1

Article type: Research Report 1

Short title: Reconstitution of heterologous RNA editing 2

Author for Contact: Ralph Bock (RBock@mpimp-golm.mpg.de) 3

4

Establishment of a Heterologous RNA Editing Event in Chloroplasts 5

Author names and affiliations: 6

F. Vanessa Loiacono1, Wolfram Thiele, Mark Aurel Schöttler, Michael Tillich, Ralph Bock* 7

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, 8

Germany 9

1 Present address: F. Vanessa Loiacono, Department of Molecular Biology and Genetics, Cornell University, 10

Ithaca, NY 14853, USA 11

12

One sentence summary: An Arabidopsis PPR protein fully edits a heterologous editing site from spinach in 13

tobacco chloroplasts. 14

15

Author contributions: 16

M.T. and R.B. conceived the project. F.V.L. performed most experiments. W.T. and M.A.S. performed 17

protein analyses and photosynthetic measurements. F.V.L. and R.B. wrote the article with input from all co-18

authors. All authors contributed to the editing and review of the manuscript. R.B serves as the author 19

responsible for contact. 20

21

This research was supported by the Max Planck Society, and by grants from the Deutsche 22

Forschungsgemeinschaft (DFG) to M.T. (TI 605/5-1) and R.B. (SFB-TRR 175). 23

24

Abstract 25

In chloroplasts and plant mitochondria, specific cytidines in mRNAs are post-transcriptionally converted to 26

uridines by RNA editing. Editing sites are recognized by nucleus-encoded RNA-binding proteins of the 27

pentatricopeptide repeat (PPR) family, which bind upstream of the editing site in a sequence-specific manner 28

Plant Physiology Preview. Published on September 13, 2019, as DOI:10.1104/pp.19.00922

Copyright 2019 by the American Society of Plant Biologists

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and direct the editing activity to the target position. Editing sites have been lost many times during evolution 29

by C-to-T mutations. Loss of an editing site is thought to be accompanied by loss or degeneration of its 30

cognate PPR protein. Consequently, foreign editing sites are usually not recognized when introduced into 31

species lacking the site. Previously, the spinach (Spinacia oleracea) psbF-26 editing site was introduced into 32

the tobacco (Nicotiana tabacum) plastid genome. Tobacco lacks the psbF-26 site and cannot edit it. 33

Expression of the “unedited” PsbF protein resulted in impaired photosystem II function. In Arabidopsis 34

(Arabidopsis thaliana), the PPR protein LPA66 is required for editing at psbF-26. Here, we show that 35

introduction of the Arabidopsis LPA66 reconstitutes editing of the spinach psbF-26 site in tobacco and 36

restores a wild-type-like phenotype. Our findings define the minimum requirements for establishing novel 37

RNA editing sites and suggest that the evolutionary dynamics of editing patterns is largely explained by co-38

evolution of editing sites and PPR proteins. 39

40

Introduction 41

C-to-U RNA editing in plants was first discovered in mitochondria (Covello and Gray, 1989; Gualberto et 42

al., 1989) and soon afterwards also in plastids (chloroplasts) (Hoch et al., 1991). Since then, C-to-U editing 43

has been detected in plastids and mitochondria of virtually all embryophytes analyzed, with the Marchantiid 44

subclade of liverworts being the only exception (Malek et al., 1996). The origin of C-to-U editing probably 45

dates back to the colonization of terrestrial habitats by early embryophytes. While green algae lack C-to-U 46

editing, hundreds or even thousands of editing sites are found in species from evolutionarily old 47

embryophyte lineages such as the hornwort Anthoceros formosae, the fern Adiantum capillus-veneris and the 48

spike moss Selaginella uncinata (Freyer et al., 1997; Kugita et al., 2003; Wolf et al., 2004; Tillich et al., 49

2006a; Smith, 2009; Oldenkott et al., 2014). By contrast, typical angiosperms edit only 30 to 40 sites in their 50

chloroplast genomes. Editing sites are frequently lost during evolution with an average speed of one site per 51

2.43 million years (Fujii and Small, 2011). Consequently, even closely related species or different ecotypes 52

of the same species can exhibit different patterns of editing sites (referred to as editotypes; (Freyer et al., 53

1995; Freyer et al., 1997; Schmitz-Linneweber et al., 2002; Sasaki et al., 2003; Tillich et al., 2005; Germain 54

et al., 2015)). For instance, deadly nightshade (Atropa belladonna) and tobacco (Nicotiana tabacum) possess 55

35 and 37 plastid editing sites, respectively, but only 32 of these occur in both species, although the two 56

species belong to the same family (Kahlau et al., 2006). 57

Because of the limited evolutionary conservation of editing sites, a number of previous studies have 58

investigated if editing activity is maintained despite the loss of an editing site by C-to-T mutation. Taking 59

advantage of the possibility to manipulate the plastid genome of tobacco plants, several heterologous editing 60

sites from other species such as spinach (Spinacia oleracea) (Bock et al., 1994), maize (Zea mays) (Reed and 61

Hanson, 1997) and tomato (Solanum lycopersicum) (Karcher et al., 2008) were introduced into tobacco 62

plastids. While in a few cases editing at the heterologous sites could be detected (especially, across short 63

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evolutionary distances, i.e., within the Solanaceae family) (Tillich et al., 2006b; Karcher et al., 2008), in 64

most cases, the heterologous sites remained unprocessed (Bock et al., 1994; Reed and Hanson, 1997). Thus, 65

loss of an editing site is usually accompanied by the loss of the capacity to edit this site. This conclusion 66

gained further support from studies in cybrids. Three editing sites from Atropa belladonna (atpA-264, ndhD-67

200 and ndhD-225) are not processed in a cybrid containing the nucleus of tobacco and the cytoplasm of 68

Atropa (Schmitz-Linneweber et al., 2005). All this evidence led to the hypothesis that the capacity to edit a 69

heterologous site depends on the retention of the corresponding site-specific trans-acting editing factor(s) 70

encoded by the nucleus. 71

The editing complex (or editosome) is comprised of several proteinaceous factors. Members of the organelle 72

RNA-recognition motif (ORRM), multiple organellar RNA editing factor/RNA editing factor interacting 73

protein (MORF/RIP) and organelle zinc-finger (OZ) families (reviewed in (Sun et al., 2016)) have been 74

identified as factors required for editing at multiple sites in both organelles. The site specificity of the editing 75

reaction is believed to be conferred by RNA-binding proteins belonging to the pentatricopeptide repeat 76

(PPR) protein family. PPRs are characterized by a modular organization of tandem repeats that fold into 77

helix-turn-helix structures similar to those found in tetratricopeptide repeats. PPRs of the pure (P-)type 78

contain only classical motifs composed of 35 amino acids, while variants of longer (L-type) or shorter (S-79

type) PPR motifs are found in proteins which belong to the so-called PLS subfamily (Lurin et al., 2004; 80

Cheng et al., 2016). In addition, PLS PPRs can contain a C-terminal extension following the PPR tract: the E 81

and/or DYW domains. While P-type PPR proteins are involved in RNA end maturation, intron splicing and 82

transcript stability, E/DYW-PLS PPRs usually mediate RNA editing in plant organelles. The PPR motifs 83

directly bind the RNA in a modular one repeat-one nucleotide fashion. Mutagenesis and crystallization 84

studies reveal the amino acids at position 5 and the last position of P- and S-type PPR motifs (formerly 85

designated as positions 6 and 1´, respectively) are directly involved in base recognition in that the amino 86

acids at these positions define which nucleotide is recognized in the target RNA (Barkan et al., 2012; Cheng 87

et al., 2016). In this way, PPR editing factors define which cytosine will undergo editing by specifically 88

binding to the adjacent sequence upstream, with the last PPR motif aligning to position -4 with respect to the 89

editing site. In chloroplasts, a given PPR protein usually recognizes a single or a few sites and is essential for 90

editing to occur. Lack of the PPR protein results in complete abolishment of editing at its cognate target 91

site(s). Because of their high specificity, PPRs are thought to tightly co-evolve with their target site(s). It is, 92

therefore, assumed that the loss of an editing site is often accompanied by the loss or degeneration of the 93

corresponding PPR gene in the nuclear genome (Hayes et al., 2012; Hein et al., 2016), consistent with the 94

lack of editing at heterologous sites introduced into the plastid genome. 95

Previously, the psbF-26 site from spinach was introduced into the plastid cytochrome b559 subunit β (psbF) 96

gene of tobacco, which lacks the corresponding editing site, by plastid transformation (Bock et al., 1994). 97

The heterologous site remained unprocessed in the transplastomic tobacco plants, causing a strong 98

impairment in photosystem II (PSII) activity. Editing at the heterologous site was partially restored by fusing 99

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protoplasts from the transplastomic mutant with spinach protoplasts (Bock and Koop, 1997), suggesting the 100

spinach nucleus supplies the missing trans-acting factor(s) required for psbF-26 editing. 101

Here, we show a single nucleus-encoded PPR protein is sufficient to direct editing at the spinach psbF-26 102

site heterologously expressed in the tobacco plastid genome. Moderate expression of the editing DYW-type 103

PPR is sufficient to restore complete editing at psbF-26 and fully complement the mutant PSII-deficient 104

phenotype caused by synthesis of “unedited” PsbF protein. 105

106

107

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Results 108

LPA66 and psbF-26: an example of the co-evolution of a plastid PPR-type editing factor and its 109

cognate target RNA 110

In several species, including the gymnosperm Ginkgo biloba (Kudla and Bock, 1999), evening primrose 111

(Oenothera berteriana), spinach (Bock et al., 1993) and Arabidopsis (Arabidopsis thaliana) (Chateigner-112

Boutin and Small, 2007), codon 26 of the psbF mRNA is converted from an UCU serine to an UUU 113

phenylalanine codon by RNA editing (Figure 1A; Supplemental Figure S1) (Cai et al., 2009). The 114

phenylalanine codon resulting from editing at this position is highly conserved in the whole green lineage 115

(Supplemental Figure S1) (Cai et al., 2009), and species that do not possess the psbF-26 editing site (e.g., 116

tobacco), encode a phenylalanine codon already at the DNA level (Figure 1A; Supplemental Figure S1). 117

LPA66 (AT5G48910) is a PPR protein belonging to the DYW-PLS subgroup that specifically recognizes the 118

psbF-26 editing site in Arabidopsis (Cai et al., 2009). Arabidopsis LPA66 is predicted to have 15 PPR 119

motifs, based on the latest PPR annotation (Cheng et al., 2016) and is the best matching Arabidopsis PPR to 120

the psbF-26 site according to the PPR code (Kobayashi et al., 2019). An in-house database (POTbase) 121

(Moreno et al., 2018) and publicly available transcriptomic datasets (Yan et al., 2016) were screened for the 122

presence of orthologs of LPA66 in a set of 17 dicotyledonous and five monocotyledonous species. The 123

presence of LPA66 orthologs strictly associated with the presence of a C at the position corresponding to the 124

psbF-26 editing site (Figure 1A; Supplemental Figure S1). Tobacco and other species that lack the psbF-125

26 site and encode a TTT phenylalanine codon in the DNA lacked a recognizable ortholog of LPA66 126

(Figures 1A; Supplemental Figure S1). Thus, the co-occurrence of LPA66 and the psbF-26 site represents 127

a striking example of the tight co-evolution of a PPR editing factor and its cognate chloroplast target site. 128

Although genetic data are currently available only for Arabidopsis, it appears very likely the orthologs of 129

LPA66 target the psbF-26 site also in the other species. 130

131

Expression of Arabidopsis LPA66 fully restores editing of spinach psbF-26 in tobacco chloroplasts 132

In a previous study (Bock et al., 1994), a 34 nt-long segment spanning the spinach psbF-26 site (from -16 to 133

+18) was used to replace the corresponding region in the tobacco plastid psbF gene. This led, in addition to 134

the introduction of the psbF-26 editing site, to two additional nucleotide changes in the tobacco psbF 135

sequence at position -2 and +10 (editing site: position 0), which are, however, silent with respect to the 136

encoded amino acid sequence (line pRB8-S6 in (Bock et al., 1994), referred to as pRB8 here; Figure 1B). 137

Plants containing only the resistance marker for selection of transplastomic plants (the aminoglycoside 3’’-138

adenylyltransferase (aadA) gene conferring resistance to streptomycin/spectinomycin) but the wild-type 139

tobacco psbF sequence were used as control (line pRB8-S5 in (Bock et al., 1994), referred to as pRB8c 140

here). 141

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To determine whether expression of Arabidopsis LPA66 was sufficient to restore editing of psbF-26, the 142

Arabidopsis LPA66 gene was expressed in the pRB8 transplastomic line (Bock et al., 1994). To this end, the 143

Arabidopsis full-length LPA66 cDNA was cloned, and the LPA66 protein was expressed from two different 144

promoters: the HYDROPEROXIDE LYASE1 (HPL) promoter and the UBIQUITIN10 (UBQ) promoter, 145

resulting in constructs HPL::LPA66 (pVL29) and UBQ::LPA66 (pVL30), respectively (Figure 1C). These 146

two promoters were chosen to assess possible effects of different expression levels of LPA66 on editing 147

efficiency at the heterologous psbF-26 site. PPR-type specificity factors for plastid RNA editing are 148

expressed at moderate levels in Arabidopsis according to the expression data available from the 149

GENEVESTIGATOR® database (Supplemental Figure S2). However, whether moderate (wild-type-like) 150

expression levels of a PPR protein are sufficient to efficiently edit its cognate site(s) in a heterologous 151

system is not known. Complementation experiments with editing mutants in Arabidopsis are usually 152

performed using strong promoters (e.g., the CaMV 35S promoter), suggesting at least some PPR proteins can 153

be overexpressed without causing evident mutant phenotypes, at least in the native (homologous) system. 154

Based on the expression data available from GENEVESTIGATOR, the HPL gene is expressed to 155

comparable levels as the native PPR editing factors in Arabidopsis (Supplemental Figure S2), and 156

therefore, the HPL promoter is referred to as a moderate promoter in this study. By contrast, UBIQUITIN10 157

has a much higher expression level throughout development than Arabidopsis genes for PPRs 158

(Supplemental Figure S2) and, therefore, the UBQ promoter was used to investigate the effects of LPA66 159

overexpression in tobacco. 160

The HPL::LPA66 and UBQ::LPA66 expression constructs were introduced in the nuclear genome of the 161

transplastomic pRB8 tobacco line (Bock et al., 1994) by stable Agrobacterium tumefaciens-mediated 162

transformation and selection for kanamycin resistance conferred by a neomycin phosphotransferase II (nptII) 163

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cassette in the transformation vector (see Methods). For each construct, several independent transgenic lines 164

were isolated and grown to maturity. Expression of the transgene was confirmed by RT-PCR (Figure 1D). 165

To assess editing at the heterologous psbF-26 site, the amplified cDNA population of the psbEFLJ transcript 166

was sequenced. These analyses revealed full editing of the spinach psbF-26 site in all lines, independent of 167

the Arabidopsis LPA66 being expressed from the moderate HPL or the strong UBQ promoter (pRB8 + 168

HPL::LPA66 or UBQ::LPA66, respectively; Figure 1E). Complete editing was observed in all analyzed 169

transgenic lines: 8 HPL::LPA66 lines, and 11 UBQ::LPA66 lines. Introduction of the Arabidopsis LPA66 170

editing factor did not affect editing at the neighboring psbE-72 and psbL-1 sites that occur in the same 171

transcript (Figure 1E). Hence, moderate expression of Arabidopsis LPA66 is sufficient to faithfully edit the 172

heterologous spinach psbF-26 site in tobacco (Figure 1E), strongly suggesting (i) LPA66 is necessary and 173

sufficient to restore editing, and (ii) LPA66 represents the missing specificity factor that had prevented 174

editing of the spinach psbF-26 site in tobacco plastids. 175

176

Complementation of the PSII defect by RNA editing 177

As previously described (Bock et al., 1994), the transplastomic pRB8 plants harboring the psbF-26 site from 178

spinach showed retarded growth and a pale green leaf phenotype when grown under photoautotrophic 179

conditions in soil (Figure 2A). By contrast, when tested in growth experiments under standard greenhouse 180

conditions, HPL::LPA66 and UBQ::LPA66 transformants (which edit the heterologous psbF-26 site; Figure 181

1E) were indistinguishable from the wild type and the pRB8c control plants (that harbor the aadA marker 182

gene but not the editing site) throughout development (Figure 2A). To confirm full complementation of the 183

mutant phenotype that was caused by the unedited psbF-26 site in pRB8 plants, a series of physiological 184

measurements were performed. As expected, based on their pale-green phenotype, the pRB8 transplastomic 185

mutants had a significantly reduced total chlorophyll content compared to the wild type. Consequently, their 186

leaf absorptance of photosynthetic active radiation was decreased (Table 1). By contrast, in the LPA66-187

expressing lines, both chlorophyll content and leaf absorptance were restored to wild-type levels, and no 188

significant difference could be detected between the complemented lines and the wild type (Table 1). The 189

maximum quantum efficiency of PSII in the dark-adapted state (FV/FM), which serves as a measure of 190

photochemical intactness of the PSII reaction center and efficient coupling of the light-harvesting complex II 191

(LHCII) antenna system with the reaction center, was strongly decreased in the pRB8 mutant but nearly 192

completely restored in the LPA66-expressing lines (which reached very similar levels as the pRB8c control 193

line; Table 1). 194

To determine if restored wild-type-like growth and chlorophyll content per leaf area were due to full 195

restoration of PSII accumulation in the complemented lines, photosynthetic complex contents were 196

quantified in isolated thylakoids based on in vitro difference absorptance measurements (Figure 2B) and 197

immunoblots (Figure 2C). Since the pRB8 transplastomic line had significantly reduced total chlorophyll 198

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content compared to the wild type, the data were normalized to leaf area (rather than equal amounts of 199

chlorophyll). As expected, PSII accumulation was severely affected in pRB8 plants and reduced to 200

approximately one fifth of that in the wild type. Cytochrome b6f complex, photosystem I (PSI) and 201

plastocyanin (PC) contents were also reduced in the pRB8 mutant. Importantly, expression of HPL::LPA66 202

or UBQ::LPA66 completely restored the amount of PSII to wild-type levels (Figure 2B). PC and PSI also 203

accumulated to wild-type levels in the complemented lines, suggesting that the reduced amounts of PC and 204

PSI observed in pRB8 plants represent secondary defects caused by the strong impairment in PSII. Defects 205

in PSII are, in fact, known to affect the accumulation and activity of the downstream complexes in the 206

electron transport chain (Krech et al., 2013). Surprisingly, the accumulation of cytochrome b6f complex did 207

not fully recover to wild-type levels, neither in HPL::LPA66 nor in UBQ::LPA66 plants. Similar to pRB8 208

plants, HPL::LPA66 and UBQ::LPA66 showed an approximately 30% reduction in cytochrome b6f complex 209

contents. However, the pRB8c line, which contains the aadA resistance gene but the wild-type tobacco psbF 210

sequence, also showed a similar reduction in cytochrome b6f complex content (but not in the contents of 211

PSII, PSI and PC; Figure 2B). This finding demonstrates that the presence of the aadA gene rather than the 212

presence of the spinach psbF sequence causes the observed reduction in cytochrome b6f complex content. 213

Consequently, wild-type levels of cytochrome b6f complex cannot be restored by the expression of LPA66. 214

The spectroscopic data were confirmed by immunoblots against diagnostic (i.e., essential) subunits of the 215

photosynthetic complexes. As expected, accumulation of both the PSII reaction center subunit PsbD (the D2 216

protein) and the cytochrome b559 subunit PsbE were strongly reduced in the pRB8 mutant, but 217

indistinguishable from the wild type in the aadA control line (pRB8c) and in both complemented lines 218

(Figure 2C). For the cytochrome b6f complex, the accumulation of the essential plastid-encoded cytochrome 219

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f (PetA) and the nucleus-encoded Rieske protein (PETC) were determined. Immunoblots with both 220

antibodies confirmed a similar reduction in the accumulation of the cytochrome b6f complex in all mutant 221

lines relative to the wild type, in line with our spectroscopic data. For PSI, the plastid-encoded reaction 222

center subunit PsaA and the nucleus-encoded stromal ridge subunit PSAD (involved in ferredoxin binding) 223

were clearly reduced in the pRB8 editing mutant but present in wild-type amounts in the pRB8c control line 224

and both complemented lines. Finally, accumulation of chloroplast ATP synthase was probed with an 225

antibody against the plastid-encoded catalytic CF1 subunit AtpB. No change in AtpB content was seen in 226

any of the mutants. This is well in line with dark-interval decay kinetics of the proton motive force across the 227

thylakoid membrane, which demonstrated a similar membrane conductivity for protons in all plants (which 228

is determined by chloroplast ATP synthase activity; Table 1). 229

Next, 77K chlorophyll-a fluorescence emission spectra were recorded to monitor the distribution of 230

excitation energy between the two photosystems. The emission maxima at 687 nm and 734 nm wavelength 231

reflect the fluorescence emission of PSII-LHCII and PSI-LHCI, respectively. While the emission spectra of 232

all other lines were similar to the wild type, the pRB8 editing mutant showed a significant reduction in 233

chlorophyll-a fluorescence emission from PSII (Figure 2D), indicating PSII accumulation is strongly 234

reduced. More importantly, the maximum emission wavelength was shifted from 687 nm to 684 nm, which 235

is indicative of the presence of free, uncoupled LHCII proteins in the thylakoid membrane and suggests 236

LHCII accumulation is less affected than that of PSII. Free LHCII complexes display an emission maximum 237

at 680 nm wavelength (Krause and Weis, 1991). The 684 nm signal measured likely represents the average 238

of the emission signals originating from intact PSII-LHCII and free LHCII complexes. 239

Finally, light response curves of chlorophyll-a fluorescence parameters were measured at room temperature 240

(Figure 2E). The light response curves of linear electron flux were corrected for differences in leaf 241

absorptance (Table 1; see above). Linear electron transport capacity was severely reduced in pRB8 plants 242

compared to wild-type plants, which is well in agreement with the reduced contents of all photosynthetic 243

complexes (Figure 2B and Figure 2C). In pRB8c, HPL::LPA66 and UBQ::LPA66, linear electron transport 244

capacity was also somewhat reduced relative to the wild type. However, this effect was much less 245

pronounced than in pRB8 plants and most likely results from the observed reduction in cytochrome b6f 246

complex contents due to insertion of the aadA marker next to the petA gene. The cytochrome b6f complex 247

normally controls linear electron transport, because it catalyzes the slowest reaction of linear electron 248

transport and usually is present in sub-stoichiometric amounts relative to both photosystems (reviewed in 249

(Anderson, 1992; Schöttler and Toth, 2014)). 250

Non-photochemical quenching (qN) dissipates excess excitation energy in the PSII antenna bed as heat. The 251

induction of this process is controlled by thylakoid lumen acidification. It depends on the balance of 252

photosynthetic electron transport, which generates the proton motive force across the thylakoid membrane, 253

and ATP synthase activity, which consumes it. The qN curves (Figure 2E) showed a distinct shift of 254

photoprotective non-photochemical quenching (qN) towards lower light intensities in the pRB8 line 255

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compared to the wild type. Also, full activation of qN was impaired in the mutant. The pRB8c, HPL::LPA66 256

and UBQ::LPA66 lines had very similar qN curves, with reduced non-photochemical quenching only at light 257

intensities above 500 μmol photons m-2 s-1. Since both linear electron transport and cytochrome b6f complex 258

contents were reduced in these lines, proton influx via linear electron transport is reduced. However, 259

chloroplast ATP synthase activity (which consumes the proton motive force generated by photosynthetic 260

electron transport) remained unaltered, strongly suggesting that an imbalance between processes generating 261

and processes consuming the proton motive force resulted in reduced thylakoid lumen acidification. This 262

behavior is typical of mutants with specific defects in cytochrome b6f complex accumulation (Price et al., 263

1995; Hojka et al., 2014). 264

The qL curve represents a measure of the redox state of the PSII acceptor side (Kramer et al., 2004; Baker et 265

al., 2007). The light response curve was shifted to lower light intensities in pRB8 plants as well as in pRB8c, 266

HPL::LPA66 and UBQ::LPA66 lines (Figure 2E), in agreement with the observed restricted linear electron 267

flux in these lines. 268

In conclusion, expression of the Arabidopsis PPR protein LPA66 in tobacco is sufficient to reconstitute 269

editing of the spinach psbF-26 site in tobacco chloroplasts. It confers complete RNA editing and rescues the 270

mutant phenotype that resulted from introduction of the spinach editing site into the tobacco psbF gene. This 271

rescue occurs through restoration of the wild-type sequence for PsbF by RNA editing which, in turn, restores 272

wild-type levels of PSII. 273

274

275

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Discussion 276

RNA editing in plant organelles is a post-transcriptional process characterized by exceptional phylogenetic 277

dynamics. The set of chloroplast editing sites (also called the editotype) differs even between closely related 278

species (Freyer et al., 1995; Schmitz-Linneweber et al., 2002; Sasaki et al., 2003). Spontaneous mutations in 279

the plastid DNA are strongly biased towards C-to-T transitions, which results in highly AT-rich chloroplast 280

genomes in vascular plants (Kusumi and Tachida, 2005; Smith, 2012). This bias likely contributes to the 281

frequent loss of individual editing sites during evolution and may provide an explanation for loss events 282

being much more frequent than the gain of new editing sites. 283

Several studies have shown heterologous RNA editing sites are not recognized when introduced into species 284

that had lost the site and encode a T in the DNA at the corresponding position (Bock et al., 1994; Reed and 285

Hanson, 1997). It has been speculated the presence of an editing capacity for a heterologous site depends on 286

the retention of trans-acting specificity factor(s) encoded in the nuclear genome. 287

Several classes of nucleus-encoded trans-acting protein factors are involved in chloroplast RNA editing 288

(reviewed in (Sun et al., 2016)). Among those, PLS PPR proteins are crucial to define the position of the 289

cytosine that will undergo editing. Due to their high binding specificity for particular RNA sequences, PPR-290

type editing factors strictly co-evolve with their target cis-element(s) and, in this way, with the 291

corresponding RNA editing site(s). Therefore, the loss of a chloroplast editing site often correlates with the 292

degeneration of the gene for the corresponding PPR protein in the nucleus (Hayes et al., 2012; Hein et al., 293

2016) and this study), especially in those cases where the PPR protein only serves a single editing site. It, 294

therefore, seemed reasonable to suspect the lack of editing at heterologous sites is due to the lack of the 295

required PPR factor. 296

In this study, we have shown expression of the Arabidopsis PPR-type editing factor LPA66 is sufficient to 297

fully edit the heterologous psbF-26 site from spinach in tobacco chloroplasts. Notably, full editing was 298

observed in all transgenic lines analyzed, largely independent of the expression strength of LPA66. By 299

editing the spinach psbF-26 site, the expression of LPA66 also completely rescued the PSII-deficient 300

phenotype of the transplastomic pRB8 lines, which had been caused by the unedited psbF-26 site. 301

The remaining minor measurable difference in photosynthetic electron transfer between the wild type and the 302

lines complemented with LPA66 (small reduction in FV/FM and in linear electron transport capacity, delayed 303

induction of non-photochemical quenching) do not translate into a growth phenotype and, importantly, are 304

also found in the pRB8c control plants. We have shown these differences are caused by a reduced 305

accumulation of the cytochrome b6f complex. The pRB8c control mutant only harbors the selectable marker 306

gene aadA, but not the psbF-26 editing site, suggesting the presence of the selectable marker gene in this 307

particular genomic position is causally responsible for the reduced accumulation of the cytochrome b6f 308

complex in the mutants. The aadA cassette in the pRB8 and pRB8c lines is inserted downstream of the 309

psbEFLJ transcriptional unit (Bock et al., 1994). Downstream of the insertion site resides the petA gene, 310

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which encodes the essential cytochrome f subunit of the cytochrome b6f complex and is transcribed in the 311

opposite direction. It, thus, seems reasonable to assume that the insertion of the aadA marker in this position 312

directly or indirectly affects the expression of PetA. The precise mechanism of this interference remains to 313

be determined. 314

Two non-PPR factors are involved in editing of psbF-26 in Arabidopsis: MORF8/RIP1 (Bentolila et al., 315

2012) and ORRM6 (Hackett et al., 2017). Although their role in the editing reaction is not yet fully 316

understood, disruption of each of these two genes is sufficient to completely abolish editing at psbF-26. 317

Using in-house transcriptomic datasets, we could identify putative orthologs of MORF8/RIP1 and ORRM6 318

in tobacco (Table 2). It, therefore, is reasonable to assume Arabidopsis LPA66 is capable of faithfully 319

interacting with the tobacco orthologs of the two other essential trans-acting editing factors. In line with the 320

high sequence specificity of RNA binding by PPR proteins, overexpression of LPA66 did not affect editing 321

at the neighboring psbE-72 and psbL-1 sites occurring in the same transcript (Figure 1E). 322

In summary, this study demonstrates the possibility to transfer editing events between species by expressing 323

a heterologous editing site in the plastid genome and its cognate site-specific factor in the nuclear genome. 324

This can be accomplished without interfering with editing activity at endogenous sites. The significance of 325

this finding is twofold. First, our successful reconstitution of heterologous editing demonstrates that, 326

although editing reactions are known to depend on multiple trans-acting protein factors, the inter-species 327

transfer of editing events can be achieved by introduction of the two rapidly evolving components of the 328

system: the editing site and the corresponding PPR protein. Second, the possibility to implement new editing 329

sites in plastid genomes provides a powerful tool for the development of inducible expression systems in 330

biotechnology and the design of efficient on/off switches in synthetic biology. For example, synthesis of a 331

gene product can be made dependent on RNA editing by correcting a missense mutation or creating a 332

translational start codon. Editing then can be induced by simply placing the corresponding PPR gene under 333

the control of a promoter that is inducible by chemical cues or responsive to an environmental trigger (e.g., a 334

biotic or abiotic stress signal). 335

336

Materials and Methods 337

Plant material and growth conditions 338

Tobacco (Nicotiana tabacum) seeds from transplastomic lines pRB8 and pRB8c (described as pRB8-S6 and 339

pRB8-S5 in (Bock et al., 1994), respectively) were surface sterilized by 70% (v/v) ethanol for 7 min, 340

followed by a 7 min treatment with 7% (v/v) hypochlorite. Seeds were then washed five times with sterile 341

water and plated on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 500 mg/L 342

spectinomycin dihydrochloride. The plates were incubated at 4°C in the dark for two days for stratification 343

before transfer to controlled environment chambers (light intensity: 50 μE m-2 s-1, diurnal cycle: 16 h light at 344

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24°C and 8 h dark at 22°C). Ten days after germination, seedlings were transferred to soil and grown under 345

standard greenhouse conditions (unless otherwise mentioned) to maturity: 16 h light at 25°C and 8 h 346

darkness at 20°C. Plants used for photosynthetic measurements were grown in controlled environment 347

chambers (Conviron, Winnipeg, Canada) at 120 μE m-2 s-1 light intensity (16 h light at 22°C, 75% relative 348

humidity, and 8 h dark at 18°C, 70% relative humidity). After approximately three weeks, plants were 349

transferred to controlled environmental chambers with the actinic light intensity set to 350 μE m-2 s-1. All 350

other environmental parameters remained unaltered. 351

Cloning and plant transformation 352

For the generation of construct HPL::LPA66, the full-length coding sequence of LPA66 (AT5G48910) was 353

amplified from Arabidopsis (Arabidopsis thaliana) Col-0 genomic DNA with primers oVL84 and oVL85 354

and cloned by Gibson Assembly into vector pORE-E2 (Coutu et al., 2007) that had been linearized with 355

KpnI. The HPL (hydroperoxide lyase) promoter was replaced by the UBIQUITIN10 promoter to generate the 356

UBQ::LPA66 construct. The UBIQUITIN10 promoter was amplified from Arabidopsis Col-0 genomic DNA 357

with primers P_UBQ10for and P_UBQ10rev and subsequently digested with XhoI and EcoRI. Each 358

construct was introduced into line pRB8 by Agrobacterium tumefaciens-mediated nuclear transformation 359

(Rosahl et al., 1987) using A. tumefaciens strain pGV2260. Transgenic lines were regenerated on RMOP 360

medium (Svab et al., 1990) supplemented with 50 mg/L kanamycin for selection and 250 mg/L cefotaxime 361

sodium salt (claforan) to prevent growth of bacteria. 362

RNA editing and expression analyses 363

Total plant RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific) following the protocol 364

of the manufacturer. Complementary DNA (cDNA) was synthesized with the help of the QuantiTect Reverse 365

Transcription Kit (Qiagen) following the manufacturer’s recommendations and using a 1:1 mixture of 366

random hexamer (Qiagen) and oligo(dT)18 primers. cDNA fragments were amplified by PCR using specific 367

oligonucleotides as primers (Supplemental Table S1) and following standard protocols (Taq DNA 368

polymerase, ThermoFisher). PCR was performed for 35 cycles, with the exception of the amplification of 369

ACTIN, where the number of cycles was reduced to 23. PCR products were purified with the NucleoBond 370

PCR Clean-up kit (Macherey-Nagel) and sequenced (Eurofins Genomics, Ebersberg, Germany). 371

Immobilization and detection of proteins by immunoblot analysis 372

Thylakoids were extracted according to (Schöttler et al., 2004) from leaf material of plants grown in the 373

conditions described for photosynthetic measurements. Thylakoid proteins were separated using the 374

PerfectBlue Dual Gel System Twin L (VWR International). Then 15% or 12% (w/v) denaturating 375

polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970). Separated 376

proteins were transferred to Hybond-P PVDF membranes (GE Healthcare) using a tank blotter (VWR 377

International). Membranes were stained by 0.25% (w/v) Coomassie Brilliant Blue R-250 (SERVA), 378

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14

destained using 100% (v/v) methanol and scanned using an EPSON Perfection V700 Photo scanner. 379

Blocking was performed in TBS-T buffer (20mM Tris-HCl pH 7.6, 137 mM NaCl, Tween20 0.1% (w/v)) in 380

the presence of 4% (w/v) skimmed milk powder and 0.5% (w/v) bovine serum albumin (BSA, Carl Roth 381

GmbH) for 1 h at room temperature under continuous shaking. After a washing step in TBS-T, membranes 382

were incubated with the primary antibody of interest in TBS-T for 1 h at room temperature with slow 383

shaking. The primary antibodies used (α-PsbD, α -PsbE, α -PetA, α -PETC, α -PsaA, α -PsaD, α -AtpB) were 384

purchased from Agrisera. Binding of the appropriate HRP conjugated-secondary antibody (Sigma; diluted 385

1:50,000 in TBS-T) was allowed in the presence of 0.5% (w/v) BSA for 1 h at room temperature with slow 386

shaking. Membranes were treated with the ECL Plus Western Blotting Detection Kit (GE Healthcare) 387

according to the manufacturer’s instruction and exposed in a G:BOX Chemi XT4 (Syngene) for signal 388

detection. 389

Spectroscopic methods 390

Leaf chlorophyll content and chlorophyll a/b ratio were determined from leaf extracts in 80% (v/v) acetone 391

according to (Porra et al., 1989) using a V-730 UV-Vis Spectrophotometer (Jasco GmbH, Groß-Umstadt, 392

Germany). Chlorophyll-a fluorescence emission at 77 K was determined on freshly isolated thylakoids 393

(Schöttler et al., 2004) equivalent to 10 μg of chlorophyll mL–1 using a F-6500 fluorometer (Jasco). The 394

sample was excited at a 430 nm wavelength (10 nm bandwidth). Emission spectra between 655 and 800 nm 395

were recorded with a bandwidth of 1 nm, and 10 spectra were averaged to improve the signal-to-noise ratio. 396

Chlorophyll-a fluorescence of intact leaves was measured at 22°C using a Dual-PAM-100 instrument (Heinz 397

Walz GmbH, Effeltrich, Germany). FV/FM and light-response curves of linear electron transport, qN and qL 398

were measured on intact leaves after at least 30 min of dark adaptation. Under light-limited conditions, each 399

actinic light intensity was measured for 150 s while under light-saturated conditions, light intensity was 400

increased every 60 s. Linear electron transport was corrected for the leaf absorptance measured with an 401

integrating sphere (ISV-722, Jasco) attached to a spectrophotometer (V-650, Jasco). Transmittance and 402

reflectance spectra of leaves were recorded between 400 and 700 nm wavelength, and leaf absorptance was 403

calculated as 100% minus transmittance of light through the leaf minus reflectance on the leaf surface. The 404

average value of the absorptance spectrum between 400 and 700 nm wavelength was used for the calculation 405

of linear electron flux. Dark-interval decay kinetics of the electrochromic absorption shift (ECS) were used 406

as in vivo probes of ATP synthase activity (Baker et al., 2007). The ECS, which is proportional to the light-407

induced proton motive force across the thylakoid membrane, was measured using a KLAS-100 LED-Array 408

Spectrophotometer (Heinz Walz GmbH), allowing the simultaneous measurement of light-induced 409

difference absorption signals at eight pairs of wavelengths in the visible range of the spectrum between 505 410

and 570 nm, as described by (Rott et al., 2011). Leaves were illuminated with saturating light (1200 µmol 411

photons m–2 s–1) for 10 min prior to each measurement to allow photosynthesis to reach the steady state. 412

Actinic illumination was interrupted by a short interval of darkness (15 s), and the dark-interval decay of the 413

ECS was measured. In pre-illuminated leaves, this decay kinetic is determined by ATP synthase activity. 414

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15

Photosynthetic complex contents of PSII, cytochrome b6f complex and PSI were quantified in isolated 415

thylakoids as described in (Hojka et al., 2014) using a V-550 spectrophotometer (Jasco) and a Dual-PAM 416

instrument (Heinz Walz). Plastocyanin contents relative to PSI were determined by in vivo difference 417

absorption spectroscopy in the far-red range of the spectrum and then recalculated based on the absolute PSI 418

quantification in isolated thylakoids (Schöttler et al., 2007). 419

Bioinformatic analyses 420

Orthologs of Arabidopsis LPA66 (AT5G48910) were extracted from an in-house database (POTbase, 421

https://chlorobox.mpimp-golm.mpg.de; (Moreno et al., 2018)). The sequence of the LPA66 ortholog from 422

spinach (Spinacia oleracea) was obtained from an RNAseq dataset from (Yan et al., 2016) (GenBank 423

databases ID: SRP051935). Nucleotide and amino acid sequences of psbF were obtained from NCBI. Sanger 424

sequencing data were visualized using SeqMan Pro 14 (DNASTAR®), and chromatograms were extracted 425

using Chromas (http://technelysium.com.au/wp/chromas/). DNA and protein alignments were produced 426

using ClustalW within BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). 427

428

Accession Numbers 429

AEF1: AT3G22150; CP31A: AT4g24770; CREF7: AT5G66520; CRR4: AT2G45350; CRR21: AT5G55740; 430

HPL: AT4G15440; LPA66: AT5G48910; MORF2/RIP2: AT2G33430; MORF8/RIP1: AT3G15000; 431

MORF9/RIP9: AT1G11430; ORRM1: AT3G20930; ORRM6: AT1G73530; OZ1: AT5G17790; QED1: 432

AT2G29760; RARE1: AT5G13270; RBCS1A: AT1G67090; UBQ10: AT4G05320. 433

Supplemental Data 434

The following supplemental figures, tables and information are included separately as a single pdf file. 435

Supplemental Figure S1. Alignment of psbF genes. 436

Supplemental Figure S2. Expression patterns of endogenous Arabidopsis PPR-type editing factors. 437

Supplemental Table S1. Oligonucleotides used in this study. 438

439

Acknowledgments 440

We thank the Max Planck Institute of Molecular Plant Physiology (MPI-MP) Green Team for plant care and 441

cultivation. This research was supported by the Max Planck Society, and by grants from the Deutsche 442

Forschungsgemeinschaft (DFG) to M.T. (TI 605/5-1) and R.B. (SFB-TRR 175). 443

444

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Table 1. Measurement of photosynthesis-related parameters in wild-type tobacco plants, transplastomic 445

pRB8 and pRB8c plants, and supertransformed pRB8 plants expressing LPA66. The number of biological 446

replicates is indicated (n). Values in bold: One-way ANOVA, Holm–Sidak method, p≤0.05. All mutants 447

were compared to each other and the wild type, with only pRB8 showing significant differences from the 448

wild type and all other mutants for chlorophyll content, leaf absorptance, and the maximum quantum 449

efficiency of PSII in the dark-adapted state (FV/FM). 450

451

Parameter WT 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

452

453

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Table 2. Chloroplast RNA editing factors required for editing at Arabidopsis site psbF-26. Editing efficiency 454

at psbF-26 is indicated as reported in the corresponding knock-out mutants (% T/C), and the reference is 455

given. Values in bold represent severe reductions in editing efficiency compared to the wild type. The 456

presence of an ortholog in the tobacco nuclear genome is indicated by +. 457

458

Co-factor 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 +

459

460 461 462

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463

Figure legends 464

465

Figure 1. Expression of Arabidopsis LPA66 in transplastomic pRB8 plants. 466

(A) Association between the presence (C) or absence (T) of the psbF-26 editing site and the presence of an 467

LPA66 ortholog in the nuclear genome of 15 selected angiosperm species. Note that the presence of 468

orthologs of Arabidopsis LPA66 (+) associates strictly with the occurrence of the psbF-26 site. 469

(B) Nucleotide sequence alignment of the region surrounding the psbF-26 editing site (from position –30 to 470

+10, with the editing site being position 0). Deviations from the consensus sequence are shaded in grey. The 471

predicted binding sequence of LPA66 according to the PPR code (Barkan et al., 2012; Cheng et al., 2016) is 472

boxed. 473

(C) Physical maps of the constructs generated for stable nuclear transformation of transplastomic pRB8 474

plants (Bock et al., 1994). Full-length Arabidopsis LPA66 is expressed under the moderate 475

HYDROPEROXIDE LYASE1 promoter (PHPL) in construct pVL29 (HPL::LPA66) or under the strong 476

UBIQUITIN10 promoter (PUBQ) in construct pVL30 (UBQ::LPA66). In all constructs, the resistance gene 477

(nptII, conferring resistance to kanamycin) is expressed from the enhanced tobacco constitutive promoter 478

(PenTCUP2) and the nos terminator (Tnos). LB: left border. RB: right border of the T-DNA. 479

(D) Expression of Arabidopsis LPA66 assessed by semiquantitative RT-PCR in tobacco wild-type plants 480

(WT) and nuclear transformants expressing LPA66 under the HPL or UBQ promoter. The expression of 481

ACTIN was analyzed as a constitutively expressed control gene. H2O: negative control. 482

(E) Editing status of the heterologously expressed spinach psbF-26 site and the tobacco psbE-72 and psbL-1 483

sites assessed by sequencing of the amplified cDNA population. C-to-U conversions are marked by asterisks. 484

485

Figure 2. Complementation of the PSII defect of pRB8 by Arabidopsis LPA66. 486

(A) Phenotype of N. tabacum wild type (WT), the pRB8c aadA control, the pRB8 transplastomic recipient 487

line and pRB8 nuclear transformants expressing LPA66 under the control of the HPL or UBQ promoter. 488

Photographs were taken 11 weeks after sowing. Scale bar: 10 cm. 489

(B) In vitro complex content quantification in isolated thylakoids for photosystem II (PSII), the cytochrome 490

b6f complex (Cyt-bf), plastocyanin (PC) and photosystem I (PSI) normalized to the leaf area. Error bars 491

indicate the standard deviation of biological replicates. All mutants were compared to the wild type and each 492

other for significant differences, with pRB8 showing significant differences from the wild type and all other 493

mutants for the contents of PSII, plastocyanin, and PSI. All mutants showed a significant reduction in the 494

content of the cytochrome b6f complex, relative to the wild type. *p≤0.05, One-way ANOVA, Holm–Sidak 495

method. The number of replicates is given in Table 1. 496

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(C) Immunoblots against diagnostic subunits of PSII, cytochrome b6f (Cyt-bf), PSI and ATP synthase 497

(AtpB). Thylakoid extracts were loaded on leaf area basis. 498

(D) 77K chlorophyll-a fluorescence measurements normalized to PSI emission at 733 nm. 499

(E) In vivo light response curves of the fraction of open PSII centers (qL), non-photochemical quenching 500

capacity (qN) and linear electron transport rate. The three graphs share the same x-axis. Error bars indicate 501

the standard deviation of the number of biological replicates indicated in Table 1. 502

503

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504

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