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Key Message: Upon loss of either its chloroplast or mitochondrial target, a uniquely dual-targeted factor for C-to-U RNA editing in angiosperms reveals low evidence for improved molecular adaptation to its remaining target. RNA-binding pentatricopeptide repeat (PPR) proteins specifically recognize target sites for C-to-U RNA editing in the transcriptomes of plant chloroplasts and mitochondria. Among more than 80 PPR-type editing factors that have meantime been characterized, AEF1 (or MPR25) is a special case given its dual targeting to both organelles and addressing an essential mitochondrial (nad5eU1580SL) and an essential chloroplast (atpFeU92SL) RNA editing site in parallel in Arabidopsis. Here, we explored the angiosperm-wide conservation of AEF1 and its two organelle targets. Despite numerous independent losses of the chloroplast editing site by C-to-T conversion and at least four such conversions at the mitochondrial target site in other taxa, AEF1 remains consistently conserved in more than 120 sampled angiosperm genomes. Not a single case of simultaneous loss of the chloroplast and mitochondrial editing target or of AEF1 disintegration or loss could be identified, contrasting previous findings for editing factors targeted to only one organelle. Like in most RNA editing factors, the PPR array of AEF1 reveals potential for conceptually "improved fits" to its targets according to the current PPR-RNA binding code. Surprisingly, we observe only minor evidence for adaptation to the mitochondrial target also after deep losses of the chloroplast target among Asterales, Caryophyllales and Poales or, vice versa, for the remaining chloroplast target after a deep loss of the mitochondrial target among Malvales. The evolutionary observations support the notion that PPR-RNA mismatches may be essential for proper function of editing factors.

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. [ABSTRACT FROM AUTHOR]

Background: C-to-U RNA editing in mitochondria and chloroplasts and the nuclear-encoded, RNA-binding PPR proteins acting as editing factors present a wide field of co-evolution between the different genetic systems in a plant cell. Recent studies on chloroplast editing factors RARE1 and CRR28 addressing one or two chloroplast editing sites, respectively, found them strictly conserved among 65 flowering plants as long as one of their RNA editing targets remained present. Results: Extending the earlier sampling to 117 angiosperms with high-quality genome or transcriptome data, we find more evidence confirming previous conclusions but now also identify cases for expected evolutionary transition states such as retention of RARE1 despite loss of its editing target or the degeneration of CRR28 truncating its carboxyterminal DYW domain. The extended angiosperm set was now used to explore CLB19, an "E+"-type PPR editing factor targeting two chloroplast editing sites, rpoAeU200SF and clpPeU559HY, in Arabidopsis thaliana. We found CLB19 consistently conserved if one of the two targets was retained and three independent losses of CLB19 after elimination of both targets. The Ericales show independent regains of the ancestrally lost clpPeU559HY editing, further explaining why multiple-target editing factors are lost much more rarely than single target factors like RARE1. The retention of CLB19 despite loss of both editing targets in some Ericaceae, Apocynaceae and in Camptotheca (Nyssaceae) likely represents evolutionary transitions. However, the retention of CLB19 after a phylogenetic deep loss in the Poaceae rather suggests a yet unrecognized further editing target, for which we suggest editing event ndhAeU473SL. Conclusion: Extending the scope of studies on plant organelle RNA editing to further taxa and additional nuclear cofactors reveals expected evolutionary transitions, strikingly different evolutionary dynamics for multiple-target editing factors like CLB19 and CRR28 and suggests additional functions for editing factor CLB19 among the Poaceae. [ABSTRACT FROM AUTHOR]