RNA editing occurs in the organellar mRNAs of all land plants but the marchantioid liverworts, making liverworts a perfect group for studying the evolution of RNA editing. Here, we profiled the RNA editing of 42 exemplars spanning the ordinal phylogenetic diversity of liverworts, and screened for the nuclear-encoded pentatricopeptide repeat (PPR) proteins in the transcriptome assemblies of these taxa. We identified 7,428 RNA editing sites in 128 organellar genes from 31 non-marchantioid liverwort species, and characterized 25,059 PPR protein sequences.

• Edera Serial Ep 1 Season 1

The abundance of organellar RNA editing sites varies greatly among liverwort lineages, genes, and codon positions, and shows strong positive correlations with the GC content of protein-coding genes, and the diversity of the PLS class of nuclear PPR proteins. IntroductionRNA editing is a co- or posttranscriptional process that changes the content of the transcripts, resulting in sequences of mature RNA transcripts deviating from that of the genomic templates (; ). In plants, this process mainly converts cytidines (C) to uridines (U) in organellar transcripts, and occasionally U to C (; ). Plant organellar RNA editing plays important roles in gene expression and functioning, by restoring start/stop codons, removing internal stop codons, maintaining proper splicing of transcripts, or modifying amino acids. RNA editing occurs in organellar genomes of all land plant lineages except the marchantioid liverworts. In general, angiosperms contain 200–500 RNA editing sites in their mitochondrial (mt) genomes, but only 30–50 editing sites in their plastid (pt) genomes.

Lycophytes possess the highest number of editing sites, with for example, the mt genome of Selaginella moellendorffii holding 2,139 C-to-U RNA editing sites in only 18 protein-coding genes (PCGs) and the pt genome of Selaginella uncinata holding 3,415 such sites in its 82 genes. Mosses have the fewest RNA editing sites, with only 11 and 1 RNA editing sites in the ) and pt genes, respectively, of Physcomitrella patens. RNA editing sites have been characterized in many vascular plants (;,; ), but only in a few nonvascular plants, that is, the liverwort Marchantia polymorpha , the hornworts Anthoceros formosae and Leiosporoceros dussii , and the moss Physcomitrella patens. Thus, empirical data are still scarce to understand the phylogenetic distribution and evolution of RNA editing among nonvascular land plants.The abundance of RNA editing in organellar genes may be determined by many factors.

The RNA editing frequency is clearly associated with the function of the gene (; ), with genes encoding membrane-bound proteins or under strong functional selections holding more RNA editing sites (; ). RNA editing frequency may also be positively linked to the genomic GC content, especially across second codon positions (;;; ), and the diversity of nuclear-encoded pentatricopeptide repeat (PPR) proteins. The PPR proteins, characterized by tandem arrays of a weakly conserved 35 amino-acid motif , form one of the largest gene families in land plants , with most angiosperms encoding 400–600 PPRs. PPR proteins are divided into two major classes, the P and PLS classes, with the latter further subdivided into five groups, namely PLS, E1, E2, E+, and DYW, based on the characteristic of the carboxyterminus.

The majority of PLS-class members may be dedicated to RNA editing processes , especially the DYW type proteins, whose functional cytidine deaminase activities have recently been confirmed. The diversity of DYW domains was reported to be well correlated to the frequency of RNA editing sites in plants (; ). Based on empirical and in silico predictive RNA editing data, the abundance of plastid RNA editing was proposed to be strongly and positively correlated (PCC = 0.9) to the diversity of PLS PPR proteins in 21 green plants. As these correlationships were inferred with simple regression methods without accounting for phylogenetic relationships, which may yield trivial implications on biological relationships. Therefore, comparative methods incorporating the phylogenetic information need to be implemented to further test such correlations.With some 7,300 extant species , liverworts compose a diverse lineage of land plants. Although their relationships to other bryophytes remains somewhat controversial , they undoubtedly arose early in the diversification of land plants, and hence might be critical to unravel the timing and subsequent evolution of various innovations such as RNA editing, a process possibly acquired by the common ancestor of land plants (; ). Here, we screened deep genomic and organellar transcripts enriched transcriptomic sequence data to critically analyze RNA editing in mt and pt genes of 42 liverworts from 13 of the 15 liverwort orders.

We also characterized the diversity of six types of PPR proteins based on transcriptome assemblies. Our study aims to contrast RNA editing site abundance and variation 1) between mt and pt genomes, and 2) among different liverwort clades, and to test for a relationship of RNA editing site abundance 3) with the GC content, 4) and the diversity of PPR proteins. Taxon Sampling and NGS ExperimentsWe sampled fresh collections of 42 liverworts (, online) representing all but 2 of the 15 orders of liverworts. Individual shoots and branches of each accession were separated under a dissecting microscope to avoid contamination from other organisms, cleaned with distilled water, and used for DNA and RNA isolations using the modified CTAB methods following and, respectively. Approximately 1 μg of genomic DNA and RNA were used to generate paired-end sequencing libraries with the insert fragment size of 300–500 and 200–300 bp, respectively.

NGS sequencing was carried out on an Illumina HiSeq 2000 platform at Majorbio (Shanghai, China). The raw sequencing data in fastq format (∼10 and ∼6 G raw data for genomic and transcriptomic sequencing, respectively) were trimmed and filtered for adaptors, low quality reads, undersized inserts, and duplicate reads using Trimmomatic. Genome Assembly and AnnotationThe filtered reads from each species were de novo assembled into contigs (≥1 kb) using the CLC Genomics Workbench v5.5 (CLC Bio, Aarhus, Denmark) using default parameters (word size = 20, bubble size = 50, minimum contig length = 1,000). All contigs were blasted to the Marchantia polymorpha pt and mt genomes (GenBank accession: and ), to retrieve the pt and mt contigs, respectively.

These contigs were elongated and assembled into organellar genome scaffolds using overlapping methods. Ultimately, we assembled 29 and 40 complete mt and pt genomes, respectively. For the remaining 13 mt genomes and the 2 pt genomes, for which corresponding contigs could not be linked due to frequent occurrences of repeated sequences, we anchored the contigs by mapping them to the organellar genomes of Marchantia polymorpha and connected them with Ns to generate the draft genomes.

Edera Serial Ep 1 Season 1

The PCGs of the organellar genome were annotated following the steps described in ). The exact gene and exon/intron boundaries were further confirmed in Geneious v10.0.2 by aligning orthologous genes with the available annotated liverwort organellar genomes (such as Pleurozia, GenBank accession no.: ) to those of the 42 liverworts. RNA Editing Site IdentificationThe DNA-seq reads and RNA-seq reads were mapped to the corresponding draft organellar genome using Bowtie2 and Tophat2 separately as described.

The resulting mapping files in bam format were sorted to generate vcf file using Samtools and Bcftools , from which, the snp file were produced using a custom perl script (Dryad Digital Repository, accession 10.5061/dryad.nzs7h44ms). The RNA mapping files were processed using Redo using default parameters. The Redo output of the RNA editing site annotation file was then manually filtered against the SNP sites by their positions on the genome sequence to remove potential genomic SNPs. The final annotation file of the RNA editing sites and the corresponding transcriptome mapping bam file were imported in Geneious v10.0.2 to manually check the bioinformatically identified editing sites and make necessary revisions to further reduce false positives and false negatives.

Correlation Tests between the Number of RNA Editing Site and PPR Protein Diversity/GC ContentTo assess the evolutionary correlationships between the abundance of RNA editing sites and the diversity of PPR proteins, as well as the GC content. We used the phylogenetic generalized least-squares (PGLS) regression, allowing λ to be fitted by maximum likelihoods. In all analyses, variables were log transformed to make them normally distributed. We calculated the variance–covariance matrix for the ML tree derived from 127 organellar genes using “corPagel” as implemented in the R package “ape” , and then fit the regression by maximizing the restricted log-likelihood using “gls” as implemented in the R package “nlme”. RNA Editing Site Abundance in LiverwortsOur genomic and transcriptomic data yielded high read coverage for both mt and pt PCGs (, online). Altogether, we identified 7,428 C-to-U RNA editing sites from the 128 organellar PCGs of 33 liverworts, including 4,694 RNA editing sites for 42 mt PCGs (, online) and 2,734 RNA editing sites for 86 pt PCGs (, online).

The average number of RNA editing sites per mt gene (3.39) is more than three times that per pt gene (0.96). No RNA editing was detected from either organellar genome of any complex thalloid (Marchantiopsida) species.The number of organellar RNA editing sites varies significantly among liverwort lineages, and even among different accessions of the same species. The Haplomitriales (with Haplomitrium sampled here), widely recognized as emerging from the deepest splits in liverworts, has the highest editing level, whereas its sister group, the Treubiales, has the fewest editing sites among all liverworts with the exception of the complex thalloids (Marchantiopsida), which completely lack editing sites. The simple thalloids, that is, Pelliidae and Metzgeriidae, exhibit the second highest RNA editing frequency, followed by the Jungermanniales, except for two Herbertus species with a low editing level. —( a) Phylogenetic tree of liverworts based on a concatenated nucleotide data set of 41 mt and 86 pt genes. Branches are maximally supported (i.e., 100% bootstrap frequencies) unless otherwise marked.

( b) Heatmap of PPR protein diversity across liverworts. ( c) Histogram of RNA editing site abundance in mt (pink) and pt (green) genomes for each liverwort accession.The abundance of RNA editing also varies among genes and functional categories within liverworts (, online), with high editing level in the respiration complex and photosynthesis related protein genes, and low editing level in ribosomal protein genes, a pattern consistent with that observed in angiosperms.

RNA editing sites show relatively higher editing efficiencies in the second and first codon positions compared with the third codon positions (, online), and occur primarily in the second codon positions of both mt and pt genes (, online), leading to amino acid changes that primarily increase the hydrophobicity of proteins (, online). Such modifications may be essential for protein folding, functioning, and interacting with other proteins. In Arabidopsis thaliana , Physcomitrella patens , and maize (; ), the disruption of RNA editing activity severely impacts plant growth at specific developmental stages. Testing the significance of RNA editing process on the fitness of liverworts may await the development of tools for genetically transforming liverworts, but the development of Marchantia as a model system (; ) contributes little to such understanding since it lacks organellar RNA editing ability. Hence, other liverwort model systems must be developed.

PPR Protein Diversity in LiverwortsWe identified 25,059 PPR protein sequences (i.e., 165 P and 19,894 PLS class proteins) in the transcriptome assembly of the 43 liverworts (including the proteome of Marchantia polymorpha downloaded from phytozome.jgi.doe.gov;, online). The diversity of PPR proteins varies considerably among different liverwort lineages. Among the five groups of PLS class PPR proteins (PLS, E1, E2, E+, and DYW), the PLS group contributes most of the diversity (∼80%), and shows greatest variations in number (2–2,213) among liverworts. The other four groups of PLS PPR proteins (, online) are generally absent in charophycean algae and complex thalloid liverworts, with the exception of the occasional presence of E1 and E2 proteins in three complex thalloid liverworts. However, the E2 type proteins from the complex thalloid group (i.e., Wiesnerella denudata and Monosolenium tenerum II) form a structure with a tandem repeat of P type motifs plus a single E2 motif, and the E1 type protein from Asterella wallichiana forms a structure of L-S-E1.

All these E-type PPR proteins from the complex thalloids lack the PLS triplet structure that characterizes the PLS PPR proteins. The absence of the functional E and DYW type PLS PPR proteins observed in complex thalloids might be associated with their exclusive absence of RNA editing sites. As have suggested, plant E and E+ type PLS proteins are likely products of serial carboxyterminal domain deletions from the DYW type proteins, the presence of a few truncated E type PLS protein sequences in complex thalloids might imply they are from relics of functional genes, thus providing new evidence for a secondary loss of RNA editing machinery in this group. Factors Correlated with the Number of RNA Editing SitesBased on the in silico prediction of RNA editing sites in three mt PCGs and PCR amplifications of DYW type protein genes in over 100 bryophyte species, the diversity of the DYW type PPR protein genes appears to be positively correlated with the abundance of RNA editing sites (; ). Such a correlation of RNA editing site abundance and PPR protein diversity is here confirmed across all organellar genes within liverworts.

In general, the number of RNA editing sites in mt and pt genes is strongly and positively correlated to the diversity of nuclear PLS class rather than P class PPR proteins. All the five types of PLS PPR proteins, but the DYW type, show significantly positive correlations with the RNA editing site abundance for both mt and pt genes. As there is an experimentally proved functional association between DYW type PPRs and RNA editing , the lack of evidence for their correlationships is unclear and should be further tested with expanded samplings. StatisticsLambda (λ)SlopeP valuesPPPR/MTE0.9963−0.PPPR/PTE0.0.5409PLSPPR/MTE0.0.0000.PLSPPR/PTE0.0.0000.PLS/MTE0.0.0000.PLS/PTE0.0.0000.E1/MTE0.0.0000.E1/PTE0.0.0000.E2/MTE0.0.0014.E2/PTE0.0.0000.E+/MTE0.0.0001.E+/PTE0.0.0000.DYW/MTE0.9967−0.DYW/PTE0.0.2383MGC/MTE0.0.0006.MGC1/MTE10.0.0369.MGC2/MTE20.90.0000.MGC3/MTE30.0.0639PGC/PTE0.0.0003.PGC1/PTE11.0.8204PGC2/PTE20.90.0000.PGC3/PTE30.0.0006. The authors are grateful to the two anonymous reviewers for their constructive suggestions on the manuscripts. We thank David Glenny for collecting Treubia lacunosa from New Zealand, Qin Zuo for collecting Fossombronia cristula. We also thank Yang Peng for assisting with the lab experiments, Pu Liang, Fujuan Song, and Manhua Lin from the Shenzhen Volunteer Association for help cleaning the liverwort materials.

This project is funded by the National Natural Science Foundation of China (NSFC-31470314 and NSFC-31600171).

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