Nuclear and cytoplasmic genes controlling synthesis of variant mitochondrial polypeptides in male-sterile maize

The polypeptides synthesized in vitro by mitochondria isolated from etiolated maize shoots of a number of different nuclear and cytoplasmic genotypes have been compared by using polyacrylamide gel electrophoresis. We have previously shown that mitochondria from maize plants carrying the T or C forms of cytoplasmically inherited male sterility (cms-T and cms-C mitochondria) can be distinguished from each other and from the mitochondria of normal (N) plants by the synthesis of a single additional or variant polypeptide species. Using lines that carry the T cytoplasm, and that differ principally in the presence or absence of nuclear “restorer” alleles that suppress the male-sterile phenotype, we find that these nuclear genes specifically suppress synthesis of the 13,000 M_r variant polypeptide. A 21,000 M_r polypeptide that is synthesized by N mitochondria is not detectable among the translation products of cms-T mitochondria from either restored or nonrestored lines. Results obtained with a number of lines possessing dominant restorer alleles from different sources indicate that it is the restorer gene at the Rf₁ locus that is primarily responsible for regulating synthesis of the 13,000 M_r polypeptide. Mitochondria from lines with the S form of cytoplasmic male sterility (cms-S) were found to synthesize a group of minor polypeptides, ranging in molecular weight from 42,000 to 88,000, which were not detected in N, cms-T, or cms-C mitochondria. In the case of the S and C forms of male sterility no differences were found between the translation products of mitochondria from restored and nonrestored lines.     

Unique plasmid-like mitochondrial DNAs from indigenous maize races of Latin America

Mitochondrial DNA from 81 races of Latin American maize were examined by agarose gel electrophoresis. Twelve South American races each contained two plasmid-like mtDNA molecules similar to those of the cytoplasmic male-sterile S type (cms-S). The plasmid-like elements from all 12 races, designated RU, appear to be identical. Both molecules appear in vitro as double-stranded linear DNAs terminated by repeated sequences arranged in reverse polarity (terminal inverted repeats). The larger molecule of the pair, R-1, contains about 7460 nucleotides. It shares considerable homology with the larger plasmid-like molecule of cms-S, S-1, but is about 1000 nucleotides longer than S-1, has a unique sequence of about 2576 nucleotides, and also contains a BamHI recognition site not present in S-1, R-2, the smaller plasmid-like element, consists of about 5450 nucleotides and appears to share complete homology with S-2, the smaller plasmid-like molecule of cms-S. Neither pollen sterility nor any other trait has been associated with the R-1 and R-2 plasmid-like mtDNAs. The BamHI restriction fragments of total mtDNA from the 12 RU cytoplasms display similar patterns, which differ only slightly but vividly from that of a normal maize standard, B73 × Mo17. BamHI restriction analysis of 22 additional races produced arrays similar to those of the RU cytoplasms, but which lacked plasmid-like mtDNAs. The taxonomic significance of this digestion pattern and of the RU cytoplasms is discussed. One Mexican race, Conico Norteño, has been shown to contain the cms-S cytoplasm.     

Maize nuclear background regulates the synthesis of a 22-kDa polypeptide in Zea luxurians mitochondria

When cytoplasm of the teosinte Zea luxurians is introduced into certain maize inbred nuclear backgrounds, the pattern of protein synthesis in the teosinte mitochondria is altered. Teosinte mitochondria purified from plants possessing a maize A619 nuclear background (Z.l.—A619 plants) synthesize a novel 22-kDa polypeptide that is associated exclusively with the membrane fraction of the organelle. Mitochondria from plants possessing a W23 nuclear background do not synthesize this protein. The F₁ hybrids Z.l.—A619 × W23 and Z.l.—W23 × A619 do not synthesize the protein. However, synthesis of the polypeptide was observed in 14 of 21 individual progeny from the backcross of the F₁ hybrid Z.l.—A619 × W23 to the pollen parent A619. These data suggest that a single nuclear gene controls the synthesis of the 22-kDa protein in mitochondria, with the recessive allele of the gene allowing expression of the polypeptide. Mitochondria from the F₁ hybrid Z.l.—A619 × Mo17 synthesize the 22-kDa protein, whereas mitochondria from Z.l.—A619 × B73 do not. Data from these outcrosses demonstrate that other maize lines also possess nuclear genes capable of regulating the synthesis of the 22-kDa Zea luxurians mitochondrial protein.     

Inheritance of selected pathotoxin resistance in maize plants regenerated from cell cultures

Texas male-sterile cytoplasm (cms-T) maize is susceptible to Helminthosporium maydis race T and its pathotoxin, whereas nonsterile cytoplasm maize is resistant. Callus cultures initiated from immature embryos of a cms-T genotype, BC₁A188(T), were susceptible to the toxin and were capable of plant regeneration. Toxin-resistant cell lines were selected by a sublethal enrichment procedure in which cms-T callus was grown for several selection cycles (subculture transfers) in the presence of progressively higher concentrations of toxin. Periodically during the selection process, plants were regenerated from the cms-T cultures to determine their susceptibility or resistance to the toxin. Plants regenerated after four cycles of selection were male-sterile and toxin-susceptible as shown by leaf bioassays. All plants regenerated from cell lines isolated from the fifth selection cycle onward, however, were toxin-resistant and 52 of 65 were fully male-fertile. The remaining 13 “male-sterile” resistant plants did not shed pollen and did not resemble cms-T plants in tassel morphology. Some “male-sterile” plants produced anthers containing a small amount of starch-filled pollen, suggesting that the sterility of these 13 plants was not the result of the cms-T trait. Leaf bioassays on F₁ progeny from regenerated resistant plants indicated that resistance to the toxin was inherited only through the female. The male-fertility trait also was inherited only through the female. After inoculation with H. maydis race T spores, leaf lesion size for progeny from regenerated resistant plants coincided with their reaction to the toxin. This result indicated that plant resistance to the pathogen was closely correlated with the toxin resistance obtained through cell culture selection.     

Cytoplasmic male sterility in barley: Evidence for the involvement of cytokinins in fertility restoration

The hypothesis of the association between an increase in cytokinin activity and restoration of anther fertility in msm1 cytoplasm was tested. The following barley lines with Hordeum vulgare cv. Adorra nuclear gene background were studied: Adorra cytoplasm without nuclear restorer gene (fertile), Adorra cytoplasm homozygous for nuclear Rfm1a gene (fertile), msm1 cytoplasm without restorer gene (male sterile), msm1 cytoplasm homozygous for nuclear Rfm1a gene (fertile). Ethanolic extracts of root exudate were fractionated and bioassayed for cytokinins. Both the biological activity and the total quantity of cytokinins appeared lowest in the unrestored male sterile line. The total biological activities of cytokinins in the three fertile lines were similar, but the quantities in the restorer gene carriers appeared lower. On the other hand, the restorer gene carriers, independent of the cytoplasm, showed 8-9 times more of a bound cytokinin. Because the bound form is evidently underestimated by the bioassay, the increase in the bound cytokinin fraction may mean even a higher total content in the Rfm1a gene carriers than in Adorra without the gene. The bound cytokinin may be translocated more readily to distal organs (e.g., the anthers) compared with unbound cytokinins. Because cytokinins are associated with various ecophysiological processes, the rise in a particular form may explain the heterogeneous distribution of the restorer gene in wild barley populations in different regions of Israel.     

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba

The cytoplasmic male sterility (CMS) trait of at least one line of Vicia faba plants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the “447” male sterile line of V.faba plants and could not be isolated from the recurrent parent (maintainer), from lines that had been fertility-restored, or from lines that had reverted from the sterile condition. We have been able to move the CMS-associated RNA from donor to fertile host plants through a dodder bridge. These hots not only contain the RNA but now exhibit a male sterile phenotype, as detected by visual examination of the flower, the pollen, morphological characteristics, and pollen staining ability.     

Purification of cowpea mosaic virus RNA replication complex: Identification of a virus-encoded 110,000-dalton polypeptide responsible for RNA chain elongation

An endogenous cowpea mosaic virus (CPMV) RNA-protein complex (CPMV replication complex) capable of elongating in vitro preexisting nascent chains to full-length viral RNAs has been solubilized from the membrane fraction of CPMV-infected cowpea leaves using Triton X-100 and purified by Sepharose 2B chromatography and glycerol gradient centrifugation in the presence of Triton X-100. Analysis of the polypeptide composition of the complex by NaDod-SO₄/PAGE and silver staining revealed major polypeptides with molecular masses of 110, 68, and 57 kilodaltons (kDa), among which the 110-kDa polypeptide was consistently found to cosediment precisely with the RNA polymerase activity. Using antisera to specific viral proteins, we found the 110-kDa polypeptide to be the only known viral polypeptide associated with the RNA replication complex, the 68- and 57-kDa polypeptides being most probably host-specific. The host-encoded 130-kDa monomeric RNA-dependent RNA polymerase, which is known to be stimulated in CPMV-infected cowpea leaves, did not copurify with the virus-specific RNA polymerase complex. Our results dispute the hypothesis that plant viral RNA replication may be mediated by the RNA-dependent RNA polymerase of uninfected plants. We tentatively conclude that the 110-kDa polypeptide encoded by the bottom component RNA of CPMV constitutes the core of the CPMV RNA replication complex.     

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 (BC₃F₃ 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.     

cDNA cloning and induction of the alcohol dehydrogenase gene (Adh1) of maize

cDNA clones of Adh1, one of two genes encoding alcohol dehydrogenase (ADH; alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1) in the maize genome, have been isolated. They were derived from mRNA extracted from anaerobically treated roots of maize seedlings. Identification was initially made on the basis of molecular weight and electrophoretic properties of the in vitro polypeptide obtained in hybridization-release-translation experiments. The identification was confirmed by antibody precipitation and by the use of maize stocks having different genetic constitutions at the Adh1 locus. The sequence of the longest cDNA segment, ≈900 base pairs, was determined and appears to code for 168 COOH-terminal amino acids and to have a 3′ nontranslated region of 364 base pairs. Reverse Southern hybridizations established that two different Adh1-S stocks produce a mRNA of 1,650 nucleotides, whereas an additional mRNA of 1,750 nucleotides is produced in three Adh1-F stocks. A 50-fold increase in Adh1 mRNA level occurs during anaerobiosis, reaching a maximum at 5 hr. Return to aerobic conditions indicates a half-life of more than 18 hr for the anaerobically induced Adh1 mRNA.     

Nucleotide sequence of the S-2 mitochondrial DNA from the S cytoplasm of maize

Mitochondria from the S male-sterile cytoplasm (cms-S) of maize contain two plasmid-like DNAs, S-1 and S-2, that appear to be prominently involved with the cytoplasmic male sterility trait. The complete nucleotide sequence of the S-2 DNA molecule was determined by the chain termination method. The linear S-2 DNA molecule contains 5,452 base pairs and is terminated by exact 208-base-pair inverted repetitions. Two large open reading frames were identified in the S-2 DNA, suggesting the possibility of protein-encoding genes. The nucleotide sequence of the S-2 termini are discussed with regard to models proposed for the replication of linear DNA molecules.     

Targeting the maize T-urf13 product into tobacco mitochondria confers methomyl sensitivity to mitochondrial respiration.

The URF13 protein, which is encoded by the maize mitochondrial T-urf13 gene, is thought to be responsible for pathotoxin and methomyl sensitivity and male sterility. We have investigated whether T-urf13 confers toxin sensitivity and male sterility when expressed in another plant species. The coding sequence of T-urf13 was fused to a mitochondrial targeting presequence, placed under the control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Plants expressing high levels of URF13 were methomyl sensitive. Subcellular analysis indicated that URF13 is mainly associated with the mitochondria. Adding methomyl to isolated mitochondria stimulated NADH-linked respiration and uncoupled oxidative phosphorylation, indicating that URF13 was imported into the mitochondria, and conferred toxin sensitivity. Most control plants, which expressed the T-urf13c construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction. Subcellular fractionation by sucrose gradient centrifugation showed that URF13 sedimented at several positions, suggesting the protein is associated with various organelles, including mitochondria. No methomyl effect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mitochondria. Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria. There was no correlation between the expression of URF13 and male sterility, suggesting either that URF13 does not cause male sterility in transgenic tobacco or that URF13 is not expressed in sufficient amounts in the appropriate anther cells.     

Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors

In response to wounding, a 48-kDa myelin basic protein (MBP) kinase is activated within 2 min, both locally and systemically, in leaves of young tomato plants. The activating signal is able to pass through a steam girdle on the stem, indicating that it moves through the xylem and does not require intact phloem tissue. A 48-kDa MBP kinase is also activated by the 18-amino acid polypeptide systemin, a potent wound signal for the synthesis of systemic wound response proteins (swrps). The kinase activation by systemin is strongly inhibited by a systemin analog having a Thr-17 → Ala-17 substitution, which is a powerful antagonist of systemin activation of swrp genes. A 48-kDa MBP kinase activity also increases in response to polygalacturonic acid and chitosan but not in response to jasmonic acid or phytodienoic acid. In def1, a mutant tomato line having a defective octadecanoid pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP kinase functions between the perception of primary signals and the DEF1 gene product. In response to wounding, the MBP kinase is phosphorylated on phosphotyrosine residues, indicating a relationship to the mitogen-activated protein kinase family of protein kinases.     

Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.     

Variation in mitochondrial translation products associated with male-sterile cytoplasms in maize

Mitochondria were isolated under sterile conditions from maize lines with normal cytoplasm and with T, C, and S sources of cytoplasmic male sterility. Protein synthesis by these mitochondria was dependent upon the presence of ADP and an oxidizable substrate or an energy generating system and displayed characteristic sensitivity towards inhibitors of protein synthesis. Approximately 20 polypeptides were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the [³⁵S]methionine-labeled mitochondrial translation products. Mitochondria from the T and C cytoplasms were characterized by the synthesis of a single additional or variant polypeptide.     

Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358.

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNA X DNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 determinant. The 2153 base pairs of the promoter-distal part of the broad-spectrum Hg2+-resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C--Hg bond of phenylmercury.     

Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase consists of large subunits (LS) and small subunits. In plants, the LS is encoded in chloroplast DNA and the small subunit, in nuclear DNA. In cyanobacteria, both subunits are thought to be encoded in chromosomal DNA because of prokaryotes. The gene for the LS of ribulose-1,5-bisphosphate carboxylase/oxygenase from a cyanobacterium, Anacystis nidulans 6301, has been cloned in pBR322 and subjected to sequence analysis. The coding region contains 1,416 base pairs (472 codons). The deduced amino acid sequence of A. nidulans LS protein shows 80% homology with sequences of maize, spinach, and tobacco LS proteins; the nucleotide sequence of A. nidulans LS gene shows 70% homology with sequences of the plant genes. Between A. nidulans LS and the plant LS proteins there is exact sequence homology around the lysine residue to which the activator CO₂ binds and around the two lysine residues to which ribulose 1,5-bisphosphate binds. The amino acid sequence where the LS binds to the small subunit is also highly conserved. From comparison of the LS proteins of A. nidulans and the three plants, the rate of amino acid substitution is estimated to be 0.25-0.5 × 10^-9 per year per site, which is far below the median value of various types of proteins (1.2 × 10^-9 for hemoglobin α). The LS protein is thus a conserved protein.     

Maize mitochondria synthesize organ-specific polypeptides.

We detected both quantitative and qualitative organ-specific differences in the total protein composition of mitochondria of maize. Labeling of isolated mitochondria from each organ demonstrated that a few protein differences are due to changes in the polypeptides synthesized by the organelle. The synthesis of developmental stage-specific mitochondrial polypeptides was found in the scutella of developing and germinating kernels. The approximately 13-kDa polypeptide synthesized by mitochondria from seedlings of the Texas (T) male-sterile cytoplasm was shown to be constitutively expressed in all organs of line B37T tested. Methomyl, an insecticide known to inhibit the growth of T sterile plants, was shown to be an effective inhibitor of protein synthesis in mitochondria from T plants.