Unveiling Chloroplast RNA Editing Events Using Next Generation Small RNA Sequencing Data

Organellar RNA editing involves the modification of nucleotide sequences to maintain conserved protein functions, mainly by reverting non-neutral codon mutations. The loss of plastid editing events, resulting from mutations in RNA editing factors or through stress interference, leads to developmental, physiological and photosynthetic alterations. Recently, next generation sequencing technology has generated the massive discovery of sRNA sequences and expanded the number of sRNA data. Here, we present a method to screen chloroplast RNA editing using public sRNA libraries from Arabidopsis, soybean and rice. We mapped the sRNAs against the nuclear, mitochondrial and plastid genomes to confirm predicted cytosine to uracil (C-to-U) editing events and identify new editing sites in plastids. Among the predicted editing sites, 40.57, 34.78, and 25.31% were confirmed using sRNAs from Arabidopsis, soybean and rice, respectively. SNP analysis revealed 58.2, 43.9, and 37.5% new C-to-U changes in the respective species and identified known and new putative adenosine to inosine (A-to-I) RNA editing in tRNAs. The present method and data reveal the potential of sRNA as a reliable source to identify new and confirm known editing sites

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

A brazilian soybean database.

Soybean is a legume with large economic importance in the international market, with a world production of almost two hundred and ten million tons in the 2008/2009 harvest. Brazil appears as the second largest producer, with about twenty-five percent of the world production. In 2007, the Brazilian Soybean Genome Consortium (GENOSOJA) was established with the goal of integrating several institutions currently working with soybean genomics in Brazil. The project has an initiative to search for new treats to improve the soybean production process, emphasizing in stresses that affect the national production, like the occurrence of droughts, pests attacks and the Asian Rust disease. Among the objectives of GENOSOJA is the creation of a relational database, integrating the results achieved by different methodologies utilized in the project. In the GENOSOJA context, we created a brazilian soybean database, integrating: (1) public data consisting of genome and predicted genes from JGI, an assembly of 1,276,813 of NCBI ESTs from several cultivars and 4,712 full-length cDNA sequences from one japanese cultivar; and (2) private data consisting of (i ) three cDNA libraries explored by SuperSAGE methodology, resulting in 4,373,053 tags of 26 bp, (ii ) 22 cDNA subctrative libraries from several brazilian cultivars under different stresses and (iii ) several libraries of soybean microRNAs under eight conditions and size between 19 and 24 bp. All these data were sequenced using Solexa/Illumina sequencing technology. This database offers to the users some features, including keywords searches, statics comparisons, automatic annotation, gene ontology classification and gene expression of the genes under certain conditions. All data are storage in a Fedora Linux machine, running the MySQL database server. The web interface is based in a combination of CGI scripts using Perl language (including BioPerl module) and the Apache Web Server

Bioinformatics analysis applied to genosoja project.

Soybean is the legume of most economic importance in the international market, with world production of almost two hundred and thirty million tons in the 2007/2008 harvest. The Brazil appears as the largest exporter of the product in the world, with about twenty-five percent of the world production. In 2007 the brazilian government started the GENOSOJA project with the main objective of discover new treats to improve the plant production process, emphasizing in stresses that affect the national production like the occurrence of droughts, pests attacks and the Asian rust disease. This work is inserted in the GENOSOJA scope and aims to generate bioinformatics tools to integrate the public soybean data like ESTS and genomic sequences, with all data generated during the project like SuperSAGE tags, MicroRNAs and subtractive libraries sequences analysis. These sequences were generated using high throughput sequencing technologies and for each analysis a specific pipeline have to be implemented. Actually, we have processed and performed a hybrid assembly of 1,276,813 ESTs available at NCBI (sequenced by sanger and pyrosequencing tecnologies) resulting in 30,809 contigs and 29,938 singlets, supersage analysis of 2,334,864 tags sequenced by Ilumina/Solexa from several libraries resulting in 54,052 unique tags, mapped into soybean genome and assembly of 11,783,331 short-sequences sequenced by Ilumina/Solexa generated by subtracted library experiments from leaf and root tissues submitted to drought stress resulting in 5,084 genes. All these results are making available to the final user in a web interface that allows searches by keywords, statistics comparisons, visualization of automatic annotation (AutoFACT pipeline) and the gene ontology classification

Small RNA profiles in soybean primary root tips under water deficit

Background: Soybean (Glycine max) production is significantly hampered by frequent droughts in many regions of the world including the United States. Identifying microRNA (miRNA)-controlled posttranscriptional gene regulation under drought will enhance our understanding of molecular basis of drought tolerance in this important cash crop. Indeed, miRNA profiles in soybean exposed to drought were studied but not from the primary root tips, which is not only a main zone of water uptake but also critical for water stress sensing and signaling.Methods: Here we report miRNA profiles specifically from well-watered and water-stressed primary root tips (0 to 8 mm from the root apex) of soybean. Small RNA sequencing confirmed the expression of vastly diverse miRNA (303 individual miRNAs) population, and, importantly several conserved miRNAs were abundantly expressed in primary root tips.Results: Notably, 12 highly conserved miRNA families were differentially regulated in response to water-deficit; six were upregulated while six others were downregulated at least by one fold (log2) change. Differentially regulated soybean miRNAs are targeting genes include auxin response factors, Cu/Zn Superoxide dismutases, laccases and plantacyanin and several others.Conclusions: These results highlighted the importance of miRNAs in primary root tips both under control and water-deficit conditions; under control conditions, miRNAs could be important for cell division, cell elongation and maintenance of the root apical meristem activity including quiescent centre whereas under water stress differentially regulated miRNAs could decrease auxin signaling and oxidative stress as well as other metabolic processes that save energy and water.Peer reviewedBiochemistry and Molecular Biolog

Salt stress affects mRNA editing in soybean chloroplasts

Abstract Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress