Identification of novel soybean microRNAs involved in abiotic and biotic stresses

<p>Abstract</p> <p>Background</p> <p>Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.</p> <p>Results</p> <p>The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.</p> <p>Conclusions</p> <p>Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.</p

Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants

Stability of the mitochondrial genome is controlled by nuclear loci. In plants, nuclear genes suppress mitochondrial DNA rearrangements during development. One nuclear gene involved in this process is Msh1. Msh1 appears to be involved in the suppression of illegitimate recombination in plant mitochondria. To test the hypothesis that Msh1 disruption leads to the type of mitochondrial DNA rearrangements associated with naturally occurring cytoplasmic male sterility in plants, a transgenic approach for RNAi was used to modulate expression of Msh1 in tobacco and tomato. In both species, these experiments resulted in reproducible mitochondrial DNA rearrangements and a condition of male (pollen) sterility. The male sterility was, in each case, heritable, associated with normal female fertility, and apparently maternal in its inheritance. Segregation of the transgene did not reverse the male sterile phenotype, producing stable, nontransgenic male sterility. The reproducible transgenic induction of mitochondrial rearrangements in plants is unprecedented, providing a means to develop novel cytoplasmic male sterile lines for release as non-GMO or transgenic materials

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.201

Variabilidade genética em biotipos de leiteiro de Londrina/PR Genetic variability among Euphorbia heterophylla

Euphorbia heterophylla, também conhecida como amendoim-bravo ou leiteira, é considerada planta invasora importante em mais de 56 países, inclusive no Brasil, tendo acarretado perdas de até 33 % na cultura da soja. Fenotipicamente, é uma espécie de características variáveis, especialmente em relação ao formato do limbo foliar. Esta variabilidade fenotípica tem sido utilizada para diferenciar e classificar as plantas, sugerindo a vários autores que a leiteira seria, de fato, constituída por diferentes espécies. Para estudar a variabilidade genética a nível de DNA entre plantas de Euphorbia heterophylla, que apresentam folhas morfologicamente diferentes, foram analisadas dez plantas diferentes coletadas em campos de soja, em Londrina/PR. As plantas foram transplantadas para casa-devegetação e o DNA das folhas foi extraído para análise pela técnica de RAPD. Vinte seis diferentes "primers", de dez nucleotídeos de sequência aleatória, geraram total de 102 bandas de DNA, sendo 38 delas polimórficas. A distância genética entre os indivíduos foi calculada em função da presença e da ausência das bandas, variando de 1 a 39% entre plantas. A análise de agrupamento dividiu as plantas em dois grupos, considerando limite de distância relativa de 22%. Os grupos gerados separaram nitidamente as plantas quanto ao formato do limbo foliar (estreito ou arredondado) e quanto á ramificação (densa ou normal).<br>Euphorbia heterophylla is an important weed affecting the performance of annual and perennial crops. It is native from tropical and subtropical regions in the American continent, and has been detected at high densities in 20 different countries worldwide, and at low densities in other 40 countries. In Brazil, it has been inclued among the ten most important weeds affecting different crops, causing yield losses up to 33% in soybean fields. Phenotypically, this species is extremely variable, especially in relation to leaf shape and size, which can vary among and within populations. This variability suggested to several authors that E. heterophylla was, in fact, formed by different species. To systematize the study of E. heterophylla and to determine if the phenotypic variability correspond to modifications at the DNA level, we analyzed 10 different plants collected in the soybean field in Londrina (Parana, Brazil). The plants were transplanted to the greenhouse and leaf DNA was extracted for RAPD technique analysis. Twenty-six RAPD "primers" different amplified 102 DNA bands, 38 of them being polymorphic. Genetic distances among the individuals were calculated based on the presence (1) or absence (0) of those bands. Cluster analyses divided the plants into two distinct groups considering an upper limit of 22% relative genetic distance. The genetic distances among the plants were between 1 and 39%, in agreement with the variability obseved at the morphological level

Differential expression of four soybean bZIP genes during Phakopsora pachyrhizi infection

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is one of most important diseases in the soybean (Glycine max (L.) Merr.) agribusiness. The identification and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes, GmbZIP62, GmbZIP105, GmbZIPE1, and GmbZIPE2, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to P. pachyrhizi infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to P. pachyrhizi infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR