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

Abstract

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.

A novel chloroplast gene reported for flagellate plants

“…Raw reads were processed as described above, and the cleaned reads were mapped to the A. shastense plastome using Tophat 2 (Kim et al., ). We then used ChloroSeq (Castandet et al., ) and custom scripts to identify RNA‐editing sites and to calculate RNA‐editing efficiencies (defined as the percentage of total mapped RNA reads that are edited).…”

The Roles of Mutation, Selection, and Expression in Determining Relative Rates of Evolution in Mitochondrial versus Nuclear Genomes

“…Our search was limited to libraries that were prepared using rRNA depletion strategies (i.e., Ribo-Zero) rather than poly-A selection because mt transcripts are often not polyadenylated and could be highly underrepresented in conventional RNA-seq libraries (Castandet et al 2016). The overall data set used to investigate transcript abundance consisted of nine plant species, one yeast, and two primates (supplementary table S1, Supplementary Material online).…”

Peer Review #3 of "ECuADOR—Easy Curation of Angiosperm Duplicated Organellar Regions, a tool for cleaning and curating plastomes assembled from next generation sequencing pipelines (v0.1)"

“…the dataset assembly); and 3) the actual phylogenetic reconstruction [15]. Current software focuses on organelle reconstruction [e.g 16,17], multiple sequence alignment (MSA) algorithms [e.g 18] and phylogenetic approaches [e.g. 19].…”