The plant mitochondrial genome has long been an enigma to molecular biologists. Even the smallest is more than 200 kilobases in size, more than 10 times the size of animal mitochondrial genomes (15-18 kb) and several times the size of mitochondrial genomes in fungi (18-78 kb) or protists (15-47 kb). In contrast to the DNA of chloroplasts which is relatively conserved, the DNA of mitochondria exhibits a wide variation in size and form.
Higher plant mtDNA can be circular or linear and varies from 200 kbp (in brassicas) up to greater than 2500 kbp (in muskmelon).The genome of plant mitochon dria is thus very large and may also be divided between one or more DNA molecules.
The largest plant mitochondrial genome studied so far is half the size of the entire E. coli genome (4,500 kb). The large size of these genomes presents several problems.On the one hand, simply determining the physical structure of such large genomes is a major challenge, especially since recombination events are known to produce several different molecular configurations.
On the other hand, the large size per se and the dramatic variation of such genomes demands an explanation.We know of few genes in plant mitochondrial genomes which are not also present in the mitochondria of yeast or animal cells.And the few additional genes we do know about do not begin to account for the additional DNA in even the smallest of plant mitochondrial genomes. Both animal and plant mitochondria encode their own ribosomal and transfer RNAs.
The number of proteins encoded in plant mitochondrial DNA is probably not much higher than the number encoded in mammalian mitochondria.Experiments have shown that most of the protein synthesis in isolated maize mitochondria can be accounted for by some 18-20 polypeptides.Although there is always a possibility that more proteins might be synthesized in mitochondria in vivo than in vitro, it seems likely that most of the mitochondrial genome is noncoding DNA.
Size and Structure of Mitochondrial Genome -
Changes in the size of the mitochondrial genome seem to occur quite rapidly in plant evolution since closely related species sometimes have quite different mitochondrial genome sizes.The best example of this phenomenon comes from studies in the laboratory of Arnold Bendich at the University of Washington.Combining measurements of mitochondrial DNA renaturation kinetics with an analysis of restriction profiles for the same DNAs, Bendich and his colleagues B. Ward and R.Anderson estimated the minimal size (complexity) of mitochondrial DNA for several species in the family Cucurbitaceae.
Their estimates, along with similar estimates for several other plant mitochondrial genomes. Within the Cucurbitaceae the size of the mitochondrial genome can vary by at least 10-fold.This variation cannot be explained by postulating rapid changes in the amount of repetitive DNA since little repeated DNA (less than 10%) was found in any of the genomes.Hence, the mitochondrial genome is like the chloroplast genome in being composed principally of single copy DNA. But it is like the nuclear genome in its highly variable size and its content of "excess" DNA which has no known function.
In several cases, including Brassica, maize, and wheat, restriction maps have been constructed for mitochondrial genomes. Although the smaller genomes (for example, Brassica) can be mapped by procedures such as those used for chloroplast DNA, the larger genomes require different techniques.The most successful approach has been to clone large fragments of mitochondrial DNA and then use a combination of hybridization and restriction mapping to identify overlapping clones and establish linkage groups. This procedure is called chromosome walking.With such a procedure it has been possible to show that most, possibly all, mitochondrial DNA can be described as one large circular linkage group.
In contrast to chloroplast DNA, in which circular molecules can be seen in an electron microscope, the physical form of the mitochondrial genome is still not well understood. Many investigators have reported the presence of circular DNAs in plant mitochondria, but these are small in relation to the total genome size. Some are mitochondrial plasmids which are not considered to be part of the main genome; others clearly contain genomic sequences.In some cases most of the DNA sequences in the mitochondrion can be found in the collection of circles, although individual circles are often much smaller , than the size of the genome. Cultured tobacco cells provide a good case in point. These cells are an excellent source of the relatively small mitochondrial DNA circles which can be purified on density gradients and analyzed with restriction enzymes.
Although none of the circles comes close to the size of the genome as a whole, the restriction profile of the DNA in the circular fraction is identical to that of total mitochondrial DNA from either cultured cells or intact plants.At least in cultured tobacco cells, therefore, it seems that the entire mitochondrial genome can exist as a population of subgenomic circles.
Mitochondrial DNA consisting predominantly of small circles may be peculiar to cultured plant cell since it is often difficult to isolate any circular DNA at all (with the exception of plasmids) from the mitochondria of mature plants.In these cases the physical form of the DNA remains unknown. Although it is logical to suppose that larger circles are present in vivo, technical difficulties make it quite difficult to test this hypothesis.
The complex and variable pattern of mitochondrial DNA organizations was difficult to rationalize until complete restriction maps became available.The relatively small Brassica mitochondrial genome was mapped by J. Palmer and C. Chields at the Carnegie Institution, Department of, Plant Biology, using relatively simple mapping techniques developed originally for chloroplast DNA. Meanwhile, D. Lonsdale and his colleagues at the Plant Breeding Institute in Cambridge, England, had been using chromosome walking techniques to map the much larger maize mitochondrial genome. The two groups reported their findings almost simultaneously.
It was discovered in both cases that the genome contained a set of repeat sequences which could be found associated with different permutations of flanking sequences, defining a set of substoichiometric restriction fragments. This data is highly consistent with a model in which site specific recombination occurs at the repeated sequence, generating a series of subgenomic circular molecules that are in conformational equilibrium with each other and with a master circle.Recombination in mitochondrial DNA has also been shown to occur in somatic hybrid cells produced by protoplast fusion techniques.
Simply maintaining cells in tissue culture can lead to variations in the restriction pattern of their mitochondrial DNA.It is difficult from our present level of knowledge to determine whether these changes reflect (recombination events or simply differential replication of different preexisting variant molecules.
However, certain somatic hybrid cells have been shown to contain mitochondrial DNA restriction fragments which are not present in either parental genome. And clones have been obtained which contain marker DNA segments from different parents. As yet it is not known whether intergenomic recombination events occur by the same mechanism as the intragenomic recombinations that produce the diverse array of molecules in a single genome, but it seems reasonable to suppose that they do.
The significance (if any) of the plasmid-like DNAs to the plant is not known, and the plasmid like DNAs can be lost without obvious effects on appearance or viability.The best studied examples of mitochondrial episomes are found in certain male sterile lines of maize.They have been studied, at least in part, in the hope that they might be involved in producing the male sterility trait.Cytoplasmic male sterility (CMS) appears to be associated with alterations in the mitochondrial DNA.Several types of CMS maize cytoplasm can be distinguished by their responses to nuclear restorer genes, which restore fertility to some types but not to others, and by their mitochondrial polypeptides and the restriction profiles of their mitochondrial DNA. A major CMS cytoplasm is the s type, which is characterized by two prominent inverted repeats 208 bp long.
These repeats are covalently linked to protein. By analogy to adenovirus and certain bacteriophage systems, it is thought that the terminal protein complexes may be involved in initiating DNA replication.The episomal DNAs 8-1 and S-2 are not detectable as free episomes in the mitochondria-from CMS-S plants that have reverted to fertility, but sequences homologous to S-1 and S-2 can be found in high molecular weight mitochondrial DNA, in normal, CMS-S, and fertile revertant cytoplasms.
Higher plant mtDNA can be circular or linear and varies from 200 kbp (in brassicas) up to greater than 2500 kbp (in muskmelon).The genome of plant mitochon dria is thus very large and may also be divided between one or more DNA molecules.
The largest plant mitochondrial genome studied so far is half the size of the entire E. coli genome (4,500 kb). The large size of these genomes presents several problems.On the one hand, simply determining the physical structure of such large genomes is a major challenge, especially since recombination events are known to produce several different molecular configurations.
On the other hand, the large size per se and the dramatic variation of such genomes demands an explanation.We know of few genes in plant mitochondrial genomes which are not also present in the mitochondria of yeast or animal cells.And the few additional genes we do know about do not begin to account for the additional DNA in even the smallest of plant mitochondrial genomes. Both animal and plant mitochondria encode their own ribosomal and transfer RNAs.
The number of proteins encoded in plant mitochondrial DNA is probably not much higher than the number encoded in mammalian mitochondria.Experiments have shown that most of the protein synthesis in isolated maize mitochondria can be accounted for by some 18-20 polypeptides.Although there is always a possibility that more proteins might be synthesized in mitochondria in vivo than in vitro, it seems likely that most of the mitochondrial genome is noncoding DNA.
Size and Structure of Mitochondrial Genome -
Changes in the size of the mitochondrial genome seem to occur quite rapidly in plant evolution since closely related species sometimes have quite different mitochondrial genome sizes.The best example of this phenomenon comes from studies in the laboratory of Arnold Bendich at the University of Washington.Combining measurements of mitochondrial DNA renaturation kinetics with an analysis of restriction profiles for the same DNAs, Bendich and his colleagues B. Ward and R.Anderson estimated the minimal size (complexity) of mitochondrial DNA for several species in the family Cucurbitaceae.
Their estimates, along with similar estimates for several other plant mitochondrial genomes. Within the Cucurbitaceae the size of the mitochondrial genome can vary by at least 10-fold.This variation cannot be explained by postulating rapid changes in the amount of repetitive DNA since little repeated DNA (less than 10%) was found in any of the genomes.Hence, the mitochondrial genome is like the chloroplast genome in being composed principally of single copy DNA. But it is like the nuclear genome in its highly variable size and its content of "excess" DNA which has no known function.
In several cases, including Brassica, maize, and wheat, restriction maps have been constructed for mitochondrial genomes. Although the smaller genomes (for example, Brassica) can be mapped by procedures such as those used for chloroplast DNA, the larger genomes require different techniques.The most successful approach has been to clone large fragments of mitochondrial DNA and then use a combination of hybridization and restriction mapping to identify overlapping clones and establish linkage groups. This procedure is called chromosome walking.With such a procedure it has been possible to show that most, possibly all, mitochondrial DNA can be described as one large circular linkage group.
In contrast to chloroplast DNA, in which circular molecules can be seen in an electron microscope, the physical form of the mitochondrial genome is still not well understood. Many investigators have reported the presence of circular DNAs in plant mitochondria, but these are small in relation to the total genome size. Some are mitochondrial plasmids which are not considered to be part of the main genome; others clearly contain genomic sequences.In some cases most of the DNA sequences in the mitochondrion can be found in the collection of circles, although individual circles are often much smaller , than the size of the genome. Cultured tobacco cells provide a good case in point. These cells are an excellent source of the relatively small mitochondrial DNA circles which can be purified on density gradients and analyzed with restriction enzymes.
Although none of the circles comes close to the size of the genome as a whole, the restriction profile of the DNA in the circular fraction is identical to that of total mitochondrial DNA from either cultured cells or intact plants.At least in cultured tobacco cells, therefore, it seems that the entire mitochondrial genome can exist as a population of subgenomic circles.
Mitochondrial DNA consisting predominantly of small circles may be peculiar to cultured plant cell since it is often difficult to isolate any circular DNA at all (with the exception of plasmids) from the mitochondria of mature plants.In these cases the physical form of the DNA remains unknown. Although it is logical to suppose that larger circles are present in vivo, technical difficulties make it quite difficult to test this hypothesis.
The complex and variable pattern of mitochondrial DNA organizations was difficult to rationalize until complete restriction maps became available.The relatively small Brassica mitochondrial genome was mapped by J. Palmer and C. Chields at the Carnegie Institution, Department of, Plant Biology, using relatively simple mapping techniques developed originally for chloroplast DNA. Meanwhile, D. Lonsdale and his colleagues at the Plant Breeding Institute in Cambridge, England, had been using chromosome walking techniques to map the much larger maize mitochondrial genome. The two groups reported their findings almost simultaneously.
It was discovered in both cases that the genome contained a set of repeat sequences which could be found associated with different permutations of flanking sequences, defining a set of substoichiometric restriction fragments. This data is highly consistent with a model in which site specific recombination occurs at the repeated sequence, generating a series of subgenomic circular molecules that are in conformational equilibrium with each other and with a master circle.Recombination in mitochondrial DNA has also been shown to occur in somatic hybrid cells produced by protoplast fusion techniques.
Simply maintaining cells in tissue culture can lead to variations in the restriction pattern of their mitochondrial DNA.It is difficult from our present level of knowledge to determine whether these changes reflect (recombination events or simply differential replication of different preexisting variant molecules.
However, certain somatic hybrid cells have been shown to contain mitochondrial DNA restriction fragments which are not present in either parental genome. And clones have been obtained which contain marker DNA segments from different parents. As yet it is not known whether intergenomic recombination events occur by the same mechanism as the intragenomic recombinations that produce the diverse array of molecules in a single genome, but it seems reasonable to suppose that they do.
Mitochondrial Plasmids -
In addition to a variety of circular mitochondrial chromosomes, mitochondria from a number of plants contain episomal or plasmid like molecules.These are generally small circular, or small linear double stranded DNAs.They are usually detected as strong discrete bands in electrophoretic separations of untreated (intact) mitochondrial DNA.In well studied cases, it has been shown that the episomal DNAs do not fit into the restriction map of the main genome.
Plasmid like DNAs have been characterized in a number of plants, including sugar beet, sorghum, and some species of Brassica. Normal maize mitochondria carry a linear episome 2.3 kb in length and a circular DNA of about 1.9 kb.This is in addition to the various subgenomic circles generated by recombination in the main genome.
The significance (if any) of the plasmid-like DNAs to the plant is not known, and the plasmid like DNAs can be lost without obvious effects on appearance or viability.The best studied examples of mitochondrial episomes are found in certain male sterile lines of maize.They have been studied, at least in part, in the hope that they might be involved in producing the male sterility trait.Cytoplasmic male sterility (CMS) appears to be associated with alterations in the mitochondrial DNA.Several types of CMS maize cytoplasm can be distinguished by their responses to nuclear restorer genes, which restore fertility to some types but not to others, and by their mitochondrial polypeptides and the restriction profiles of their mitochondrial DNA. A major CMS cytoplasm is the s type, which is characterized by two prominent inverted repeats 208 bp long.
These repeats are covalently linked to protein. By analogy to adenovirus and certain bacteriophage systems, it is thought that the terminal protein complexes may be involved in initiating DNA replication.The episomal DNAs 8-1 and S-2 are not detectable as free episomes in the mitochondria-from CMS-S plants that have reverted to fertility, but sequences homologous to S-1 and S-2 can be found in high molecular weight mitochondrial DNA, in normal, CMS-S, and fertile revertant cytoplasms.
Their arrangement with respect to adjacent genomic sequences differs between CMS-S and revertants, however, and it is thought that this rearrangement may somehow be involved in the process of reversion to fertility. C, Schardl, in collaboration with D.Lonsdale at the Plant Breeding Institute in Cambridge, England, and with D. Pring and K. Rose at the University of Florida, analyzed cosmid clones containing sequences homologous to S-1 and S-2 from CMS and revertant plants.Data from restriction analysis of many such cosmids was consistent with a model in which recombination between the terminal inverted repeats of the S-1 and S-2 episomes and homologous sequences in the mitochondrial chromosomes would generate linear mitochondrial DNA molecules containing S-1 or S-2 at their end.
The relationship of this linearization of the otherwise circular mitochondrial genome to the CMS phenotype is still not clear, although the fact that the same arrangements are seen in the DNA of both CMS plants in which the CMS phenotype has been corrected by nuclear genes indicates that linearization per se is not sufficient to cause male sterility.
Chloroplast Sequences in Mitochondrial DNA -
The observation that chloroplast DNA sequences are contained in the mitochondrial genome provided another surprise for plant molecular biologists.The initial observations came from Cambridge, England, where D.Stern and D. Lonsdale of the Plant Breeding Institute reported that mitochondrial DNA from maize contained a 12 kb sequence from the maize chloroplast genome.
When labelled chloroplast DNA was reacted with restriction digests of mitochondrial DNA, this sequence hybridized preferentially.Investigations further showed that the preferentially hybridizing sequence could be cloned on a cosmid (a plasmid packaged in a lambda virus coat) that contained mitoshy;chondrial DNA on either side of the chloroplast segment It was found that the 12 kb sequence contained a portion of the chloroplast inverted repeat with genes for several tRNAs and the 16S ribosomal RNA.
By restriction mapping, the mitochondrial version of the 12 kb sequence appeared virually identical to its presumed progenitor sequence in the chloroplast, the only differences occurring at the conjunction sites of the ends of the inserted segment and the mitochondrial DNA sequences.
Two other segments of chloroplast DNA have been characterized in the maize mitochondrial genome.The first of these segments includes the 3 end of the chloroplast 23S ribosomal RNA gene, the genes for 4.5S and 5S ribosomal RNAs, and two tRNA genes. The other segment contains the rbcL gene and its flanking sequences on both the 3/- and 5/ -ends. The gene is functional in the E. coli transcription/translation system and its protein product can be precipitated with antibodies to RuBP carboxylase.
However, the mitochondrial gene produces truncated polypeptide of 21,000 daltons instead of the 54,000 dalton protein synthesized by the chloroplast gene. Whether this gene or any other chloroplast gene actually functions in the mitochondrion is not known. However, the genetic code is slightly different in mitochondria and there are also likely to be important differences in transcriptional and translational control signals between the two organelles.In view of these differences it seems unlikely that chloroplast genes could be functional in the mitochondrial environment. The presence in mitochondrial DNA of sequences that hybridize to chloroplast DNA has since been shown to be a widespread phenomenon, which is not restricted to maize. In collaboration with J. Palmer, who had been studying evolutionary relationships among the chloroplast DNAs of a wide variety of plants.
D. Stern showed homologies between cloned segments of chloroplast DNA and mitochondrial DNA fragments from several species, including pea, mung bean, spinach, and four different species of cucurbits. Adding up the segments of chloroplast DNA that seemed to be represented in the mitochondrial DNA of one or more of these plants gave the impression that almost the entire chloroplast genome might be subject to random transfer.In addition, different degrees of homology were seen, suggesting that transfer events have occurred at different times during evolution. Although some of the homologies observed in these survey experiments might be the result of cross hybridization between chloroplast and mitochondrial genes of similar function, this is not always the case, and it appears that there are too many cross reacting fragments to be easily accounted for by this hypothesis.
The overall picture is most consistent with a series of events in which random sequences from the chloroplast genome appear at random positions in the mitochondrial genome.These events would be frequent in an evolutionary sense. There is no direct evidence concerning the mechanism by which DNA transfer occurs between organelles.However, it is easier to reconcile the present indirect evidence with an essentially random process than with directed transfer by some kind of vector.The fusion of organelles of the uptake by one organelle of DNA released by lysis of another organelle might occur often enough to explain the foregoing observations.This view is also consistent with the demonstration that chloroplast DNA fragments can be found in the nuclear DNA of higher plants.Sequences transferred between organelles in this way have been called promiscuous DNA, a term designed to highlight the random nature of the process.
Transfers from a mitochondrion to chloroplast have not yet been demonstrated and may not occur with significant frequency. The chloroplast genome simply may not tolerate random insertions of foreign DNA. As noted previously, although rearrangements have occurred in a few chloroplast genomes, chloroplast DNA generally shows a high degree of conservation in both size and sequence arrangementThose insertion and deletion events which do occur seem mostly to involve rather small segments of DNA, which would not be easy to detect by hybridization techniques. In contrast, the large and highly variable mitochondrial and nuclear genomes probably contain many regions in which relatively large pieces of foreign DNA can be inserted with minimal effect.
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