A mutation causing variegation and abnormal development in tobacco is associated with an altered mitochondrial DNA

A variegated mutation appeared in the leaves of a tobacco cybrid plant resulting from fusion of protoplasts from tobacco with Petunia . The mutation was inherited maternally. The light green coloration of leaf sectors resulted from a substitution of spongy parenchyma for palisade parenchyma. No defects were detected in the chloroplasts of the plants, which were derived from Petunia . The mitochondria, as judged by the electrophoretic pattern of their DNA after digestion with restriction endonucleases, were very similar to mitochondria of tobacco, although with some unique cybrid-specific fragments. A second round of fusions was performed to confirm that mitochondria, rather than chloroplasts, were associated with the variegated phenotype. In these fusions, the Petunia chloroplasts of the variegated plants were replaced by tobacco chloroplasts. The mitochondria, according to the DNA restriction pattern, retained all or some of the unique cybrid-specific fragments found in the original variegated tobacco cybrid. Since the variegated phenotype remained after the chloroplast exchange, the chloroplast DNA cannot be the site of the mutation which is responsible for the mutant phenotype. This result eliminates the chloroplast and confirms that the mitochondrial genome is associated with the mutant phenotype.     

Altered mitochondrial gene expression in a maternal distorted leaf mutant of Arabidopsis induced by chloroplast mutator.

Chloroplast mutator (chm) of Arabidopsis is a recessive nuclear mutation that causes green and white variegation in leaves and is inherited in a non-Mendelian fashion. In this study, we have identified and characterized a mutant observed in F1 and backcrossed BC1 populations from a cross between chm1-3 and ecotype Columbia. This mutant, maternal distorted leaf (MDL), grows very poorly and is distinguished by distorted rough leaves and aborted flowering organs. Electron microscopic observation showed that in MDL plants, a significant portion of mitochondria are abnormal and appear to be nonfunctional. DNA gel blot and sequence analysis of the MDL mitochondrial DNA (mtDNA) revealed rearrangements in two mtDNA fragments associated with rps3-rpl16 genes (encoding ribosomal proteins S3 and L16, respectively). One rearrangement resulted in the insertion of the rps3-rpl16 operon downstream of atp9. An independent deletion in this region had eliminated the majority of rps3. In contrast, another rearrangement deleted part of rpl16, whereas rps3 remained intact. RNA gel blot analysis indicated that expression of these genes is also altered as a consequence of the mtDNA rearrangements. Thus, a mutation at the CHM locus affects mitochondrial gene expression, and impaired mitochondrial function may result in the distorted phenotype.     

A second acromelanistic allelomorph at the albino locus of the Mongolian gerbil (Meriones unguiculatus)

A new autosomal recessive coat color mutant in the Mongolian gerbil (Meriones unguiculatus) is described: chinchilla medium (symbol c(chm)). The mutant has typical acromelanistic features similar to those of several acromelanistic c locus mutants of other species of mammals. Previously a more severe form of acromelanism (c(h)c(h)) has been described in the Mongolian gerbil. The new allele shows to be allelic with this form. On a nonagouti background compound heterozygotes (aac(chm)c(h)) show an intermediate phenotype that is very similar to that of the Siamese mouse (Mus musculus) and rat (Rattus norvegicus). Homozygotes (aac(chm)c(chm)) display a very dark acromelanistic phenotype reminiscent of that of the sable rabbit (Oryctolagus cuniculus). The gray phenotype (gg) in the Mongolian gerbil resembles the albino locus phenotype chinchilla (c(ch)c(ch)) in mice. We show that the new mutant is not allelic with gray. Fertility and viability of the new mutant are within normal range.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.     

An Arabidopsis pentatricopeptide repeat protein, SUPPRESSOR OF VARIEGATION7, is required for FtsH-mediated chloroplast biogenesis

The Arabidopsis (Arabidopsis thaliana) yellow variegated2 (var2) mutant has green- and white-sectored leaves due to loss of VAR2, a subunit of the chloroplast FtsH protease/chaperone complex. Suppressor screens are a valuable tool to gain insight into VAR2 function and the mechanism of var2 variegation. Here, we report the molecular characterization of 004-003, a line in which var2 variegation is suppressed. We found that the suppression phenotype in this line is caused by lack of a chloroplast pentatricopeptide repeat (PPR) protein that we named SUPPRESSOR OF VARIEGATION7 (SVR7). PPR proteins contain tandemly repeated PPR motifs that bind specific RNAs, and they are thought to be central regulators of chloroplast and mitochondrial nucleic acid metabolism in plants. The svr7 mutant has defects in chloroplast ribosomal RNA (rRNA) processing that are different from those in other svr mutants, and these defects are correlated with reductions in the accumulation of some chloroplast proteins, directly or indirectly. We also found that whereas var2 displays a leaf variegation phenotype at 22°C, it has a pronounced chlorosis phenotype at 8°C that is correlated with defects in chloroplast rRNA processing and a drastic reduction in chloroplast protein accumulation. Surprisingly, the cold-induced phenotype of var2 cannot be suppressed by svr7. Our results strengthen the previously established linkage between var2 variegation and chloroplast rRNA processing/chloroplast translation, and they also point toward the possibility that VAR2 mediates different activities in chloroplast biogenesis at normal and chilling temperatures.     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels

Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.     

Cucumber: a model angiosperm for mitochondrial transformation?

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.     

Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea

In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea.     

Molecular and ultrastructural analysis of a nonchromosomal variegated mutant. Tomato mitochondrial mutants that cause abnormal leaf development.

Mutants were recovered in a population of cybrids formed following protoplast fusion between tomato (Lycopersicon esculentum Mill.) cv UC82 and Lycopersicon pennellii Corr. The cybrids were identified as individuals with recombinant cytoplasmic genomes but only tomato nuclear genomes. The mutants were identified based on two features, a variegated sectoring of light and dark green regions on their leaves, stems, and fruit, and reduced growth in the field. The mutants produced 50% of the shoot fresh weight and 20% of the fruit fresh weight of the parental type, UC82. The variegated sectoring was maternally inherited. The chloroplast genome in the mutants was indistinguishable from the chloroplast genome in UC82, when distribution of restriction endonuclease sites was used as an assay. The mitochondrial genome in the mutants, however, was recombinant, containing genes from UC82 and L. pennellii. Light microscopic analysis of the leaves of the mutants demonstrated an absence of the palisade layer in the variegated sectors. Electron microscopic analysis of these same regions demonstrated an absence of normal inner membranes in the mitochondria of these cells.     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

Summary The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using ³²P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Summary Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Chloroplast genome evolution in the genus Avena

The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.     

A multi-recombination model for the mtDNA rearrangements seen in maize cmsT regenerated plants

Regeneration of plants from maize cytoplasmic male sterile type T (cmsT) callus tissue culture promotes, in some instances, genetic variability in their mitochondrial genomes. These mutations have been analyzed in various cmsT regenerated plants that have or have not regained the male fertile phenotype. A unique multi-recombination model explains the various mitochondrial genome rearrangements. First, recombination involving two different sets of direct repeats gives rise to subgenomic recombinant circles. Second, intermolecular recombination between some selected subgenomes gives rise to a new rearranged master chromosome. The consequence of these events is the formation of a new master chromosome containing sequence deletions and duplications when compared to the progenitor. This new mitochondrial genome seems stable, although it does not contain the entire genetic complexity of the progenitor.