Full length transcriptome highlights the coordination of plastid transcript processing

Abstract

Plastid gene expression involves many post‐transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short‐read RNA‐Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full‐length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand‐specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post‐transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2,500 nucleotides were missing, the study of the co‐occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.