Frequent chloroplast RNA editing in early-branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors

Abstract

Background: RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30-50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles.

Results: With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs.

Conclusion: Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

Fig. 1

Map of the Amborella trichopoda plastome drawn with the OGDRAW tool [75]. Different colors indicate functional gene categories as indicated in the legend. Numbers of non-silent (bold) and silent (behind the plus symbol) C-to-U RNA-editing sites identified in the respective cDNAs are indicated next to each protein-coding gene. Italics indicate genes transcribed clockwise (inner circle) and normal font indicating genes transcribed counterclockwise (outer circle). Larger than (>) and smaller than (<) labels indicate the creation of start or stop codons by RNA editing, respectively

Fig. 2

RNA editing in the chloroplast ndhD locus. Arrowheads indicate editing events in Amborella trichopoda (top) and Arabidopsis thaliana (bottom) reconstituting conserved amino acid identities (shaded, bold). Further editing events identified in other taxa (Table 1) are indicated by the pipe symbol (|) and likewise reconstitute conserved amino acids. Two further candidate edits ndhDeU145HY and ndhDeU1424TI (italics) in Zingiber spectabile remain to be investigated

Fig. 3

Venn diagram showing the occurrence of chloroplast RNA editing sites in Amborella trichopoda, Arabidopsis thaliana and Cucumis sativus. Editing position numbering refers to Amborella for shared sites. Underlining indicates RNA editing sites in Cucumis predicted and confirmed in the course of this study to extend the cucumber cp editome reported previously [32]. Highlighted in bold are sites ndhBeU467PL and ndhDeU878SL previously shown to be affected by editing factor CRR28 [37] and accDeU923SL, corresponding to Arabidopsis thaliana accDeU794SL, shown to be affected by editing factor RARE1 [39]

Fig. 4

Cladogram of 65 selected angiosperms for which reliable protein model and/or genome data are available, based on current insights on angiosperm phylogeny with orders indicated. No chloroplast genome data are currently available for taxa marked with an asterisk and for Cajanus cajan (pigeon pea). The ndhB and ndhD genes of the latter have been analyzed individually in this study, however. Chloroplast editing sites ndhBeU467PL and ndhDeU878SL and their cognate specificity co-factor CRR28 are widely distributed among angiosperms except cases where labels are attached. Editing event ndhDeU878SL has been lost independently (filled squares) in Eucalyptus, Fragaria, Cicer, Nicotiana and in the palms (Arecales). Editing event ndhBeU467PL has been lost independently (filled circles) in Linum and in a subclade of Fabales including chickpea (Cicer arietinum). CRR28 homologues are identified in all taxa (Additional file 3) except in chickpea where both editing sites are absent

Fig. 5

Cladogram of 65 flowering plants as in Fig. 4. Chloroplast RNA editing site accDeU923SL (or the accD gene altogether) is lost at least 14 times independently during angiosperm evolution (no plastome sequences are available for species marked with an asterisk). The loss of the accDeU923SL editing event is consistently accompanied by an apparent absence of RARE1 (black triangle) whereas RARE1 orthologues are always identified in the other taxa (Additional file 4)

Fig. 6

Matching key PPR residues with target sequences for RARE1 and CRR28. Relevant key residues in PPR positions 3, 6 and 1’ were selected from the total alignments of 35 RARE 1 (A) and 64 CRR28 (B) orthologues in angiosperms to obtain weblogo conservation plots at http://weblogo.berkeley.edu/logo.cgi [76]. Target sequences are aligned with the terminal S-type PPR (which contributes position 1’ to the preceding L-type PPR) juxtaposed with nucleotide -4 in front of the C-to-U editing site (underlined). Ambiguities are indicated where represented at least twice in the corresponding targets. Green shading indicates perfect matching of positions 6 and 1’ in P- and S-type repeats with corresponding nucleotides according to the proposed core binding code (T + N:A, N + N:C, T + D:G, N + D:U). Blue matching indicates distinction of pyrimidines vs. purines in position 6 only and grey shading indicates mismatches. Allowing for replacement of threonine by serine matches RARE1 PPR S4 (S + D) with G in its target and CRR28 (S + N) with A in its targets, but also mismatches RARE1 P4 (S + N) with U