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. 2019 Sep 13;181(3):891–900. doi: 10.1104/pp.19.00922

Establishment of a Heterologous RNA Editing Event in Chloroplasts1,[OPEN]

Filomena Vanessa Loiacono 1,2, Wolfram Thiele 1, Mark Aurel Schöttler 1, Michael Tillich 1, Ralph Bock 1,4,3
PMCID: PMC6836845  PMID: 31519789

An Arabidopsis PPR protein fully edits a heterologous editing site from spinach in tobacco chloroplasts.

Abstract

In chloroplasts and plant mitochondria, specific cytidines in mRNAs are posttranscriptionally converted to uridines by RNA editing. Editing sites are recognized by nucleus-encoded RNA-binding proteins of the pentatricopeptide repeat (PPR) family, which bind upstream of the editing site in a sequence-specific manner and direct the editing activity to the target position. Editing sites have been lost many times during evolution by C-to-T mutations. Loss of an editing site is thought to be accompanied by loss or degeneration of its cognate PPR protein. Consequently, foreign editing sites are usually not recognized when introduced into species lacking the site. Previously, the spinach (Spinacia oleracea) psbF-26 editing site was introduced into the tobacco (Nicotiana tabacum) plastid genome. Tobacco lacks the psbF-26 site and cannot edit it. Expression of the “unedited” PsbF protein resulted in impaired PSII function. In Arabidopsis (Arabidopsis thaliana), the PPR protein LPA66 is required for editing at psbF-26. Here, we show that introduction of the Arabidopsis LPA66 reconstitutes editing of the spinach psbF-26 site in tobacco and restores a wild-type-like phenotype. Our findings define the minimum requirements for establishing new RNA editing sites and suggest that the evolutionary dynamics of editing patterns is largely explained by coevolution of editing sites and PPR proteins.

C-to-U RNA editing in plants was first discovered in mitochondria (Covello and Gray, 1989; Gualberto et al., 1989) and soon afterward also in plastids (chloroplasts; Hoch et al., 1991). Since then, C-to-U editing has been detected in plastids and mitochondria of virtually all embryophytes analyzed, with the Marchantiid subclade of liverworts being the only exception (Malek et al., 1996). The origin of C-to-U editing probably dates back to the colonization of terrestrial habitats by early embryophytes. Although green algae lack C-to-U editing, hundreds or even thousands of editing sites are found in species from evolutionarily old embryophyte lineages such as the hornwort Anthoceros formosae, the fern Adiantum capillus-veneris, and the spike moss Selaginella uncinata (Freyer et al., 1997; Kugita et al., 2003; Wolf et al., 2004; Tillich et al., 2006a; Smith, 2009; Oldenkott et al., 2014). By contrast, typical angiosperms edit only 30 to 40 sites in their chloroplast genomes. Editing sites are frequently lost during evolution with an average speed of one site per 2.43 million years (Fujii and Small, 2011). Consequently, even closely related species or different ecotypes of the same species can exhibit different patterns of editing sites (referred to as editotypes; Freyer et al., 1995; Freyer et al., 1997; Schmitz-Linneweber et al., 2002; Sasaki et al., 2003; Tillich et al., 2005; Germain et al., 2015). For instance, deadly nightshade (Atropa belladonna) and tobacco (Nicotiana tabacum) possess 35 and 37 plastid editing sites, respectively, but only 32 of these occur in both species, although the two species belong to the same family (Kahlau et al., 2006).

Because of the limited evolutionary conservation of editing sites, a number of previous studies have investigated if editing activity is maintained despite the loss of an editing site by C-to-T mutation. To take advantage of the possibility to manipulate the plastid genome of tobacco plants, several heterologous editing sites from other species such as spinach (Spinacia oleracea; Bock et al. 1994), maize (Zea mays; Reed and Hanson, 1997), and tomato (Solanum lycopersicum; Karcher et al., 2008) were introduced into tobacco plastids. Although in a few cases editing at the heterologous sites could be detected (especially, across short evolutionary distances, i.e. within the Solanaceae family; Tillich et al., 2006b; Karcher et al., 2008), in most cases, the heterologous sites remained unprocessed (Bock et al., 1994; Reed and Hanson, 1997). Thus, loss of an editing site is usually accompanied by the loss of the capacity to edit this site. This conclusion gained further support from studies in cybrids. Three editing sites from Atropa belladonna (atpA-264, ndhD-200, and ndhD-225) are not processed in a cybrid containing the nucleus of tobacco and the cytoplasm of Atropa (Schmitz-Linneweber et al., 2005). All this evidence led to the hypothesis that the capacity to edit a heterologous site depends on the retention of the corresponding site-specific transacting editing factor(s) encoded by the nucleus.

The editing complex (or editosome) comprises several proteinaceous factors. Members of the organelle RNA-recognition motif (ORRM), multiple organellar RNA editing factor/RNA editing factor interacting protein (MORF/RIP), and organelle zinc-finger (OZ) families (for review, see Sun et al., 2016) have been identified as factors required for editing at multiple sites in both organelles. The site specificity of the editing reaction is believed to be conferred by RNA-binding proteins belonging to the pentatricopeptide repeat (PPR) protein family. PPRs are characterized by a modular organization of tandem repeats that fold into helix-turn-helix structures similar to those found in tetratricopeptide repeats. PPRs of the pure (P-)type contain only classical motifs composed of 35 amino acids, whereas variants of longer (L-type) or shorter (S-type) PPR motifs are found in proteins, which belong to the so-called PLS subfamily (Lurin et al., 2004; Cheng et al., 2016). In addition, PLS PPRs can contain a C-terminal extension following the PPR tract: the E and/or DYW domains. Although P-type PPR proteins are involved in RNA end maturation, intron splicing, and transcript stability, E/DYW-PLS PPRs usually mediate RNA editing in plant organelles. The PPR motifs directly bind the RNA in a modular one repeat-one nucleotide fashion. Mutagenesis and crystallization studies reveal the amino acids at position 5 and the last position of P- and S-type PPR motifs (formerly designated as positions 6 and 1′, respectively) are directly involved in base recognition in that the amino acids at these positions define which nucleotide is recognized in the target RNA (Barkan et al., 2012; Cheng et al., 2016). In this way, PPR editing factors define which cytosine will undergo editing by specifically binding to the adjacent sequence upstream, with the last PPR motif aligning to position −4 with respect to the editing site. In chloroplasts, a given PPR protein usually recognizes a single or a few sites and is essential for editing to occur. Lack of the PPR protein results in complete abolishment of editing at its cognate target site(s). Because of their high specificity, PPRs are thought to tightly coevolve with their target site(s). It is, therefore, assumed that the loss of an editing site is often accompanied by the loss or degeneration of the corresponding PPR gene in the nuclear genome (Hayes et al., 2012; Hein et al., 2016), consistent with the lack of editing at heterologous sites introduced into the plastid genome.

Previously, the psbF-26 site from spinach was introduced into the plastid cytochrome b559 subunit beta (psbF) gene of tobacco, which lacks the corresponding editing site, by plastid transformation (Bock et al., 1994). The heterologous site remained unprocessed in the transplastomic tobacco plants, causing a strong impairment in PSII activity. Editing at the heterologous site was partially restored by fusing protoplasts from the transplastomic mutant with spinach protoplasts (Bock and Koop, 1997), suggesting the spinach nucleus supplies the missing transacting factor(s) required for psbF-26 editing.

Here, we show a single nucleus-encoded PPR protein is sufficient to direct editing at the spinach psbF-26 site heterologously expressed in the tobacco plastid genome. Moderate expression of the editing DYW-type PPR is sufficient to restore complete editing at psbF-26 and fully complements the mutant PSII-deficient phenotype caused by synthesis of “unedited” PsbF protein.

RESULTS

LPA66 and psbF-26: An Example of the Coevolution of a Plastid PPR-type Editing Factor and its Cognate Target RNA

In several species, including the gymnosperm Ginkgo biloba (Kudla and Bock, 1999), evening primrose (Oenothera berteriana), spinach (Bock et al., 1993), and Arabidopsis (Arabidopsis thaliana; Chateigner-Boutin and Small, 2007), codon 26 of the psbF mRNA is converted from an UCU Ser to an UUU Phe codon by RNA editing (Fig. 1A; Supplemental Fig. S1; Cai et al., 2009). The Phe codon resulting from editing at this position is highly conserved in the whole green lineage (Supplemental Fig. S1; Cai et al., 2009), and species that do not possess the psbF-26 editing site (e.g. tobacco) encode a Phe codon already at the DNA level (Fig. 1A; Supplemental Fig. S1).

Figure 1.

Figure 1.

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Expression of Arabidopsis LPA66 in transplastomic pRB8 plants. A, Association between the presence (C) or absence (T) of the psbF-26 editing site and the presence of an LPA66 ortholog in the nuclear genome of 15 selected angiosperm species. Note that the presence of orthologs of Arabidopsis LPA66 (+) associates strictly with the occurrence of the psbF-26 site. B, Nucleotide sequence alignment of the region surrounding the psbF-26 editing site (from position –30 to +10, with the editing site being position 0). Deviations from the consensus sequence are shaded in gray. The predicted binding sequence of LPA66 according to the PPR code (Barkan et al., 2012; Cheng et al., 2016) is boxed. C, Physical maps of the constructs generated for stable nuclear transformation of transplastomic pRB8 plants (Bock et al., 1994). Full-length Arabidopsis LPA66 is expressed under the moderate HYDROPEROXIDE LYASE1 promoter (PHPL) in construct pVL29 (HPL::LPA66) or under the strong UBIQUITIN10 promoter (PUBQ) in construct pVL30 (UBQ::LPA66). In all constructs, the resistance gene (nptII, conferring resistance to kanamycin) is expressed from the enhanced tobacco constitutive promoter (PenTCUP2) and the nos terminator (Tnos). LB, left border; RB, right border of the T-DNA. D, Expression of Arabidopsis LPA66 assessed by semiquantitative RT-PCR in tobacco wild-type (WT) plants and nuclear transformants expressing LPA66 under the HPL or UBQ promoter. The expression of ACTIN was analyzed as a constitutively expressed control gene. H2O: negative control. E, Editing status of the heterologously expressed spinach psbF-26 site and the tobacco psbE-72 and psbL-1 sites assessed by sequencing of the amplified cDNA population. C-to-U conversions are marked by asterisks.

LPA66 (AT5G48910) is a PPR protein belonging to the DYW-PLS subgroup that specifically recognizes the psbF-26 editing site in Arabidopsis (Cai et al., 2009). Arabidopsis LPA66 is predicted to have 15 PPR motifs, based on the latest PPR annotation (Cheng et al., 2016) and is the best matching Arabidopsis PPR to the psbF-26 site according to the PPR code (Kobayashi et al., 2019). An in-house database (POTbase; Moreno et al., 2018) and publicly available transcriptomic datasets (Yan et al., 2016) were screened for the presence of orthologs of LPA66 in a set of 17 dicotyledonous and five monocotyledonous species. The presence of LPA66 orthologs strictly associated with the presence of a C at the position corresponding to the psbF-26 editing site (Fig. 1A; Supplemental Fig. S1). Tobacco and other species that lack the psbF-26 site and encode a TTT Phe codon in the DNA lacked a recognizable ortholog of LPA66 (Fig. 1A; Supplemental Fig. S1). Thus, the co-occurrence of LPA66 and the psbF-26 site represents a striking example of the tight coevolution of a PPR editing factor and its cognate chloroplast target site. Although genetic data are currently available only for Arabidopsis, it appears very likely the orthologs of LPA66 target the psbF-26 site also in the other species.

Expression of Arabidopsis LPA66 Fully Restores Editing of Spinach psbF-26 in Tobacco Chloroplasts

In a previous study (Bock et al., 1994), a 34 nt-long segment spanning the spinach psbF-26 site (from −16 to +18) was used to replace the corresponding region in the tobacco plastid psbF gene. This led, in addition to the introduction of the psbF-26 editing site, to two additional nucleotide changes in the tobacco psbF sequence at position −2 and +10 (editing site: position 0), which are, however, silent with respect to the encoded amino acid sequence (line pRB8-S6 in Bock et al. [1994], referred to as pRB8 here; Fig. 1B). Plants contained only the resistance marker for selection of transplastomic plants (the aminoglycoside 3′’-adenylyltransferase [aadA] gene conferring resistance to streptomycin/spectinomycin), but the wild-type tobacco psbF sequence was used as control (line pRB8-S5 in Bock et al. [1994], referred to as pRB8c here).

To determine whether expression of Arabidopsis LPA66 was sufficient to restore editing of psbF-26, the Arabidopsis LPA66 gene was expressed in the pRB8 transplastomic line (Bock et al., 1994). To this end, the Arabidopsis full-length coding sequence of LPA66 was cloned, and the LPA66 protein was expressed from two different promoters: the HYDROPEROXIDE LYASE1 (HPL) promoter and the UBIQUITIN10 (UBQ) promoter, resulting in constructs HPL::LPA66 (pVL29) and UBQ::LPA66 (pVL30), respectively (Fig. 1C). These two promoters were chosen to assess possible effects of different expression levels of LPA66 on editing efficiency at the heterologous psbF-26 site. PPR-type specificity factors for plastid RNA editing are expressed at moderate levels in Arabidopsis according to the expression data available from the GENEVESTIGATOR database (Supplemental Fig. S2). However, whether moderate (wild-type-like) expression levels of a PPR protein are sufficient to efficiently edit its cognate site(s) in a heterologous system is not known. Complementation experiments with editing mutants in Arabidopsis are usually performed using strong promoters (e.g. the CaMV 35S promoter), suggesting at least some PPR proteins can be overexpressed without causing evident mutant phenotypes, at least in the native (homologous) system. Based on the expression data available from GENEVESTIGATOR, the HPL gene is expressed to comparable levels as the native PPR editing factors in Arabidopsis (Supplemental Fig. S2), and therefore, the HPL promoter is referred to as a moderate promoter in this study. By contrast, UBIQUITIN10 has a much higher expression level throughout development than Arabidopsis genes for PPRs (Supplemental Fig. S2), and, therefore, the UBQ promoter was used to investigate the effects of LPA66 overexpression in tobacco.

The HPL::LPA66 and UBQ::LPA66 expression constructs were introduced in the nuclear genome of the transplastomic pRB8 tobacco line (Bock et al., 1994) by stable Agrobacterium tumefaciens–mediated transformation and selection for kanamycin resistance conferred by a neomycin phosphotransferase II (nptII) cassette in the transformation vector (see “Materials and Methods”). For each construct, several independent transgenic lines were isolated and grown to maturity. Expression of the transgene was confirmed by reverse transcription (RT)-PCR (Fig. 1D). To assess editing at the heterologous psbF-26 site, the amplified cDNA population of the psbEFLJ transcript was sequenced. These analyses revealed full editing of the spinach psbF-26 site in all lines, independent of the Arabidopsis LPA66 being expressed from the moderate HPL or the strong UBQ promoter (pRB8 + HPL::LPA66 or UBQ::LPA66, respectively; Fig. 1E). Complete editing was observed in all analyzed transgenic lines: eight HPL::LPA66 lines and 11 UBQ::LPA66 lines. Introduction of the Arabidopsis LPA66 editing factor did not affect editing at the neighboring psbE-72 and psbL-1 sites that occur in the same transcript (Fig. 1E). Hence, moderate expression of Arabidopsis LPA66 is sufficient to faithfully edit the heterologous spinach psbF-26 site in tobacco (Fig. 1E), strongly suggesting (1) LPA66 is necessary and sufficient to restore editing and (2) LPA66 represents the missing specificity factor that had prevented editing of the spinach psbF-26 site in tobacco plastids.

Complementation of the PSII Defect by RNA Editing

As previously described (Bock et al., 1994), the transplastomic pRB8 plants harboring the psbF-26 site from spinach showed retarded growth and a pale green leaf phenotype when grown under photoautotrophic conditions in soil (Fig. 2A). By contrast, when tested in growth experiments under standard greenhouse conditions, HPL::LPA66 and UBQ::LPA66 transformants (which edit the heterologous psbF-26 site; Fig. 1E) were indistinguishable from the wild type and the pRB8c control plants (that harbor the aadA marker gene but not the editing site) throughout development (Fig. 2A). To confirm full complementation of the mutant phenotype that was caused by the unedited psbF-26 site in pRB8 plants, a series of physiological measurements were performed. As expected, based on their pale-green phenotype, the pRB8 transplastomic mutants had a significantly reduced total chlorophyll content compared with the wild type. Consequently, their leaf absorptance of photosynthetic active radiation was decreased (Table 1). By contrast, in the LPA66-expressing lines, both chlorophyll content and leaf absorptance were restored to wild-type levels, and no significant difference could be detected between the complemented lines and the wild type (Table 1). The maximum quantum efficiency of PSII in the dark-adapted state (FV/FM), which serves as a measure of photochemical intactness of the PSII reaction center and efficient coupling of the light-harvesting complex II (LHCII) antenna system with the reaction center, was strongly decreased in the pRB8 mutant but nearly completely restored in the LPA66-expressing lines (which reached very similar levels as the pRB8c control line; Table 1).

Figure 2.

Figure 2.

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Complementation of the PSII defect of pRB8 by Arabidopsis LPA66. A, Phenotype of N. tabacum wild type (WT), the pRB8c aadA control, the pRB8 transplastomic recipient line, and pRB8 nuclear transformants expressing LPA66 under the control of the HPL or UBQ promoter. Photographs were taken 11 weeks after sowing. Scale bar = 10 cm. B, In vitro complex content quantification in isolated thylakoids for PSII, the cytochrome b6f complex (Cyt-bf), PC, and PSI normalized to the leaf area. Error bars indicate the sd of biological replicates. All mutants were compared with the wild type and each other for significant differences, with pRB8 showing significant differences from the wild type and all other mutants for the contents of PSII, plastocyanin, and PSI. All mutants showed a significant reduction in the content of the cytochrome b6f complex, relative to the wild type. *P ≤ 0.05, One-way ANOVA, Holm–Sidak method. The number of replicates is given in Table 1. C, Immunoblots against diagnostic subunits of PSII, cytochrome b6f (Cyt-bf), PSI, and ATP synthase (AtpB). Thylakoid extracts were loaded on leaf area basis. D, 77K chlorophyll-a fluorescence measurements normalized to PSI emission at 733 nm. E, In vivo light response curves of the fraction of open PSII centers (qL), nonphotochemical quenching capacity (qN), and linear electron transport rate. The three graphs share the same x axis. Error bars indicate the sd of the number of biological replicates indicated in Table 1.

Table 1. Measurement of photosynthesis-related parameters in wild-type tobacco plants, transplastomic pRB8 and pRB8c plants, and supertransformed pRB8 plants expressing LPA66.

The number of biological replicates is indicated (n). Values in bold: One-way ANOVA, Holm–Sidak method, P ≤ 0.05. All mutants were compared with each other and the wild type, with only pRB8 showing significant differences from the wild type and all other mutants for chlorophyll content, leaf absorptance, and FV/FM.

Parameter Wild Type pRB8 pRB8c HPL::LPA66 UBQ::LPA66
Chlorophyll [mg m−2] 463.9 ± 60.5 255.5 ± 55.4 539.6 ± 71.0 516.7 ± 21.2 526.0 ± 20.9
Leaf absorptance (%) 86.2 ± 2.0 77.2 ± 3.0 86.9 ± 1.4 86.5 ± 0.5 86.3 ± 1.2
Fv/Fm 0.80 ± 0.01 0.40 ± 0.03 0.78 ± 0.01 0.79 ± 0.01 0.78 ± 0.01
Membrane conductivity [s−1] 42.6 ± 2.5 46.8 ± 5.2 42.9 ± 2.8 41.8 ± 3.5 43.9 ± 3.4
n 9 8 8 5 6
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To determine if restored wild-type–like growth and chlorophyll content per leaf area were due to full restoration of PSII accumulation in the complemented lines, photosynthetic complex contents were quantified in isolated thylakoids based on in vitro difference absorptance measurements (Fig. 2B) and immunoblots (Fig. 2C). Because the pRB8 transplastomic line had significantly reduced total chlorophyll content compared with the wild type, the data were normalized to leaf area (rather than equal amounts of chlorophyll). As expected, PSII accumulation was severely affected in pRB8 plants and reduced to approximately one fifth of that in the wild type. Cytochrome b6f complex, PSI, and plastocyanin (PC) contents were also reduced in the pRB8 mutant. Importantly, expression of HPL::LPA66 or UBQ::LPA66 completely restored the amount of PSII to wild-type levels (Fig. 2B). PC and PSI also accumulated to wild-type levels in the complemented lines, suggesting that the reduced amounts of PC and PSI observed in pRB8 plants represent secondary defects caused by the strong impairment in PSII. Defects in PSII are, in fact, known to affect the accumulation and activity of the downstream complexes in the electron transport chain (Krech et al., 2013). Surprisingly, the accumulation of cytochrome b6f complex did not fully recover to wild-type levels, neither in HPL::LPA66 nor in UBQ::LPA66 plants. Similar to pRB8 plants, HPL::LPA66 and UBQ::LPA66 showed an ∼30% reduction in cytochrome b6f complex contents. However, the pRB8c line, which contains the aadA resistance gene but the wild-type tobacco psbF sequence, also showed a similar reduction in cytochrome b6f complex content (but not in the contents of PSII, PSI, and PC; Fig. 2B). This finding demonstrates that the presence of the aadA gene rather than the presence of the spinach psbF sequence causes the observed reduction in cytochrome b6f complex content. Consequently, wild-type levels of cytochrome b6f complex cannot be restored by the expression of LPA66.

The spectroscopic data were confirmed by immunoblots against diagnostic (i.e. essential) subunits of the photosynthetic complexes. As expected, accumulation of both the PSII reaction center subunit PsbD (the D2 protein) and the cytochrome b559 subunit PsbE were strongly reduced in the pRB8 mutant, but indistinguishable from the wild type in the aadA control line (pRB8c) and in both complemented lines (Fig. 2C). For the cytochrome b6f complex, the accumulation of the essential plastid-encoded cytochrome f (PetA) and the nucleus-encoded Rieske protein (PETC) were determined. Immunoblots with both antibodies confirmed a similar reduction in the accumulation of the cytochrome b6f complex in all mutant lines relative to the wild type, in line with our spectroscopic data. For PSI, the plastid-encoded reaction center subunit PsaA and the nucleus-encoded stromal ridge subunit PSAD (involved in ferredoxin binding) were clearly reduced in the pRB8 editing mutant but present in wild-type amounts in the pRB8c control line and both complemented lines. Finally, accumulation of chloroplast ATP synthase was probed with an antibody against the plastid-encoded catalytic CF1 subunit AtpB. No change in AtpB content was seen in any of the mutants. This is well in line with dark-interval decay kinetics of the proton motive force across the thylakoid membrane, which demonstrated a similar membrane conductivity for protons in all plants (which is determined by chloroplast ATP synthase activity; Table 1).

Next, 77K chlorophyll-a fluorescence emission spectra were recorded to monitor the distribution of excitation energy between the two photosystems. The emission maxima at 687- and 734-nm wavelength reflect the fluorescence emission of PSII-LHCII and PSI-LHCI, respectively. Although the emission spectra of all other lines were similar to the wild type, the pRB8 editing mutant showed a significant reduction in chlorophyll-a fluorescence emission from PSII (Fig. 2D), indicating PSII accumulation is strongly reduced. More importantly, the maximum emission wavelength was shifted from 687 to 684 nm, which is indicative of the presence of free, uncoupled LHCII proteins in the thylakoid membrane and suggests LHCII accumulation is less affected than that of PSII. Free LHCII complexes display an emission maximum at 680-nm wavelength (Krause and Weis, 1991). The 684-nm signal measured likely represents the average of the emission signals originating from intact PSII-LHCII and free LHCII complexes.

Finally, light response curves of chlorophyll-a fluorescence parameters were measured at room temperature (Fig. 2E). The light response curves of linear electron flux were corrected for differences in leaf absorptance (Table 1; see above). Linear electron transport capacity was severely reduced in pRB8 plants compared to wild-type plants, which is well in agreement with the reduced contents of all photosynthetic complexes (Fig. 2, B and C). In pRB8c, HPL::LPA66, and UBQ::LPA66, linear electron transport capacity was also somewhat reduced relative to the wild type. However, this effect was much less pronounced than in pRB8 plants and most likely results from the observed reduction in cytochrome b6f complex contents due to insertion of the aadA marker next to the petA gene. The cytochrome b6f complex normally controls linear electron transport, because it catalyzes the slowest reaction of linear electron transport and usually is present in substoichiometric amounts relative to both photosystems (for review, see Anderson, 1992; Schöttler and Tóth, 2014).

Nonphotochemical quenching (qN) dissipates excess excitation energy in the PSII antenna bed as heat. The induction of this process is controlled by thylakoid lumen acidification. It depends on the balance of photosynthetic electron transport, which generates the proton motive force across the thylakoid membrane, and ATP synthase activity, which consumes it. The qN curves (Fig. 2E) showed a distinct shift of photoprotective qN toward lower light intensities in the pRB8 line compared with the wild type. Also, full activation of qN was impaired in the mutant. The pRB8c, HPL::LPA66, and UBQ::LPA66 lines had very similar qN curves, with reduced nonphotochemical quenching only at light intensities above 500 μmol photons m−2 s−1. Because both linear electron transport and cytochrome b6f complex contents were reduced in these lines, proton influx via linear electron transport is reduced. However, chloroplast ATP synthase activity (which consumes the proton motive force generated by photosynthetic electron transport) remained unaltered, strongly suggesting that an imbalance between processes generating and processes consuming the proton motive force resulted in reduced thylakoid lumen acidification. This behavior is typical of mutants with specific defects in cytochrome b6f complex accumulation (Price et al., 1995; Hojka et al., 2014).

The qL curve represents a measure of the redox state of the PSII acceptor side (Kramer et al., 2004; Baker et al., 2007). The light response curve was shifted to lower light intensities in pRB8 plants as well as in pRB8c, HPL::LPA66, and UBQ::LPA66 lines (Fig. 2E), in agreement with the observed restricted linear electron flux in these lines.

In conclusion, expression of the Arabidopsis PPR protein LPA66 in tobacco is sufficient to reconstitute editing of the spinach psbF-26 site in tobacco chloroplasts. It confers complete RNA editing and rescues the mutant phenotype that resulted from introduction of the spinach editing site into the tobacco psbF gene. This rescue occurs through restoration of the wild-type sequence for PsbF by RNA editing, which, in turn, restores wild-type levels of PSII.

DISCUSSION

RNA editing in plant organelles is a posttranscriptional process characterized by exceptional phylogenetic dynamics. The set of chloroplast editing sites (also called the editotype) differs even between closely related species (Freyer et al., 1995; Schmitz-Linneweber et al., 2002; Sasaki et al., 2003). Spontaneous mutations in the plastid DNA are strongly biased toward C-to-T transitions, which results in highly AT-rich chloroplast genomes in vascular plants (Kusumi and Tachida, 2005; Smith, 2012). This bias likely contributes to the frequent loss of individual editing sites during evolution and may provide an explanation for loss events being much more frequent than the gain of new editing sites.

Several studies have shown heterologous RNA editing sites are not recognized when introduced into species that had lost the site and encode a T in the DNA at the corresponding position (Bock et al., 1994; Reed and Hanson, 1997). It has been speculated the presence of an editing capacity for a heterologous site depends on the retention of trans-acting specificity factor(s) encoded in the nuclear genome.

Several classes of nucleus-encoded trans-acting protein factors are involved in chloroplast RNA editing (for review, see Sun et al., 2016). Among those, PLS PPR proteins are crucial to define the position of the cytosine that will undergo editing. Due to their high binding specificity for particular RNA sequences, PPR-type editing factors strictly coevolve with their target cis-element(s) and, in this way, with the corresponding RNA editing site(s). Therefore, the loss of a chloroplast editing site often correlates with the degeneration of the gene for the corresponding PPR protein in the nucleus (Hayes et al., 2012; Hein et al., 2016; this study), especially in those cases where the PPR protein only serves a single editing site. It, therefore, seemed reasonable to suspect the lack of editing at heterologous sites is due to the lack of the required PPR factor.

In this study, we have shown expression of the Arabidopsis PPR-type editing factor LPA66 is sufficient to fully edit the heterologous psbF-26 site from spinach in tobacco chloroplasts. Notably, full editing was observed in all transgenic lines analyzed, largely independent of the expression strength of LPA66. Because the spinach psbF-26 site was edited, the expression of LPA66 also completely rescued the PSII-deficient phenotype of the transplastomic pRB8 lines, which had been caused by the unedited psbF-26 site.

The remaining minor measurable difference in photosynthetic electron transfer between the wild type and the lines complemented with LPA66 (small reduction in FV/FM and in linear electron transport capacity, delayed induction of nonphotochemical quenching) do not translate into a growth phenotype and, importantly, are also found in the pRB8c control plants. We have shown these differences are caused by a reduced accumulation of the cytochrome b6f complex. The pRB8c control mutant only harbors the selectable marker gene aadA, but not the psbF-26 editing site, suggesting the presence of the selectable marker gene in this particular genomic position is causally responsible for the reduced accumulation of the cytochrome b6f complex in the mutants. The aadA cassette in the pRB8 and pRB8c lines is inserted downstream of the psbEFLJ transcriptional unit (Bock et al., 1994). Downstream of the insertion site resides the petA gene, which encodes the essential cytochrome f subunit of the cytochrome b6f complex and is transcribed in the opposite direction. It, thus, seems reasonable to assume that the insertion of the aadA marker in this position directly or indirectly affects the expression of PetA. The precise mechanism of this interference remains to be determined.

Two non-PPR factors are involved in editing of psbF-26 in Arabidopsis: MORF8/RIP1 (Bentolila et al., 2012) and ORRM6 (Hackett et al., 2017). Although their role in the editing reaction is not yet fully understood, disruption of each of these two genes is sufficient to completely abolish editing at psbF-26. Using in-house transcriptomic datasets, we could identify putative orthologs of MORF8/RIP1 and ORRM6 in tobacco (Table 2). It, therefore, is reasonable to assume Arabidopsis LPA66 is capable of faithfully interacting with the tobacco orthologs of the two other essential transacting editing factors. In line with the high sequence specificity of RNA binding by PPR proteins, overexpression of LPA66 did not affect editing at the neighboring psbE-72 and psbL-1 sites occurring in the same transcript (Fig. 1E).

Table 2. Chloroplast RNA editing factors required for editing at Arabidopsis site psbF-26.

Editing efficiency at psbF-26 is indicated as reported in the corresponding knock-out mutants (% T/C), and the reference is given. Values in bold represent severe reductions in editing efficiency compared with the wild type. The presence of an ortholog in the tobacco nuclear genome is indicated by +.

Cofactor psbF-26 Reference Tobacco
MORF2/RIP2 0 Takenaka et al., 2012 +
MORF8/RIP1 86 ± 1 Bentolila et al., 2012 +
MORF9/RIP9 70 Takenaka et al., 2012 +
ORRM1 98 Sun et al., 2013 +
ORRM6 0 Hackett et al., 2017 +
OZ1 88 ± 1 Sun et al., 2015 +
CP31A 100 Tillich et al., 2009 +
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In summary, this study demonstrates the possibility to transfer editing events between species by expressing a heterologous editing site in the plastid genome and its cognate site-specific factor in the nuclear genome. This can be accomplished without interfering with editing activity at endogenous sites. The significance of this finding is twofold. First, our successful reconstitution of heterologous editing demonstrates that, although editing reactions are known to depend on multiple trans-acting protein factors, the interspecies transfer of editing events can be achieved by introduction of the two rapidly evolving components of the system: the editing site and the corresponding PPR protein. Second, the possibility to implement new editing sites in plastid genomes provides a powerful tool for the development of inducible expression systems in biotechnology and the design of efficient on/off switches in synthetic biology. For example, synthesis of a gene product can be made dependent on RNA editing by correcting a missense mutation or creating a translational start codon. Editing, then, can be induced by simply placing the corresponding PPR gene under the control of a promoter that is inducible by chemical cues or responsive to an environmental trigger (e.g. a biotic or abiotic stress signal).

MATERIALS AND METHODS

Plant Material and Growth Conditions

Tobacco (Nicotiana tabacum) seeds from transplastomic lines pRB8 and pRB8c (described as pRB8-S6 and pRB8-S5 in Bock et al., 1994, respectively) were surface sterilized by 70% (v/v) ethanol for 7 min, followed by a 7-min treatment with 7% (v/v) hypochlorite. Seeds were then washed five times with sterile water and plated on Murashige and Skoog (MS) medium supplemented with 3% (w/v) Suc and 500 mg/L spectinomycin dihydrochloride. The plates were incubated at 4°C in the dark for 2 d for stratification before transfer to controlled environment chambers (light intensity: 50 μE m−2 s−1, diurnal cycle: 16 h light at 24°C and 8 h dark at 22°C). At 10 d after germination, seedlings were transferred to soil and grown under standard greenhouse conditions (unless otherwise mentioned) to maturity: 16 h light at 25°C and 8 h darkness at 20°C. Plants used for photosynthetic measurements were grown in controlled environment chambers (Conviron) at 120 μE m−2 s−1 light intensity (16 h light at 22°C, 75% relative humidity, and 8 h dark at 18°C, 70% relative humidity). After ∼3 weeks, plants were transferred to controlled environmental chambers with the actinic light intensity set to 350 μE m−2 s−1. All other environmental parameters remained unaltered.

Cloning and Plant Transformation

For the generation of construct HPL::LPA66, the full-length coding sequence of LPA66 (AT5G48910) was amplified from Arabidopsis (Arabidopsis thaliana) Col-0 genomic DNA with primers oVL84 and oVL85 and cloned by Gibson Assembly into vector pORE-E2 (Coutu et al., 2007) that had been linearized with KpnI. The HPL (hydroperoxide lyase) promoter was replaced by the UBIQUITIN10 promoter to generate the UBQ::LPA66 construct. The UBIQUITIN10 promoter was amplified from Arabidopsis Col-0 genomic DNA with primers P_UBQ10for and P_UBQ10rev and subsequently digested with XhoI and EcoRI. Each construct was introduced into line pRB8 by Agrobacterium tumefaciens–mediated nuclear transformation (Rosahl et al., 1987) using A. tumefaciens strain pGV2260. Transgenic lines were regenerated on RMOP medium (Svab et al., 1990) supplemented with 50 mg/L kanamycin for selection and 250 mg/L cefotaxime sodium salt (claforan) to prevent growth of bacteria.

RNA Editing and Expression Analyses

Total plant RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific) following the protocol of the manufacturer. cDNA was synthesized with the help of the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s recommendations and using a 1:1 mixture of random hexamer (Qiagen) and oligo(dT)18 primers. cDNA fragments were amplified by PCR using specific oligonucleotides as primers (Supplemental Table S1) and following standard protocols (Taq DNA polymerase, ThermoFisher). PCR was performed for 35 cycles, with the exception of the amplification of ACTIN, where the number of cycles was reduced to 23. PCR products were purified with the NucleoBond PCR Clean-up kit (Macherey-Nagel) and sequenced (Eurofins Genomics).

Immobilization and Detection of Proteins by Immunoblot Analysis

Thylakoids were extracted according to Schöttler et al. (2004) from leaf material of plants grown in the conditions described for photosynthetic measurements. Thylakoid proteins were separated using the PerfectBlue Dual Gel System Twin L (VWR International). Then 15% or 12% (w/v) denaturating PAGE (SDS-PAGE) was performed according to Laemmli (1970). Separated proteins were transferred to Hybond-P polyvinylidene difluoride membranes (GE Healthcare) using a tank blotter (VWR International). Membranes were stained by 0.25% (w/v) Coomassie Brilliant Blue R-250 (SERVA), destained using 100% (v/v) methanol, and scanned using an EPSON Perfection V700 Photo scanner. Blocking was performed in TBS-T buffer [20 mm Tris-HCl (pH 7.6), 137 mm NaCl, Tween 20 0.1% (w/v)] in the presence of 4% (w/v) skimmed milk powder and 0.5% (w/v) bovine serum albumin (Carl Roth GmbH) for 1 h at room temperature under continuous shaking. After a washing step in TBS-T, membranes were incubated with the primary antibody of interest in TBS-T for 1 h at room temperature with slow shaking. The primary antibodies used (α-PsbD, α-PsbE, α-PetA, α-PETC, α-PsaA, α-PsaD, α-AtpB) were purchased from Agrisera. Binding of the appropriate HRP conjugated-secondary antibody (Sigma; diluted 1:50,000 in TBS-T) was allowed in the presence of 0.5% (w/v) bovine serum albumin for 1 h at room temperature with slow shaking. Membranes were treated with the ECL Plus western Blotting Detection Kit (GE Healthcare) according to the manufacturer’s instruction and exposed in a G:BOX Chemi XT4 (Syngene) for signal detection.

Spectroscopic Methods

Leaf chlorophyll content and chlorophyll a/b ratio were determined from leaf extracts in 80% (v/v) acetone according to Porra et al. (1989) using a V-730 UV-Vis Spectrophotometer (Jasco GmbH). Chlorophyll-a fluorescence emission at 77 K was determined on freshly isolated thylakoids (Schöttler et al., 2004) equivalent to 10 μg of chlorophyll mL–1 using a F-6500 fluorometer (Jasco). The sample was excited at a 430-nm wavelength (10-nm bandwidth). Emission spectra between 655 and 800 nm were recorded with a bandwidth of 1 nm, and 10 spectra were averaged to improve the signal-to-noise ratio. Chlorophyll-a fluorescence of intact leaves was measured at 22°C using a Dual-PAM-100 instrument (Heinz Walz GmbH). FV/FM and light-response curves of linear electron transport, qN and qL, were measured on intact leaves after at least 30 min of dark adaptation. Under light-limited conditions, each actinic light intensity was measured for 150 s while under light-saturated conditions, and light intensity was increased every 60 s. Linear electron transport was corrected for the leaf absorptance measured with an integrating sphere (ISV-722, Jasco) attached to a spectrophotometer (V-650, Jasco). Transmittance and reflectance spectra of leaves were recorded between 400- and 700-nm wavelength, and leaf absorptance was calculated as 100% minus transmittance of light through the leaf minus reflectance on the leaf surface. The average value of the absorptance spectrum between 400- and 700-nm wavelength was used for the calculation of linear electron flux. Dark-interval decay kinetics of the electrochromic absorption shift (ECS) were used as in vivo probes of ATP synthase activity (Baker et al., 2007). The ECS, which is proportional to the light-induced proton motive force across the thylakoid membrane, was measured using a KLAS-100 LED-Array Spectrophotometer (Heinz Walz GmbH), allowing the simultaneous measurement of light-induced difference absorption signals at eight pairs of wavelengths in the visible range of the spectrum between 505 and 570 nm, as described by Rott et al. (2011). Leaves were illuminated with saturating light (1200 µmol photons m–2 s–1) for 10 min before each measurement to allow photosynthesis to reach the steady state. Actinic illumination was interrupted by a short interval of darkness (15 s), and the dark-interval decay of the ECS was measured. In preilluminated leaves, this decay kinetic is determined by ATP synthase activity. Photosynthetic complex contents of PSII, cytochrome b6f complex, and PSI were quantified in isolated thylakoids as described in Hojka et al. (2014) using a V-550 spectrophotometer (Jasco) and a Dual-PAM instrument (Heinz Walz). Plastocyanin contents relative to PSI were determined by in vivo difference absorption spectroscopy in the far-red range of the spectrum and then recalculated based on the absolute PSI quantification in isolated thylakoids (Schöttler et al., 2007).

Bioinformatic Analyses

Orthologs of Arabidopsis LPA66 (AT5G48910) were extracted from an in-house database (POTbase, https://chlorobox.mpimp-golm.mpg.de; Moreno et al., 2018). The sequence of the LPA66 ortholog from spinach (Spinacia oleracea) was obtained from an RNA sequencing dataset from Yan et al. (2016; GenBank databases ID: SRP051935). Nucleotide and amino acid sequences of psbF were obtained from National Center for Biotechnology Information. Sanger sequencing data were visualized using SeqMan Pro 14 (DNASTAR), and chromatograms were extracted using Chromas (http://technelysium.com.au/wp/chromas/). DNA and protein alignments were produced using ClustalW within BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

Accession Numbers

Sequence data used in this article can be found in the GenBank/EMBL database under accession numbers: AT3G22150 (AEF1); AT4g24770 (CP31A); AT5G66520 (CREF7); AT2G45350 (CRR4); AT5G55740 (CRR21); AT4G15440 (HPL); AT5G48910 (LPA66); AT2G33430 (MORF2/RIP2); AT3G15000 (MORF8/RIP1); AT1G11430 (MORF9/RIP9); AT3G20930 (ORRM1); AT1G73530 (ORRM6); AT5G17790 (OZ1); AT2G29760 (QED1); AT5G13270 (RARE1); AT1G67090 (RBCS1A); AT4G05320 (UBQ10).

Supplemental Data

The following supplemental figures, tables, and information are included separately as a single pdf file.

ACKNOWLEDGMENTS

We thank the Max Planck Institute of Molecular Plant Physiology (MPI-MP) Green Team for plant care and cultivation.

Footnotes

1

This work was supported by the Max Planck Society and by grants from the Deutsche Forschungsgemeinschaft (DFG) (TI 605/5-1 to M.T.; SFB-TRR 175 to R.B.).

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