The most fundamental step in the evolution of the eukaryotic cell was the endosymbiotic acquisition of organelles. The association of formerly free-living prokaryotes with the host cell was followed by a dramatic reorganization of the genomes of both the host and the symbionts. Among other processes, a substantial part of the genetic information of the prokaryotic endosymbionts was transferred into the nuclear genome (Dyall et al., 2004). Many products of these transferred genes have to be reimported into the organelles, and once there, they interact with organellar gene products. This type of interaction requires tight spatiotemporal and quantitative regulation and leads to coevolution of the interacting compartments (Blier et al., 2001; Herrmann et al., 2003). To date, it has not been clear which interactions are evolutionarily conserved and which diverge more rapidly. Are there interactions that are specific to a certain taxon, or are most, if not all, interactions common among most species? A way to study these questions is to determine what happens when organelles are exchanged between species. It is interesting that interspecific organelle exchange often leads to malfunctions in cellular development and differentiation, a phenomenon referred to as nuclear–organellar incompatibility.
The initial characterization of nuclear–organellar incompatibility dates back to 1929, when Renner described aberrant pigment phenotypes of interspecies hybrids in the higher plant genus Oenothera (Renner, 1929, 1934; Stubbe, 1989). Similar studies throughout the plant kingdom showed that in many genera of angiosperms, interspecies hybrids and alloplasmic lines (cybrids) generated either by sexual crosses or by the fusion of somatic cells exhibit defective chloroplast proteins (Babiychuk et al., 1995), chlorophyll deficiencies, and defects in chloroplast development (Hagemann, 1964; Kirk and Tilney-Bassett, 1987; Zubko et al., 2001; Levin, 2002). Nuclear–organellar incompatibility is also well documented in metazoans. In crustaceans, subpopulations of copepods exhibit differences in mitochondrial genes that cause a pronounced decline in cytochrome oxidase activity in interpopulation hybrids (Edmands and Burton, 1999). Similarly, cybrid cells containing human nuclei and mitochondria from different primate species vary in fitness (Kenyon and Moraes, 1997). The loss of normal organelle development in these models has been interpreted as a failure of the chondriome to function in a foreign genetic background. In molecular terms, the exchange of organelles between species can disrupt interactions between coadapted macromolecules, which can lead to cellular malfunction.
It has been estimated that up to 5000 unique nuclear genes encode proteins that are targeted to plastids (Martin and Herrmann, 1998; Peltier et al., 2000). Many nucleus-encoded plastid proteins participate in stable or transient interactions with plastid-encoded proteins or nucleic acids (Barkan and Goldschmidt-Clermont, 2000; Nickelsen, 2003). Intertaxonomic variability between such partners could trigger nuclear–organellar incompatibility. To locate differences in plastid genomes that potentially could be responsible for nuclear–organellar incompatibility, a genomic approach is inevitable. Comparative genomic analyses of plastomes can be used to identify all species-specific polymorphisms between different species that are able to produce natural or artificial cytoplasmic hybrids. Subsequently, the functional role of the identified polymorphisms in nuclear–organellar incompatibility can be tested by reverse genetics of the plastome, which of course requires a plant species amenable to plastid transformation. In addition, a forward genetic approach can be applied to isolate revertants from incompatibility, which requires easily distinguishable wild-type and nuclear–organellar incompatibility phenotypes.
Model organisms that meet these requirements and allow the study of the molecular mechanisms of nuclear–organellar incompatibility in higher plants are the cytoplasmic hybrids (cybrids) of Atropa belladonna (deadly nightshade) and Nicotiana tabacum (tobacco). Tobacco and nightshade are sexually incompatible species of the family Solanaceae but readily produce hybrids and cybrids in somatic cell hybridization experiments. Ab(Nt) cybrids with a nuclear genome Ab from nightshade and a plastome (Nt) from tobacco display an albino phenotype and are viable only when grown under heterotrophic conditions in vitro (Kushnir et al., 1991) (Figure 1A, Table 1). Plastids in mesophyll cells of Ab(Nt) plants are smaller than those in the wild type, have no normal thylakoid membrane system, and contain instead large vesicular structures (Herrmann et al., 2003). The easily scorable phenotype facilitates both direct and reverse genetic analyses. More importantly, targeted genetic manipulation of the plastome of one of the parental species, tobacco, is straightforward (Svab and Maliga, 1993). This, in combination with the somatic cell genetics available for both plant species, allows testing of the functional significance of species-specific polymorphisms for nuclear–organellar incompatibility. We sequenced the entire plastid chromosome from nightshade and compared it with the corresponding molecule from tobacco (Schmitz-Linneweber et al., 2002). The two chromosomes turned out to be 96% identical but were distinguished by >100 insertion/deletions in intergenic regions and several insertion/deletions and numerous point mutations in the coding regions of genes. Other polymorphisms affected nucleotide positions that are subject to RNA editing in corresponding transcripts (Schmitz-Linneweber et al., 2002). The wide variety of differences discovered suggested initially that multiple defects should underlie nuclear–organellar incompatibility. Here, we show that the albino phenotype in Ab(Nt) plants is caused primarily by the failure to edit a particular tobacco-specific plastid RNA editing site in the ATPase α-subunit (atpA) transcript. Further editing defects of species-specific sites were found in cybrids and may contribute to nuclear–organellar incompatibility to a lesser extent. These results emphasize an important role for the organellar RNA editing machinery in the evolutionary coadaptation of plastids and nuclei in higher plants.
To determine whether genetic suppressor mutation(s) are cytoplasmically inherited, plastids from the randomly chosen Abm(Ntm) line AG7 were first transferred into tobacco cells and then back to nightshade. The albino cytoplasmic tobacco mutant A15 (Table 1) served as a recipient of the AG7 plastids. In a protoplast fusion experiment, numerous green calli that regenerated tobacco-like shoots were selected. Tobacco plants of the presumed Nt(Ntm) genotype were healthy, fertile, and produced only green progeny after backcrossing with wild-type tobacco. This established that putative mutations in the plastome of the Abm(Ntm) line AG7 do not affect its ability to cooperate with the tobacco nuclear genome in chloroplast development. Next, plastids from one of the Nt(Ntm) lines (L3/7; Table 1) were combined with the nuclear genome of the original Ab(Nt) cybrid albino line Abw3. The resulting plants of the expected genotype Ab(Ntm) were green, photoautotrophic, and displayed normal vegetative development as well as nightshade morphology (Figure 1B). However, Ab(Ntm) plants were male sterile. Upon pollination with wild-type nightshade, normal-looking nightshade-like green progeny plants were obtained. Analysis of the Ab(Ntm) plants with regard to species-specific nuclear markers, such as tobacco-specific repetitive elements and isoenzyme types of peroxidases and amylases, did not reveal the presence of tobacco nuclear genes (see Supplemental Figure 1 online). Based on the results of plastid transfer experiments and because the cytoplasmic genomes of both species are maternally inherited in sexual crosses, we concluded that suppressor mutations are borne by the cytoplasmic genome, most likely the plastome, and that these mutations enable cooperation between the tobacco plastome and the nightshade nuclear genome to develop functional chloroplasts.
To understand how the identified mutation may contribute to the suppression of the albino phenotype of Ab(Nt) plants, we next analyzed the editing of the atpA transcript. We found that Ab(Nt) plants fail to edit the atpA transcript (Table 2). As all previous evidence suggests that editing factors are encoded in the nucleus (Bock, 2000), these data indicate that the nightshade nuclear genome lacks the gene that codes for an appropriate editing factor for this site. Thus, codon 264 remains CCC in the atpA mRNA, suggesting that the nascent AtpA polypeptide in Ab(Nt) cybrids contains a L264P amino acid substitution. Consequently, the characterized suppressor mutations remove the atpA editing site, which should restore the Leu in the AtpA protein, thus compensating for nightshade's lack of tobacco-specific nucleus-encoded editing factors. These data suggested that the Leu-264 residue in the AtpA polypeptide is indispensable for the proper development of chloroplasts, because all three independently isolated suppressor lines possessed an identical mutational change affecting this codon.
Next, a panel of asymmetric nuclear hybrids with a full chromosome set of nightshade and a partial genomic complement of tobacco was generated. In this somatic hybridization experiment, cells of herbicide (BASTA)-resistant tobacco were irradiated with a lethal dose (500 Gy) of γ-rays and fused with protoplasts of the albino cybrid Ab(Nt) (Table 1). BASTA-resistant green colonies were selected, and regenerating shoots were screened for the absence of tobacco-specific morphological traits. The morphology of symmetric tobacco/nightshade hybrids served as a reference. Specifically, the counterselected traits included the absence of (1) abundant trichome development (i.e., hirsuteness, a typical tobacco trait); (2) characteristic tobacco leaf shape; and (3) typical morphology of the tobacco root system. The selected line Bar103 with the expected genotype AbNt(Nt) displayed nightshade shoot and leaf morphology (Figure 1C, Table 1) and developed greenish adventitious roots, a nightshade trait. Pollination of flowers of Bar103 plants with wild-type nightshade pollen yielded only progeny that were purely albino. Most likely, the tobacco nuclear genome fragments were not transmitted to the F1 progeny plants as a result of their loss during meiosis. The albino phenotype of F1 progeny plants further suggests that the tobacco plastome in Bar103 is wild type, as it is still incompatible with the nightshade nuclear genome. AbNt(Nt) plants displayed a fully edited atpA codon 264 (Table 2). This established that the introduction of tobacco nuclear DNA can complement the albinism of Ab(Nt) plants and that this complementation correlates with the plant's ability to edit the atpA transcript.
In the control protoplast fusion experiment using a kanamycin-resistant nightshade strain and line KA7, 16 Ab(Nt:CCC) albino lines were recovered that were indistinguishable from Ab(Nt) plants generated previously (Table 3, Figure 2E). By contrast, four independent fusion experiments with protoplasts of CAT6 and CAT10 tobacco lines led to the generation of 32 lines of cybrids that were all green and essentially indistinguishable from Ab(Ntm) plants (Table 3, Figure 2E). They grew on soil with growth rates similar to those of nightshade wild-type plants, demonstrating photoautotrophy. Sequence and restriction analyses of the atpA gene in newly generated cybrids confirmed the presence and absence of the point mutation in Ab(Nt:CTC) and Ab(Nt:CCC) plants, respectively. When we used tobacco line CAT8 as a plastid donor, a line that was heteroplasmic for the mutation (Figure 2C), both green and albino nightshade cybrids were recovered. Sequence and restriction analyses showed that only the green cybrids contained the C-to-T point mutation (Figure 2D; see also Supplemental Figure 4 online).
Whereas cybrid plants did not differ significantly from wild-type nightshade in their vegetative growth under our standard greenhouse conditions, independent lines varied in fertility. Of 12 lines tested, only 2 were self-fertile. Other lines were female fertile (i.e., developed seeds after pollination with pollen of wild-type nightshade) but male sterile. The male sterility of cybrids varied. Plants of some lines had normal flowers with developed anthers that, however, did not contain viable pollen upon dehiscence. In other lines, petaloid anthers were common. Such abnormalities in flower development are the basis for so-called cytoplasmic male sterility. They are common among cytoplasmic hybrids of plants and are thought to be caused by disturbed nuclear–mitochondrial interactions (Nivison and Hanson, 1989; Yesodi et al., 1997). However, the recovery of two fully fertile, photoautotrophic Ab(Nt:CTC) cybrid lines demonstrates that nuclear–plastidial incompatibility between the nightshade nuclear genome and the tobacco plastid genome manifested as pigment deficiency can be directly overcome by a single point mutation.
To test a possible role of the interspecies rpoC2 polymorphism for nuclear–organellar incompatibility in planta, we implemented the same experimental strategy that was used to assess the role of the tobacco atpA-264 editing site. First, we generated two types of transplastomic tobacco plants. Plants with the genome composition Nt(Nt:AbC2), lines C2A-2 and C2A-9, had a tobacco plastome in which the rpoC2 gene carried the same three-codon deletion as the rpoC2 gene of nightshade and was linked with the aadA selection cassette inserted 30 bp downstream of the rpoC2 stop codon. Plants with the genome composition Nt(Nt:NtC2), line C2N, possessed a control tobacco plastome that carried the selectable marker aadA linked to the rpoC2 gene containing the three tobacco-specific codons (Figure 3A). Both types of transplastomic tobacco plants grew photoautotrophically in soil but were slightly paler green than the wild-type plants (see Supplemental Figure 5 online). This suggested that the aadA cassette at this chromosomal position has some negative effect on chloroplast development, for example, by compromising the proper termination, stability, or translation of the rpoC2 transcript. Notwithstanding this slight pigment deficiency, we used these transplastomic plants as donors of plastids in protoplast fusion experiments with nightshade as the plastid recipient. All recovered lines of cybrids were albino regardless of genome composition, which was either Ab(Nt:AbC2) or Ab(Nt:NtC2), as verified by sequence analysis (Figure 3B, Table 3). These results suggested that the interspecific divergence in rpoC2 is not responsible for the albino phenotype of Ab(Nt) cybrids.
It is interesting that the asymmetric hybrid AbNt(Nt) showed differential rescue of editing of the sites affected in Ab(Nt). In addition to atpA-264, editing at site rps14-50 was fully restored, whereas editing at site ndhD-225 was restored only partially, and no restoration occurred at site ndhD-200 (Table 2). Whether this means that several independent editing factors have been introduced in AbNt(Nt) plants, or alternatively, that one factor serves several sites, remains to be determined. In contrast with AbNt(Nt) plants, no alteration in editing of additional sites took place in the rescued Ab(Nt:CTC) cybrids. Here, editing of rps14 was still partial and no editing of the two ndhD sites was observed (Table 2). This shows that editing of these additional sites is not dependent on the status of the atpA site and that the editing defect of these sites does not contribute significantly to albinism in the Ab(Nt) plants.
We also investigated editing of the three nightshade-specific sites ndhA-189, ndhD-293, and rpoB-809 in the reciprocal Nt(Ab) cybrid (Figure 1A) and found that site rpoB-809 is not processed in these green plants (see Supplemental Figure 2C online). We concluded that both tobacco and nightshade nuclear genomes lack some, but not all, factors to support the editing of heterologous plastid transcripts, which makes RNA editing defects a general and, in the case of the pigment deficiency of Ab(Nt) cybrids, a causative feature of nuclear–plastidial incompatibility.
Although the editing defect of the atpA-264 site is clearly the most important component of incompatibility in the Ab(Nt) cybrids, other editing defects encountered in cybrids analyzed here might contribute to incompatibility on a smaller scale. AbNt(Nt), Ab(Ntm), and Ab(Nt:CTC) plants all exhibited different combinations of editing defects at the tobacco-specific sites rps14-50, ndhD-200, and ndhD-225. Although all of these plants grew photoautotrophically, we cannot exclude the possibility that their RNA editing defects result in more subtle phenotypic alterations, such as a reduction in photosynthetic efficacy under certain stress conditions. The ndhD gene codes for a subunit of the Ndh complex, which participates in the reduction of plastoquinones and cyclic electron flow around photosystem I. The Ndh complex supplies extra ATP for photosynthesis, particularly under environmental stress conditions, but it is not essential for basic photosynthetic competence, because loss-of-function mutants are viable and their growth does not differ significantly from that of wild-type plants (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998; Horvath et al., 2000; Kotera et al., 2005). The plastid rps14 gene encodes a subunit of the ribosome. A knockout mutation of rps14 is lethal (Ahlert et al., 2003), and thus even the partial rps14-50 editing defect might impinge on normal plastid function in some subtle manner. Similarly, the rpoB-809 defect found in the Nt(Ab) cybrid could play a role in the incompatibility syndrome observed in these plants (Babiychuk et al., 1995; Peter et al., 1999). This is supported by the finding that null mutants of rpoB, which encodes the β subunit of the plastid RNA polymerase, are albino plants (Allison et al., 1996; De Santis-Maciossek et al., 1999), although the amino acid Leu-809 is in a poorly conserved region of the protein and its functional importance remains to be validated (Corneille et al., 2000). Together, the results of our plastome-wide analysis of RNA editing in cybrids are consistent with a complex polygenic nature of nuclear–organellar incompatibility in plants (Yao and Cohen, 2000; Zubko et al., 2001). It will be important to understand the functional role of rps14-50, ndhD-200, ndhD-225, and rpoB-809 editing sites for plant growth and development. An extreme nuclear–organellar incompatibility reaction like the pigment deficiency of Ab(Nt) plants is rare in sexual hybrids. If RNA editing defects decrease the fitness of hybrids in natural environments, they most likely do so in a more subtle way, for example, by slightly decreasing the overall efficiency of photosynthesis.
Are editing defects expected to be a general feature of nuclear–organellar incompatibility? The loss of editing in a nightshade nuclear background can be explained by the absence of genes coding for species-specific editing factor. As all available evidence suggests a proteinaceous nature of editing factors (Miyamoto et al., 2002; Kotera et al., 2005), it follows that nuclear–organellar incompatibility in Ab(Nt) cybrids is the result of impaired protein–RNA interactions. Several studies suggest that these interactions may be evolutionarily labile. Editing sites evolve with extreme rapidity (Shields and Wolfe, 1997; Sasaki et al., 2003). In addition, the responsible nucleus-encoded RNA editing factors are evolutionarily labile as well (Bock et al., 1994; Bock and Koop, 1997; Reed and Hanson, 1997). This rapid evolution leads to interspecific divergence of editing sites and factors; thus, an exchange of organelles between species should be followed by editing defects. This prediction can be further tested in interspecific hybrids exhibiting nuclear–organellar incompatibility, for example, in the genera Oenothera and Epilobium (Renner, 1934; Michaelis, 1954; Kirk and Tilney-Bassett, 1987).
In conclusion, the combination of experimental approaches implemented in this study helped to unravel the molecular mechanism behind pigment deficiency as a manifestation of nuclear–organellar incompatibility in Ab(Nt) plants. In general, these techniques should be applicable to the study of molecular processes that initiate nuclear–cytoplasmic incompatibilities in various families of higher plants and will advance our understanding of the role that cytoplasmic genomes play in the life cycle and evolution of plants.
The glufosinate ammonium–resistant transgenic SR1 tobacco line BarD was generated by the standard leaf disc transformation procedure using Agrobacterium tumefaciens C1Rifr (pGV2260; pGSFR890). To generate asymmetric nuclear hybrids (Table 1), leaf protoplasts of BarD plants were irradiated with γ-rays from a Co60 source. The ionizing radiation dose used, 500 Gy, completely prevents cell division (i.e., is lethal). Irradiated BarD protoplasts were fused with nonirradiated leaf protoplasts of the albino nightshade Abw3 line. Such pretreatment usually results in the elimination of the donor nuclear genome after the first divisions of heterokaryocytes (Menczel et al., 1982). Glufosinate ammonium–resistant green calli were selected and induced to regenerate shoots.
To generate nightshade cybrids with plastids from transplastomic tobacco lines, we fused leaf protoplasts of the kanamycin-resistant nightshade line Ab5 with leaf protoplasts of tobacco lines as summarized in Table 3. Three-week-old colonies obtained after protoplast fusion were then cultured on media supplemented with kanamycin (100 μg/mL) and spectinomycin (1200 μg/mL). Nightshade-like shoots regenerated from independent colonies that developed under such selection were excised and micropropagated without selection. In the protoplast fusion Abw3+CAT6, only kanamycin was present during selection and green colonies were selected, because the parental nightshade cybrid line was albino.
Plants were grown aseptically with 8 h/16 h dark/light cycles at 0.5 to 1 W/m2 (Osram L85 W/25 Universal White fluorescent lamps; Munich, Germany) and 25°C on synthetic MS medium that was supplemented with sucrose (30 g/L) and solidified with 0.6% agar. Species-specific polymorphic markers of nuclear genomes, such as isoenzymes of peroxidases, catalase, malate dehydrogenase, and genes encoding plant tubulins, were assayed in somatic hybrids as described (Kushnir et al., 1991). Karyotypes of somatic hybrids were analyzed by acetoorceine staining of chromosome spreads from dividing cells in root tips (Gleba et al., 1988). Plastome types were identified by RFLP analysis of purified chloroplast DNA or by direct DNA sequencing of relevant PCR-amplified DNA fragments.
Plastid transformants generated with pC2A were identified by PCR using primer pairs rpoC2for and rpoC2rev. Sequencing of the PCR product made with primers rpoC2rev identified the presence or absence of the introduced nightshade-specific deletion in rpoC2. Several lines were recovered that had the nightshade-specific deletion in rpoC2. These lines were designated C2A. Others carried the tobacco sequence instead of the nightshade-specific deletion. The aadA cassette was present in all lines. The distance between the insertion/deletion and the aadA cassette is 1320 bp, providing ample space for recombination, as observed in other plastid transformation studies (Kavanagh et al., 1999). These recombinant lines were designated C2N and served as controls during subsequent experiments. Other polymorphic sites tested by PCR with primers EatpAfor/rev and ndhAfor/rev were tobacco-like in all lines, as expected.
Sequence data from this article have been deposited previously with the EMBL/GenBank data libraries under accession number AJ316582.
We thank Sabine Kemp for skillful technical assistance and Michael Tillich for helpful discussions. We also thank Yuri Gleba, Marc Van Montagu, Dirk Inze, and Uwe Maier for the use of laboratory space and fruitful discussions. We thank Beatrice Grabowski and the anonymous reviewers for valuable suggestions and comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB TR1 to R.M.M. and R.G.H., fellowship to C.S.-L.) and the Fonds der Chemischen Industrie (to R.G.H.). In memory of Rainer M. Maier, 1961–2004. We lost a colleague, mentor, and friend.