ChloroSeq, an Optimized Chloroplast RNA-Seq Bioinformatic Pipeline, Reveals Remodeling of the Organellar Transcriptome Under Heat Stress.

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsis thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.

Castandet B ，Hotto AM ，Strickler SR ，Stern DB ... -  《G3-Genes Genomes Genetics》

A Guide to the Chloroplast Transcriptome Analysis Using RNA-Seq.

Michel EJS ，Hotto AM ，Strickler SR ，Stern DB ，Castandet B ... -  《-》

Unraveling chloroplast transcriptomes with ChloroSeq, an organelle RNA-Seq bioinformatics pipeline.

Online sequence repositories are teeming with RNA sequencing (RNA-Seq) data from a wide range of eukaryotes. Although most of these data sets contain large numbers of organelle-derived reads, researchers tend to ignore these data, focusing instead on the nuclear-derived transcripts. Consequently, GenBank contains massive amounts of organelle RNA-Seq data that are just waiting to be downloaded and analyzed. Recently, a team of scientists designed an open-source bioinformatics program called ChloroSeq, which systemically analyzes an organelle transcriptome using RNA-Seq. The ChloroSeq pipeline uses RNA-Seq alignment data to deliver detailed analyses of organelle transcriptomes, which can be fed into statistical software for further analysis and for generating graphical representations of the data. In addition to providing data on expression levels via coverage statistics, ChloroSeq can examine splicing efficiency and RNA editing profiles. Ultimately, ChloroSeq provides a well-needed avenue for researchers of all stripes to start exploring organelle transcription and could be a key step toward a more thorough understanding of organelle gene expression.

Smith DR ，Sanitá Lima M 《-》

Strand-specific RNA sequencing uncovers chloroplast ribonuclease functions.

Organellar gene expression incorporates ribonucleases as indispensable participants. Here, we explored the capacity of strand-specific RNA sequencing (RNA-Seq) as a tool to analyze chloroplast ribonuclease functions using the 3'→5' exoribonuclease polynucleotide phosphorylase (PNPase) as a proof of concept. The role of PNPase in transcript 3' end maturation was easily monitored, and additional targeted mRNAs were discovered. Moreover, a role in tRNA precursor degradation was predicted and validated. These results, together with previously published data, suggest that RNA-Seq represents a unique opportunity to decipher the roles of organellar ribonucleases and deepen our understanding of chloroplast gene expression.

Castandet B ，Hotto AM ，Fei Z ，Stern DB ... -  《-》

The chloroplast RNA helicase ISE2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana.

INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is a chloroplast-localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE2 expression have previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE2's role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE2 from Arabidopsis thaliana leaves resulted in defects in C-to-U RNA editing, altered accumulation of chloroplast transcripts and chloroplast-encoded proteins, and defective processing of chloroplast ribosomal RNAs. Potential ISE2 substrates were identified by RNA immunoprecipitation followed by next-generation sequencing (RIP-seq), and the diversity of RNA species identified supports ISE2's involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE2 is a non-canonical Ski2-like RNA helicase that represents a separate sub-clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE2's evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression.

Bobik K ，McCray TN ，Ernest B ，Fernandez JC ，Howell KA ，Lane T ，Staton M ，Burch-Smith TM ... -  《-》