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S-type cytoplasmic male sterility (CMS-S) i,a maize is associated with high levels of a 1.6-kb RNA in mitochondria. This RNA contains two chimeric open reading frames (ORFs), orf355 and orf77. The previously described nuclear restorer-of-fertility allele RJ3causes the processing of all transcripts that contain these chimeric ORFs. The Lancaster Surecrop-derived inbred line A619 carries a restorer that is distinct from Rf3 in that it selectively reduces only the CMS-S-specific 1.6-kb RNA. We have found that 10 additional Lancaster lines carry, a single restoring allele traceable to either of two inbred lines, C103 and Oh40B. The C103 and Oh40B restorers are allelic to each other, but not to Rf3. Thus, this restoring allele, designated Rf9, represents a second naturally occurring CMS-S restorer in maize. R]9 is a less effective restorer of fertility than is Rf3; its expression is influenced by both inbred nuclear background and temperature. Rf9 acts to reduce the amounts of orf355/orf77-containing linear mitochondrial subgenomes, which are generated by recombination of circular subgenomes with CMS-S-specific linear plasmids. The 1.6-kb RNA, which is transcribed only from linear ends, is correspondingly reduced. DOI: 10.1534/genetics.108.099895

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, Mol 7, 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.

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 535,895 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-190 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.

Sequencing, DNA and RNA gel blot analyses, PCR and primer extension experiments of the NCS2 mutant DNA help study the molecular foundations of the maternally inherited heteroplasmic NCS2 mutant of maize. NCS2 mutation in the first mitochondrial electron transport chain complex elements is due to a rearrangement that interferes in the NADH dehydrogenase subunit 4 (nad4) coding sequence. The rearrangement between intron sequences of nad4 and nad7 genes, spanning 16 base pairs of homology, indicates that it results from recombination at a tiny repeat. Longer transcripts arise due to the substitution of the 3' end of the nad4 gene with sequences from nad7 gene during rearrangement.

Each mitochondrion possesses a genome that encodes some of its own components. The nucleus encodes most of the mitochondrial proteins, including the polymerases and factors that regulate the expression of mitochondrial genes. Little is known about the number or location of these nuclear factors. B-A translocations were used to create dosage series for 14 different chromosome arms in maize plants with normal cytoplasm. The presence of one or more regulatory factors on a chromosome arm was indicated when variation of its dosage resulted in the alteration in the amount of a mitochondrial transcript. We used quantitative Northern analysis to assay the transcript levels of three mitochondrially encoded components of the cytochrome c oxidase complex (cox1, cox2, and cox3). Data for a nuclearly encoded component (cox5b) and for two mitochondrial genes that are unrelated to cytochrome c oxidase, ATP synthase [Alpha]-subunit and 18S rRNA, were also determined. Two tissues, embryo and endosperm, were compared and most effects were found to be tissue specific. Significantly, the array of dosage effects upon mitochondrial genes was similar to what had been previously found for nuclear genes. These results support the concept that although mitochondrial genes are prokaryotic in origin, their regulation has been extensively integrated into the eukaryotic cell.

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F1 hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis. [ABSTRACT FROM AUTHOR]

Investigates the molecular basis of the maternally inherited, heteroplasmic NCS2 mutant of maize. Resemblance between NCS2 mitochondrial DNA and cmsT mitochondrial genome; Transcripts containing exon 4 of the nad4 gene from heteroplasmic NCS2 plants; Deletion of the transcription termination signal of nad4 in mutant DNA.

The transfer of mitochondrial DNA (mtDNA) into nuclear genomes is a regularly occurring process that has been observed in many species. Few studies, however, have focused on the variation of nuclear-mtDNA sequences (NUMTs) within a species. This study examined mtDNA insertions within chromosomes of a diverse set of Zea mays ssp. mays (maize) inbred lines by the use of fluorescence in situ hybridization. A relatively large NUMT on the long arm of chromosome 9 (9L) was identified at approximately the same position in four inbred lines (B73, M825, HP301, and Oh7B). Further examination of the similarly positioned 9L NUMT in two lines, B73 and M825, indicated that the large size of these sites is due to the presence of a majority of the mitochondrial genome; however, only portions of this NUMT (~252 kb total) were found in the publically available B73 nuclear sequence for chromosome 9. Fiber-fluorescence in situ hybridization analysis estimated the size of the B73 9L NUMT to be ~1.8 Mb and revealed that the NUMT is methylated. Two regions of mtDNA (2.4 kb and 3.3 kb) within the 9L NUMT are not present in the B73 mitochondrial NB genome; however, these 2.4-kb and 3.3-kb segments are present in other Zea mitochondrial genomes, including that of Zea mays ssp. parviglumis, a progenitor of domesticated maize., (Copyright © 2015 Lough et al.)