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

ABSTRACT: 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.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.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.

SUBMITTER: Hein A

PROVIDER: S-EPMC4727281 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Hein Anke A Polsakiewicz Monika M Knoop Volker V

BMC evolutionary biology 20160125

<h4>Background</h4>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 pentatricop ...[more]

PMID: 26809609

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Project description:BackgroundGene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process.ResultsUsing tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein).ConclusionsEditing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.

Dataset Information

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

Publications

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

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