A Comprehensive Evolutionary Study of Chloroplast RNA Editing in Gymnosperms: A Novel Type of G-to-A RNA Editing Is Common in Gymnosperms

Abstract

Although more than 9100 plant plastomes have been sequenced, RNA editing sites of the whole plastome have been experimentally verified in only approximately 21 species, which seriously hampers the comprehensive evolutionary study of chloroplast RNA editing. We investigated the evolutionary pattern of chloroplast RNA editing sites in 19 species from all 13 families of gymnosperms based on a combination of genomic and transcriptomic data. We found that the chloroplast C-to-U RNA editing sites of gymnosperms shared many common characteristics with those of other land plants, but also exhibited many unique characteristics. In contrast to that noted in angiosperms, the density of RNA editing sites in ndh genes was not the highest in the sampled gymnosperms, and both loss and gain events at editing sites occurred frequently during the evolution of gymnosperms. In addition, GC content and plastomic size were positively correlated with the number of chloroplast RNA editing sites in gymnosperms, suggesting that the increase in GC content could provide more materials for RNA editing and facilitate the evolution of RNA editing in land plants or vice versa. Interestingly, novel G-to-A RNA editing events were commonly found in all sampled gymnosperm species, and G-to-A RNA editing exhibits many different characteristics from C-to-U RNA editing in gymnosperms. This study revealed a comprehensive evolutionary scenario for chloroplast RNA editing sites in gymnosperms, and reported that a novel type of G-to-A RNA editing is prevalent in gymnosperms.

Keywords: G-to-A; GC content; RNA editing; chloroplast; gymnosperms.

Figure 1

Examples of identification and validation of G-to-A and C-to-U RNA editing sites. The left three panels represent G-to-A RNA editing sites (a–c), and the last two panels indicate C-to-U RNA editing sites with low RNA-seq read coverage (d,e). Editing sites are indicated by arrows. From top to bottom shows species name; genic and plastomic positions of RNA editing sites; RNA editing sites and their upstream and downstream sequences; the mapping of DNA-seq and RNA-Seq data on plastomes, numbers on the left-top show read coverage; sequences of cDNA clones; and Sanger sequencing of some examples of cDNA clones.

Figure 2

Phylogenetic tree of 19 gymnosperms with associated information of RNA editing sites. (a) Cladogram of 19 gymnosperms based on Ran et al. [27,34]. (b) Histogram of chloroplast RNA editing site abundance across gymnosperms.

Figure 3

Comparative analyses of editing efficiency, distribution and codon position of chloroplast C-to-U (a) and G-to-A (b) RNA editing sites among five gymnosperm lineages.

Figure 4

Comparative characterization of RNA editing sites of chloroplast C-to-U and G-to-A RNA editing sites among five gymnosperm lineages. (a) Non-synonymous and synonymous changes; (b) amino acid alteration.

Figure 5

Context (123 sites) flanking the focal C-to-U (a) and G-to-A RNA editing sites (b) among five gymnosperm lineages. The sequence logos were generated using the “ggseqlogo” package [35].

Figure 6

UpsetR plot illustrating the intersection of chloroplast C-to-U RNA editing sites of different gymnosperm lineages. The bottom-left bar plot shows the number of RNA editing sites in the CDS.

Figure 7

Ancestral state reconstruction of chloroplast C-to-U RNA editing sites during the evolution of gymnosperms. Numbers above the branches denote number of gain and loss events of C-to-U RNA editing sites in the CDS regions, and numbers below the branches are numbers of genes (14 genes containing C-to-U RNA editing sites in at least ten species in 19 gymnosperm species, details see Table S3) gained and lost C-to-U RNA editing sites, respectively. Values in parentheses are the number of C-to-U RNA editing sites detected in the CDS regions.

Figure 8

Correlation between the number of chloroplast RNA editing sites and some factors. (a) Plastomic size; (b) GC content; (c) C content; (d) dN (nonsynonymous substitution rates); (e) dS (synonymous substitution rates); (f) RN × 1000 (RN: absolute nonsynonymous substitution rates per branch); and (g) RS × 1000 (RS: absolute synonymous substitution rates per branch).