One C-to-U RNA Editing Site and Two Independently Evolved Editing Factors: Testing Reciprocal Complementation with DYW-Type PPR Proteins from the Moss Physcomitrium (Physcomitrella) patens and the Flowering Plants Macadamia integrifolia and Arabidopsis

Abstract

Cytidine-to-uridine RNA editing is a posttranscriptional process in plant organelles, mediated by specific pentatricopeptide repeat (PPR) proteins. In angiosperms, hundreds of sites undergo RNA editing. By contrast, only 13 sites are edited in the moss Physcomitrium (Physcomitrella) patens Some are conserved between the two species, like the mitochondrial editing site nad5eU598RC. The PPR proteins assigned to this editing site are known in both species: the DYW-type PPR protein PPR79 in P. patens and the E+-type PPR protein CWM1 in Arabidopsis (Arabidopsis thaliana). CWM1 also edits sites ccmCeU463RC, ccmBeU428SL, and nad5eU609VV. Here, we reciprocally expressed the P. patens and Arabidopsis editing factors in the respective other genetic environment. Surprisingly, the P. patens editing factor edited all target sites when expressed in the Arabidopsis cwm1 mutant background, even when carboxy-terminally truncated. Conversely, neither Arabidopsis CWM1 nor CWM1-PPR79 chimeras restored editing in P. patens ppr79 knockout plants. A CWM1-like PPR protein from the early diverging angiosperm macadamia (Macadamia integrifolia) features a complete DYW domain and fully rescued editing of nad5eU598RC when expressed in P. patens. We conclude that (1) the independently evolved P. patens editing factor PPR79 faithfully operates in the more complex Arabidopsis editing system, (2) truncated PPR79 recruits catalytic DYW domains in trans when expressed in Arabidopsis, and (3) the macadamia CWM1-like protein retains the capacity to work in the less complex P. patens editing environment.

Figure 1.

Characterization of Arabidopsis CWM1 and P. patens PPR79. (A) and (B) Models of the DYW-type and E+-type PPR proteins P. patens PPR79 (A) and Arabidopsis CWM1 (B) aligned to the assigned targets of both species. The nomenclature of RNA editing sites is based on Rüdinger et al., (2009) starting with the gene name (nad5), RNA editing from C-to-U (eU), the position of the C in the transcript counted from the first position of the start codon, and the amino acid before and after editing, here Arg (R) to Cys (C). Both PPR proteins feature an array of PLS-repeats downstream of the N-terminal mitochondrial target peptide (red). The last PLS triplet, differing in amino acid conservation, is labeled P2L2S2 according to Cheng et al., (2016). The PPR array is numbered backward starting from the terminal S2 repeat (Hein and Knoop, 2018). The respective fifth and last amino acid positions, critical for PPR-RNA interaction, are displayed below each PPR. DYW, DYW domain; E1 and E2, E motifs. Shown below the PPR protein model are aligned pre-mRNA targets of nad5eU598RC from P. patens and Arabidopsis and ccmBeU428SL, ccmCeU463RC, and nad5eU609VV from Arabidopsis. Ribonucleotides are shaded according to the PPR-RNA binding code proposed by Barkan et al. (2012) based on amino acid identities at the fifth and last amino acid positions: T/S+N: A, T/S+D: G, N+D: U, N+S: C, N+N: Y. Colored backgrounds indicate a match in green, a transition among pyrimidines (Y) or purines (R) in light blue, and a mismatch in magenta shading, respectively. The L-motifs and juxtaposed ribonucleotides are indicated by light gray shading. The ribonucleotides are numbered backward, starting from the editing site (underlined and capitalized C). RNA editing efficiencies in the wild type (WT) and mutant lines for Arabidopsis and P. patens are displayed next to the RNA stretch. The slash indicates editing efficiencies of different plant lines (Arabidopsis: cwm1-1 [left], cwm1-2 [right]; P. patens: ecotype Gransden [left], ecotype Reute [right]). Mean values are displayed for silent site nad5eU609VV, as variability in RNA editing has been observed in Col-0 (100%/80%/78%) and in the mutant lines (cwm1-1: 0%/42%/40%; cwm1-2: 0%/36%/39%). Additional Cs in the RNA targets undergoing C-to-U RNA editing are underlined.

Figure 2.

Phenotypic Analysis of P. patens ppr79 ko. (A) Comparison of 4-week old gametophytes for the wild type (WT) and ppr79 ko of the two P. patens ecotypes Gransden and Reute grown on Knop agar. Bar = 1 mm. Both the wild-type Reute and ko Reute ppr79 can produce viable sporophytes. Bar = 1 mm. (B) Boxplots displaying the average number of mature (postmeiotic, white) and immature (premeiotic, dark gray) sporophytes developed per individual gametophyte (n = 35 plantlets). Center lines, medians; box limits, 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range; individual data points are plotted as open gray circles. ppr79 Reute ko plants show a significant reduction of mature (P < 3.7 × 10⁻¹⁶) and immature (P < 1.1 × 10⁻⁰³) sporophytes per plant.

Figure 3.

Comparative Analysis of the Mitochondrial Proteomes of the Wild-Type P. patens Ecotype Reute and ppr79 Reute ko. Proteins were separated by 2D BN/SDS polyacrylamide gel electrophoresis and stained with silver. The molecular masses of standard proteins are given in between the two 2D gels. The identities of the OXPHOS complexes are indicated above the gels: V, complex V, ∼650 kD; III₂, dimeric complex III, ∼500 kD; I+III₂, supercomplex formed of complex I and dimeric complex III, ∼1500 kD; F1, F1-part of complex V, ∼300 kD; IV, complex IV, ∼200 kD; and II, complex II, ∼160 kD. Complexes F1, III₂, V, and supercomplex I+III₂ were identified based on visual comparison to Arabidopsis 2D BN/SDS gels (Klodmann et al., 2011) and by mass spectrometric analysis (Supplemental Figure 2). Chloroplast contaminants, that are subunits of PSI, PSII, and the light-harvesting complex II (LHCII), are indicated by magenta circles. Supercomplex I+III₂ position is indicated by a blue frame in the wild-type (WT) Reute and the corresponding position in ppr79 Reute ko by a gray frame.

Figure 4.

Evolution of RNA Editing Factor CWM1 in Angiosperms. Cladogram of 123 angiosperms. Asterids, Proteales, Ranunculales, Caryophyllales, and Liliopsida have been collapsed in the left panel and Rosids in the right panel. The cladogram is based on the current understanding of angiosperm phylogeny (Open Tree of Life; The Angiosperm Phylogeny Group, 2009; Hinchliff et al., 2015). Putative CWM1 orthologs were identified for all species except the ones marked by red branches. Most identified putative orthologs belong to the E+-subclass of PPR proteins (Cheng et al., 2016). Only species in the early-branching Caryophyllales, Proteales, and Ranunculales feature C-terminal DYW domains (marked in blue). A remaining DYW coding sequence was also identified downstream of the CWM1 stop codon in P. americana (underlined).

Figure 5.

Overview of CWM1-Associated RNA Editing Targets in Selected Angiosperms. Overview of CWM1-associated RNA editing targets (nomenclature based on Arabidopsis CDS) in selected angiosperms with (+) or without a putative CWM1 ortholog (–). Related proteins with an apparent complete DYW domain are indicated by a red +. Black dots: RNA editing verified by RT-PCR at the respective position. Gray dots, potential RNA editing sites (cytidines at DNA level); open circles, unedited cytidines; X, pre-edited state with a thymidine in the mitochondrial genome.

Figure 6.

Phylogenetic Analysis of the PPR Proteins CWM1 from Arabidopsis and PPR79 from P. patens. Phylogram of 103 angiosperm putative CWM1 orthologs, including Arabidopsis CWM1 (collapsed; see Supplemental Figure 5B for the full angiosperm branch), 35 putative orthologs of P. patens PPR79 (collapsed for 21 Hypnales; see Supplemental Figure 5A for the uncondensed moss branch), 27 Arabidopsis DYW-type PPR proteins identified as editing factors (collapsed; listed in Methods), and all 9 DYW-type PPR P. patens paralogs (Schallenberg-Rüdinger et al., 2013). Accession numbers for the moss proteins are displayed behind the species name (e.g., VBMM-2051258 for OneKP accessions or c24535 g1 i1 m22706 for accessions from Johnson et al., 2016). The Maximum Likelihood phylogenetic tree was calculated with IQ-tree (http://iqtree.cibiv.univie.ac.at/; Trifinopoulos et al., 2016) using the JTT+F+R6 model of sequence evolution. The alignment of the terminal domains (P2L2S2-E1-E2-DYW) used for tree construction contained 283 parsimony informative sites (Supplemental Data Set 5). Node confidence was determined from 1000 bootstrap replicates and is shown at nodes where exceeding 70%.

Figure 7.

Functional Complementation Test of Arabidopsis cwm1 Mutant Lines. (A) RNA editing in the Arabidopsis cwm1 mutants can be restored by transformation with the Arabidopsis CWM1 gene, different versions of P. patens PPR79, and chimeras between the two editing factor genes. Red and blue backgrounds indicate species origins for creation of chimeric proteins. All constructs are listed in Supplemental Table 4. (B) Chromatopherograms of all known CWM1 targets in the respective top transgenic lines (with highest editing efficiencies for nad5eU598RC) in the Arabidopsis cwm1 mutant background. Percentages of C-to-U conversion are given below. The editing positions under consideration are highlighted by underlining and red shading. Partially edited sites are labeled as pyrimidine ambiguities (Y). Full editing data for all transgenic lines are listed in Supplemental Table 2.

Figure 8.

Functional Complementation Test of the P. patens ppr79 ko. (A) Physcomitrium patens PPR79, the Funaria hygrometrica putative PPR79 ortholog, Arabidopsis CWM1, and chimeric constructs between P. patens PPR79 and Arabidopsis CWM1 (background coloring as in Figure 7) were introduced into the ppr79 ko line. All constructs are listed in Supplemental Table 4. (B) Only full-length PPR79 and its putative ortholog from F. hygrometrica complement the ppr79 ko. RNA editing efficiencies of nad5eU598RC of the ppr79 transgenic lines are given. Results of one independent transgenic line each are displayed as chromatopherograms (others are listed in Supplemental Table 3), which are labeled according to Figure 7. Single gametophores of ppr79 ko transgenic lines were grown on non-selective Knop media for 8 weeks in long-day conditions. Bars = 1 mm. Transgenic lines with restored RNA editing at nad5eU598RC display the wild-type phenotype, while transgenic lines expressing CWM1 or CWM1+PPR79 fusion constructs display the mutant phenotype (Supplemental Figure 8).

Figure 9.

CWM1 Ortholog from Macadamia Can Reconstitute RNA Editing in Arabidopsis and P. patens. (A) Macadamia CWM1 PPR array compared to Arabidopsis CWM1. The fifth and last positions differing from Arabidopsis CWM1 are indicated by blue letters. The N-terminal sequence of Arabidopsis CWM1 used for transformation is highlighted with red shading. PPR binding code matches of macadamia CWM1 and candidate RNA targets of macadamia, Arabidopsis (nad5eU598RC, ccmBeU428SL, ccmCeU463RC, and nad5eU609VV), and P. patens (nad5eU598RC) are shown with shading and labeling of PPR-RNA interactions as in Figure 1. (B) RNA editing efficiencies of the respective CWM1 targets in macadamia and transgenic lines in the Arabidopsis cwm1 mutant or P. patens ppr79 ko expressing macadamia CWM1, displayed as chromatopherograms and percentage of C-to-U conversion as in Figures 7 and 8.

Figure 10.

PPR79 Variants with Mutations Expected to be Relevant for RNA Binding Can Fully Rescue RNA Editing in the ppr79 ko. Individually modified PPRs are shown in color in the PPR79 model. Protein modifications and resulting change in PPR-RNA interaction are displayed below the respective native PPR79 repeats. The color code follows the PPR-RNA binding code proposed by Barkan et al. (2012) as in Figure 1. The resulting RNA editing efficiencies remain 100% at the nad5eU598RC site in at least two independent lines for each of the six mutants.