Higher plant mitochondrial and chloroplast DNAs are known to share extensive sequence homologies. The present work addresses issues raised by these initial observations: (1) what is the distributive pattern of ctDNA sequences among different mitochondrial genomes, (2) what is the frequency of DNA transposition between the two organelles, (3) are the transposed ctDNA sequences transcribed? The results to be presented demonstrate that many ctDNA sequences, including identified genes, are widespread in mitochondrial genomes and in some cases are highly conserved. However, the distribution of any one particular sequence is sporadic, even within a plant family. Preliminary data, obtained in studies of watermelon, raise the possibility that some mtDNA transcripts share homology with ctDNA sequences.

Significant DNA sequence homologies exist between higher plant mitochondrial and chloroplast DNAs (Stern & Lonsdale, 1982; Lonsdale et al. 1983; Stern et al. 1983; Stern & Palmer, 1984a; Dron et al. 1985; Whisson & Scott, 1985). These studies have provoked speculation that interorganellar DNA transfer is a continuing phenomenon. They also provide evidence that certain DNA segments in these two organelles may have a common origin (Ellis, 1982; Timmis & Scott, 1984; Stern & Palmer, 1984a).

The mechanism by which exogenous DNA enters the mitochondrion and becomes stably integrated in its genome has not been elucidated, although physical associations between organelles (references enumerated in Stern & Palmer, 1984a) may provide a ‘bridge’ to facilitate the movement of DNA across membrane barriers. Both chloroplasts (Palmer, 1985) and mitochondria (Palmer & Shields, 1984; Lonsdale et al. 1984; Stern & Palmer, 19846) appear to possess active recombination systems that could be instrumental in the excision or integration of DNA. The direction of most interorganellar DNA exchange is probably from chloroplast to mitochondrion. This is suggested by the presence of known chloroplast genes in mtDNA, and the absence of mitochondrial genes in ctDNA. The relatively constrained size and uniform organization of chloroplast genomes (Palmer, 1985) contrasts with the large and variable plant mitochondrial genome (Ward et al. 1981; Leaver & Gray, 1982), and is consistent with a unidirectional transposition.

To understand the nature and function of this ‘promiscuous’ DNA, I have investigated the specific segments of ctDNA transferred, their conservation at the sequence level, and evidence for their expression in the mitochondrion. Results are described bearing on these questions, including an analysis of the distribution of certain chloroplast genes among different plant mitochondrial genomes, nucleotide sequence data for a transposed corn ctDNA sequence, and a preliminary analysis to detect transcription of promiscuous DNA in watermelon mitochondria.

Table 1 lists 14 higher plant mitochondrial genomes known to exhibit homology to ctDNA. These species encompass both monocots and dicots, and their mitochondrial genomes range from 218 kb (turnip) to approximately 2400 kb (muskmelon) in size. The method by which such homologies are identified is illustrated in Fig. 1. Mitochondrial and chloroplast DNAs from four members of the plant family Cucurbitaceae were digested with the restriction endonuclease PvuII, electrophoresed in agarose gels, transferred to nitrocellulose and hybridized with 32P-labelled cloned ctDNA segments from spinach (Fig. 1). The ctDNA is included as a control, since mtDNA preparations are inevitably contaminated with minor amounts of ctDNA. Hybridizing fragments appearing only in the mtDNA lane represent bona fide mtDNA fragments with homology to the ctDNA probe (e.g. Fig. 1 (left), lane Z(m)), while fragments hybridizing in both mtDNA and ctDNA lanes indicate contamination, and are disregarded (e.g. Fig. 1 (right), lanes M(m) and M(c)). Since the percentage of ctDNA contaminating each mtDNA preparation is variable, for certain plants (e.g. watermelon and zucchini, Fig. 1 (left)), very little ctDNA is present on the filter, and hybridization between ctDNA and the 32P-labelled probe is only evident upon prolonged exposure of the filter.

Table 1.

Plant species known to have mtDNA-ctDNA sequence homologies

Plant species known to have mtDNA-ctDNA sequence homologies
Plant species known to have mtDNA-ctDNA sequence homologies
Fig. 1.

Homologies between ctDNA and cucurbit mtDNAs. Mitochondrial (m) and choroplast (c) DNAs of watermelon (W), zucchini (Z), cucumber (C) and muskmelon (M) were digested with Pvull, electrophoresed in 0· 7% agarose gels, transferred to nitrocellulose and probed with the 32P-labelled cloned ctDNA fragments indicated on the restriction map (Zurawski et al. 1982) below the figure. Size standards, in kb, were calculated from ÈcoRI, Sall and 7/mdIII-digested phage λ DNA.

Fig. 1.

Homologies between ctDNA and cucurbit mtDNAs. Mitochondrial (m) and choroplast (c) DNAs of watermelon (W), zucchini (Z), cucumber (C) and muskmelon (M) were digested with Pvull, electrophoresed in 0· 7% agarose gels, transferred to nitrocellulose and probed with the 32P-labelled cloned ctDNA fragments indicated on the restriction map (Zurawski et al. 1982) below the figure. Size standards, in kb, were calculated from ÈcoRI, Sall and 7/mdIII-digested phage λ DNA.

The results show that a gene encoding the large subunit of ribulose bisphosphate carboxylase (rbcL, Fig. 1 (left)) is homologous to zucchini and cucumber mtDNAs, but not to those of watermelon and muskmelon, and that genes encoding the beta (atpB, Fig. 1 (middle)) and epsilon (atpE, Fig. 1 (right)) subunits of the chloroplast ATPase hybridize with all the mtDNAs except that of muskmelon. Analogous experiments were performed with gene-containing ctDNA clones for rbcL, atpB, atpE and the chloroplast rRNA genes and additional plant mtDNAs. These results, together with those shown in Fig. 1, are summarized in Table 2. Only the cucumber mitochondrial genome possesses sequences homologous with each of the chloroplast genes tested, whereas none of the protein-coding genes tested hybridize with either spinach or pea mtDNAs. Furthermore, the largest genome (muskmelon, Table 1) has no homology to any of the protein-coding genes, in contrast to cucumber, a member of the same genus (Cucumis). It should be noted that neither atpB nor atpE has been identified as a bona fide plant mitochondrial gene. Thus, the chloroplast atpB and atpE probes would not be expected to cross-hybridize with a mitochondrial counterpart. This contrasts with the situation for the rRNA genes, for which the chloroplast and mitochondrial versions are homologous (Stern et al. 1984). In this instance, prior knowledge of the mtDNA fragments containing the bona fide mitochondrial rRNA genes is required, before a transposed region in the mitochondrial genome can be assigned using a heterologous chloroplast rRNA gene probe. Lastly, it is important to realize that the presence of specific chloroplast gene sequences in mtDNA is significant only to the extent that a short, identifiable sequence is being tested. The large probes used in previous studies (Stern & Palmer, 1984a, 1986) preclude analysis of distributive patterns of specific ctDNA sequences in different mitochondrial genomes. The results presented in Table 2 have been extended for four members of the plant family Cucurbitaceae, by using approximately 35 short (<4kb) segments of cloned ctDNA from mung bean, to investigate the distribution of their sequences in the mitochondrial genomes. The results of these experiments will be published separately.

Table 2.

Occurrence of selected chloroplast gene sequences in plant mitochondrial genomes

Occurrence of selected chloroplast gene sequences in plant mitochondrial genomes
Occurrence of selected chloroplast gene sequences in plant mitochondrial genomes

The strong homology between the chloroplast genes and the mtDNAs (Figs 1, 4; Lonsdale et al. 1983; Stern & Lonsdale, 1982), taken together with the apparently random phylogenetic distribution of transposed sequences, suggests that many of the homologies are a result of recent evolutionary events. The data presented Jjelow, however, indicate that at least in spinach mtDNA, a complex pattern of ctDNA integration precludes a single, recent occurrence of DNA transposition. The distribution of ctDNA sequences in the spinach mitochondrial genome has been analysed with the aid of a complete physical map of the mtDNA (Stern & Palmer, 1986). A simplified version of the results (Fig. 2) shows that some regions of ctDNA appear to hybridize to multiple loci in the mitochondrial genome (e.g. open arrows, Fig. 2). This interpretation is supported by the cross-hybridization of the two mtDNA fragments (data not shown). We have also found that a 1670bp AcoRI fragment of spinach ctDNA hybridizes with three zucchini mtDNA fragments (Fig. 1 (right)) of >2 kb, suggesting that sequences within Eco 1670 occur more than once in the zucchini mitochondrial genome. At least three scenarios can be drawn to explain these data (Fig. 3), only one of which invokes multiple interorganellar DNA transfers. The ambiguity between multiple transfers, and an instance in which the ctDNA probe spans two separately transferred regions (Fig. 3, top and bottom), cannot be resolved by filter hybridizations. Ultimately, nucleotide sequence data and comparisons between closely related mitochondrial genomes will aid in unravelling the mechanism and frequency of DNA transfer.

Fig. 2.

Pattern of DNA homologies between spinach chloroplast and mitochondrial DNAs. Restriction maps of the 152kb spinach chloroplast genome (top; ‘solid lines’= Pstl, ‘broken lines’ = Xhol) and the 327kb mitochondrial genome (bottom; ‘solid lines’ = Sall) are shown, with solid lines connecting segments of ctDNA used as probes with the hybridizing region in the mitochondrial genome. For details and mapping references, see Stern & Palmer (1986). The arrows (▹) indicate ctDNA sequences present in more than one location in the mtDNA. For simplicity, homology with only one of the large chloroplast inverted repeat sequences is shown.

Fig. 2.

Pattern of DNA homologies between spinach chloroplast and mitochondrial DNAs. Restriction maps of the 152kb spinach chloroplast genome (top; ‘solid lines’= Pstl, ‘broken lines’ = Xhol) and the 327kb mitochondrial genome (bottom; ‘solid lines’ = Sall) are shown, with solid lines connecting segments of ctDNA used as probes with the hybridizing region in the mitochondrial genome. For details and mapping references, see Stern & Palmer (1986). The arrows (▹) indicate ctDNA sequences present in more than one location in the mtDNA. For simplicity, homology with only one of the large chloroplast inverted repeat sequences is shown.

Fig. 3.

Mechanistic interpretations of multiple homologous regions in the mitochondrial genome with a single ctDNA probe. Top, multiple transfers of a given segment of ctDNA. Middle, transfer of ctDNA to the mitochondrial genome (1), followed by intragenomic dispersal (2). Bottom, the ctDNA probe (‘probe’) may span two individually transferred regions, that are unrelated in nucleotide sequence.

Fig. 3.

Mechanistic interpretations of multiple homologous regions in the mitochondrial genome with a single ctDNA probe. Top, multiple transfers of a given segment of ctDNA. Middle, transfer of ctDNA to the mitochondrial genome (1), followed by intragenomic dispersal (2). Bottom, the ctDNA probe (‘probe’) may span two individually transferred regions, that are unrelated in nucleotide sequence.

Fig. 4.

Sequence of a transposed segment of ctDNA in the corn mitochondrial genome. Restriction maps of a portion of the corn ctDNA inverted repeat (top and middle) and the corresponding region of the mitochondrial genome (bottom) are shown. Below, the maps, the nucleotide sequence of the ctDNA (Koch et al. 1981) and the nonidentical bases in the mtDNA are shown. (−) indicates deleted bases. Restriction sites are Bam III (B) and Sad (T). Chloroplast genes are 23S and 16S, subunits of the rRNAs; AR, AL, IR, IL, right and left exons of the split tRNA genes for alanine and isoleucine, respectively. V, tRNAval gene. For more detailed mapping data see Stern & Lonsdale (1982).

Fig. 4.

Sequence of a transposed segment of ctDNA in the corn mitochondrial genome. Restriction maps of a portion of the corn ctDNA inverted repeat (top and middle) and the corresponding region of the mitochondrial genome (bottom) are shown. Below, the maps, the nucleotide sequence of the ctDNA (Koch et al. 1981) and the nonidentical bases in the mtDNA are shown. (−) indicates deleted bases. Restriction sites are Bam III (B) and Sad (T). Chloroplast genes are 23S and 16S, subunits of the rRNAs; AR, AL, IR, IL, right and left exons of the split tRNA genes for alanine and isoleucine, respectively. V, tRNAval gene. For more detailed mapping data see Stern & Lonsdale (1982).

To determine the extent of conservation of a portion of a 12 kb sequence shared by corn ctDNA and mtDNA, which contains most of the chloroplast rrn operon (Stern & Lonsdale, 1982), Sau3A subfragments of the mtDNA sequence were cloned into the BamHA site of M13mp7 (Messing et al. 1981), sequenced by the dideoxy chain termination method (Sanger et al. 1977), and compared to the previously published corn ctDNA sequence (Koch et al. 1981). Fig. 4 shows that 249 of 255 positions examined are identical (98%). The mitochondrial sequence has undergone two substitutions, three deletions and one insertion (of 5 bp). The fidelity of this sequence in the mitochondrial genome is remarkable, inasmuch as it derives from a ctDNA intron and is unlikely to function in the mitochondrion. This result can be interpreted as evidence for very recent transfer, but a slow rate of base substitutions, and/or nonclassical mechanisms of genetic fixation and maintenance (Dover, 1982; A. Wilson, personal communication) could also account for the lack of sequence divergence.

An initial assay to detect transcription of ctDNA-homologous sequences in watermelon mitochondria is presented in Fig. 5. Lane 1 shows which mtDNA restriction fragments are homologous with mtRNA and therefore contain transcribed regions. Lane 2 shows which mtDNA fragments have regions homologous with ctDNA, and lane 3 serves as a control, so that ctDNA fragments contaminating the mtDNA in lanes 1 and 2 can be disregarded. Any mtDNA fragment identified in both lanes 1 and 2 then, contains regions both homologous to ctDNA and mtRNA. Three such fragments are indicated (▸) to the left of Fig. 5, lane 1. The mitochondrial rRNA genes are expected to cross-react with the ctDNA probe by virtue of homologies between the bona fide mitochondrial 26S and 18S rRNA genes and the chloroplast 23S and 16S rRNA genes, respectively (Fig. 5, ▹ and ◊; Stern et al. 1984).

Fig. 5.

Expression of mtDNA fragments with homology to ctDNA, in watermelon. The indicated DNAs (top) were digested with PuwII, electrophoresed in a 0·7 % agarose gel, transferred to GeneScreen and hybridized with 32P-labelled probes (below). Solid arrows (▸) indicate mtDNA fragments that have homology with both mtRNA and ctRNA (see text). Fragments identified with open arrows (▹) or an open diamond (◊) contain the mitochondrial or chloroplast rRNA genes, respectively. Size markers are in kb, and were calculated as for Fig. 1.

Fig. 5.

Expression of mtDNA fragments with homology to ctDNA, in watermelon. The indicated DNAs (top) were digested with PuwII, electrophoresed in a 0·7 % agarose gel, transferred to GeneScreen and hybridized with 32P-labelled probes (below). Solid arrows (▸) indicate mtDNA fragments that have homology with both mtRNA and ctRNA (see text). Fragments identified with open arrows (▹) or an open diamond (◊) contain the mitochondrial or chloroplast rRNA genes, respectively. Size markers are in kb, and were calculated as for Fig. 1.

Although the mtDNA fragments of interest have not yet been shown to have a single region within them that hybridizes with both ctDNA and a transcript found exclusively within the mitochondrion, it must be allowed that functions for transposed DNA could operate even in the absence of transcription. The sequence could function, for example, as a spacer, an origin of replication, or could be transcribed in an organ-specific or developmentally-regulated manner. A simple interpretation of DNA transfer from chloroplast to mitochondrion views the phenomenon as merely a ‘footprint’ of a more or less random insertional event into the large and ‘permissive’ mitochondrial genome. This interpretation considers the transposed ctDNA as irrelevant to mitochondrial function, a viewpoint that should not be accepted without reservation. The close biochemical interplay of the two organelles in the plant cell, their common dependence on nuclear gene products, and the necessity to maintain an energetic balance between photosynthesis and respiration may require closely linked regulatory functions, some of which may be specified in shared DNA sequences. A continued and detailed analysis of promiscuous DNA will provide a basis for studying these interorganellar interactions.

Some of this work was performed in the laboratories of Dr D. Lonsdale at the Plant Breeding Institute, Trumpington, Cambridge, England and Dr W. Thompson at the Carnegie Institution of Washington, Department of Plant Biology, Stanford, California, USA. I am grateful for their guidance, for that of Jeffrey Palmer and Mike Saul, to Herbert Stern and Helen Jones for critical readings of this manuscript, and to Loretta Tayabas for indispensable secretarial assistance. This is CIW-DPB Publication #959.

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