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Plant Cell Advance Publication. Published on April 6, 2016, doi:10.1105/tpc.15.00879 1 Spontaneous chloroplast mutants mostly occur by replication slippage and 2 show a biased pattern in the plastome of Oenothera 3 4 Authors: Amid Massouh1), Julia Schubert1), Liliya Yaneva-Roder1), Elena S. 5 Ulbricht-Jones1), Arkadiusz Zupok1), Marc T. J. Johnson2), Stephen I. Wright3), 6 Tommaso Pellizzer1), Johanna Sobanski1), Ralph Bock1), Stephan Greiner1,4) 7 8 9 1) Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D- 14476 Potsdam-Golm, Germany 10 11 12 2) Department of Biology, University of Toronto-Mississauga, Mississauga, ON, L5L 1C6, Canada 13 14 15 3) Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, M5S 3B2, Canada 16 17 4) Corresponding author: Stephan Greiner [email protected] 18 19 20 The author responsible for distribution of materials integral to the findings presented 21 in this article in accordance with the policy described in the Instructions for Authors 22 (www.plantcell.org) is: Stephan Greiner ([email protected]). 23 24 Short title: Spontaneous plastome mutants in Oenothera 25 26 Synopsis: Spontaneous chloroplast mutants mostly occur by replication slippage 27 and display a mutation spectrum very different from that in the eukaryotic nucleus 28 and that of bacteria. 29 1 ©2016 American Society of Plant Biologists. All Rights Reserved. 30 Abstract 31 Spontaneous plastome mutants have been used as a research tool since the 32 beginnings of genetics. However, technical restrictions have severely limited their 33 contributions to research in physiology and molecular biology. Here, we used full 34 plastome sequencing to systematically characterize a collection of 51 spontaneous 35 chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a 36 single mutation. Unexpectedly, the vast majority of mutations do not represent single 37 nucleotide polymorphisms, but are insertions/deletions originating from DNA 38 replication slippage events. Only very few mutations appear to be caused by 39 imprecise double-strand break repair, nucleotide misincorporation during replication, 40 or incorrect nucleotide excision repair following oxidative damage. U-turn inversions 41 were not detected. Replication slippage is induced at repetitive sequences that can 42 be very small and tend to have high A/T content. Interestingly, the mutations are not 43 distributed randomly in the genome. The underrepresentation of mutations caused 44 by faulty double-strand break repair might explain the high structural conservation of 45 seed plant plastomes throughout evolution. In addition to providing a fully 46 characterized mutant collection for future research on plastid genetics, gene 47 expression and photosynthesis, our work identified the spectrum of spontaneous 48 mutations in plastids and reveals that this spectrum is very different from that in the 49 nucleus. 50 2 51 Introduction 52 Mutations in the chloroplast genome (plastome) are typically recognized by pale or 53 bleached leaf areas in variegated plants (Kirk and Tilney-Bassett, 1978; Greiner, 54 2012; Figure 1A). Such material has been used as a genetic tool since the beginning 55 of genetics. For example, the use of plastome mutants by Correns and Baur formed 56 the foundation of cytoplasmic genetics (Baur, 1909; Correns, 1909; Baur, 1910; 57 Hagemann, 2010). Plastome mutants continue to play an important role in 58 elucidating the rules of non-Mendelian inheritance (Chiu and Sears, 1985; Azhagiri 59 and Maliga, 2007; Ruf et al., 2007) and in studying chloroplast gene expression and 60 photosynthesis (Schaffner et al., 1995; Landau et al., 2009). Moreover, plastid 61 mutants provide selectable markers for antibiotic or herbicide resistance that are 62 widely used in molecular biology, biotechnology, and agriculture (e.g., Schmitz- 63 Linneweber et al., 2005; Powles and Yu, 2010). 64 Over several decades, a substantial number of chlorotic chloroplast mutants 65 have been identified and characterized from multiple plant species (Kirk and Tilney- 66 Bassett, 1978; Börner and Sears, 1986; Somerville, 1986; Hagemann, 1992; 67 Greiner, 2012). Importantly, phenotypes of plastome mutants go beyond simple 68 bleaching. 69 developmentally impaired or sensitive to environmental factors, show mottled 70 phenotypes and/or bleach reversibly (e.g., Kirk and Tilney-Bassett, 1978; Stubbe 71 and Herrmann, 1982; Chia et al., 1986; Archer and Bonnett, 1987; Colombo et al., 72 2008; Hirao et al., 2009; Landau et al., 2009; Greiner, 2012; Figure 1B-D, Table 1). A 73 number of mutant lines have been described that display unexpected phenotypes, 74 such as mutant plastids having a phenotypic effect on wild-type plastids in the same 75 cell (Michaelis, 1957), or mutations that confer drought and temperature tolerance 76 (Usatov et al., 2004; Mashkina et al., 2010). Collectively these works show that 77 although chlorosis is a common feature of plastome mutants, plastid mutations can 78 have diverse effects on the plant phenotype. Some plastome mutants display mild chlorotic effects, are 79 Although chloroplast mutants can provide insight into chloroplast gene 80 function and regulation, technical limitations have limited wider application in plant 81 research. Disregarding mutants resistant to herbicides or antibiotics, only 12 82 plastome mutations have been identified in vascular plants (Greiner, 2012). Since 83 such mutations cannot be identified by conventional linkage mapping, direct 84 sequence analysis is required to pinpoint a mutated locus. Past approaches relied on 3 85 physiological and biochemical analyses of the mutant, which allowed predictions 86 about the mutated gene. This strategy was applied to most of the plastome 87 mutations that have been identified by molecular approaches (Greiner, 2012), and is 88 still successfully used in screens of plastid-encoded herbicide resistance (Thiel et al., 89 2010). However, the approach of combining physiological characterization with local 90 sequence analysis suffers from limitations: First, the candidate gene needs to be 91 predictable, which implies that its biochemical and physiological attributes are 92 already well characterized. Consequently, identifying mutations in genes of unknown 93 function is very difficult. Second, the presence of a second site mutation cannot be 94 ruled out. Full chloroplast genome sequencing by next-generation sequencing can 95 overcome these technical constraints. 96 Another reason why chloroplast mutants are currently understudied is the 97 challenge of maintaining plastome mutants in standard model species with 98 uniparental plastid inheritance. Many plastome mutants are not viable on soil. They 99 must therefore be kept in as variegated plants during propagation, because wild-type 100 plastomes are essential to nourish the mutant tissue (Figure 1A). Variegation occurs 101 because of random chloroplast sorting during cell division, which gives rise to 102 homoplastic mutants and wild-type tissue. Sorting-out is a statistical process and 103 very rapid (Michaelis 1955, Kirk and Tilney-Bassett, 1978). It is typically completed 104 early in leaf development (Schötz 1954). Hence, in most plant species, variegation 105 cannot be maintained through sexual reproduction because it is challenging to 106 identify egg cells that are heteroplasmic and could give raise to variegated plants in 107 the next generation (Kirk and Tilney-Bassett, 1978; Greiner 2012). Therefore, 108 chloroplast mutants are typically maintained by cutting variegated stems or in tissue 109 culture, but these approaches are infeasible for many vascular plants. Using a model 110 organism displaying biparental plastid inheritance, like Oenothera, overcomes the 111 technical limitation. 112 The genus Oenothera (evening primrose, Onagraceae) is an excellent model 113 for the study of chloroplast mutants, including their mutation spectrum, their 114 molecular basis and phenotypic effects. Past research shows that spontaneous 115 chloroplast mutants occur with a frequency of 0.3% in Oenothera (Kutzelnigg and 116 Stubbe, 1974). Biparental transmission allows the directed generation of variegated 117 plants by crossing, using individuals with mutated and wild-type plastomes as 118 crossing partners. Thus, plastome mutants can be reliably propagated from seeds 4 119 and kept as variegated plants in soil (Figure 1 A, Methods). This approach has 120 allowed the generation of a large collection of spontaneous chloroplast mutants. 121 Based on chlorotic phenotypes, a total of 51 spontaneous plastome mutants were 122 isolated and maintained through efforts of Wilfried Stubbe during the past century 123 (Kutzelnigg and Stubbe, 1974; Stubbe and Herrmann, 1982; Table 1; Supplemental 124 Table 1). All mutations arose in the shoot apical meristem, since they entered the 125 germline and are sexually transmitted. Hence, the collection represents the spectrum 126 of heritable mutations that lead to visibly impaired chloroplast function. Although the 127 collection has been characterized extensively at the phenotypic level, technical 128 limitations have prevented molecular analysis (Greiner, 2012). 129 Here we describe the mutation spectrum of disruptive spontaneous 130 chloroplast mutations using the Oenothera plastome mutant collection. Based on full 131 plastome sequencing we suggest a molecular mechanism of their occurrence. 132 Employing statistical methods, we test to what extent mutation events and recovery 133 are biased toward the mutated target gene, the local sequence context, and the 134 location of a mutation on the chloroplast genome. 135 136 Results 137 Phenotypes of chloroplast mutants in homoplasmic leaf tissue 138 The 51 mutants of the collection show various types of chlorophyll deficiency, which 139 typically occurred in an age-dependent manner. Some of the phenotypes are 140 sensitive to environmental factors (Figure 1, Table 1). Non-uniform leaf phenotypes 141 in homoplasmic mutant tissue occur either constitutively or are restricted to a certain 142 developmental stage in 16 mutants. Three of those display a pigmentation gradient 143 from the leaf base to the tip (Figure 1B). In mature rosette leaves, 40 mutants fully 144 bleach to a nearly white or yellowish creamed-colored phenotype, whereas light 145 green tissue remains throughout development in eleven mutants. Two of those are 146 viable in soil and need no nourishment from wild-type tissue (Table 1, cf. Methods). 147 Particularly interesting phenotypes are seen in the mutants I-tau and I-iota. In the 148 mutant I-iota, an age-dependent mosaic pattern of pigmentation is observed where 149 young leaves are white and slightly mottled. The green spots increase in size and 150 number during development, leading to nearly green mature rosette leaves (Figure 151 1C). I-tau displays a reversible bleaching (virescent) phenotype (Figure 1D). It can 5 152 grow photoautotrophically and forms bleached and green leaves in an alternating 153 order. 154 155 Wild-type plastomes 156 For identification of the underlying mutations, the full sequence of the corresponding 157 wild-type reference plastome was obtained. The mutants of the collection are derived 158 from 13 wild-type strains (Supplemental Tables 1 and 2). The complete plastome 159 sequences of four of the wild-type lines had been determined previously (Greiner et 160 al., 2008a). In this study, we re-sequenced all five previously determined Oenothera 161 plastomes 162 (EMBL/GenBank accessions: AJ271079.4, EU262887.2, EU262889.2, EU262890.2, 163 EU262891.2). In addition to correcting a few sequencing errors (mostly single 164 nucleotides) we detected two larger insertions of 147 and 24 bps compared to the 165 previously published chloroplast genome of Oenothera elata ssp. hookeri strain 166 johansen Standard. This likely reflects genetic variation in our seed stocks within 167 unstable highly repetitive genomic regions (Figure 2, Greiner et al. 2008a, and 168 below). Newly determined were the complete chloroplast genome sequence of eight 169 reference strains and an additional line to address plastome stability (see below; 170 GenBank accession numbers KT881169.2 - KT881177.1; for summary see 171 Supplemental Table 3). The wild-type reference strain of one mutant (V-alpha) is no 172 longer present in our collections (see Table 1 for details). Overall, the Oenothera 173 chloroplast genome is well conserved in structure and size (spanning 163,367 - 174 166,545 bp) and harbors an identical gene set of 113 unique genes, as described 175 earlier (Greiner et al., 2008a; Greiner et al., 2008b). using Illumina next-generation DNA sequencing technology 176 177 Identification of mutated alleles and second-site mutations in the plastome mutant 178 collection 179 From the 51 spontaneous mutants, 46 unique chloroplast genomes were recovered 180 (Table 1), and five mutant pairs share an identical sequence. It is conceivable that 181 contamination during propagation of the collection is responsible for these cases of 182 seemingly identical chloroplast genomes (the collection was maintained for decades 183 without the benefit of molecular markers for tracking). However, since the distribution 184 of mutated alleles was found to be biased within the Oenothera plastome (Figure 3 185 and below), we cannot exclude the possibility that at least some of the identical 6 186 mutations might have arisen independently. This view is supported by the fact that in 187 lines with different wild-type plastomes, the same locus sometimes experienced 188 independent mutations. However, those resulted in distinct mutated alleles (mutant 189 pairs I-iota/III-delta and I-xi/III-alpha; Supplemental Figure 1 - 3). All mutants sharing 190 an identical mutation also share the same wild-type background. Also, in previous 191 analyses, their phenotypic descriptions were essentially identical (Tables 1 and 192 Methods for details). In this context, it is noteworthy that the oldest viable seed lots 193 that we could investigate for potential propagation errors date back to 1985. 194 Within the 46 unique mutated chloroplast genomes of the collection, 37 195 genomes carry a mutation at a single site (Table 1). The identification of a single 196 mutation in V-alpha is tentative, since the original wild-type reference is no longer 197 present in our strain collection (see above; Table 1). Two mutated sites are present 198 in each of the remaining nine mutants. Two of these mutants represent known 199 second-site mutations in that they were isolated based on a phenotypic change 200 arising in a pre-existing chloroplast mutant. Thus, in total 53 (37 + 9 + 7) unique 201 mutated sites could be detected in the entire collection (Table 1 and Supplemental 202 Data Set 1). The second-site mutations of the remaining seven mutants are located 203 in regions of intrinsic instability associated with larger tandem repeats or oligo(N) 204 stretches in intergenic regions (Figure 2). Their phenotypic relevance is discussed 205 below. 206 207 Types of mutations in the collection 208 The 53 mutant sites in the collection essentially fall into five classes: (i) single-bp 209 transversions (two mutations), (ii) single-bp, double-bp and micro insertions/deletions 210 (indels, < 10 bp) at oligo(N) stretches (six mutations), (iii) micro indels at micro 211 repeats (< 10 bp; 32 mutations; 21 duplications, eleven deletions), (iv) macro indels 212 (mostly between 12 bp and 70 bp, one exceptional deletion of 153 bp) at macro 213 repeats (> 10 bp; twelve mutations; five duplications, six deletions; one mutation 214 event most likely caused by an insertion and a deletion), and (v) one large deletion 215 (1,364 bp), which could not be associated with a repetitive element (see 216 Supplemental Data Set 1 and Supplemental Figures 1 - 6 for detailed description of 217 the individual mutations). Within the micro indels (< 10 bp), the major class of 218 mutations detected in this study, a bias towards duplications is not present (χ2: p = 219 0.3113). However, a clear bias to indels conferred by tandem/direct repeats is 7 220 present (χ2: p = 0.0003; 32 mutations, 30 associated with tandem/direct repeats, two 221 with palindrome/inverted repeats; Supplemental Data Set 1). In summary, nearly all 222 chloroplast mutations are associated with repetitive elements. These repeats can be 223 very small, in some cases with two repetitive elements of 3 bp (Supplemental 224 Figures 1 - 6; Supplemental Data Set 1). 225 226 Molecular cause of mutations and phenotypic relevance of the second site mutations 227 The detected chloroplast mutations have a bias toward coding sequence. Most 228 mutations are located in protein-coding regions (44 of 53) of which 37 induce 229 frameshifts and six represent in-frame indels. One mutation is conferred by a 230 transversion, leading to a single amino acid change (Table 1). At least five of the 231 mutations affect chloroplast mRNA metabolism. Three of these occur in introns 232 where they result in splicing defects. The mutations in I-psi and II-kappa are located 233 in intron 1 and intron 2 of ycf3, respectively, and affect splicing of these introns (cf. 234 Landau et al., 2009). The gene is involved in photosystem I assembly (Ruf et al., 235 1997). A similar situation is found for the mutant II-theta where the mutation is 236 located in the intron of petB. The pet genes encode subunits of the cytochrome b6f 237 complex. Here, unspliced transcripts overaccumulate. The mutant II-delta occurs in 238 the intergenic region of the ndhE-psaC spacer (ndh genes encode subunits of the 239 chloroplast NADH dehydrogenase complex; psa genes encode subunits of 240 photosystem I). The mutation in the ndhE-psaC spacer is the sole mutation in this 241 line and it is not located in a known chloroplast promoter (see Greiner et al., 2008a 242 for searched motifs). The mutant has an mRNA processing defect in the ndhH-ndhA- 243 ndhI-ndhG-ndhE-psaC-ndhD operon (Figure 4). Interestingly, defects in mRNA 244 metabolism were not only found in the four mutants where one might have expected 245 them (i.e., mutants with mutations in introns or intergenic regions). The mutant II-eta, 246 harboring a mutation in exon 2 of petB, was found to be affected in processing 247 and/or stability of the psbB (petB) operon transcripts. The primary transcripts from 248 this operon undergo a series of processing and maturation steps, resulting in a 249 highly complex pattern of partially processed transcripts and mature mRNAs 250 (Westhoff and Herrmann, 1988). As judged from transcript sizes and the previous 251 characterization of RNA species (Westhoff and Herrmann, 1988), a major spliced 252 RNA species comprising psbH, petB, and petD is hardly detectable in II-eta (psbH 8 253 encodes a subunit of photosystem II), whereas a transcript likely containing the 254 spliced petB/petD dicistron overaccumulates (Figure 4). 255 The remaining five mutations of the collection are in non-coding regions and 256 are second-site mutations. Two are located within oligo(N) stretches in intergenic 257 regions; three are upstream of ycf1 (tic214) or accD in regions of intrinsic instability 258 due to the presence of large complex tandem repeats (Figure 2 and below). The 259 accD gene encodes a subunit of the plastidic acetyl-CoA carboxylase; ycf1 (tic214) 260 is currently considered to encode a subunit of the chloroplast protein import 261 machinery (Kikuchi et al., 2013). 262 Among the mutations in genic regions, three are associated with ATP 263 synthase, twelve with photosystem I (nine of them within psaA, exclusively 264 representing duplications; Figure 3), eleven with photosystem II, and six within the 265 cytochrome b6f complex. Further, four mutations were found in ycf3 and one in ycf4 266 (as ycf3 involved in photosystem I assembly; Ruf et al., 1997; Krech et al., 2012), 267 three within rbcL (encoding the large subunit of RuBisCO), and four within ccsA (a 268 gene involved in cytochrome c biogenesis; Xie and Merchant, 1996). These 269 functional gene classes affect photosynthesis and are considered to be non- 270 essential for cell viability (Scharff and Bock, 2014). An additional mutation occurred 271 in rps7, which is likely an essential gene in dicots encoding a protein for the small 272 (30S) subunit of the plastid ribosome (Scharff and Bock, 2014; Tiller and Bock, 273 2014). Viability of this mutation is likely due to the particular mutated allele, which 274 modifies the very C-terminal sequence of the protein. Additional second site 275 mutations include in-frame indels in the intrinsically unstable regions of accD and 276 ycf1 (tic214). Those are again associated with large complex tandem repeats (Figure 277 2). 278 The phenotypic relevance of the second site mutations is not entirely clear. 279 Mutations within oligo(N) stretches in intergenic regions are likely to be 280 phenotypically neutral. By contrast, the contribution to the phenotype is uncertain for 281 the mutations in the intrinsically unstable repetitive regions: The location of these 282 mutations is upstream or in the coding regions of accD and ycf1 (tic214), 283 respectively, which are both essential in dicots (Drescher et al., 2000; Kode et al., 284 2005; Supplemental Data Set 2). However, the phenotype of all mutants carrying 285 such mutations can be fully explained by the second mutated allele present in these 286 lines (Table 1 and Supplemental Data Set 2). This is likely also the case with 9 287 mutated sites in repetitive regions that represent indels within the reading frames of 288 accD and ycf1 (tic214; Figure 2). These indels are in frame and the amino acid 289 sequences in these regions are poorly conserved among Oenothera species 290 (Greiner et al., 2008a). In addition, very similar polymorphisms, including in-frame 291 indels within the coding region of ycf1 (tic214), were also detected upon comparison 292 of very closely related wild-type strains, as described below. 293 294 Statistical distribution of mutant alleles within the chloroplast genome 295 An important question addressed by our study is whether mutations are randomly or 296 non-randomly distributed within the plastome, and whether these patterns reflect real 297 mutational biases or biases related to our selection process. We tested for biological 298 and methodological biases by assessing the influence of the expected phenotype, 299 gene length, and the location of the gene in the plastome on the probability of 300 isolating a mutation. We assigned the genes of the Oenothera plastome to one of 301 three classes based on phenotypes observed in systematic knock-out analyses of 302 chloroplast genes in Nicotiana tabacum (Supplemental Data Set 2). “Class I” genes 303 included those in which spontaneous mutations are easily detected. This class 304 combines genes that are non-essential for heterotrophic growth and whose knock- 305 outs display either white or pale phenotypes (subgroups: “White” and “Pale”). 306 Examples for such genes are psaA and rbcL. “Class II” genes do not display a visible 307 phenotype (e.g., ndhB and all other subunits of the plastid NDH complex) and 308 unsurprisingly the collection does not contain any mutants in that class, which 309 excludes it from the following analyses. “Class III” genes are essential for cellular 310 viability [e.g., rps7 or ycf1 (tic214)]. Only a limited number of specific (non-lethal) 311 mutations could be isolated from this class (see above). Class I genes are 312 exclusively located in the two single-copy regions of the plastome [large single-copy 313 (LSC) or small single-copy (SSC) region]. Class III genes, however, are also located 314 in the inverted repeat (IR; Figure 2). The IR is a typical structure of chloroplast 315 genomes and contains exclusively Class II and Class III genes. Due to 316 recombination and gene conversion between the two large inverted repeats (Palmer, 317 1983), mutations in the IR are constantly purged. Hence, Class III genes need to be 318 subdivided based on their physical location on the plastome (LSC and SSC vs. IR). It 319 also should be noted that regions of intrinsic instability are found only in Class III 320 genes (Figure 2). 10 321 The first assumption we tested was if mutations in non-genic regions (52,922 322 bp in the genome; six mutations in the collection) were more common than mutations 323 within genes (112,977 bp in the genome; 47 mutations in the collection). 324 Unsurprisingly, this was the case (χ2: p = 0.0022). Second, we confirmed our 325 expectation that mutations in Class I genes were more common than mutations in 326 Class III genes (Class I: 44 mutations within 37,080 bp of genic sequence; Class III: 327 three mutations within 56,920 bp; χ2: p < 0.0001). We found no difference in the 328 abundance of mutations between the two subgroups of Class III genes (IR: one 329 mutation within 33,672 bp; LSC and SSC: two mutations within 23,248 bp; χ2: p = 330 0.7470), but this non-significance may reflect the small sample size. 331 Next, we asked if gene length affected the recovery of mutations in Class I 332 genes, represented by 44 mutations within 37,080 bp of genic sequence. χ2 test 333 yielded p = 0.0002 for the likelihood of accumulating nine mutations in psaA (2,253 334 bp), p = 0.0254 for four mutations in ccsA (960 bp), but p = 0.5287 for three 335 mutations in rbcL (1,428 bp). This strongly suggests a gene-specific bias for 336 mutation recovery beyond the differences observed between gene classes (Figure 337 3). The lack of relationship with gene length is further supported by a large cluster of 338 Class I genes in the LSC (13,969 bp) that lack any mutation. The cluster contains 339 ATP synthase genes (atpA, F, H and I) and genes for the chloroplast bacterial-type 340 RNA polymerase (rpoB, C1, C2; Figure 2, Supplemental Data Set 2). The joint 341 random probability that among 44 independent mutations not a single one is located 342 in this cluster is p = 9.2 x 10-10. 343 Finally, we assessed whether bias in mutant recovery can be explained by the 344 strength of the chlorotic effect of a mutation, since strong (white) phenotypes might 345 be easier to detect by eye during the selection procedure. Of the 51 mutant lines, 40 346 displayed an essentially white phenotype and eleven a pale phenotype 347 (Supplemental Table 4). Surprisingly, compared to the combined lengths of the 348 coding sequences of “White” and “Pale” genes (35,119 bp and 1,961 bp, 349 respectively), white phenotypes are significantly under-represented whereas pale 350 phenotypes are over-represented (χ2: p < 0.0001). This difference is most likely 351 explained by many mutations not representing full knock-outs (see Discussion). 352 353 Addressing plastome stability 11 354 The finding that chloroplast mutations are biased towards replication slippage and 355 spatially clustered in the genome raises the question: To what extent is the natural 356 mutation spectrum of the chloroplast represented by our collection? To address this 357 point, we sequenced and compared four wild-type Oenothera plastomes from O. 358 glazioviana (=O. lamarckiana; Cleland, 1972; Harte, 1994; GenBank accessions: 359 EU262890.2, KT881171.1, KT881172.1, and KT881174.1). This is a young species 360 that arose in Europe in the mid-19th century by hybridization (Cleland, 1972; Dietrich 361 et al., 1997). We investigated the Swedish strain of O. lamarckiana, a line that has 362 been cultivated since 1906, as well as three independent descendants of O. 363 lamarckiana de Vries (strains blandina, deserens and vetaurea). Those were isolated 364 in 1908, 1913, and 1921 from the original line of de Vries, collected in the 365 Netherlands in 1886 (see Supplemental Table 2 for details on the O. lamarckiana 366 lines). Sequence comparison of the plastome of O. lamarckiana Sweden with those 367 of the three descendants of O. lamarckiana de Vries revealed one transition in the 368 intergenic spacer between trnL-UAA and trnT-UGU, one 48 bp indel in the region of 369 larger complex repeats upstream of accD, and a single bp insertion at a tandem 370 oligo(C+T) stretch between trnL-UAG and ccsA. The three O. lamarckiana de Vries 371 plastomes differ by two 21 bp indels in the repetitive region of the coding frame of 372 ycf1 (tic214). These arose independently at different sites in blandina and deserens. 373 Only the vetaurea strain fully resembles the ycf1 (tic214) sequence of O. 374 lamarckiana Sweden. Lastly, the deserens strain has a 6-bp deletion in the 375 intergenic spacer between rbcL and atpB that is associated with a micro repeat. The 376 mutation is not present in the corresponding plastome mutant III-alpha. Hence, it 377 must have occurred in the deserens plastome after isolation of the mutant in 1954 378 (cf. Table 1). 379 Further evidence that certain regions in the chloroplast genome of Oenothera 380 can evolve very rapidly was provided by comparison of plastome sequences of O. 381 elata ssp. hookeri strain johansen Standard (assembled in 2007; Greiner et al., 382 2008a) with that obtained in this work (EMBL accessions: AJ271079.3 vs. 383 AJ271079.4). A 147-bp insertion was detected in the region of large complex repeats 384 upstream of accD, and a 24-bp insertion was identified at the 3’ end of rpoA, 385 associated with a larger tandem repeat. Inspection of the original Sanger sequencing 386 chromatograms revealed that our 2007 sequence lacked these features. The current 387 version of our johansen Standard plastome is based on multiple sequencing 12 388 methods (Illumina, 454, PacBio, and Sanger) and about 50 individuals. It is further 389 supported by the independent chloroplast mutants I-zeta, I-xi and I-fa, which had 390 been isolated in 1962, 1968, and 2011, respectively. It is therefore clear that the 391 particular material sequenced in 2007 must have been polymorphic for the two 392 plastomes. 393 In summary, all but one mutations detected in the closely related Oenothera 394 plastome under study have arisen by replication slippage. They are found at very 395 similar locations and/or sequence contexts as the detect mutations of the collection 396 and are often located in regions of intrinsic instability associated with oligo(N) 397 stretches or large repetitive elements (Figure 2). 398 399 Discussion 400 Spontaneous mutations are largely limited to replication slippage-based events 401 Strikingly, nearly all detected mutations could be associated with repetitive elements 402 (Supplemental Data Set 1). Therefore they must have occurred by a replication 403 slippage-based mechanism, conferred by misalignment or formation of hairpin 404 structures (Levinson and Gutman, 1987; Wolfson et al., 1991; Leach, 1994; Lovett, 405 2004; Figure 5). Consistent with this reasoning is the observation that, with only one 406 exception, all duplications are perfect tandem duplications, which strongly implicates 407 replication slippage as a template-based mechanism responsible for the occurrence 408 of spontaneous chloroplast mutations. Although tandem, direct, inverted, or 409 palindrome repeats can induce indels in the chloroplast genome (cf. Lovett, 2004; 410 Darmon and Leach, 2014), tandem/direct repeats dominated as mutation-inducing 411 sequence motives. Interestingly these repeats can be very small, spanning only two 412 repetitive elements, sometimes no larger than 3 bp (cf. Kondrashov and Rogozin, 413 2004; Tanay and Siggia, 2008; Supplemental Figures 1 - 6; Supplemental Data Set 1 414 and Results). 415 We only rarely detected mutations suggestive of DNA damage by reactive 416 oxygen species (ROS; resulting in transversions; Imlay, 2008) or ultraviolet (UV) light 417 (likely causing transitions; Pfeifer et al., 2005). This also holds true for the types of 418 mutations that are caused by double-strand breaks (DSB) followed by non- 419 homologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ) or 420 microhomology-mediated break-induced replication (MMBIR) pathways (McVey and 13 421 Lee, 2008; Hastings et al., 2009). NHEJ typically results in small indels of 1 to 4 bp 422 and require 0 - 5 bp microhomologies at the joining ends. Deletions of variable size 423 are common for MMEJ (between 3.1 kb and 4.6 kb, as determined for the chloroplast 424 genome) and microhomologies are utilized for end joining (6 - 12 bp for perfect and 425 10 - 16 bp for imperfect homology in the chloroplast; Kwon et al., 2010). However, 426 such signatures are not found in the indels identified in this study (Supplemental 427 Data Set 1). Furthermore, no plastome rearrangements suggesting MMBIR repair 428 (Cappadocia et al., 2010) or U-turn-like inversions (Zampini et al. 2015) were found. 429 One possible exception is the large 1,364 bp deletion in the mutant V-delta, which is 430 not associated with a repetitive element (Supplemental Figure 6). It might have 431 arisen from a NHEJ-like pathway (cf. Kwon et al., 2010). 432 Our data strongly suggest that mutations caused by processes other than 433 replication slippage are rare in the germline and largely absent from our collection. 434 This observation might reflect protection of the plastid DNA (ptDNA) from ROS and 435 UV light mutagenesis in the shoot apical meristem (Greiner et al., 2015). Harder to 436 explain is the nearly complete lack of signatures resulting from the repairing of DSB 437 in the chloroplast genome. Appropriate pathways exist for DSB repair (Maréchal et 438 al., 2009; Cappadocia et al., 2010; Kwon et al., 2010), but they are likely of minor 439 biological relevance. Similar arguments apply to U-turn-like rearrangements. NHEJ- 440 like repair mechanisms, if present in chloroplasts at all, occur at very low frequencies 441 (Kohl and Bock, 2009; Cappadocia et al., 2010; Kwon et al., 2010). However, neither 442 MMEJ nor MMBRI repair nor U-turn-like rearrangements appear to have produced 443 mutations in our material. This is in line with the observation that extensive X-ray 444 radiation experiments failed to cause recoverable chloroplast mutations (Greiner, 445 2012). Therefore, it seems reasonable to propose an integrity-sensing mechanism 446 for ptDNA in the germline. This could explain the strong structural conservation of 447 higher plant chloroplast genomes during evolution, as reflected in the rarity of large 448 sequence inversions or rearrangements (Ruhlman and Jansen, 2014). 449 450 Mutation spectrum of the chloroplast mutant collection 451 The mutation spectrum in the Oenothera plastome is in sharp contrast to that 452 detected in the nuclear genome of Arabidopsis (Ossowski et al., 2010; Yang et al., 453 2015) or Escherichia coli (Lee et al., 2012). Mutations in nuclear genomes are 454 biased towards single base pair G:C to A:T transitions (with a lower frequency of 14 455 transversions) that are explained by deamination of methylated cytosine and UV 456 light-induced DNA damage. If single base pair substitutions were common in plastid 457 genomes we would expect more than just the two single-nucleotide mutations 458 present in our collection. The paucity of single-nucleotide mutations in the plastome 459 compared to the nuclear genome may reflect that substitution rates in chloroplast 460 DNA are substantially lower than in the nuclear genome (Wolfe et al., 1987; Drouin 461 et al., 2008). Our results clearly show that chloroplast ptDNA replication machinery is 462 more prone to mutations from replication slippage than misincorporation of single 463 nucleotides. Small indels in E. coli also arise in repetitive sequences but they 464 represent only 10% of spontaneous mutations (Lee et al., 2012). Hence, the 465 chloroplast DNA replication and/or repair machinery differs in its error spectrum from 466 that of bacteria. Although the chloroplast originated from an endosymbiotic event, the 467 difference mutation spectrum likely reflects the fact that the DNA replication system 468 is a mosaic of proteobacterial, cyanobacterial and eukaryotic components (Moriyama 469 and Sato, 2014). The fact that indels in chloroplast genomes are generated with 470 much higher frequency than single base substitutions should be taken into account 471 when performing phylogenetic analyses based on chloroplast sequences. The two 472 classes of mutations cannot be weighted equally as phylogenetically informative 473 characters. 474 475 Reasons for biased mutation recovery 476 Some regions in the chloroplast genome of Oenothera are more prone to mutation 477 than others. Although oligo(N) stretches in coding regions are rare, they are 478 preferred targets of mutation (Supplemental Figure 1, Supplemental Data Set 1). 479 Other hotspots reside in large tandem repeats (Figure 2). Strikingly, compared to the 480 second site mutations found in the plastome mutant collection, very similar mutations 481 could be detected between the closely related wild type plastomes of O. glazioviana. 482 We therefore conclude that specific repetitive regions in the Oenothera chloroplast 483 genomes are intrinsically unstable and prone to mutation. This finding is in line with 484 earlier studies on Oenothera plastome organization, since these reparative regions 485 are major sources of phylogenetic divergence in evening primrose (cf. Gordon et al., 486 1982; Greiner et al., 2008a; Greiner et al., 2008b). Many mutations in these regions 487 might be phenotypically neutral and can be fixed over experimental time frames, at 488 least in cultured evening primroses. 15 489 Our work also shows a strong mutation bias towards certain non-essential 490 genes. Most mutations appear to be mediated by micro repeats, but several lines of 491 evidence point to additional factors contributing to the unequal distribution of mutated 492 alleles. First, many of the micro repeats are very small (Supplemental Data Set 1), 493 and they are abundant throughout the chloroplast DNA. From a statistical 494 perspective, they might occur at random. For example, the nested micro tandem 495 repeat TATAT independently induced an insertion and a deletion at exactly the same 496 site in the mutants I-xi and III-alpha (Supplemental Figures 2 and 3). However, this is 497 the only TATAT site in the genome where mutations were found, although the same 498 motif is present altogether 114 times in the sequences of Class I genes. Within the 499 atp-rpo cluster of Class I genes, this motif occurs 45 times and again with no 500 mutations. In psaA, nine mutations could be recovered independently of this element 501 (Figure 3, Supplemental Data Set 1, Supplemental Figure 2 and 4). It is present 502 there three times. Hence, although this element is distributed randomly in the 503 genome (χ2: p = 0.4942 for the full set of Class I genes vs. the atp-rpo subset 504 cluster), its potential to induce a recoverable mutation seems to be confined to 505 particular positions in the plastome. It seems conceivable that the surrounding 506 sequence context governs whether TATAT motifs are susceptible to DNA 507 polymerase slippage (cf. Figure 3). 508 We were not able to deduce a consensus sequence for the mutation-inducing 509 micro repeats. The 32 identified sites with mutations consist of many different types 510 of repeats [perfect or imperfect, (nested) tandem, direct or inverted/palindromic 511 repeats]. Also, in a few cases, a combination of repeat types is present. The 512 individual repeat elements are 3 - 9 bp long and the spacer regions, if present, are 513 between 1 bp and 9 bp (Supplemental Data Set 1). A much larger dataset would be 514 needed to deduce any consensus that may exist. Nevertheless, we noticed that 515 mutation-inducing micro repeats tend to be more A/T rich (mutation-inducing sites in 516 total: 364 bp, with 252 bp A+T; “Class I genes”: 37,080 bp, with 22,031 bp A+T; χ2: p 517 = 0.0694; cf. Tanay and Siggia, 2008). However, this tendency cannot explain the 518 lack of mutations in the atp-rpo cluster, when compared to Class I genes (13,969 bp, 519 with 8,424 bp A+T; χ2: p = 0.3631). 520 Beside the sequence context, the relative positions of genes to each other or 521 within the chloroplast genome might generate mutational hotspots. For example, 522 DNA polymerase processivity could vary with distance relative to the origins of 16 523 replication. Furthermore, collision of the replication with the transcription complexes, 524 or other DNA-binding proteins might differ depending on gene position, or the usage 525 of the nuclear- or plastid-encoded RNA polymerase (Börner et al. 2014). Whereas 526 the interference of the DNA replication complex with other DNA binding proteins is a 527 matter of speculation, the relative position of a gene to an origin of replication does 528 not seem to have an influence on a mutational hotspot. For example, the hotspot in 529 ccsA is rather close to the origin of replication, where the one in psaA is not. On the 530 other hand, the atp-rpo gene cluster, fully lacking mutations, is most distantly located 531 from the replication origins (Figure 2). 532 Differences in gene expression and mutation rate have been reported to be 533 correlated in E. coli (Martincorena et al., 2012; but also see Lee et al., 2012). 534 However, this observation is unlikely to explain the patterns of mutation in our 535 material. Since all chloroplast mutations are present in the germ line, they must have 536 arisen in the meristem of the plants. Although gene expression data from proplastids 537 in meristematic tissues are largely lacking, it is extremely unlikely that the expression 538 rates of photosynthesis genes differ substantially in meristems. We, therefore, favor 539 the alternative explanation that mutations in the atp-rpo gene cluster are more 540 difficult to recover than genes with elevated mutation rates such as psaA or ccsA 541 (see above). Possible reasons include biased gene conversion (Khakhlova and 542 Bock, 2006) 543 recombination repair by homologous recombination with an unmutated genome 544 copy. Such hotspots of recombination were reported for the chloroplast DNA of the 545 green alga Chlamydomonas (Newman et al., 1992). It seems conceivable that 546 molecular mechanisms like biased gene conversion or biased recombination repair 547 eliminate de novo mutations in some plastome regions more efficiently than in 548 others. or recombinational hotspots that increase the efficiency of 549 550 Mutation rates in the chloroplast genome 551 Comparison of the closely related O. glazioviana plastomes allows a rough 552 estimation of the mutation rates for chloroplast genomes. We used the reasonable 553 assumptions that O. glazioviana is monophyletic. The species arose by hybridization 554 in the middle of the 19th century (Cleland, 1972; Dietrich et al., 1997). Hence, we can 555 estimate the maximum number of generations separating the Swedish strain from 556 the lines of Hugo de Vries to be about 150 generations with annual reproduction. 17 557 Since three to five polymorphisms distinguish these lines (see Results), we can 558 assume one fixed mutation per 30 - 50 generations, equivalent to 2.0 – 3.3 x 10-2 559 mutations per generation and genome. Per site (~165,000, with each bp 560 representing a site), the mutation rate would be 1.2 - 2.0 x 10-7 per site and 561 generation. Interestingly, compared to the nuclear genome of Arabidopsis thaliana (7 562 x 10-9; Ossowski et al., 2010), and the genome of E. coli (2.2 × 10-10; Lee et al., 563 2012), this rate is rather high. Nevertheless, taking into account the small genome 564 size of the plastid, the actual number of mutations that effectively accumulate is still 565 small. 566 These approximations should be treated with caution for three reasons. First, 567 the number of mutations is low and thus the precision of our estimate is 568 compromised. Second, a previous study assessing intraspecific plastome variation in 569 Solanum lycopersicum (tomato) over the timespan of approximately one century 570 found no polymorphisms (Kahlau et al., 2006). Third, substitution rates calculated 571 based on phylogenetic data suggest a lower mutation rate in chloroplast genomes 572 than in plant nuclear genomes (Wolfe et al., 1987; Drouin et al., 2008). However, it is 573 also known that organellar mutation rates can vary significantly in different lineages 574 of seed plant evolution, even within a genus (e.g., Cho et al., 2004; Guisinger et al., 575 2008; Sloan et al., 2009). Last, it should be noted that three of the mutations in the 576 O. glazioviana lines are associated with large repetitive elements repeats. These 577 regions are a major source of phylogenetic divergence (Greiner et al. 2008a) and 578 evolve relatively quickly. Therefore, an accurate mutation rate for plastid DNA in 579 Oenothera remains to be determined by genome sequencing at a larger scale. 580 581 Chloroplast mutants as a tool to understand chloroplast gene functions and gene 582 regulation 583 The finding that most chloroplast mutants in our collection show only one mutated 584 site makes them a valuable resource to understand chloroplast gene function and 585 regulation. Although not all genes in the genome are covered, chloroplast mutants 586 for all photosynthetic complexes and also for the chloroplast translation machinery 587 are represented. For the psaA, psbA, and ccsA genes, allelic series are available, 588 with nine, five, and four mutants, respectively (Table 1, Figure 3). Although rare, the 589 collection also contains mutations in essential genes, such as the mutant I-tau, which 590 carries a defect in rps7. Since this mutant displays a reversible bleaching (virescent) 18 591 phenotype (Figure 1D) its characterization should provide new insights into the 592 developmental regulation of chloroplast translation. Also of particular interest are the 593 five mutants identified so far that display defects in chloroplast RNA metabolism 594 (Figure 4). Such mutants are not readily obtainable by targeted approaches. A 595 particularly interesting finding is this respect was that mutations in the protein-coding 596 region can influence transcript processing, as discovered in the mutant II-eta (Figure 597 4). The molecular basis of these effects needs to be further investigated. 598 It is also important to note that many of the mutants (I-alpha, I-delta, I-iota, I- 599 tau, I-omega, I-do, II-delta, II-theta, II-kappa, II-lambda, III-alpha, III-beta, IV-beta, 600 and V-alpha) do not harbor full gene knock-outs, as judged by their phenotype and 601 genotype (Table 1). This renders them a rich platform for functional analyses. Most 602 null mutants of photosynthesis genes are essentially white (Schaffner et al., 1995; 603 Ruf et al., 1997; Hager et al., 1999; Supplemental Data Set 2 and Supplemental 604 Table 4), lacking any photosynthetic activity and thus of limited value for functional 605 studies. The mutations in our Oenothera collection that affect chloroplast mRNA 606 metabolism are located in photosynthesis genes. At least two of them (II-kappa and 607 II-delta) display mild phenotypes. This is due to reduced abundance of the mature 608 transcript (Figure 4), leading to a less severe phenotype than a frameshift mutation 609 in the coding region (Table 1). An additional set of five mutants that are affected in 610 the coding region of photosynthesis genes (I-alpha, I-delta, I-iota, II-lambda and III- 611 beta) are pale green rather than white. With one exception (II-lambda), the mutations 612 cause a frameshift. In some of these cases, the pale phenotype of the mutants are 613 likely caused by the position of the frame-shifting indel within the gene. For example, 614 the mutants I-alpha and I-delta carry a 5-bp duplication just downstream of the 615 translation initiation codon of psbD (Table 1). We hypothesize that these mutations 616 are compensated by an alternative in-frame start codon present downstream of the 617 mutation. By contrast, in the mutant III-beta, a 5 bp duplication exists in the middle of 618 psaB (encoding an essential reaction center protein of photosystem I) (Table 1), 619 which makes its pale green phenotype more difficult to understand (cf. Table 1). A 620 possible clue may be provided by one of the most striking mutant phenotypes 621 observed in the collection: the mottled phenotype of the mutant I-iota, which largely 622 disappears with age, giving rise to a nearly green phenotype (Figure 1C). The 623 underlying mutation is a single A insertion located in a oligo(A) stretch very early in 624 atpB (Table 1; Supplemental Data Set 1). The recovery of the mutant over time 19 625 strongly points to the presence of a frame-correcting mechanism in chloroplasts, 626 such as ribosomal frameshifting (Kohl and Bock, 2009). 627 In summary, the mild chlorotic and developmental-stage dependent 628 phenotypes of several mutants in the collection provide interesting new avenues for 629 the study of plastid gene function and expression. 630 631 Methods 632 Plant material 633 Throughout this work, the terms Oenothera or evening primrose(s) refer to 634 subsection Oenothera (genus Oenothera section Oenothera; Dietrich et al., 1997). 635 Plant material used here is derived from the Oenothera germplasm collection 636 harbored at the Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, 637 Germany (Greiner and Köhl, 2014). Part of this collection is represented by the 51 638 spontaneous plastome mutants that were collected systematically during the past 639 century in Germany (Kutzelnigg and Stubbe, 1974; Stubbe and Herrmann, 1982; 640 Stubbe, 1989; Supplemental Table 1). The plastome mutants were mostly compiled 641 by Wilfried Stubbe and co-workers, maintained by Reinhold G. Herrmann since the 642 end of the 1980s and later taken over by the authors. The mutants are designated 643 according to their basic plastome genotype (I - V) and Greek letters, in order of the 644 respective isolation date (i.e., I-alpha, I-beta, II-alpha, II-beta, etc.). In the case of 645 basic plastome I, of which more mutants were isolated than Greek letters exist, 646 further mutants are designated by the seven tone syllables. To maintain the material, 647 a substitution of the mutant chloroplast by introgression into the suitable johansen 648 Standard strain of O. elata ssp. hookeri was performed by W. Stubbe. Except for the 649 lines containing plastome V, which are still maintained in the background of O. 650 argillicola, the collection has been propagated in the johansen Standard background 651 since then (Greiner 2012). Due to the introgression of most of the lines into the 652 johansen Standard strain, chloroplast substitution lines with this nuclear background 653 and the wild-type plastomes of the investigated mutant chloroplasts were used for 654 RNA gel blot analyses as wild-type controls. For a summary of the genetic stocks, 655 see Supplemental Tables 1 and 2. For further details see Kutzelnigg and Stubbe 656 (1974), Stubbe (1989), Rauwolf et al. (2008), and Greiner (2012). 657 20 658 Plant cultivation, maintenance, tissue harvest, and phenotyping of chloroplast 659 mutants 660 Chloroplast mutants, wild type, and introgression strains were cultivated as 661 described previously (Greiner and Köhl, 2014). In Oenothera, photosynthetically 662 incompetent chloroplast mutants can be kept as variegated plants, containing green 663 wild type as well as mutated chloroplasts (Figure 1A). These plants are viable in soil, 664 because the green tissue feeds the bleached tissue. The combination of two 665 chloroplast types in one plant is possible by taking advantage of biparental 666 chloroplast transmission, which is the rule in evening primroses. In such 667 experiments, a maintainer strain containing a green nursery plastome is crossed to 668 the chloroplast mutant. This results in variegated offspring, because sorting-out of 669 the two chloroplast types leads to tissues homoplasmic for either wild-type or mutant 670 chloroplasts (Figure 1A,B; also see Introduction). Homoplasmic mutant tissue 671 generated this way was used for sequence and molecular analysis, as well as 672 phenotyping in this work. For details on the genetics, see Kutzelnigg and Stubbe 673 (1974), Kirk and Tilney-Bassett (1978), Stubbe and Herrmann (1982) and Greiner 674 (2012). 675 Phenotyping of mutants was performed in an unheated polyethylene 676 greenhouse. Plants were analysed after the appearance of the first two true leaves 677 (young), during the early rosette stage (medium) and during the mature rosette stage 678 (old). Observed phenotypes largely resembled those reported in the laboratory 679 documentation of W. Stubbe that was based on field-grown material. However, minor 680 discrepancies were observed. Different degrees of bleaching were assigned to a 681 numerical code (1 = nearly white, 2 = yellowish cream-colored, 3 = light green, 4 = 682 nearly green). Numbers in parentheses in Table 1 indicate mottled or heterogeneous 683 chlorotic phenotypes. 684 685 Chloroplast isolation 686 An improved chloroplast isolation protocol for Oenothera was developed for this 687 study. It overcomes severe technical limitations caused by the high amounts of 688 mucilage present in Oenothera leaf tissue and does not require ultra-centrifugation 689 steps. As judged from sequencing analysis, ptDNA can be enriched to up to 90% by 690 employing the following procedure at 4°C: 50 g of leaf material were homogenized in 691 a blender adding 1 L of BoutHomX buffer [0.4 M sucrose, 50 mM Tris, 25 mM boric 21 692 acid, 10 mM EGTA, 10 mM KH2PO4; 1% (w/v) BSA, 0.1% (w/v) PVP-40; pH 7.6 with 693 KOH; 5 mM freshly added β-mercaptoethanol]. Aliquots of the homogenate were 694 filtered through a double layer of cheese cloth (Hartmann, Heidenheim, Germany), 695 followed by filtering through a single layer of Miracloth (Merck, Darmstadt, Germany). 696 The filtrate was then centrifuged for 15 min at 5,000 g. The chloroplast pellet was 697 resuspended in 150 ml ChloroWash (2 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, 50 698 mM HEPES, 330 mM sorbitol; pH 7.6 with KOH; 5 mM freshly added sodium 699 ascorbate) using a 30 cm3 Potter homogenizer (mill chamber tolerance: 0.15 - 0.25 700 mm; VWR, Darmstadt, Germany). The suspension was again filtered in aliquots 701 through a double layer of Miracloth and adjusted with ChloroWash to a volume of 702 about 700 mL followed by another filtering step. Next, chloroplasts were pelleted at 703 2,400 g, re-suspended in 9 mL of ChloroWash and transferred to three 85%/45% 704 Percoll step gradients [PBF-Percoll stock solution: 3% (w/v) PEG 6000, 1% (w/v) 705 BSA, 1% (w/v) Ficoll 400, dissolved in Percoll; 45%/85% Percoll: 45%/85% PBF- 706 Percoll stock solution, 330 mM sorbitol, 50 mM HEPES, 2 mM EDTA, 1 mM MgCl2; 707 pH 7.6 with KOH; Percoll step gradient: 8 ml 85% Percoll, 16 ml 45% Percoll in a 30 708 ml Corex tube]. The gradients were centrifuged at 10,000 g for 10 min. Intact 709 chloroplasts were recovered from the gradient’s interphase and washed in 200 ml 710 ChloroWash. After repeating the washing steps in smaller volumes, the isolated 711 chloroplasts were re-suspended in a suitable buffer for further use. 712 713 DNA isolation 714 Total DNA for Illumina sequencing was isolated from rosette leaves using a phenol- 715 based DNA isolation protocol developed for Oenothera (Ågren et al., 2015). In 716 contrast to the previously described protocol, we used 100 µl of a 10 mg/mL RNAse 717 A solution (50 U/mg; Roche Diagnostics GmbH, Manheim, Germany). For PCR 718 analyses, we used total DNA obtained with the DNeasy Plant Mini Kit (Qiagen, 719 Hilden, Germany; Rauwolf et al., 2008). For certain Oenothera strains, especially 720 when older material was used, DNA was additionally purified by an anion-exchange 721 column (Nucleobond AX 20; Macherey-Nagel, Düren, Germany). This procedure 722 was found to be very effective in removing remaining phenolic components, 723 mucilage and/or polysaccharides that potentially inhibit enzymatic reactions from 724 Oenothera DNA isolations in general. 22 725 Total high molecular weight DNA for PacBio sequencing was obtained from 726 etiolated seedlings. To this end, seeds were sown on a wet sterile filter paper in Petri 727 dishes and supplemented with 0.05% (v/v) of Plant Preservative Mixture (Plant Cell 728 Technology Store, Washington, DC, USA) to prevent fungal contamination. 729 Subsequently, seeds were incubated at 27°C in the light. Immediately after 730 germination (when root tips became visible), Petri dishes were wrapped with 731 aluminium foil and after three days, material was harvested and frozen in liquid 732 nitrogen. 100 mg of seedlings were ground in liquid nitrogen, subsequently 733 supplemented with 1550 µL CTAB buffer [130 mM Tris/HCl (pH 8.0), 1.8 M NaCl, 734 1.3% (w/v) PVP-40, 2.6% (w/v) CTAB] and 250 µL 1-thioglycerol. Throughout the 735 procedure, samples were mixed by gentle inversion. The aqueous DNA solution was 736 pipetted with a cut tip. After adding 100 µL of a 10 mg/mL RNase A solution (see 737 above), samples were incubated for 30 min at 60°C. Afterwards, 100 µL of 738 proteinase K (10 mg/mL; Sigma-Aldrich, Steinheim, Gemany) was added and 739 samples were incubated at the same conditions as described above. To remove cell 740 debris, samples were centrifuged at 20,000 g for 5 min at room temperature. The 741 supernatant was treated with 2 mL phenol/chloroform/isoamyl alcohol (25:25:1, pH 742 7.5) and 2 mL chloroform/isoamyl alcohol (24:1). DNA was precipitated with 1/10 743 volume of 5 M NH4-acetate and 1 volume of isopropanol. Further purification was 744 achieved using QIAGEN Genomic-tip 20/G columns (Qiagen, Hilden, Germany) and 745 (optionally) with a “High Salt Phenol Chloroform Cleanup” recommended by Pacific 746 Biosciences (Menlo Park, CA, USA). Deviating from the Pacific Biosciences protocol, 747 we omitted EDTA from our buffers. As judged from pulsed-field gel electrophoresis, 748 our procedure yields DNA fragments between 20 kb and 200 kb, with ODs of ~1.8 749 (A260/A280) and ~2.1 (A260/A230). Prior to library preparation, the DNA was subjected 750 to an additional AMPure PB purification step (Pacific Biosciences, Menlo Park, CA, 751 USA). 752 To obtain ptDNA for 454 sequencing, isolated chloroplasts were re- 753 suspended in 400 µL of TENTS buffer [100 mM Tris/HCl (pH 8.0), 50 mM EDTA, 0.5 754 M NaCl, 0.2% (v/v) Triton X-100, 1% (w/v) SDS] and incubated at 60˚C for 15 min. 755 Next, the lysate was supplemented with 100 µL RNase A (10 mg/mL, see above) 756 and incubated for 1.5 h at 37°C. Subsequently, 100 µL of 10 mg/mL proteinase K 757 (see above) solution was added and samples were placed overnight at room 758 temperature. After extraction with phenol/chloroform/isoamyl alcohol (25:24:1, pH 23 759 7.5) and with chloroform/isoamylalcohol (24:1), the DNA was precipitated at -20°C 760 overnight as described above. 761 762 RNA isolation 763 Total RNA for RNA gel blot analyses was isolated from Oenothera leaves using the 764 Spectrum Plant Total RNA Kit (Sigma, Steinheim, Germany). Columns and buffers 765 used were included by the manufacturer. To cope with mucilage and high amounts 766 of phenolic components in the tissue, minor modifications were introduced to the 767 provided protocol: Up to 25 mg of frozen tissue was ground in liquid nitrogen. 768 Material was lysed in 750 μL of freshly prepared Lysis Solution (50 μL β- 769 mercaptoethanol for 1 mL of supplied Lysis Solution). The lysate was incubated at 770 room temperature for 5 min and filtrated on a Filtration Column. Centrifugation time 771 was extended up to 5 minutes. 750 μL of Binding Solution was mixed with the 772 clarified lysate, mixed gently through pipetting and centrifuged on a Binding Column. 773 An additional elution step was performed to increase of RNA yields. 774 775 Detection of RNA via RNA gel blot 776 1.5 - 2.0 µg of total RNA was separated on 1% agarose gels (w/v) under denaturing 777 condition. RNA was transferred to a nylon membrane (Hybond–XL, GE Healthcare, 778 Buckinghamshire, UK) by capillary transfer using 10x SSC buffer (1x SSC: 0.015 M 779 sodium citrate, 0.15 M NaCl). The RNA was covalently linked to the membrane using 780 a UV cross linker (Vilber Lourmat, Ebertharfzell, Germany). Radiolabelled probes 781 were used for strand-specific detection of transcripts in RNA gel blot hybridization. 782 For this purpose transcription of gene specific PCR products was performed with the 783 MAXIscript T7 Kit (Ambion, Darmstadt, Germany) using radiolabelled 784 (Hartman Analytic GmbH, Braunschweig, Germany). Gene specific PCR products for 785 ycf3, petB and psaC were obtained employing the following primers in a PCR with 786 total Oenothera DNA as a template: ycf3ex2_north_fr: 5’-ggatgtcggctcagtcag-3’, 787 ycf3ex2_north_rv: 788 petBex2_north_fr: 789 taatacgactcactatagggaatacagctagaaccacgcctgtg-3’, 790 gtcggatattcattctcgtatctggagc-3’, 791 taatacgactcactatagggagttgctcgttccaggcctc-3’, 792 taatacgactcactatagggacccatacttcgagttgtttcatgc-3’, 32 P dUTP 5’-taatacgactcactataggggtaagaacgggtttcgttctagtg-3’, 5’-gtctcgagattcaggcgattgc-3’, petBex2_north_rv: 5’- petBint_north_fr: 5’- petBint_north_rv: 24 psaC_north_fr: and psaC_north_rv: 5’5’5’- 793 atgtcgcattcagtaaagatttatgatacctg-3’. RNA gel blots were performed according to a 794 standard protocol. Nylon membrane with RNA was incubated in Church buffer [0.5 M 795 NaH2PO4, 7% SDS (w/v), 1 mM EDTA; pH 7.2] for one hour. After this time 796 radioactive probes were transferred to hybridization tube and incubated overnight in 797 65°C. Next day three washing steps were performed: once in 1x SSC supplemented 798 with 0.2% SDS (w/v) for 20 min and twice in 0.5x SSC supplemented with 0.2% SDS 799 (w/v) for 20 min each. Visualization of radioactive signals was performed at different 800 time points, depending on the strengths of the mRNA signal. 801 802 DNA sequencing 803 Sanger sequencing was performed with PCR products using standard methods (at 804 Eurofins MWG Operon, Ebersberg, Germany). Primers were obtained from the same 805 source. 806 Illumina paired-end sequencing (375 bp insert size) was performed as 807 described previously (Ågren et al., 2015). To improve sequence quality at very long 808 oligo(N) stretches and to avoid a bias in the Illumina libraries against certain (TA)n 809 rich sequences in some regions of the Oenothera chloroplast genome, “PCR free” 810 Illumina libraries were employed at a later stage of the project. In most cases, about 811 10% to 30% of the sequenced DNA was found to be ptDNA. The observed variance 812 results from the developmental stage of the leaf tissue used and/or the particular 813 genotype of the line (cf. Rauwolf et al., 2010). From these data, we could deduce 814 that roughly 5 million total DNA 100 bp Illumina paired-end reads are sufficient to 815 sequence a chloroplast genome of Oenothera at 500x - 1500x coverage. In this 816 context, it is relevant to mention that the mutant tissue (obtained from random 817 sorting-out of mutant and nursery plastomes) used in this work for Illumina 818 sequencing (see above), was confirmed to be fully homoplasmic for the mutant 819 chloroplast genome. 820 Prior to 454 sequencing, 2 - 5 μg of isolated ptDNA were pre-amplified to 20 821 µg using the GenomiPhi HY DNA Amplification Kit (GE Healthcare, Chalfont St 822 Giles, UK). Single-end read libraries were sequenced on a Roche/GS FLX Titanium 823 platform to an approximate coverage of 20x per chloroplast genome. 454 824 sequencing was performed at Eurofins MWG Operon, Ebersberg, Germany. To 825 correct sequence uncertainties that are frequent in 454 datasets and a potential bias 25 826 in the template DNA introduced by the rolling cycle amplification with the GenomiPhi 827 Kit, all chloroplast genomes obtained from 454 data were re-sequenced by Illumina. 828 PacBio sequences were obtained from a 7 kb cut-off insert library using P4- 829 C2 chemistry. Size selection, library preparation and sequencing were performed at 830 the Max Planck-Genome-Centre in Cologne, Germany. PacBio sequencing is part of 831 the Oenothera genome project and will be communicated elsewhere. 832 833 Plastome assembly 834 In total, the complete sequences of 65 chloroplast genomes were determined in this 835 study, many of them through multiple methods. For a summary of the different 836 sequencing and assembly methods used, see Supplemental Tables 3 and 5. De 837 novo assemblies of the chloroplast genomes and reference-guided mutation 838 mapping were performed using the standard parameters of SeqMan NGen v.12.1.0 839 or earlier (DNASTAR, Madison, WI, USA) and/or CLC Genomic Workbench v.7.5.1 840 or earlier (CLC bio, Aarhus, Denmark). First we determined whether Illumina libraries 841 obtained from total DNA contain plastome sequences translocated to the nuclear 842 genome (NUPTs; Kleine et al., 2009). Translocated sequences, potentially 843 containing polymorphisms compared to the true cytoplasmic ptDNA, might interfere 844 with the assembly/mutation identification. Therefore, we independently de novo 845 assembled 454 reads from ptDNA of isolated chloroplasts and Illumina reads from 846 total DNA. For this, we used three wild-type lines and eleven chloroplast mutants. 847 Both approaches (Illumina and 454 of purified ptDNA from the same materials) gave 848 comparable results and NUPTs could not be detected with certainty in the Illumina 849 sequences. Hence, the remaining chloroplast genomes were solely obtained from 850 Illumina reads of total DNA. However, both assemblers and sequencing techniques 851 did not yield satisfactory results in the de novo assemblies of the notoriously 852 repetitive regions of the Oenothera plastome, such as upstream and within accD and 853 ycf1 (tic214), within ycf2 and in the rrn16-trnI-GAU spacer. In these regions, 854 individual repetitive elements can span more than the read length and/or the insert 855 size of a paired-end library. In the wild-type reference plastomes, those gaps were 856 closed by Sanger sequencing and even consistent assemblies were routinely verified 857 with the same technique. In the johansen Standard line, these regions were 858 additionally verified by single PacBio reads, fully spanning the repetitive regions. All 859 repetitive regions discussed in this work are supported by Sanger reads. Chloroplast 26 860 mutations, if not identified from a de novo assembly, were identified by a reference- 861 guided assembly using 862 Misassemblies, indicating larger indels, were identified by paired-end mapping. 863 Smaller mutations could be directly identified in the reference-guided assembly. The 864 notoriously repetitive regions as well as some of the mutant alleles, especially those 865 harbouring larger indels, were verified at random by Sanger sequencing. the chloroplast genome of the reference strain. 866 867 Sequence annotation 868 Wild-type chloroplast genomes were annotated using GeSeq v.0.2.1, GenBank 869 submission files were generated by GenBank 2 Sequin (https://chlorobox.mpimp- 870 golm.mpg.de). These software tools were developed for this project to annotate and 871 process organelle sequences (manuscripts in preparation). The existing annotation 872 of the reference Oenothera chloroplast genome from O. elata ssp. hookeri strain 873 johansen Standard (AJ271079) was used as a reference. Also, in the course of this 874 work, the annotation of our previously sequenced Oenothera plastomes (Greiner et 875 al., 2008a) was revised, and the exon/intron borders of protein-coding genes are 876 now verified by cDNA sequences. Further, some names of genes and gene products 877 were updated (see AJ271079.4, EU262887.2, EU262889.2, EU262890.2 and 878 EU262891.2). For Illumina transcriptome sequencing, total RNA extractions, 879 sequencing and assemblies, see Johnson et al. (2012) and Hollister et al. (2015). 880 881 Repeat analysis 882 Longer repetitive elements were identified manually or using the EMBOSS suite 883 (Rice et al., 2000). Larger imperfect palindromes were identified with the standard 884 parameters of mfold (Zuker, 2003). For macro repeat analyses incorporated into 885 Figure 2 refer to Greiner et al. 2008a. Since we found that repetitive elements of no 886 larger than 3 bp are able to induce replication slippage in the chloroplast, a 887 sequence inspection by eye was necessary to identify these elements. For this 888 reason, the sequence context 20 bp upstream and downstream of a micro indel (< 889 10 bp) was inspected for (perfect/imperfect/nested) micro (<10 bp) tandem, direct, 890 palindrome and inverted repeats or combinations thereof. 891 892 Global sequence analyses and visualization of the Oenothera plastome 27 893 For global sequence analyses, including statistical analysis, GC content 894 determination, repeat analyses and search for sequence motif abundance, the 895 reference Oenothera chloroplast genome of the johansen Standard strain (O. elata 896 ssp. hookeri) was used (AJ271079.4). Especially coding regions of the Oenothera 897 plastome are largely identical between Oenothera species (Greiner et al., 2008a). 898 Chloroplast genome maps were drawn by OrganellarGenomeDRAW (Lose et al. 899 2007) and edited in CorelDarw X5 (Corel Corporation, Ottawa, ON, Canada). 900 901 Statistical analyses 902 χ2 tests were performed using GraphPad (http://www.graphpad.com) with Yates' 903 correction. Two-tailed p-value equals are given. 904 905 Accession Numbers: 906 Sequence data from this article can be found in the EMBL/GenBank data libraries under 907 accession numbers AJ271079.4, EU262887.2, EU262889.2 - EU262891.2, and KT881169.2 908 - KT881177.1. 909 910 Supplemental Data 911 Supplemental Figure 1. Sequence context of the detected mutations at oligo(N) stretches. 912 Supplemental Figure 2. Sequence context of the detected micro tandem duplications. 913 Supplemental Figure 3. Sequence context of the detected micro deletions. 914 Supplemental Figure 4. Sequence context of the detected macro tandem duplications. 915 Supplemental Figure 5. Sequence context of the detected macro tandem duplication and 916 deletion. 917 Supplemental Figure 6. Sequence context of the detected macro deletions. 918 919 Supplemental Table 1. Origin of the mutant lines in the Oenothera plastome mutant 920 collection. 921 Supplemental Table 2. Oenothea wild type, nuclear genome mutants and chloroplast 922 substitution lines used this work. 923 Supplemental Table 3. Summary on the wild type Oenothera chloroplast genomes 924 determined de novo or re-sequenced in this work. 925 Supplemental Table 4. Major phenotypic classes of the Oenothera plastome mutants. 926 Supplemental Table 5. Sequencing and assembly methods of the full chloroplast genomes 927 determined in this study. 28 928 929 Supplemental Data Set 1. Summary of the sequence context of the identified mutations in 930 the chloroplast mutant collection. 931 Supplemental Data Set 2. Macroscopic phenotypes of deletion mutants in the chloroplast 932 genome. 933 934 Acknowledgments 935 Dr. Wilfried Stubbe is acknowledged for compiling and previously maintaining 936 Oenothera plastome mutant collection, and Dr. Reinhold G. Herrmann, Dr. Herbert 937 Hopf and Elisabeth Gerick for maintaining the material after the retirement of Dr. 938 Wilfried Stubbe. Thanks go to Dr. Dirk Walther for advice in statistical analysis, Drs. 939 Jörg Meurer and Serena Schwenkert for sharing information on the macroscopic 940 phenotypes of psbI and psbK knock-out plants. We wish to thank Dr. Barbara B. 941 Sears for providing her mutant material, which served as control samples. She 942 obtained her material earlier from Dr. Wilfried Stubbe’s collection. Dr. Barbara B. 943 Sears and two anonymous reviewers are acknowledged for fruitful discussion and 944 helpful comments on the manuscript. Transcriptome sequencing was made possible 945 through Dr. Gane Ka-Shu Wong, the 1kp initiative (1kp.org) and BGI-Shenzhen. The 946 work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; 947 GR 4193/1-1) to S.G. and R.B. (BO 1482/17-1), and by the Max Planck Society. 948 M.T.J.J. is supported by an NSERC Discovery Grant and an Early Researcher 949 Award from the Ontario Government. S.I.W. is funded by a Natural Sciences and 950 Engineering Research Council of Canada (NSERC) grant. 951 952 Author Contributions 953 A.M., J.S., L.Y.-R., E.U.-J., A.Z., T.P., J.S., and S.G. performed experiments. All 954 authors analyzed and discussed the data. S.G. designed the study and wrote the 955 manuscript. R.B. participated in writing. 956 29 957 References 958 Ågren, J.A., Greiner, S., Johnson, M.T.J., and Wright, S.I. (2015). No evidence 959 that sex and transposable elements drive genome size variation in evening 960 primroses. 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Phenotypes, major defects and mutated loci of the mutant lines in the Oenothera plastome mutant collection Mutant1) Defect in Gene or locus M1/M4 I-alpha/ I-delta PS II psbD M2 I-beta Cytb6f petD Leaf phenotype2) Young Medium 4 3-4 4 M2 I-beta M3/M5 I-gamma/ I-epsilon - PS II 3 Old 3 2-3 5 bp duplication (CTTGG), + 18 to + 22 psbD, truncated PsbD, alternative start codon [?] 1 old leaves often with small green spots 4 bp duplication (ATTT), + 998 to + 1001 petD, truncated PetD 66 bp deletion (AATTT…TATGG), - 258 to - 323 ycf1 (tic214)3) 1 older leaves with enhanced greening close to vascular tissue 2 bp duplication (GT), + 82 to + 83 psbA, truncated PsbA 4 M6 I-zeta PS I psaA 1-2 1 1 - M7 I-eta PS I psaA 2 1-2 1 - M8 I-theta PS I psaB 2 1-2 1 - M9 I-iota ATP synthase atpB (1) (1-4) (4) 38 Mutation and predicted molecular consequence very old leaves with narrow whitish zones, older leaves sometimes darker green SSC-IRA junction psbA Description mottled, young leaves white, old leaves nearly green 5 bp duplication (GTTTT), + 1350 to + 1354 psbA, truncated PsaA 4 bp duplication (GATT), + 1088 to + 1090 psaA, truncated PsaA 32 bp duplication (TTCCA…ATTAG), + 2132 to + 2163 psaB, truncated PsaB 1 bp insertion (A), + 58 atpB, truncated AtpB [?] Table 1. (continued) Mutant1) Defect in Gene or locus Leaf phenotype2) Young Medium Old Description M10/M15 I-kappa/ I-omikron PS I psaA 1-2 1-2 1-2 - M11 I-lambda PS II psbA 3-4 1-2 1 - M12 I-my Cytb6f ccsA 4 3-4 1 old leaves often with small green spots M13 I-ny PS I psaB 2 1-2 1 - M14 I-xi PS II psbD 4 3-4 1 - M16 I-pi PS II psbA 3-4 2-3 1 - M17 I-rho PS II psbA 3-4 2 1 greening declining from leaf base to tip 4 4 4 - M17 I-rho - upstream accD M184) I-sigma RuBisCO rbcL 39 Mutation and predicted molecular consequence 70 bp duplication (ACGAT…CCCGT), + 1437 to + 1506 psaA, truncated PsaA 4 bp deletion (GCTT), + 859 to + 862 psbA, truncated PsbA 31 bp duplication (GAATT…TTAAA), + 540 to + 570 ccsA; truncated CcsA 4 bp deletion (AGAT), + 399 to +402 psaB, truncated PsaB 5 bp deletion (CAGCT), + 530 to + 534 psbD, truncated PsbD 5 bp deletion (TCTCT), + 208 to + 212 psbA, truncated PsbA 5 bp deletion (ACAAC), + 797 to + 801 psbA, truncated PsbA 30 bp deletion (CCTTT…CTTTA), -30 to -1 accD3) 5 bp deletion (TTAAC), +808 to +812 rbcL, truncated RbcL Table 1. (continued) Mutant1) M19 M20 Defect in I-tau translation I-ypsilon ATP synthase Gene or locus rps7 Description Mutation and predicted molecular consequence periodical bleaching/regreening (virescent), viable on soil at homoplasmic state, retarded in cell growth 4 bp duplication (ATTT), + 462 to + 465 rps7, last 2 aa of Rps7 modified and 2 aa added Leaf phenotype2) Young (1-4) Medium (1-4) Old (1-4) atpB (1) (1) (1) mottled, but spots very rare M20 I-ypsilon - ycf1 (tic214) M21 I-phi Cytb6f ccsA 3-4 2 2 - M22 I-chi PS I psaA 1-2 1 1 - 40 34 bp duplication (GATAC…AATTA), + 461 to + 494 atpB, truncated AtpB 21 bp deletion (AAAAG…ACGTC), +2404 to + 2424 ycf1 (tic 214), 21 bp duplication (TCAAA…CGAAA), +2486 to + 2406 ycf1 (tic214), 3 aa exchanges in the variable part of Ycf1 (Tic214)3) 4 bp duplication (ATTT), + 10 to + 13 ccsA, truncated CcsA 5 bp duplication (CCGCT), + 1420 to + 1424 psaA, truncated PsaA Table 1. (continued) Mutant1) Defect in Gene or locus Leaf phenotype2) Young Medium Old Description M23 I-psi PS I ycf3 (intron) 1-2 2 2 - M24 I-omega PS I psaA 1-2 1 1 - M25 I-do Cytb6f ccsA 3 2 2 - M26/M27 I-re/I-mi Cytb6f petA 2-3 1-2 1-2 - M28 II-alpha M29 III-epsilon M304) M31 PS I 5) ycf4 3 2-3 1 PS I ycf3 2 1 1 II-gamma PS II psbE 4 2-3 1-2 II-delta - ndhE-psaC spacer 4 4 4 41 old leaves of field grown plants with characteristic yellowish/whitish tip old leaves often with small green spots no change during development Mutation and predicted molecular consequence 5 bp duplication (ATTGA), + 316 to + 320 ycf3 (intron), splicing deficiency 4 bp duplication (TCTT), + 2226 to + 2229 psaA, changed/extended PsaA 6 bp deletion (ACAATA), + 298 to + 303 ccsA, deletion of 2 aa in CcsA 1 bp insertion (G), + 667 petA, truncated PetA 4 bp duplication (AAAA), + 442 to + 445 ycf4; truncated Ycf4 29 bp duplication (ACAAG…GCTTT), + 996 to + 1024 ycf3, truncated Ycf3 5 bp duplication (ATTAT), + 42 to + 46 psbE, truncated PsbE 24 bp deletion (TATAT…AATCA), - 60 to - 83 psaC, transcript processing affected Table 1. (continued) Mutant1) M32 M33 II-epsilon II-zeta Defect in Gene or locus PS I psaA PS II psbB Leaf phenotype2) Description Young Medium Old 2 1 1 - 1 old leaves with enhanced greening close to vascular tissue 4 2-3 M34 II-eta Cytb6f petB 4 3 1 old leaves often with small green spots M354) II-theta Cytb6f petB (intron) 4 3 1 old leaves often with small green spots M36/M47 II-iota/ IV-delta PS I psaA 2 2 2 - M37 II-kappa PS I ycf3 (intron) old leaves in parts darker than younger leaves, viable on soil at homoplasmic state M37 II-kappa - ndhF-rpl32 spacer M38 II-lambda Cytb6f petA 3-4 3-4 4 4 3-4 2-3 42 - Mutation and predicted molecular consequence 4 bp duplication (TGAG), + 124 to + 127 psaA, truncated PsaA 4 bp duplication (CTTT), + 117 to + 120 psbB, truncated PsbB 5 bp duplication (ATTAC), + 1346 to + 1350 petB, truncated PetB, transcript processing/stability affected 2 bp deletion (AC), + 430 to + 431 petB (intron), splicing deficiency 5 bp duplication (TTAAC), + 667 to + 671 psaA, truncated PsaA transversion G/T, + 1438 ycf3 (intron), splicing deficiency single bp insertion/deletion, results in a transversion (C/T), 511 rpl323) 39 bp deletion (GGTTT...CCGAG), + 337 to + 375 petA, 13 aa deleted in PetA Table 1. (continued) Mutant1) M39 II-my Defect in Gene or locus Cytb6f petA M39 II-my - ndhG-ndhI spacer M40 II-ny PS I ycf4 M40 II-ny PS II psbB M417) III-alpha PS II psbD M42 M43 III-beta III-gamma PS I PS I M43 III-gamma - M44 III-delta M44 III-delta PS I ATP synthase psaB Leaf phenotype2) Description Young Medium Old 3-4 3-4 1-2 - 2 1-2 1 - 4 3-4 1 - 1-3 1-3 1-3 bleaches under higher light conditions, sometimes inhomogeneous psaA 1-28) 1-2 1 - 1 1 1 - trnQ-UUGaccD spacer psaB atpB 43 Mutation and predicted molecular consequence 5 bp duplication (TAGCC), + 352 to + 356 petA, truncated PetA single bp deletion (A), 310 ndhG3) mutation of M28 (IIalpha) 2 bp deletion (CT), + 791 to + 792 psbB, truncated PsbB6) 6 bp duplication (TATATT), + 540 to + 545 psbD, 2 additional aa in PsbD 5 bp duplication (ATAAA), + 1246 to + 1250 psaB, truncated PsaB 8 bp duplication (AATTTAGT), + 1540 to + 1547 psaA, truncated PsaA 12 bp deletion (CTCGGCTGATGA), 689 to - 678 accD3) mutation of M42 (III-beta) 1 bp deletion (A), + 57 atpB, truncated AtpB6) Table 1. (continued) Mutant1) Gene or locus Leaf phenotype2) Young Medium Old Description M45 IV-alpha PS I psaA 3 1-2 1 - M464) IV-beta RuBisCO rbcL 4 4 3-4 - M48 IV-epsilon PS II psbA 3 2-3 1-2 greening declining from leaf base to tip V-alpha RuBisCO9) (tentative) rbcL9) (tentative) M49 M50 1222 1223 1224 Defect in V-delta PS I 4 4 4 grayish green phenotype, older leaves in parts darker ycf3 M50 V-delta - accD M51 I-fa Cytb6f ccsA 4 3 1 - 3 2-3 1-2 - 1) Mutation and predicted molecular consequence 5 bp duplication (GTACT), + 2173 to + 2177 psaA, truncated PsaA transversion (G/C), + 337 rbcL, single aa exchange 8 bp deletion (AGTGGGAA), + 389 to + 396 psbA, truncated PsbA 6 bp deletion (GATGAA), + 750 to + 755 rbcL, deletion of 2 aa in RbcL 1,364 bp deletion in ycf3 affecting 11 bp of the 3'end of exon 1, the full intron 1, the full exon 2, and 403 bp of the 5'-end of intron 2 153 bp deletion +208 to + 360 accD, deletion of 51 aa in the variable part of AccD3) 5 bp duplication (TTAAC), + 702 to + 705 ccsA, truncated CcsA Mutants are designated according to their basic plastome genotype (I – V) and Greek letters indicating the sequence of mutant isolation (with alpha representing the first mutant isolated). In the case of plastome I, for which more mutants have been isolated than 44 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 letters exist in the Greek alphabet, additional mutants are designated by the seven tone syllables. For details, see Kutzelnigg and Stubbe (1974), and Stubbe (1989). 2) 1 = nearly white, 2 = yellowish cream-colored, 3 = light green, 4 = nearly green 3) Potentially phenotypically neutral second site mutation. For details, see text. 4) Mutations in the mutants M18 (I-sigma), M30 (II-gamma), M35 (II-theta) and M46 (IV-beta) were determined previously and could be verified in this study. For summary and references, see Greiner (2012). 5) This mutant was originally obtained under the name II-beta, but re-designated in this work. For details, see Supplemental Table 1. 6) Second site mutation isolated based on a phenotypic change in the primary mutant. For details, see text. 7) With respect to its reference plastome (deserens de Vries; GenBank accession number: KT881172.1), the mutant M41 (III-alpha) carries a 6 bp deletion (TAATCA) in the rbcL-atpB intergenic spacer, - 113 to - 118 of rbcL. However, this mutation must have arisen in the wild type reference after the isolation of M41 (III-alpha). Hence, it is not treated as second site mutation in M41 (III-alpha). For details, see text. 8) The phenotype of the mutant M43 (III-gamma) was described as “lighter green (3)” in young tissue by W. Stubbe. However, in our hands the mutant appeared to be nearly white to yellowish (1-2) at most developmental stages. It is possible that this phenotypic change is due to a second site mutation upstream of accD which was not present when W. Stubbe initially assessed the phenotypes. For details, see text. 9) The corresponding wild-type strain (argillicola Douthat 4b) of the mutant M50 (V-alpha) is no longer present in our genetic stocks. For mutant allele identification, the two available O. argillicola plastomes (EU262887.2 and KT881177.1) were used. See Stubbe and Herrmann (1982), for assignment of this mutant to a physiological defect in RuBisCO. 45 1253 1254 Figure Legends 1255 1256 Figure 1. Representative phenotypes in the Oenothera chloroplast mutant collection. 1257 For details, see text and Table 1. (A) I-ny, variegated individual with completed sorting- 1258 out giving raise to homoplasmic white (mutated) and green (nursery plastome) sectors. 1259 (B) Developmental series of homoplasmic IV-epsilon leaves, showing age-dependent 1260 bleaching. (C) Age-dependent greening and mottled pattern of leaves of mutant I-iota. 1261 From left to right: homoplasmic young nearly white leaf, homoplasmic medium-age 1262 mottled leaves and older green leaf. Note that the oldest leaf displays variegation. As a 1263 result of a completed sorting-out, the leaf blade is homoplasmic for the nearly re- 1264 greened I-iota plastome. The margin is homoplasmic for the wild type chloroplast 1265 (indicated by the arrow). (D) I-tau, growing photoautotrophically in soil as homoplasmic 1266 plant. It displays a reversible bleaching (virescent) phenotype, alternatingly forming 1267 bleached and green leaves. The homoplasmic state of all mutants was confirmed by 1268 Illumina deep sequencing. For molecular causes of the mutations, see Table 1. Scale 1269 bars: 4 cm for (A), (B) and (D); leaf sizes in (C) from left to right: ~5 cm, 10 cm, 12 cm 1270 and 20 cm. 1271 1272 Figure 2. Distribution of the mutated alleles of the plastome mutant collection across 1273 the Oenothera plastome. The physical map of the plastome is shown and the origins of 1274 replication (oriA and oriB), the GC content (grey inner circle) and the macro repeats (red 1275 inner circle) are indicated. Blue circles denote mutations conferring a mutant phenotype; 1276 orange circles denote second site mutations which may be phenotypically neutral. 1277 Green circles refer to phenotypically neutral mutations in the closely related O. 1278 glazioviana and O. elata strains. Note clustering of these mutations in the highly 1279 repetitive regions around accD and ycf1 (tic214). Numbers refer to the number of 1280 individual mutations identified for a gene or locus. Class I genes are indicated in color, 1281 Class II genes and Class III genes in grey or white. For details on the definition of the 1282 gene classes, see text. For macro repeat identification, see Greiner et al. (2008a), for 1283 location of the origins of replication, see Chiu and Sears (1992). 46 1284 1285 Figure 3. Distribution of mutations in the mutational hotspots psaA, psbA and ccsA. 1286 Note that solely duplications could be identified in psaA. Also note the uneven 1287 distribution of mutations within the genes. For molecular causes of the mutations and 1288 associated repetitive elements, see Table 1, Supplemental Data Set 1, and 1289 Supplemental Figures 2 - 4. 1290 1291 Figure 4: Defects in mRNA maturation due to mutations in introns, a coding region, or 1292 an intergenic region in the chloroplast mutants I-psi, II-kappa, II-eta, II-theta and II-delta 1293 (A). Schematic overview of the ycf3, psaC and petB regions of the plastome. Positions 1294 of the mutations are marked with stars, hybridization probes are indicated as green 1295 bars. (B) RNA gel blot analyses of mRNA accumulation and RNA processing patterns. 1296 Upper row, left and middle panel: Splicing defects in ycf3 as found in the mutants I-psi 1297 and II-kappa, respectively. In I-psi, a 5 bp duplication in intron 1 leads to loss of splicing 1298 of this intron, correlating with a severe bleaching phenotype (Table 1). In II-kappa, a 1299 transversion in intron 2 results in a reduced splicing activity. Note that some mature 1300 transcript (0.7 kb) is still produced, consistent with the homoplasmic mutant being viable 1301 upon cultivation in soil (Table 1). Upper row, right panel: Processing defect in the psaC 1302 operon of the mutant II-delta. The mutant harbors a 24-bp deletion in the psaC-ndhE 1303 intergenic spacer. Whereas a precursor transcript of about 8.0 kb overaccumulates in 1304 the mutant, accumulation of processed mRNA species is strongly reduced. Some 1305 mature monocistronic mRNA (0.6 kb) is produced in the mutant, consistent with its mild 1306 phenotype (Table 1). Lower row: Splicing and processing defects in transcripts from the 1307 psbB operon (containing the petB gene) in the mutants II-eta and II-theta. A 2-bp 1308 deletion is present in the petB intron in II-theta. In II-eta, a 5 bp duplication is present in 1309 the second exon of petB. Left panel: hybridization to a petB exon probe, right panel: 1310 intron probe. In II-theta, a petB splicing defect is clearly visible. Transcripts 1311 overaccumulating in the mutant (i.e., the 2.2 kb and 1.5 kb RNA species) likely 1312 represent the unspliced petB/petD dicistron and the unspliced petB, respectively. The 1313 putative mature transcript (0.8 kb) does not accumulate in the mutant, in agreement with 1314 its severe phenotype (Table 1). Interestingly, in II-eta, the mutation in exon 2 leads to 47 1315 loss of the spliced psbH/petB/petD tricistron (1.7 kb in the WT; Westhoff and Herrmann, 1316 1988), and instead to accumulation of a 1.4 kb transcript likely containing the spliced 1317 petB/petD dicistron. The mature transcript (0.8 kb) seems to accumulate which, 1318 however, carries a frameshift. This leads to a largely identical phenotype as II-theta 1319 (Table 1). For details on the mutations, see Table 1. 1 and 2 represent biological 1320 replicates of all plant lines analyzed here. 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Johnson, Stephen Wright, Tommaso Pellizzer, Johanna Sobanski, Ralph Bock and Stephan Greiner Plant Cell; originally published online April 6, 2016; DOI 10.1105/tpc.15.00879 This information is current as of July 31, 2017 Supplemental Data /content/suppl/2016/04/06/tpc.15.00879.DC1.html Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY