The large mitochondrial genomes of angiosperms are unusually dynamic because of recombination activities involving repeated sequences. These activities generate subgenomic forms and extensive genomic variation even within the same species. Such changes in genome structure are responsible for the rapid evolution of plant mitochondrial DNA and for the variants associated with cytoplasmic male sterility and abnormal growth phenotypes. Nuclear genes modulate these processes, and over the past decade, several of these genes have been identified. They are involved mainly in pathways of DNA repair by homologous recombination and mismatch repair, which appear to be essential for the faithful replication of the mitogenome. Mutations leading to the loss of any of these activities release error-prone repair pathways, resulting in increased ectopic recombination, genome instability, and heteroplasmy. We review the present state of knowledge of the genes and pathways underlying mitochondrial genome stability.

The NB mitochondrial genome found in most fertile varieties of commercial maize (Zea mays subsp. mays) was sequenced. The 569,630-bp genome maps as a circle containing 58 identified genes encoding 33 known proteins, 3 ribosomal RNAs, and 21 tRNAs that recognize 14 amino acids. Among the 22 group II introns identified, 7 are trans-spliced. There are 121 open reading frames (ORFs) of at least 300 bp, only 3 of which exist in the mitochondrial genome of rice (Oryza sativa). In total, the identified mitochondrial genes, pseudogenes, ORFs, and cis-spliced introns extend over 127,555 bp (22.39%) of the genome. Integrated plastid DNA accounts for an additional 25,281 bp (4.44%) of the mitochondrial DNA, and phylogenetic analyses raise the possibility that copy correction with DNA from the plastid is an ongoing process. Although the genome contains six pairs of large repeats that cover 17.35% of the genome, small repeats (20-500 bp) account for only 5.59%, and transposable element sequences are extremely rare. MultiPip alignments show that maize mitochondrial DNA has little sequence similarity with other plant mitochondrial genomes, including that of rice, outside of the known functional genes. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly three-fourths of the maize NB mitochondrial genome is still of unknown origin and function.

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding ''native'' mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.

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We have examined the expression of three alternative oxidase ( aox ) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2 , whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3 . Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3 . AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.

The genetic analysis of higher plant mitochondria has been limited by a scarcity of identified mutations with known progenitors. Correspondingly, few molecular studies have been directed at types of plant mitochondrial variation other than cytoplasmic male sterility. The maternally inherited nonchromosomal stripe (NCS) mutants of maize have profound deleterious effects on plant growth and yield. We report specific alterations in mitochondrial DNA (mtDNA) for two independent, phenotypically distinct NCS mutants. NCS2 plants have a distinctive 21-kilobase Xho I mtDNA band and very reduced amounts of DNA in an 8-kilobase band that is present in the progenitor. NCS3 plants have a distinctive 20-kilobase Xho I band and a reduction in a 16-kilobase band. Our studies confirm that the affected organelle in NCS plants is the mitochondrion. Because NCStype plants appear with a certain frequency in a particular line (WF9), this line is a potential source ofadditional mutations for functional and molecular analyses of maize mitochondrial genes.It is -now well established that phenotypic variability can result from alterations in organelle genomes. Several examples have been described for lower eukaryotes, such as yeast and fungi. They include mitochondrial DNA (mtDNA) changes that correlate with premature senescence, altered colony size and morphology, and aberrant patterns ofgrowth (1-4).Previously, much of the research on mitochondrial variability in higher plants has focussed on cytoplasmic male sterility (CMS), a trait usually associated with mtDNA rearrangements (5, 6). In contrast with the work on CMS in maize, little effort has yet been expended on the study of other types of putative mitochondrial mutations. Nonchromosomal stripe (NCS) describes a set of maternally inherited mutations in maize, characterized by striking and variable phenotypic effects including poor growth, abnormal morphologies, and leaf striping. First extensively studied by Shumway and Bauman (7), NCS arose in a particular line of maize, WF9, carrying the T-type male-sterile cytoplasm (cms-T). Although the original NCS mutation has been lost, two other mutations, known as NCS2 and NCS3, were discovered by Bauman in T-cytoplasmic versions of inbred WF9 (NCS3) and the related line, H49 (NCS2) (8).The results from crosses using NCS plants as either the male or female parent showed that NCS traits are maternally inherited (7,8). Although the generation of NCS mutations may depend on the nuclear genotype, the mutations are subsequently inherited strictly through the female parent. The phenotypic expression ofthe NCS2 and NCS3 mutations was not suppressed in crosses with several inbred lines:Ky2l, K55, W23, Mol7, Tr, Oh5la, and A619 (8). The effects of fertility-restoring genes on NCS phenotypes were tested by crossing NCS2 and NCS3 plants by pollen from the Ky2l line, which carries dominant nuclear genes that restore pollen shedding to cms-T plants. The nuclear genes that suppress the male-sterile phenotype had no effect on the expression of e...

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.