspects of the soybean genome: I. Project management - responsible for the project administration, organization of meetings, group integration and research reports, among others. II. Structural Genomics - includes research activities related to the genomic physical architecture, including BAC anchoring (in the cultivar Conquista), promoter analysis and sequencing of gene-rich regions, also in comparison with other wild relatives of the genus Glycine, allowing studies of synteny and indication of regions important for ressequencing. This component is also responsible for the identification of single base polymorphisms (SNPs), very important for mapping purposes and marker assisted selection. III. Transcriptomics -comprises the largest research group, responsible for various expression profiling approaches using different strategies to access transcripts generated under different biotic (Asian rust: Phakopsora pachyrhizi, CPMMV: Cowpea mild mottle virus, nematodes: Meloydogyne javanica and Pratylenchus brachyurus) and abiotic (water deficit) stresses. In this workgroup different strategies were used, including: a) Subtractive cDNA libraries (76 bp tags, Solexa Illumina® sequencing) using contrasting materials submitted to biotic interactions, including diseases (~40 million tags; Asian rust and virus inoculation) and beneficial interactions (~10 million tags; inoculation with Bradyrhizobium japonicum), as well as water deficit (~42 million tags, comparing tolerant and susceptible accessions). b) SuperSAGE comprising ~3,2 Solexa Illumina® 26-bp tags distributed in six libraries generated under biotic (water deficit) and abiotic (Asian rust) stress comparisons. c) MicroRNA libraries (Solexa Illumina®, 1924 bp) including four libraries regarding water deficit ( 4,8 millions miRNAs) and other four regarding Asian rust (~7,9 million miRNAs). d) cDNA sequences (2,112 sequences, Sanger method) from roots infested with the nematode M. javanica compared with non stressed control. The three first above mentioned experiments were carried out using the same experimental conditions, generating an extensive comparable dataset to allow the understanding of the gene expression dynamic (Subtractive cDNA and SuperSAGE libraries), including biotic and abiotic cross-talk responses as well as the post transcriptional control (miRNA). IV. Proteomics -aimed to study the protein profile of soybean plants, low-mass protein and peptides identification and protein-protein interactions, using the same accessions and biological conditions established for the transcriptomic analyses to ensure complementarity and reduction of experimental variability, and thus, allowing the integration of both datasets in the functional characterization of the soybean genome. V. Expression Assays (transgenesis) -considering the results of transcriptomics and proteomics, most valuable gene candidates are being transformed in order to infer about their effects or biological function. Members of this group are also evaluating the vicinity of genes (UTRs) for the identification of regulatory regions (promoters, enhancers, cis-elements, etc.) that control their expression. VI. Bioinformatics -this workgroup developed the GENOSOJA database (see web resource) that includes a set of tools integrating the entire project data as compared with available sequences from other public data banks. The present issue represents the starting point of an extensive catalogue of products generated by the GENO-SOJA consortium, since all members agree that many additional inferences will be soon mature for publication and application to breeding projects. Thousands of candidate genes differentially expressed have been identified and are being validated using quantitative real time PCR, many regarding strongly induced genes in contrasting libraries (e.g. stressed against control or tolerant against sensible in the same condition). Many of them refer to uncharacterized genes, with no given function, representing relevant data to be worked out in future functional studies, since they may represent not yet described genes, some possibly unique to legumes and important for plant breeding. Finally, the present volume does not represent a milestone for completion of the GENOSOJA project, but an announcement of its birth, crowned with solid growth, integration and consolidation prospects. The data generated by the GENOSOJA consortium will also join the worldwide effort to study the soybean genome through the participation in the International Soybean Genome Consortium (ISGC). In this sense, the next step involves the public release of the generated data, which shall be available for the world community, allowing the effective integration with other networks throughout the world

Cytogenetics and cytotaxonomy of Velloziaceae.

Chromosome number and other cytological features are reported from 35 species of Velloziaceae, including several african and brazilian populations. All analyzed species show areticulate interphase nuclei and prophase/prometaphase chromosomes with proximal early condensation. Most heteropycnotic blocks do not seem to correspond to heterochromatin since, at least in Vellozia patens, they do not stain differentially after C-banding procedures. Regarding the chromosome number, three main groups could be identified. The first comprised diploid species of the genera Nanuza, Vellozia and the brazilian species of Xerophyta with 2n=14 or 16; the second comprised tetraploid species with 2n=34, and included all brazilian species of subfam. Barbacenioideae; the third group, of hexaploid species, comprised the African representatives of the genus Xerophyta. A single population of Vellozia, possibly of hybrid origin, had 2n~=32. A basic number of x=8 is proposed for the family. The karyological information supports the hypothesis that the Velloziaceae originated on the South American, rather than on the African continent

Caracterização molecular por ISSR de acessos de melancia do Banco Ativo de Germoplasma de Cucurbitáceas.

Suplemento. Edição dos Anais do 52 Congresso Brasileiro de Olericultura, Salvador, jul. 201

Distribuição de microssatélites no genoma de leguminosas mediante hibridização in situ fluorescente.

Os microssatélites consistem em unidades de repetição (1 a 5pb) distribuídas em tandem, que podem estar dispersas ao longo dos genomas ou associadas a regiões heterocromáticas. Tais características têm permitido sua utilização na construção de mapas cromossômicos mediante hibridização in situ fluorescente (FISH), auxiliando no entendimento da organização genômica entre espécies proximamente relacionadas. O presente trabalho caracterizou a distribuição de oligonucleotídeos sintéticos (AG)8, (AAG)5, (ACC)5, (CTC)5 e (TGA)6 em Phaseolus vulgaris, P. lunatus, Vigna unguiculata, V. radiata e Glycine max, identificando marcas cromossômicas para a comparação destas leguminosas. Lâminas foram hibridizadas com sondas de oligonucleotídeos e rehibridizadas com DNAr 5S e 45S, provenientes de Lotus japonicus e Arabidopsis thaliana, respectivamente. Em soja, esta análise estendeu-se a uma investigação genômica, a partir de sequências disponibilizadas no banco de dados SoyBase (http://www.soybase.org/), usando o programa BLASTN com os seguintes parâmetros: formato de saída de alinhamentos sem lacunas, matriz de comparação blossum62, valor W padrão, e-value 0.1 ou menor e filtro de baixa complexidade desligado. Os oligonucleotídeos [(AAG)5]10, [(AAC)5]10, [(AG)8]15, [(CTC)5]10 e [(TGA)6]10 foram utilizados como sondas, adotando como ponto de corte valores de identidade de pareamento inferiores a 77%, objetivando caracterizar número, tamanho e localização destas unidades de repetições no genoma. A FISH com oligonucleotídeos evidenciaram marcações pericentroméricas e proximais, embora um padrão de distribuição disperso tenha prevalecido ao longo dos cromossomos das espécies analisadas, com exceção de (AG)8 em soja que não revelou marcações visíveis. O oligonucleotídeo (AAG)5 revelou uma marcação pericentromérica evidente em P. lunatus, tratando-se de um marcador cromossômico para tal espécie. Na hibridização com (ACC)5 notou-se a presença de seis marcações fortes em V. unguiculata, estando uma delas localizada no par cromossômico 10, portador do sítio de DNAr 45S. O microssatélite (TGA)6 revelou uma marcação bem evidente no cromossomo 10 de P. lunatus, identificado pela presença de DNAr 5S. Algumas divergências observadas no padrão de distribuição e na intensidade dos sinais entre os genomas das espécies confirmam a hipótese de que as sequências de DNA repetitivo apresentam uma composição heterogênea. Na análise genômica de soja, foram observados 92, 84, 83, 142 e 103 sítios de repetições para os oligonucleotídeos (AG)8, (AAG)5, (ACC)5, (CTC)5 e (TGA)6, respectivamente, de tamanhos variáveis entre 30 a 454 pb, localizados, principalmente, em regiões de alta a moderada densidade gênica, por vezes associados a genes e elementos transponíveis. Considerando o pequeno tamanho, fica evidente que estas sequências não correspondem aos sítios observados pela FISH, uma vez que esta técnica evidencia apenas sequências com comprimentos acima de 1-3kb. Sugere-se que as marcações localizadas por FISH estejam associadas a regiões de heterocromatina e que sejam constituintes dos mais de 1.000 scaffolds existentes no sequenciamento da soja, não sendo, por essa razão, detectadas pela análise in silico

Genetic diversity among cowpea cultivars as revealed by ISSR and DAF markers.

BIOLATINA 2010. Autoria: DAMASCENO-SILVA, K. J. [i.e. SILVA, K. J. D. e]

Transcriptoma supersage de feijão-caupi sob desidratação radicular.

O feijão-caupi é uma planta de ampla plasticidade fenotípica, podendo produzir mesmo em condições não satisfatória para outras culturas. Assim, identificar genes estresse responsivo nesta cultura é de vital importância, tanto para a espécie quanto para outras leguminosas. Estudos em transcriptômica, proporcionados pela técnica SuperSAGE, origina tags de 26 pb que podem ser quantificadas e estatisticamente analisadas, sendo classificadas em superexpressas ou reprimidas. Unitags (tags diferentes) de raízes de feijao-caupi submetidas a estresse de desidratação radicular (de até 150 min, após exposição ao ar) foram analisadas procurando-se identificar unitags diferencialmente expressas nas respostas dos genótipos analisados, considerados tolerante e sensível a seca. As anotações de gene/ função das unitags, associadas à regulação (induzida ou reprimida nas bibliotecas sob estresse em relação às bibliotecas controle) permitiram identificar alvos moleculares com potencial para desenvolvimento de marcadores moleculares e ou estudos de transgenia, visando uso futuro em programas de melhoramentos da espécie e de leguminosas relacionadas

Transcriptoma supersage de feijão-caupi sob desidratação radicular.

O feijão-caupi é uma planta de ampla plasticidade fenotípica, podendo produzir mesmo em condições não satisfatória para outras culturas. Assim, identificar genes estresse responsivo nesta cultura é de vital importância, tanto para a espécie quanto para outras leguminosas. Estudos em transcriptômica, proporcionados pela técnica SuperSAGE, origina tags de 26 pb que podem ser quantificadas e estatisticamente analisadas, sendo classificadas em superexpressas ou reprimidas. Unitags (tags diferentes) de raízes de feijao-caupi submetidas a estresse de desidratação radicular (de até 150 min, após exposição ao ar) foram analisadas procurando-se identificar unitags diferencialmente expressas nas respostas dos genótipos analisados, considerados tolerante e sensível a seca. As anotações de gene/ função das unitags, associadas à regulação (induzida ou reprimida nas bibliotecas sob estresse em relação às bibliotecas controle) permitiram identificar alvos moleculares com potencial para desenvolvimento de marcadores moleculares e ou estudos de transgenia, visando uso futuro em programas de melhoramentos da espécie e de leguminosas relacionadas.CONAC 2012. Disponível em: http://www.conac2012.org/resumos/pdf/337a.pdf. Acesso em: 09 ago. 2013

Mineração de motivos SSR em sequências expressas de feijão-caupi contendo TAGS supersage diferecialmente expressas sob desidratação radicular.

O feijão-caupi é uma planta importante na nutrição e na economia de pequenos produtores das regiões Norte e Nordeste do Brasil. Entretanto, sua produtividade ainda é pequena, quando comparada com seu potencial e o de outras leguminosas. Uma das alternativas para auxiliar o melhoramento genético da cultura e associar tecnologias modernas, como o desenvolvimento de marcadores moleculares do tipo microssatélites (SSR) e a identificação de genes importantes em resposta a estresses, via estudos de transcriptômica, proporcionados pela técnica SuperSAGE. Nesse sentido, tags SuperSAGE de 26 pb, de genótipo tolerante a desidratação radicular, que foram observadas sendo induzidas após aplicação do estresse (até 150 min), em relação ao controle sem estresse, foram ancoradas em ESTs de feijão-caupi. Essas ESTs contendo as tags induzidas foram mineradas quanto à presença de motivos SSR. Os motivos identificados em 135 ESTs permitiram desenhar pares de primers para desenvolvimento de marcadores moleculares SSR, agora baseados em DNA, e não mais anônimos, visando aproveitar esse tipo de polimorfismos para genotipagem de materiais do melhoramento. Sua associação com resposta a estresses está sendo investigada.CONAC 2012. Disponível em: http://www.conac2012.org/resumos/pdf/344a.pdf. Acesso em: 09 ago. 2013