Publications by authors named "Toru Terachi"

21 Publications

  • Page 1 of 1

Intraspecific variations of the cytoplasmic male sterility genes and in Brassica maurorum and Moricandia arvensis, and the specificity of the mRNA processing.

Genome 2021 Jun 15. Epub 2021 Jun 15.

Dept. Biotech., Fac. Eng., Kyoto Sangyo Univ., Motoyama, Kamigamo, Kyoto, Kyoto, Japan, 603-8555;

The mitochondrial gene co-transcribed with and causes cytoplasmic male sterility in crops, is widely distributed across wild species and genera of . However, intraspecific variations in the presence of have not yet been studied, and the mechanisms for the wide distribution of the gene remain unclear. We analyzed the presence and sequence variations of in two wild species, and . After polymerase chain reaction amplification of the 5' region of and the coding sequence of , we determined the DNA sequences. and showed variations for the presence of or () both between and within accessions, and were not fixed to the mitochondrial type having the male sterile genes. Sequencing of the amplicons clarified that has instead of . Sequencing also indicated mitochondrial heteroplasmy in the two species; particularly, in , one plant possessed both the and sequences. The results suggested that substoichiometric shifting of the mitochondrial genomes leads to the acquisition or loss of . Furthermore, fertility restorer genes of the two species were involved in the processing of the mRNA of the male sterility genes at different sites.
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http://dx.doi.org/10.1139/gen-2021-0011DOI Listing
June 2021

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish.

Mol Genet Genomics 2021 May 26;296(3):705-717. Epub 2021 Mar 26.

Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo, Kita, Kyoto, 603-8555, Japan.

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.
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http://dx.doi.org/10.1007/s00438-021-01777-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144145PMC
May 2021

Appearance of male sterile and black radishes in the progeny of cross between and .

Breed Sci 2020 Dec 5;70(5):637-641. Epub 2020 Nov 5.

Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo, Kita, Kyoto 603-8555, Japan.

In addition to Ogura cytoplasmic male sterility (CMS), which is used extensively for F hybrid seed production in Brassicaceae crops, two other CMS systems, NWB CMS and DCGMS, have also been identified. The causal gene for the latter two CMS systems has been identified as a novel chimeric gene, . We previously reported that is specific to black radish cultivars and that it is present in line 'RS-5' of ; however, the sequence in 'RS-5' differed from that of black radish cultivars. Though, with an sequence identical to that found in black radish cultivars was recently identified. We therefore sought to determine whether the gene in line 'RS-5' induces CMS in radishes. We crossed 'RS-5' as a female parent with a cultivated radish, 'Uchiki-Gensuke', as a male parent, and examined the gross plant morphology and pollen fertility of the resulting progeny. The F population contained both male sterile plants and plants with black roots. The findings showed that contains two types of genes that induce CMS, and that the origin of black radishes could be attributed to having gene.
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http://dx.doi.org/10.1270/jsbbs.20081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878931PMC
December 2020

Multichromosomal structure of the onion mitochondrial genome and a transcript analysis.

Mitochondrion 2019 05 10;46:179-186. Epub 2018 Jul 10.

Plant Organelle Genome Research Center, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan; Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan. Electronic address:

The structures of plant mitochondrial genomes are more complex than those of animals. One of the reasons for this is that plant mitochondrial genomes typically have many long and short repeated sequences and intra- and intermolecular recombination may create various DNA molecules in this organelle. Recombination may sometimes create a novel gene that causes cytoplasmic male sterility (CMS). The onion has several cytoplasm types, with some causing CMS while others do not. The complete mitochondrial genome sequence of the onion was reported for an inbred line with CMS-S cytoplasm; however, the number of differences between onion strains remains unclear, and studies on purified mitochondrial DNA (mtDNA) have not yet been performed. Furthermore, analyses of transcripts in the mitochondrial genome have not been conducted. In the present study, we examined the mitochondrial genome of the onion variety "Momiji-3" (Allium cepa L.) possessing CMS-S-type cytoplasm using next-generation sequencing (NGS). The "Momiji-3" mitochondrial genome mainly exists as three circles as a result of recombination through repeated sequences and we herein succeeded for the first time in visualizing its structure using pulsed field gel electrophoresis (PFGE). The ability to clarify the structure of the mitochondrial genome is rare in plant mitochondria; therefore, "Momiji-3" represents a good example for elucidating complex plant mitochondrial genomes. We also mapped transcript data to the mitochondrial genome in order to identify the RNA-editing positions in all gene-coding regions and estimate the expression levels of genes. We identified 635 editing positions in gene-coding regions. Start and stop codons were created by RNA editing in six genes (nad1, nad4L, atp6, atp9, ccmFC, and orf725). The transcript amounts of novel open reading frames (ORFs) were all markedly lower than those of functional genes. These results suggest that a new functional gene was not present in the mitochondrial genome of "Momiji-3", and that the candidate gene for CMS is orf725, as previously reported.
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http://dx.doi.org/10.1016/j.mito.2018.05.001DOI Listing
May 2019

Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination.

BMC Biol 2017 09 19;15(1):86. Epub 2017 Sep 19.

Japan International Research Center for Agricultural Sciences, Tsukuba, Japan.

Background: Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected "orphan" crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop.

Results: To accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage.

Conclusions: Guinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.
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http://dx.doi.org/10.1186/s12915-017-0419-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604175PMC
September 2017

Mitochondrial genome sequences from wild and cultivated barley (Hordeum vulgare).

BMC Genomics 2016 10 24;17(1):824. Epub 2016 Oct 24.

Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama, 710-0046, Japan.

Background: Sequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. Barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of 5.1 Gbp. Wild barley (Hordeum vulgare ssp. spontaneum) and cultivated barley (H. vulgare ssp. vulgare) have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes.

Results: We determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley. Two independent circular maps of the 525,599 bp barley mitochondrial genome were constructed by de novo assembly of high-throughput sequencing reads of barley lines H602 and Haruna Nijo, with only three SNPs detected between haplotypes. These mitochondrial genomes contained 33 protein-coding genes, three ribosomal RNAs, 16 transfer RNAs, 188 new ORFs, six major repeat sequences and several types of transposable elements. Of the barley mitochondrial genome-encoded proteins, NAD6, NAD9 and RPS4 had unique structures among grass species.

Conclusions: The mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was highly differentiated from that of other grass species. Mitochondrial genome sequencing is essential for annotating the barley nuclear genome; our mitochondrial sequencing identified a significant number of fragmented mitochondrial sequences in the reported nuclear genome sequences. Little polymorphism was detected in the barley mitochondrial genome sequences, which should be explored further to elucidate the evolution of barley.
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http://dx.doi.org/10.1186/s12864-016-3159-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078923PMC
October 2016

Complete mitochondrial genome sequence of black mustard (Brassica nigra; BB) and comparison with Brassica oleracea (CC) and Brassica carinata (BBCC).

Genome 2014 Nov 13;57(11-12):577-82. Epub 2015 Feb 13.

Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo, Kita, Kyoto 603-8555, Japan.

Crop species of Brassica (Brassicaceae) consist of three monogenomic species and three amphidiploid species resulting from interspecific hybridizations among them. Until now, mitochondrial genome sequences were available for only five of these species. We sequenced the mitochondrial genome of the sixth species, Brassica nigra (nuclear genome constitution BB), and compared it with those of Brassica oleracea (CC) and Brassica carinata (BBCC). The genome was assembled into a 232 145 bp circular sequence that is slightly larger than that of B. oleracea (219 952 bp). The genome of B. nigra contained 33 protein-coding genes, 3 rRNA genes, and 17 tRNA genes. The cox2-2 gene present in B. oleracea was absent in B. nigra. Although the nucleotide sequences of 52 genes were identical between B. nigra and B. carinata, the second exon of rps3 showed differences including an insertion/deletion (indel) and nucleotide substitutions. A PCR test to detect the indel revealed intraspecific variation in rps3, and in one line of B. nigra it amplified a DNA fragment of the size expected for B. carinata. In addition, the B. carinata lines tested here produced DNA fragments of the size expected for B. nigra. The results indicate that at least two mitotypes of B. nigra were present in the maternal parents of B. carinata.
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http://dx.doi.org/10.1139/gen-2014-0165DOI Listing
November 2014

The complete mitochondrial genome sequence of Brassica oleracea and analysis of coexisting mitotypes.

Curr Genet 2014 Nov 12;60(4):277-84. Epub 2014 Jun 12.

Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto, 603-8555, Japan,

The complete mitochondrial genome sequences of Brassica species have provided insight into inter- and intraspecific variation of plant mitochondrial genomes. However, the size of mitochondrial genome sequenced for Brassica oleracea hitherto does not match to its physical mapping data. This fact led us to investigate B. oleracea mitochondrial genome in detail. Here we report novel B. oleracea mitochondrial genome, derived from var. capitata, a cabbage cultivar ''Fujiwase''. The genome was assembled into a 219,952-bp circular sequence that is comparable to the mitochondrial genomes of other Brassica species (ca. 220-232 kb). This genome contained 34 protein-coding genes, 3 rRNA genes and 17 tRNA genes. Due to absence of a large repeat (140 kb), the mitochondrial genome of ''Fujiwase'' is clearly smaller than the previously reported mitochondrial genome of B. oleracea accession ''08C717'' (360 kb). In both mitotypes, all genes were identical, except cox2-2, which was present only in the Fujiwase type. At least two rearrangement events via large and small repeat sequences have contributed to the structural differences between the two mitotypes. PCR-based marker analysis revealed that the Fujiwase type is predominant, whereas the 08C717 type coexists at low frequency in all B. oleracea cultivars examined. Intraspecific variations in the mitochondrial genome in B. oleracea may occur because of heteroplasmy, coexistence of different mitotypes within an individual, and substoichiometric shifting. Our data indicate that the Fujiwase-type genome should be used as the representative genome of B. oleracea.
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http://dx.doi.org/10.1007/s00294-014-0433-2DOI Listing
November 2014

A possible breakage of linkage disequilibrium between mitochondrial and chloroplast genomes during Emmer and Dinkel wheat evolution.

Genome 2013 Apr 8;56(4):187-93. Epub 2013 Apr 8.

The 31st Laboratory, Department of Bioresources and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan.

In wheat (Triticum) and Aegilops, chloroplast and mitochondrial genomes have been studied for over three decades to clarify the phylogenetic relationships among species, and most of the maternal lineages of polyploid species have been clarified. Mitochondrial genomes of Emmer (tetraploid with nuclear genome AABB) and Dinkel (hexaploid with AABBDD) wheat are classified into two different types, VIIa and VIIb, by the presence-absence of the third largest HindIII fragment (named H3) in the mitochondrial DNA. Although the mitochondrial genome in the genera often provides useful information to clarify the phylogenetic relationship among closely related species, the phylogenetic significance of this dimorphism has yet not been clarified. In this study, to facilitate analysis using a large number of accessions, a sequence characterized amplified region (SCAR) marker that distinguishes the type VIIb mitochondrial genome from type VIIa was first developed. Mitochondrial genome type was determined for each of 30 accessions of wild and cultivated Emmer wheat and 25 accessions of Dinkel wheat. The mitochondrial genome type for each accession was compared with the plastogroup that had been determined using chloroplast microsatellite markers. Unexpectedly, the distribution of mitochondrial genome type was not in accordance with that of the plastogroups, suggesting occasional paternal leakage of either the mitochondrial or chloroplast genome during speciation and differentiation of Emmer and Dinkel wheat. An alternative possibility that substoichiometric shifting is involved in the observed dimorphism of the mitochondrial genome is also discussed.
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http://dx.doi.org/10.1139/gen-2012-0153DOI Listing
April 2013

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.).

BMC Genomics 2012 Jul 31;13:352. Epub 2012 Jul 31.

31 Laboratory, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto 603-8555, Japan.

Background: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138.

Results: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences.

Conclusions: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.
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http://dx.doi.org/10.1186/1471-2164-13-352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3473294PMC
July 2012

PKL01, an Ndr kinase homologue in plant, shows tyrosine kinase activity.

J Biochem 2012 Oct 29;152(4):347-53. Epub 2012 Jun 29.

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.

Protein phosphorylation by protein tyrosine (Tyr) kinases plays important roles in a variety of signalling pathways in cell growth, differentiation and oncogenesis in animals. Despite the absence of classical Tyr kinases in plants, a similar ratio of phosphotyrosine residues in phosphorylated proteins was found in Arabidopsis thaliana as in human. However, protein kinases responsible for tyrosine phosphorylation in plants except some dedicated dual-specificity kinases still remain unclear. In this study, we found that PKL01, a nuclear Dbf2-related (Ndr) kinase homologue in Lotus japonicus, was autophosphorylated at a tyrosine residue when it was expressed in Escherichia coli, but kinase-dead mutant of PKL01 was not. Tyrosine phophorylation site in PKL01 was identified as Tyr-56 by LC-MS/MS analysis. Recombinant PKL01, which had been dephosphorylated by an alkaline phosphatase, could be phosphorylated again at the Tyr residue when it was incubated with ATP. Furthermore, other Ndr kinases in plants and PKL01 phosphorylated on Tyr residues in the exogenous substrates such as poly(Glu, Tyr)(4:1) and casein. Therefore, the Ndr kinases in plants, which had been assumed as protein serine (Ser)/threonine (Thr) kinases, turned out to be dual-specificity kinases responsible for phosphorylation of Tyr residues and Ser/Thr residues in plant proteins.
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http://dx.doi.org/10.1093/jb/mvs075DOI Listing
October 2012

Polymorphic minisatellites in the mitochondrial DNAs of Oryza and Brassica.

Curr Genet 2011 Aug 12;57(4):261-70. Epub 2011 May 12.

Laboratory of Genetic Engineering, Research Faculty of Agriculture, Hokkaido University, N-9, W-9, Kita-ku, Sapporo, 060-8589, Japan.

Polymorphic analyses of angiosperm mitochondrial DNA are rare in comparison with chloroplast DNA, because few target sequences in angiosperm mitochondrial DNA are known. Minisatellites, a tandem array of repeated sequences with a repeat unit of 10 to ~100 bp, are popular target sequences of animal mitochondria, but Beta vulgaris is the only known angiosperm species for which such an analysis has been conducted. From this lack of information, it was uncertain as to whether polymorphic minisatellites existed in other angiosperm species. Ten plant mitochondrial DNAs were found to contain minisatellite-like repeated sequences, most of which were located in intergenic regions but a few occurred in gene coding and intronic regions. Oryza and Brassica accessions were selected as models for the investigation of minisatellite polymorphism because substantial systematic information existed. PCR analysis of 42 Oryza accessions revealed length polymorphisms in four of the five minisatellites. The mitochondrial haplotypes of the 16 Oryza accessions with chromosomal complement (genome) types of CC, BBCC and CCDD were identical but were clearly distinguished from BB-genome accessions, a result consistent with the notion that the cytoplasmic donor parent of the amphidiploid species might be the CC-genome species. Twenty-nine accessions of six major cultivated species of Brassica were classified into five mitochondrial haplotypes based on two polymorphic minisatellites out of six loci. The haplotypes of Brassica juncea and Brassica carinata accessions were identical to Brassica rapa and Brassica nigra accessions, respectively. The haplotypes of Brassica napus accessions were heterogeneous and unique, results that were consistent with previous studies.
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http://dx.doi.org/10.1007/s00294-011-0345-3DOI Listing
August 2011

Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion.

Plant Cell Rep 2009 Nov 2;28(11):1633-40. Epub 2009 Sep 2.

Department of Biotechnology, Kyoto Sangyo University, Japan.

Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.
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http://dx.doi.org/10.1007/s00299-009-0763-6DOI Listing
November 2009

A novel Rf gene controlling fertility restoration of Ogura male sterility by RNA processing of orf138 found in Japanese wild radish and its STS markers.

Genome 2009 Jun;52(6):495-504

Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto 603-8555, Japan.

To reveal the molecular and genetic mechanism of fertility restoration in Ogura male sterility in Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides), we investigated fertility restoration of a plant that lacks the dominant type of orf687, a previously identified fertility restorer gene. A total of 100 F2 plants were made from the cross between a male-sterile strain with the Ogura cytoplasm, 'MS-Gensuke', and a Japanese wild radish plant. Segregation of pollen fertility in the F2 plants led us to assume that 2 dominant complementary genes controlled the fertility restoration of the plants. However, the fertility of 27 of 59 male-fertile plants was not completely restored, resulting in a group of plants with partial male fertility. Northern blot analysis of the CMS-associated gene orf138 indicated that one restorer allele (termed Rft) was involved in the processing of orf138 RNA. Rapid amplification of cDNA ends (RACE) and subsequent Northern blot analysis confirmed that the orf138 transcript lost a 5' part of the coding region of the orf138 gene in the restored plants. The accumulation of ORF138 protein was significantly reduced by Rft, but trace amounts of the protein were recognized in both partially male-fertile and male-sterile plants with Rft. The relationship of pollen fertility and segregation of co-dominant sequence tagged site (STS) markers in the F2 generation suggested that the penetrance of Rft was so low that Rft needs suitable conditions to function sufficiently for the complete restoration of fertility.
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http://dx.doi.org/10.1139/g09-026DOI Listing
June 2009

Inhibition of chalcone synthase expression in anthers of Raphanus sativus with Ogura male sterile cytoplasm.

Ann Bot 2008 Oct 14;102(4):483-9. Epub 2008 Jul 14.

Department of Biotechnology, Kyoto-Sangyo University, Kyoto 603-8555, Japan.

Background And Aims: Expression of the mitochondrial gene orf138 causes Ogura cytoplasmic male sterility (CMS) in Raphanus sativus, but little is known about the mechanism by which CMS takes place. A preliminary microarray experiment revealed that several nuclear genes concerned with flavonoid biosynthesis were inhibited in the male-sterile phenotype. In particular, a gene for one of the key enzymes for flavonoid biosynthesis, chalcone synthase (CHS), was strongly inhibited. A few reports have suggested that the inhibition of CHS causes nuclear-dependent male sterile expression; however, there do not appear to be any reports elucidating the effect of CHS on CMS expression. In this study, the expression patterns of the early genes in the flavonoid biosynthesis pathway, including CHS, were investigated in normal and male-sterile lines.

Methods: In order to determine the aberrant stage for CMS expression, the characteristics of male-sterile anthers are observed using light and transmission electron microscopy for several stages of flower buds. The expression of CHS and the other flavonoid biosynthetic genes in the anthers were compared between normal and male-sterile types using real time RT-PCR.

Key Results: Among the flavonoid biosynthetic genes analysed, the expression of CHS was strongly inhibited in the later stages of anther development in sterility cytoplasm; accumulation of putative naringenin derivatives was also inhibited.

Conclusions: These results show that flavonoids play an important role in the development of functional pollen, not only in nuclear-dependent male sterility, but also in CMS.
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http://dx.doi.org/10.1093/aob/mcn116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701772PMC
October 2008

[Production of alloplasmic wheats: the ultimate way of organelle genome engineering].

Authors:
Toru Terachi

Tanpakushitsu Kakusan Koso 2005 Nov;50(14 Suppl):1808-12

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November 2005

Variations in a hotspot region of chloroplast DNAs among common wheat and Aegilops revealed by nucleotide sequence analysis.

Genes Genet Syst 2005 Aug;80(4):277-85

Laboratory of Genetics, Faculty of Biology, Harbin Normal University, Heilongjiang, PR China.

The second largest BamHI fragment (B2) of the chloroplast DNA in Triticum (wheat) and Aegilops contains a highly variable region (a hotspot), resulting in four types of B2 of different size, i.e. B2l (10.5kb), B2m (10.2kb), B2 (9.6kb) and B2s (9.4kb). In order to gain a better understanding of the molecular nature of the variations in length and explain unexpected identity among B2 of Ae. ovata, Ae. speltoides and common wheat (T. aestivum), the nucleotide sequence between a stop codon of rbcL and a HindIII site in cemA in the hotspot was determined for Ae. ovata, Ae. speltoides, Ae. caudata and Ae. mutica. The total number of nucleotides in the region was 2808, 2810, 3302, and 3594 bp, for Ae. speltoides, Ae. ovata, Ae. caudata and Ae. mutica, respectively, and the sequences were compared with the corresponding ones of Ae. crassa 4x, T. aestivum and Ae. squarrosa. Compared with the largest B2l fragment of Ae. mutica, a 791bp and a 793 bp deletion were found in Ae. speltoides and Ae. ovata, respectively, and the possible site of deletion in the two species is the same as that of T. aestivum. However, a deleted segment in Ae. ovata is 2 bp longer than that of Ae. speltoides (and T. aestivum), demonstrating that recurrent deletions had occurred in the chloroplast genomes of both species. Comparison of the sequences from Ae. caudata and Ae. crassa 4x with that of Ae. mutica revealed a 289 bp and a 61 bp deletion at the same site in Ae. caudata and Ae. crassa 4x, respectively. Sequence comparison using wild Aegilops plants showed that the large length variations in a hotspot are fixed to each species. A considerable number of polymorphisms are observed in a loop in the 3' of rbcL. The study reveals the relative importance of the large and small indels and minute inversions to account for variations in the chloroplast genomes among closely related species.
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http://dx.doi.org/10.1266/ggs.80.277DOI Listing
August 2005

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome.

Nucleic Acids Res 2005 31;33(19):6235-50. Epub 2005 Oct 31.

Faculty of Agriculture, Kyoto Prefecture University Sakyo-ku, Kyoto 606-8522, Japan.

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon-intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.
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http://dx.doi.org/10.1093/nar/gki925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1275586PMC
November 2005

Cloning and characterization of a novel radish protein kinase which is homologous to fungal cot-I like and animal Ndr protein kinases.

Genes Genet Syst 2004 Oct;79(5):283-91

Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto 603-8555, Japan.

According to the similarity of the amino acid sequences in their catalytic domains, eukaryotic protein kinases have been classified into the five main groups: 'AGC', 'CaMK', 'CMGC', 'PTK' and 'other'. The AGC group, represented by the cyclic nucleotide-dependent kinases (PKA and PKG), the calcium-phospholipid-dependent kinases (PKC) and the ribosomal S6 protein kinases, are poorly characterized in plants except for a few cases. In this study, in order to gain a better understanding of plant protein kinases in the AGC group, three cDNAs encoding novel protein kinases, RsNdr1 and RsNdr2a/b, were cloned from radish and characterized by molecular and biochemical methods. The deduced amino acid sequences of RsNdr1 and RsNdr2a/b contained all 12 conserved catalytic subdomains which are characteristic of the eukaryotic Ser/Thr protein kinases. A cell lysate from E. coli overexpressing RsNdr1 fusion protein had protein kinase activity toward a conventional protein substrate (myelin basic protein), whereas that from E. coli harboring a fusion plasmid encoding kinase-dead RsNdr1 or RsNdr2 did not show any protein kinase activity. A phylogenetic tree for 17 protein kinases from various organisms showed that the RsNdrs are more closely related to the protein kinases in a particular subgroup of the 'AGC' (fungal cot1-like and animal Ndr kinases) than to the authentic 'AGC' protein kinases, such as PKA, PKC or ribosomal S6 kinase.
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http://dx.doi.org/10.1266/ggs.79.283DOI Listing
October 2004

Cospeciation analysis of an obligate pollination mutualism: have Glochidion trees (Euphorbiaceae) and pollinating Epicephala moths (Gracillariidae) diversified in parallel?

Evolution 2004 Oct;58(10):2201-14

Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Nihonmatsucho, Sakyo, Kyoto 606-8501, Japan.

Species-specific obligate pollination mutualism between Glochidion trees (Euphorbiaceae) and Epicephala moths (Gracillariidae) involves a large number of interacting species and resembles the classically known fig-fig wasp and yucca-yucca moth associations. To assess the extent of parallel cladogenesis in Glochidion-Epicephala association, we reconstruct phylogenetic relationships of 18 species of Glochidion using nuclear ribosomal DNA sequences (internal and external transcribed spacers) and those of the corresponding 18 Epicephala species using mitochondrial (the cytochrome oxidase subunit I gene) and nuclear DNA sequences (the arginine kinase and elongation factor-1alpha genes). Based on the obtained phylogenies, we determine whether Glochidion and Epicephala have undergone parallel diversification using several different methods for investigating the level of cospeciation between phylogenies. These tests indicate that there is generally a greater degree of correlation between Glochidion and Epicephala phylogenies than expected in a random association, but the results are sensitive to selection of different phylogenetic hypotheses and analytical methods for evaluating cospeciation. Perfect congruence between phylogenies is not found in this association, which likely resulted from host shift by the moths. The observed significant discrepancy between Glochidion and Epicephala phylogenies implies that the one-to-one specificity between the plants and moths has been maintained through a complex speciation process or that there is an underestimated diversity of association between Glochidion trees and Epicephala moths.
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http://dx.doi.org/10.1111/j.0014-3820.2004.tb01598.xDOI Listing
October 2004

Multiple origins of cultivated radishes as evidenced by a comparison of the structural variations in mitochondrial DNA of Raphanus.

Genome 2003 Feb;46(1):89-94

Department of Biotechnology, Institute for Comprehensive Research, Kyoto Sangyo University, Kamigamo, Kita-ku, Kyoto, 603-8555 Japan.

Configurations of mitochondrial coxI and orfB gene regions were analysed by polymerase chain reaction (PCR) in three wild and one cultivated species of Raphanus. A total of 207 individual plants from 60 accessions were used. PCR with five combinations of primers identified five different amplification patterns both in wild and cultivated radishes. While the mitochondrial DNA (mtDNA) type of Ogura male-sterile cytoplasm was distinguishable from the normal type, the mtDNAs of normal radishes were further classified into four types. The variations were common to wild and cultivated radishes, although contrasting features were found depending on the region of cultivation. These results provide evidence that cultivated radishes have multiple origins from various wild plants of Raphanus.
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http://dx.doi.org/10.1139/g02-110DOI Listing
February 2003
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