y . Cell Sci. Suppl. 7, 145-154 (1987)Printed in Great Britain © The Company o f Biologists Limited 1987

145

D N A TR AN SPOSITION BETWEEN PLANT

ORGANELLAR GENOMES

D A V ID B. S T E R NDepartment of Botany, University of California, Berkeley, CA 94720, USA

S U M M A R Y

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.

I N T R O D U C T I O N

Significant DN A sequence homologies exist between higher plant mitochondrial and chloroplast DNAs (Stern & Lonsdale, 1982; Lonsdale et al. 1983; Stern et al. 1983; Stern & Palmer, 1984a; D ron et al. 1985; Whisson & Scott, 1985). These studies have provoked speculation that interorganellar DNA transfer is a continuing phenom enon. T hey also provide evidence that certain DNA segments in these two organelles may have a common origin (Ellis, 1982; Tim m is & Scott, 1984; Stern & Palmer, 1984a).

T he mechanism by which exogenous D NA 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 D N A 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 instrum ental in the excision or integration of D NA . The direction of most interorganellar DN A exchange is probably from chloroplast to mitochondrion. This is suggested by the presence of known chloroplast genes in m tD N A , and the absence of mitochondrial genes in ctDN A. T he 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 ctDN A transferred, their conservation at the sequence level, and evidence for their expression in the mitochondrion. Results are

Table 1. Plant species known to have m tDNA-ctD NA sequence homologies

146 D. B. Stern

Reference*

mtDNA-Plant species Genome size (kb) Size ctDNA homology

Turnip (Brassica campestris) 218 5 13Cauliflower (Brassica oleracea) 219 13 1Atriplex halimus 270 13 14Atriplex ?osea 270 13 14Pokeweed (Phytolacca heterotepala) 320 14 14Spinach (Spinacia oleracea) 327 10 9, 10, 12Pea (Pisum sativum) 380 13 9Mung bean (Vigna radiata) 400 13 9Wheat (Triticum aestivum) 430 6 14Corn (Zea mays) 570 3 2, 4, 7, 9Zucchini (Cucurbita pepo) 840 11 8Cucumber (Cucumis sativa) 1500 11 8Muskmelon (Cucumis melo) 2400 11 8

* 1 - Dron, Hartmann, Rode & Sevignac (1985).2 - Lonsdale, Hodge, Howe & Stern (1983).3 - Lonsdale, Hodge & Fauron (1984).4 - Lonsdale (1985).5 - Palmer & Shields (1983).6 - Quetier, Lejeune, Delorme, Falconet & Jubier (1985).7 - Stern & Lonsdale (1982).8 - Stern, Palmer, Thompson & Lonsdale (1983).9 - Stern & Palmer (1984a).

10 - Stern & Palmer (1986).11 - Ward, Anderson & Bendich (1981).12 - Whisson & Scott (1985).13 - J. D. Palmer (personal communication).14 - D. B. Stern & ]. D. Palmer (unpublished data).

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 ctD N A sequence, and a prelim inary analysis to detect transcription of promiscuous DNA in watermelon mitochondria.

D I S T R I B U T I O N O F T R A N S P O S E D D N A I N P L A N T M I T O C H O N D R I A L G E N O M E S

Table 1 lists 14 higher plant mitochondrial genomes known to exhibit homology to ctD N A . These species encompass both monocots and dicots, and their m itochon­drial genomes range from 218 kb (turnip) to approximately 2400 kb (muskmelon) in size. T he m ethod by which such homologies are identified is illustrated in Fig. 1. M itochondrial and chloroplast DNAs from four members of the plant family Cucurbitaceae were digested with the restriction endonuclease P vuW , electrophor- esed in agarose gels, transferred to nitrocellulose and hybridized with 32P-labelled cloned ctD N A segments from spinach (Fig. 1). T he ctDN A is included as a control, since m tD N A preparations are inevitably contaminated with m inor amounts of ctD N A . Hybridizing fragments appearing only in the m tD N A lane represent bona

Interorganellar DNA transposition 147

fide m tD N A fragments with homology to the etD N A probe (e.g. Fig. 1 (left), lane Z (m )), while fragments hybridizing in both m tD N A and ctDN A lanes indicate contam ination, and are disregarded (e.g. Fig. 1 (right), lanes M(m) and M (c)). Since the percentage of ctD N A contaminating each m tD NA preparation is variable, for certain plants (e.g. watermelon and zucchini, Fig. 1 (left)), very little ctD N A is present on the filter, and hybridization between ctD N A and the j2P-labelled probe is only evident upon prolonged exposure of the filter.

T he 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 (.a tpB , Fig. 1 (middle)) and epsilon (alpE , Fig. 1 (right)) subunits of the chloroplast ATPase hybridize with all the m tDNAs except that of muskmelon. Analogous experim ents were performed with gene-containing ctDN A clones for rbcL, atpB,

W Z C M W Z C M W Z C M kb me m c m c m e m c m c m c m c m c m c m c m c 20

10

4 --------------------- -------------------------------k~ rbcL atpB atpE r

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 PvulI, electrophoresed in 0-7 % agarose gels, transferred to nitrocellulose and probed with the j2P-labelled cloned ctDNA fragments indicated on the restriction map (Zurawski el al. 1982) below the figure. Size standards, in kb, were calculated from Eco R I, Sal I and H/«dIII-digested phage A DNA.

148 D. B. Stern

Table 2. Occurrence o f selected chloroplast gene sequences in p lan t mitochondrialgenomes

Chloroplast Munggene* Corn bean Spinach Pea Watermelon Zucchini Cucumber Muskm

rm 16 + N T N T N T + + + +rrn 23 + N T N T N T - - + +rbcL + - - - + + -atpB - ■ - - + + + -atpE - - - - + + + -

N T = not tested.* Gene designations are rrn 16 and rrn 23, 16S and 23S rRNAs, rbcL, large subunit of ribulose

bisphosphate carboxylase, atpB and atpE, beta and epsilon subunits, respectively, of the chloroplast ATPase. Probes were rm lb , 3-5 kb Sac I fragment (mung bean), rrnZi, 3 ■ 0 kb Xho I fragment (spinach), rbcL, 1750bp EcoKl fragment (spinach), atpB, 1980bp EcoRI fragment (spinach) and atpE, 1250 bp E coK l-X bal fragment (spinach, contains only sequences 3' to atpE, see Stern & Palmer, 1984a).

atpE and the chloroplast rRNA genes and additional plant m tDNAs. These results, together with those shown in Fig. 1, are summarized in Table 2. Only the cucumber m itochondrial 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 m tD N A s. Furtherm ore, the largest genome (muskmelon, Table 1) has no homology to any of the protein-coding genes, in contrast to cucumber, a m em ber 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. T his contrasts with the situation for the rRN A genes, for which the chloroplast and mitochondrial versions are homologous (Stern et al. 1984). In this instance, prior knowledge -of the m tD N A fragments containing the bona fid e m itochondrial rRN A genes is required, before a transposed region in the m itochon­drial genome can be assigned using a heterologous chloroplast rRN A gene probe. Lastly, it is im portant to realize that the presence of specific chloroplast gene sequences in m tD N A is significant only to the extent that a short, identifiable sequence is being tested. T he large probes used in previous studies (S tern & Palmer, 1984a, 1986) preclude analysis of distributive patterns of specific ctD N A sequences in different mitochondrial genomes. T he results presented in Table 2 have been extended for four members of the plant family Cucurbitaceae, by using approxi­mately 35 short (< 4 k b ) segments of cloned ctD N A from mung bean, to investigate the distribution of their sequences in the mitochondrial genomes. T he results of these experiments will be published separately.

T he strong homology between the chloroplast genes and the m tD N A s (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. T he data presented Jbelow, however, indicate that at least in spinach m tD N A , a complex pattern of ctD N A

Interorganellar DNA transposition 149

c t DN A

Fig. 2. Pattern of DNA homologies between spinach chloroplast and mitochondrial DNAs. Restriction maps of the 152kb spinach chloroplast genome (top; ‘solid lines’=PstI, ‘broken lines’ =A7zoI) and the 327kb mitochondrial genome (bottom; ‘solid lines’ = Sal I) 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 (O ) 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.

integration precludes a single, recent occurrence of D N A transposition. The distribution of ctD N A sequences in the spinach mitochondrial genome has been analysed with the aid of a complete physical map of the m tD N A (Stern & Palmer, 1986). A simplified version of the results (Fig. 2) shows that some regions of ctDN A appear to hybridize to multiple loci in the mitochondrial genome (e.g. open arrows, Fig. 2). T his interpretation is supported by the cross-hybridization of the two m tD N A fragments (data not shown). We have also found that a 1670bp LcoRI fragm ent of spinach ctD N A hybridizes with three zucchini m tD N A 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. T he ambiguity between multiple transfers, and an instance in which the ctD N A 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.

C O N S E R V A T I O N O F A P R O M I S C U O U S D N A S E Q U E N C E

T o determ ine the extent of conservation of a portion of a 12 kb sequence shared by corn ctD N A and m tD N A , which contains most of the chloroplast rrn operon (Stern & Lonsdale, 1982), S a n 3A subfragments of the m tD N A sequence were cloned into the Bam W \ site of M13mp7 (Messing et al. 1981), sequenced by the dideoxy chain term ination method (Sanger et al. 1977), and compared to the previously published corn ctD N A sequence (Koch et al. 1981). Fig. 4 shows that 249 of 255 positions examined are identical (98% ). T he mitochondrial sequence has undergone two

150 ID. B. Stern

substitutions, three deletions and one insertion (of 5 bp). T he fidelity of this sequence in the mitochondrial genome is remarkable, inasmuch as it derives from a ctD N A intron and is unlikely to function in the mitochondrion. T his result can be interpreted as evidence for very recent transfer, but a slow rate of base substitutions, and /o r nonclassical mechanisms of genetic fixation and maintenance (Dover, 1982; A. Wilson, personal communication) could also account for the lack of sequence divergence.

A R E T R A N S P O S E D S E Q U E N C E S E X P R E S S E D I N T H E M I T O C H O N D R I O N ?

An initial assay to detect transcription of ctDNA-homologous sequences in watermelon m itochondria is presented in Fig. 5. Lane 1 shows which m tD NA restriction fragments are homologous with mtRN A and therefore contain transcribed regions. Lane 2 shows which m tD N A fragments have regions homologous with ctD N A , and lane 3 serves as a control, so that ctDN A fragments contaminating the m tD N A in lanes 1 and 2 can be disregarded. Any m tD N A fragment identified in both lanes 1 and 2 then, contains regions both homologous to ctD N A and m tRNA. T hree such fragments are indicated (►) to the left of Fig. 5, lane 1. T he mitochondrial rRN A genes are expected to cross-react with the ctD N A probe by virtue of homologies between the bona fide mitochondrial 26S and 18S rRN A genes

ct

Mult iple t ransfers

mt

o =

0 =

i mt

mtSingle t ransfer /dispersal

o o

No dupl icat ion

mt

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.

3' A r a l i r

23S H W :3'

16S - > c t DNA- I R

23S o - ~ rT

TB

23401 I

- > m t D N A

H100 bp

1615Ct gatctagtat ggatcgtaca tggacgatag ttggagtcgg cggctctcct aggcttccct catctgggat ccctggggaa gaggatca

mt

c AGTTGGCCCT TGCGAATAGC TTGATGCACT ATCTCCCTTC AACCCTTTGA GCGAAATGTG GCAAAAGGAA GGAAAATCCA TGGACCGAmt GACACCTTTC -fCCATTTAGTl CGGACTGGTA m

T 1870CCCCATTGTC TC^CCCCGT AGGAACTACG AGATCACCCC AAGGACGCCT TCGGJGGGTC TATCGGACCG ACCATAGATC

CAGGA-_ r

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 Bamill (B) and SacI (T). Chloroplast genes are 23S and 16S, subunits of the rRNAs; AR, Aj_, I r , I i ,, right and left exons of the split tRNA genes for alanine and isoleucine, respectively. V, tR N A 'al gene. For more detailed mapping data see Stern & Lonsdale (1982).

Ik

Interorganellar DNA

transposition

152 D. B. S tem

30

20

DNAmt mt c t

>

1 0 ^

►

O

2

< < <z z zGC Q Q-t—' E ♦ 1 • 1 o oPROBE

Fig. 5. Expression of mtDNA fragments with homolog}' to ctDNA, in watermelon. The indicated DNAs (top) were digested with Pvull, 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 (O ) or an open diamond (•O) contain the mitochondrial or chloroplast rRNA genes, respectively. Size markers are in kb, and were calculated as for Fig. 1.

Interorganellar DNA transposition 153

and the chloroplast 23S and 16S rRNA genes, respectively (Fig. 5, l> and <̂ >; Stern et al. 1984).

Although the m tD N A fragments of interest have not yet been shown to have a single region within them that hybridizes with both ctDN A and a transcript found exclusively within the mitochondrion, it m ust be allowed that functions for transposed D N A could operate even in the absence of transcription. T he 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 D N A transfer from chloroplast to mitochondrion views the phenom enon as merely a ‘footprint’ of a more or less random insertional event into the large and ‘permissive’ mitochondrial genome. T his interpretation considers the transposed ctD N A as irrelevant to mitochondrial function, a viewpoint that should not be accepted without reservation. T he 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 D NA sequences. A continued and detailed analysis of pro­miscuous D N A 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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