RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats.

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM

Plants regenerated from tissue culture contain stable epigenome changes in rice.

Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:http://dx.doi.org/10.7554/eLife.00354.001

Comparative expression profiling in grape (Vitis vinifera) berries derived from frequency analysis of ESTs and MPSS signatures

<p>Abstract</p> <p>Background</p> <p><it>Vitis vinifera </it>(<it>V. vinifera</it>) is the primary grape species cultivated for wine production, with an industry valued annually in the billions of dollars worldwide. In order to sustain and increase grape production, it is necessary to understand the genetic makeup of grape species. Here we performed mRNA profiling using Massively Parallel Signature Sequencing (MPSS) and combined it with available Expressed Sequence Tag (EST) data. These tag-based technologies, which do not require <it>a priori </it>knowledge of genomic sequence, are well-suited for transcriptional profiling. The sequence depth of MPSS allowed us to capture and quantify almost all the transcripts at a specific stage in the development of the grape berry.</p> <p>Results</p> <p>The number and relative abundance of transcripts from stage II grape berries was defined using Massively Parallel Signature Sequencing (MPSS). A total of 2,635,293 17-base and 2,259,286 20-base signatures were obtained, representing at least 30,737 and 26,878 distinct sequences. The average normalized abundance per signature was ~49 TPM (Transcripts Per Million). Comparisons of the MPSS signatures with available <it>Vitis </it>species' ESTs and a unigene set demonstrated that 6,430 distinct contigs and 2,190 singletons have a perfect match to at least one MPSS signature. Among the matched sequences, ESTs were identified from tissues other than berries or from berries at different developmental stages. Additional MPSS signatures not matching to known grape ESTs can extend our knowledge of the <it>V. vinifera </it>transcriptome, particularly when these data are used to assist in annotation of whole genome sequences from <it>Vitis vinifera</it>.</p> <p>Conclusion</p> <p>The MPSS data presented here not only achieved a higher level of saturation than previous EST based analyses, but in doing so, expand the known set of transcripts of grape berries during the unique stage in development that immediately precedes the onset of ripening. The MPSS dataset also revealed evidence of antisense expression not previously reported in grapes but comparable to that reported in other plant species. Finally, we developed a novel web-based, public resource for utilization of the grape MPSS data <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Plant MPSS databases: signature-based transcriptional resources for analyses of mRNA and small RNA

MPSS (massively parallel signature sequencing) is a sequencing-based technology that uses a unique method to quantify gene expression level, generating millions of short sequence tags per library. We have created a series of databases for four species (Arabidopsis, rice, grape and Magnaporthe grisea, the rice blast fungus). Our MPSS databases measure the expression level of most genes under defined conditions and provide information about potentially novel transcripts (antisense transcripts, alternative splice isoforms and regulatory intergenic transcripts). A modified version of MPSS has been used to perform deep profiling of small RNAs from Arabidopsis, and we have recently adapted our database to display these data. Interpretation of the small RNA MPSS data is facilitated by the inclusion of extensive repeat data in our genome viewer. All the data and the tools introduced in this article are available at

Multiple RNA recognition patterns during microRNA biogenesis in plants

MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.Fil: Bologna, Nicolas Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Schapire, Arnaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Zhai, Jixian. University of Delaware. Department of Plant & Soil Sciences; Estados Unidos. Delaware Biotechnology Institute; Estados UnidosFil: Chorostecki, Uciel Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Boisbouvier, Jerome. Institut de Biologie Structurale Jean-Pierre Ebel; FranciaFil: Meyers, Blake C.. University of Delaware. Department of Plant & Soil Sciences; Estados Unidos. Delaware Biotechnology Institute; Estados UnidosFil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentin

Bioaccumulation and histopathological effects of oil on a stony coral

Colonies of the shallow-water Caribbean coral Manicina areolata incorporated petroleum hydrocarbons into their tissues during exposure to water accommodated fractions of No. 2 fuel oil for three months. This contamination was not removed after depuration periods of up to two weeks. Although these corals remained alive, evidence of pathological responses was found which included impaired development of reproductive tissues, degeneration and loss of symbiotic zooxanthellae, and atrophy of mucous secretory cells and muscle bundles.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24242/1/0000505.pd

A spatial dissection of the Arabidopsis floral transcriptome by MPSS

<p>Abstract</p> <p>Background</p> <p>We have further characterized floral organ-localized gene expression in the inflorescence of <it>Arabidopsis thaliana </it>by comparison of massively parallel signature sequencing (MPSS) data. Six libraries of RNA sequence tags from immature inflorescence tissues were constructed and matched to their respective loci in the annotated <it>Arabidopsis </it>genome. These signature libraries survey the floral transcriptome of wild-type tissue as well as the floral homeotic mutants, <it>apetala1, apetala3, agamous</it>, a <it>superman/apetala1 </it>double mutant, and differentiated ovules dissected from the gynoecia of wild-type inflorescences. Comparing and contrasting these MPSS floral expression libraries enabled demarcation of transcripts enriched in the petals, stamens, stigma-style, gynoecia, and those with predicted enrichment within the sepal/sepal-petals, petal-stamens, or gynoecia-stamens.</p> <p>Results</p> <p>By comparison of expression libraries, a total of 572 genes were found to have organ-enriched expression within the inflorescence. The bulk of characterized organ-enriched transcript diversity was noted in the gynoecia and stamens, whereas fewer genes demonstrated sepal or petal-localized expression. Validation of the computational analyses was performed by comparison with previously published expression data, <it>in situ </it>hybridizations, promoter-reporter fusions, and reverse transcription PCR. A number of well-characterized genes were accurately delineated within our system of transcript filtration. Moreover, empirical validations confirm MPSS predictions for several genes with previously uncharacterized expression patterns.</p> <p>Conclusion</p> <p>This extensive MPSS analysis confirms and supplements prior microarray floral expression studies and illustrates the utility of sequence survey-based expression analysis in functional genomics. Spatial floral expression data accrued by MPSS and similar methods will be advantageous in the elucidation of more comprehensive genetic regulatory networks governing floral development.</p

Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.</p> <p>Results</p> <p>We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg <it>erecta </it>(L<it>er</it>) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of <it>R </it>gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied.</p> <p>Conclusion</p> <p>Transcripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.</p