Inheritance of organelle DNA sequences in a citrus-poncirus intergeneric cross.

Many land plants deviate from the maternal pattern of organelle inheritance. In this study, heterologous mitochondrial and chloroplast probes were used to investigate the inheritance of organelle genomes in the progeny of an intergeneric cross. The seed parent was LB 1-18 (a hybrid of Citrus reticulata Blanco cv. Clementine x C. paradisi Macf. cv. Duncan) and the pollen parent was the cross-compatible species Poncirus trifoliata (L.) Raf. All 26 progeny examined exhibited maternal inheritance of plastid petA and petD loci. However, 17 of the 26 progeny exhibited an apparent biparental inheritance of mitochondrial atpA, cob, coxII, and coxIII restriction fragment length polymorphisms (RFLPs) and maternal inheritance of mitochondrial rrn26 and coxI RFLPs. The remaining nine progeny inherited only maternal mitochondrial DNA (mtDNA) configurations. Investigations of plant mitochondrial genome inheritance are complicated by the multipartite structure of this genome, nuclear gene control over mitochondrial genome organization, and transfer of mitochondrial sequences to the nucleus. In this study, paternal mtDNA configurations were not detected in purified mtDNA of progeny plants, but were present in progeny DNA preparations enriched for nuclear genome sequences. MtDNA sequences in the nuclear genome therefore produced an inheritance pattern that mimics biparental inheritance of mtDNA.

The tobacco apocytochrome b gene predicts sensitivity to the respiratory inhibitors antimycin A and myxothiazol.

The potential for use of the cytochrome-pathway electron-transfer inhibitors antimycin A and myxothiazol in the selection of plant mitochondrial genome transformants was investigated. The net growth of Nicotiana tabacum L. (tobacco) suspension-culture cells was reduced by these inhibitors, but complete repression of cell growth occurred only in the presence of both cytochrome and alternative electron-transfer-pathway inhibitors. Antimycin A and myxothiazol bind to and block electron transfer through different sites in the cytochrome b (COB) subunit of the mitochondrial bc1 respiratory complex (complex III). The nucleotide sequence of the tobacco cob gene was determined and found to predict highly conserved glycine and phenylalanine residues that are associated with sensitivity to antimycin A and myxothiazol, respectively. These residues are altered by mutations that confer resistance to antimycin A or myxothiazol in diverse organisms. Tobacco cob cDNA clones were constructed and sequenced, revealing eight full and 11 partial RNA-editing sites. RNA editing did not, however, alter codons for the conserved glycine and phenylalanine residues associated with sensitivity to the respiratory inhibitors. Antimycin A or myxothiazol, in conjunction with a modified cob gene, may therefore be useful in the selection of tobacco cells carrying a genetically transformed mitochondrial genome.

Pleiotropic effects of a nuclear restorer-of-fertility locus on mitochondrial transcripts in male-fertile and S male-sterile maize.

Cytoplasmic male sterility (CMS) is encoded by the plant mitochondrial genome and can be reversed by nuclear restorer-of-fertility(Rf) alleles. In the CMS-S system of maize, reproductive failure and fertility restoration are gametophytic, occurring during the starch-filling stages of pollen development. Transcripts of the CMS-S-associated mitochondrial open reading frames (orf355 and orf77) are present from the early stages of microspore development through the aborted pollen stage. To investigate the molecular basis of fertility restoration, we compared mitochondrial-transcript accumulation in aborting CMS-S pollen and in CMS-S pollen restored to fertility by the Rf3 nuclear allele. In the presence of the Rf3 allele, novel, shorter transcripts of the orf355-orf77, cob and atp6 mitochondrial genes were created, and the relative abundance of larger transcripts was decreased for each of these loci. The altered transcript patterns cosegregated with male fertility conditioned by the Rf3 allele. The novel cob and atp6 transcripts were also observed in leaf-tissues of both normal and S-cytoplasm plants carrying the Rf3 allele. These observations support the hypothesis that the Rf3 allele encodes, or regulates, a modifier of mitochondrial transcript (Mmt) activity that affects both CMS and essential mitochondrial gene transcripts.

Gametophyte genetics in Zea mays L.: dominance of a restoration-of-fertility allele (Rf3) in diploid pollen.

In Zea mays L. plants carrying the S-type of sterility-inducing cytoplasm, male fertility is determined by a gametophytic, nuclear restoration-of-fertility gene. Haploid pollen carrying the fertility-restoring allele (historically designated Rf3) is starch-filled and functional, whereas pollen carrying the nonrestoring allele (historically designated rf3) is shrunken and nonfunctional. Because restoration of fertility occurs in haploid tissue, the dominance relationship of restoring and nonrestoring alleles is unknown. We have tested the dominance relationship of the restoring and nonrestoring alleles at the rf3 locus in diploid pollen. The meiotic mutant elongate was used to generate tetraploid plants carrying both Rf3 and rf3 alleles in the S cytoplasm. These plants shed predominantly starch-filled pollen, consistent with dominance of the restoring allele. Restriction fragment length polymorphisms linked to the rf3 locus demonstrated cotransmission of rf3 and Rf3 alleles through heterozygous diploid pollen, providing conclusive genetic evidence that the restoring allele is the dominant or functional form of this restoration-of-fertility gene. We suggest that other S-cytoplasm restorers result from loss-of-function mutations and propose analysis of unreduced gametes as a test of this model.

Purification of a Zn-binding phloem protein with sequence identity to chitin-binding proteins.

In citrus blight, a decline disorder of unknown etiology, the tree canopy exhibits symptoms of Zn deficiency while Zn accumulates in the trunk phloem. We have purified a Zn-binding protein (ZBP) from phloem tissue of healthy and blight-affected citrus (Citrus sinensis [L.] Osbeck on Citrus jambhiri [L.]). The molecular weight of the ZBP was estimated to be 5000 by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion-exchange chromatography at pH 8.0 demonstrated the 5-kD ZBP to be anionic. A partial N-terminal amino acid sequence revealed a cysteine-, glycine-rich domain with 45 to 80% identity with the chitin-binding domain of hevein, wheat germ agglutinin, and several class I chitinases. That the abundance of this protein increased 2.5-fold in association with Zn accumulation in the phloem is characteristic of citrus blight. Tissue mass changes of the phloem suggests that altered tissue structure accompanies blight. Phloem accumulation of the 5-kD ZBP may be in response to wounding or other stress of blight-affected citrus.

Expression of CMS-unique and flanking mitochondrial DNA sequences in Phaseolus vulgaris L.

The expression of mitochondrial DNA sequences unique to a cytoplasmically male-sterile (CMS) line of Phaseolus vulgaris was investigated. RNA-blot hybridizations with strand-specific probes demonstrated CMS-unique transcripts (7.0, 6.8, 4.7, 3.3 and 2.8 kb) to be in the sense orientation with respect to the longest open reading frames within the CMS-unique region. Hybridizations revealed co-transcription of CMS-unique and upstream, atpA-coding sequences to generate the 6.8-kb RNA. However, hybridizations with CMS-unique and flanking DNA probes accounted for only 4.9 kb of the longest and most abundant (7.0 kb) CMS-unique transcript, providing indirect evidence for the involvement of a splicing process in the generation of this transcript. Sedimentation experiments demonstrated the association of 7.0- and 6.8-kb CMS-unique transcripts with polyribosomes in seedlings and floral buds of a CMS line and a line restored to fertility by the nuclear gene Fr2. However, steady-state levels of the 7.0- and 6.8-kb transcripts were decreased in the restored line relative to the CMS line.

Sorghum mitochondrial atp6: divergent amino extensions to a conserved core polypeptide.

Sorghum mitochondrial atp6 occurs as one copy in the line Tx398 and as two copies in IS1112C. In IS1112C a repeated sequence diverged within the atp6 open reading frames. The two open reading frames (1137 bp, atp6-1; 1002 bp, atp6-2) share an identical conserved region of 756 bp but are flanked 5' by divergent extensions of 246 (atp6-1) or 381 bp (atp6-2). Tx398 carried only atp6-2. The breakpoint of the repeated sequence of the conserved core region corresponds to the amino acid sequence Ser-Pro-Leu-Asp, which is the amino terminus of the proteolytically processed yeast ATP6. The 5' extensions of atp6-1 and atp6-2 were similar to those of rice and maize, respectively. Each open reading is transcribed, however nuclear background influenced transcriptional patterns of atp6-2 in IS1112C.

Organization of ATPA coding and 3' flanking sequences associated with cytoplasmic male sterility in Phaseolus vulgaris L.

A region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atpA) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3' ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3' coding and 3' flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5' end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.

A molecular marker-based linkage map of Phaseolus vulgaris L.

A seed and flower color marker (P), nine seed protein, nine isozyme and 224 restriction fragment length polymorphism marker loci were used to construct a linkage map of the common bean, Phaseolus vulgaris L. (n = 11). The mapping population consisted of a backcross progeny between the Mesoamerican breeding line 'XR-235-1-1' and the Andean cultivar 'Calima'; the former was used as the recurrent parent. A bean PstI genomic library enriched for single copy sequences (95%) was the source of DNA probes. Sixty percent of the probes tested detected polymorphisms between the parental genotypes with at least one of the four restriction enzymes used here (DraI, EcoRI, EcoRV and HindIII). The computer software Mapmaker was used to determine the linkage relationships and linear order of segregating markers. These markers assorted into 11 linkage groups covering 960 cM of the bean genome. Partial linkage data were used to estimate the total length of the genome at 1200 cM. This estimate and that for the physical size of the genome yield an average ratio of 530 kb/cM. The relatively small size of the genome makes this crop species a good candidate for the isolation of genes via chromosome walking techniques.

Extended map for the phaseolin linkage group ofPhaseolus vulgaris L.

The linkage relationship of 11 bean (Phaseolus vulgaris) seed proteins (including phaseolin), 9 enzyme loci, and theP locus were analyzed in backcross and F2 progenies by use of the software package "Mapmaker." The progenies were obtained by crossing the breeding line 'XR-235-1' and the cultivar 'Calima'. Allelic differences for seed protein loci were detected with SDS-PAGE and those for enzyme loci with starch gel electrophoresis and activity stains. The seed coat color of 'Calima' is a red/beige mottled pattern and that of 'XR-235-1' is white. Segregation at theP locus was followed by recording the phenotype of the BC1S1 and F3 seed. A linkage group comprising ca. 90 cM was detected with the following gene order:Est-2 - 11 -Pha - 8 - (Spe/Spg) - 24 - P - 9 - (Spa/Spv) - 16 -Spba - 22 -Mdh-1. In addition, another linkage group was detected: (Spd/Spf/Sph) - 5 -Spca. Therefore, the seed proteins appear to be organized in clusters in the bean genome.

Linkage between isozyme markers and a locus affecting seed size in Phaseolus vulgaris L.

Backcross and F2 progenies were produced between two bean genotypes, 'XR-235' and 'Calima,' which differ in seed weight by a factor of two. The small-seeded 'XR-235' was used as the pistillate and recurrent parent. These genotypes showed polymorphisms at nine isozyme loci and at the phaseolin locus. Seed size parameters (weight, length, width, and thickness) were determined for each BC1 and F2 individual, i.e., for seeds harvested from 'XR-235' after pollination with F1 and from the F1 after selfing, respectively. A combination of starch gel electrophoresis and enzyme activity staining was used to determine the genotype of each BC1 and F2 individual at the segregating loci. SDS-PAGE and Coomassie blue staining were used to determine geno-type at the phaseolin locus. Tests for independent assortment using two-way contingency and maximum likelihood tables revealed three linkage pairs: Aco-1 - 20 cM - Dia-1; Adh-1 - 2 cM - Got-2; and Est-2 - 11 cM - Pha. Statistical comparisons were made between the means of genotype classes at each segregating locus for all seed size parameters. The results from two independently obtained BC1s and the F2 consistently indicated that the Adh-1-Got-2 segment was linked to a locus that affected seed size and overcame maternal control over seed size. This locus has been designated Ssz-1. This gene exhibited additive gene action and accounted for 30-50% of the seed size difference between the parents.

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.

Fertility Restoration Is Associated with Loss of a Portion of the Mitochondrial Genome in Cytoplasmic Male-Sterile Common Bean.

Restoration of pollen fertility to cytoplasmic male-sterile common bean by nuclear gene Fr is accompanied by mitochondrial (mt) DNA rearrangements within restored plants. These rearrangements are also observed upon spontaneous cytoplasmic reversion to fertility. An mtDNA fragment of at least 25 kilobases was lost from the genome upon restoration or reversion. This fragment contained DNA segments that were not repeated elsewhere in the genome and, therefore, were not detected within the genome upon fertility restoration. This result suggested that the particular mtDNA configuration absent from restored plants could not be maintained by a constant process of recombination but rather by autonomous replication. No evidence of excision of this region from the mt genome, in the form of a junction fragment associating flanking DNA regions, was detected in fertile restored plants. DNA gel blot hybridization of this mtDNA region, compared with hybridization to related regions of the mitochondrial genome that shared sequence homology, indicated that the mtDNA region associated with sterility was present in lower copy number. These observations, as well as the occurrence of similar or identical rearrangements upon spontaneous cytoplasmic reversion, indicate that the restoration of pollen fertility may be accompanied by loss of an independently replicating subgenomic DNA molecule from the mitochondrial genome.

Mitochondrial DNA rearrangement associated with fertility restoration and cytoplasmic reversion to fertility in cytoplasmic male sterile Phaseolus vulgaris L.

Restoration of pollen fertility to cytoplasmic male sterile (CMS) Phaseolus vulgaris by a nuclear restorer gene provides a system for studying nuclear-cytoplasmic interactions. Introduction of a nuclear restorer gene to this CMS line of P. vulgaris (CMS-Sprite) results in a mitochondrial genome rearrangement similar to that observed upon spontaneous cytoplasmic reversion to fertility. Three spontaneous heritable cytoplasmic revertants were derived from CMS-Sprite. Five fully fertile restored lines were also produced by using restorer line R-351 (BC(3)F(3) populations). Comparison of the mitochondrial DNA restriction patterns of CMS-Sprite, the three fertile revertants, and the five restored lines revealed loss of a 6.0-kilobase (kb) Pst I fragment in all restored and revertant lines. Southern hybridizations with a 1.3-kb BamHI clone, internal to the 6.0-kb Pst I fragment, as a probe revealed two configurations of 6.0-kb homologous sequences in the sterile cytoplasm; one of the configurations was lost upon reversion or restoration. Mitochondrial DNA rearrangement has thus been observed upon restoration by a nuclear restorer gene in this CMS system.

Properties of the linear N1 and N2 plasmid-like DNAs from mitochondria of cytoplasmic male-sterile Sorghum bicolor.

The linear N1 and N2 plasmid-like DNAs were recovered from mitochondria of the IS1112C line of cytoplasmic male-sterile (CMS) Sorghum bicolor (S. bicolor). Molecular clones containing internal sequences of these plasmids were constructed. These clones were used to probe Southern blots of mitochondrial genomes from six CMS and five male-fertile (MF) lines of S. bicolor, as well as Southern blots of IS1112C chloroplast, IS1112C nuclear and kafir nuclear genomes. We found no evidence for integrated copies of N1 or N2 in any of the mitochondrial, chloroplast or nuclear genomes probed in this study. Our clones did detect an N1-homologous transcript of 3.1 kb and N2-homologous transcripts of 3.9 and 1.4 kb in IS1112C mitochondrial RNA prepared from lines with and without nuclear, fertility-restoring genes.N1 and N2 DNAs were degraded by exonuclease III but were resistant to lambda exonuclease, presumably due to the presence of 5' terminal proteins. We detected multimeric forms of N1 and N2 in Southern blots of unrestricted, IS1112C mitochondrial DNA (mtDNA). These forms apparently also had associated protein molecules.

Circular plasmid DNAs from mitochondria of Sorghum bicolor.

Agarose gel electrophoresis of mitochondrial DNA (mtDNA) from the IS1112C male-sterile cytoplasm of Sorghum bicolor (S. bicolor) revealed plasmid-like DNAs additional to the linear N1 and N2 molecules. Mitochondrial plasmids were separated from the principal mitochondrial genome and used in the construction of molecular clones. Clones with EcoRI inserts of 1.7 and 2.3 kb were recovered. Hybridization of these clones to Southern blots of unrestricted and EcoRI-digested IS1112C mitochondrial plasmids indicated the cloned inserts were complete or nearly-complete copies of minicircular DNA molecules. These clones were used to probe Southern blots of mitochondrial genomes from six cytoplasmic male-sterile (CMS) and five male-fertile (MF) lines of S. bicolor, as well as Southern blots of IS1112C chloroplast, kafir chloroplast, IS1112C nuclear, and kafir nuclear genomes. The 2.3 and 1.7 kb plasmids had a very limited distribution among the sorghum entries we examined. We found no evidence for integrated copies of these sequences in any of the mitochondrial, chloroplast, or nuclear genomes probed in this study. However, the 2.3 kb sorghum minicircle did hybridize to the 1.94 kb minicircle from maize mitochondria. Hybridization of the 1.7 and 2.3 kb clones to IS111L2C mitochondrial RNA reveal a transcript of 1.1 kb from the 1.7 kb minicircle and transcripts of 1.9 and 1.4 kb from the 2.3 kb molecule.

Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex.

We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome.