Koh-ichi Kadowaki

Targeting presequence acquisition after mitochondrial gene transfer to the nucleus occurs by duplication of existing targeting signals.

We have cloned a gene for mitochondrial ribosomal protein S11 (RPS11), which is encoded in lower plants by the mitochondrial genome, in higher plants by the nuclear genome, demonstrating genetic information transfer from the mitochondrial genome to the nucleus during flowering plant evolution. The sequence s11–1 encodes an N‐terminal extension as well as an organelle‐derived RPS11 region. Surprisingly, the N‐terminal region has high amino acid sequence similarity with the presequence of the beta‐subunit of ATP synthase from plant mitochondria, suggesting a common lineage of the presequences. The deduced N‐terminal region of s11–2, a second nuclear‐encoded homolog of rps11, shows high sequence similarity with the putative presequence of cytochrome oxidase subunit Vb. The sharing of the N‐terminal region together with its 5′ flanking untranslated nucleotide sequence in different proteins strongly suggests an involvement of duplication/recombination for targeting signal acquisition after gene migration. A remnant of ancestral rps11 sequence, transcribed and subjected to RNA editing, is found in the mitochondrial genome, indicating that inactivation of mitochondrial rps11 gene expression was initiated at the translational level prior to termination of transcription.

A chimeric gene containing the 5' portion of atp6 is associated with cytoplasmic male-sterility of rice

SummaryThree ATPase subunit 6 (atp6) genes of rice mitochondria were isolated, one from normal and two from cms-Bo male-sterile cytoplasms, in order to determine whether the extra atp6 gene in cms-Bo rice plays a role in cytoplasmic male-sterility (CMS). The nucleotide sequences of all three genes were determined and analysis showed a chimeric atp6 gene (urf-rmc) as well as a normal atp6 gene in cms-Bo cytoplasm, but only the normal atp6 gene in normal cytoplasm. The urf-rmc gene is completely homologous to the normal atp6 gene from at least position − 426 in the 5′ flanking region to position + 511 downstream from the initiation codon ATG; however, the following downstream sequence shows no homology with the normal rice atp6 gene, or any other reported sequence. Introduction of the restorer of fertility gene altered transcription of the urf-rmc gene but not the atp6 gene, indicating participation of the chimeric gene in the expression of CMS. Southern blot analysis showed that the urf-rmc gene was generated by an intramolecular recombination event in mitochondrial DNA, and the homologous recombination point between the atp6 gene and the opposite ancestral sequence was identified as 5′-TTCCCTC-3′.

Substitution of the Gene for Chloroplast RPS16 Was Assisted by Generation of a Dual Targeting Signal

Organelle (mitochondria and chloroplasts in plants) genomes lost a large number of genes after endosymbiosis occurred. Even after this major gene loss, organelle genomes still lose their own genes, even those that are essential, via gene transfer to the nucleus and gene substitution of either different organelle origin or de novo genes. Gene transfer and substitution events are important processes in the evolution of the eukaryotic cell. Gene loss is an ongoing process in the mitochondria and chloroplasts of higher plants. The gene for ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher plants but not in Medicago truncatula and Populus alba. Here, we show that these 2 species have compensated for loss of the rps16 from the chloroplast genome by having a mitochondrial rps16 that can target the chloroplasts as well as mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa, whose chloroplast genomes encode the rps16, we show that the product of the mitochondrial rps16 has dual targeting ability. These results suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts emerged before the divergence of monocots and dicots (140-150 MYA). The gene substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher plants is the first report about ongoing gene substitution by dual targeting and provides evidence for an intermediate stage in the formation of this heterogeneous organelle.

An integrated high-density linkage map of soybean with RFLP, SSR, STS, and AFLP markers using A single F2 population.

Abstract Soybean [Glycine max (L.) Merrill] is the most important leguminous crop in the world due to its high contents of high-quality protein and oil for human and animal consumption as well as for industrial uses. An accurate and saturated genetic linkage map of soybean is an essential tool for studies on modern soybean genomics. In order to update the linkage map of a F2 population derived from a cross between Misuzudaizu and Moshidou Gong 503 and to make it more informative and useful to the soybean genome research community, a total of 318 AFLP, 121 SSR, 108 RFLP, and 126 STS markers were newly developed and integrated into the framework of the previously described linkage map. The updated genetic map is composed of 509 RFLP, 318 SSR, 318 AFLP, 97 AFLP-derived STS, 29 BAC-end or EST-derived STS, 1 RAPD, and five morphological markers, covering a map distance of 3080 cM (Kosambi function) in 20 linkage groups (LGs). To our knowledge, this is presently the densest linkage map developed from a single F2 population in soybean. The average intermarker distance was reduced to 2.41 from 5.78 cM in the earlier version of the linkage map. Most SSR and RFLP markers were relatively evenly distributed among different LGs in contrast to the moderately clustered AFLP markers. The number of gaps of more than 25 cM was reduced to 6 from 19 in the earlier version of the linkage map. The coverage of the linkage map was extended since 17 markers were mapped beyond the distal ends of the previous linkage map. In particular, 17 markers were tagged in a 5.7 cM interval between CE47M5a and Satt100 on LG C2, where several important QTLs were clustered. This newly updated soybean linkage map will enable to streamline positional cloning of agronomically important trait locus genes, and promote the development of physical maps, genome sequencing, and other genomic research activities.

Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes

Simple sequence repeats (SSR) and their flanking regions in the mitochondrial and chloroplast genomes were sequenced in order to reveal DNA sequence variation. This information was used to gain new insights into phylogenetic relationships among species in the genus Oryza. Seven mitochondrial and five chloroplast SSR loci equal to or longer than ten mononucleotide repeats were chosen from known rice mitochondrial and chloroplast genome sequences. A total of 50 accessions of Oryza that represented six different diploid genomes and three different allopolyploid genomes of Oryza species were analyzed. Many base substitutions and deletions/insertions were identified in the SSR loci as well as their flanking regions. Of mononucleotide SSR, G (or C) repeats were more variable than A (or T) repeats. Results obtained by chloroplast and mitochondrial SSR analyses showed similar phylogenetic relationships among species, although chloroplast SSR were more informative because of their higher sequence diversity. The CC genome is suggested to be the maternal parent for the two BBCC genome species (O. punctata and O. minuta) and the CCDD species O. latifolia, based on the high level of sequence conservation between the diploid CC genome species and these allotetraploid species. This is the first report of phylogenetic analysis among plant species, based on mitochondrial and chloroplast SSR and their flanking sequences.

The involvement of a PPR protein of the P subfamily in partial RNA editing of an Arabidopsis mitochondrial transcript.

C-to-U RNA editing (i.e., alteration of a C in the genomic sequence to U in the transcript) has been confirmed widely in angiosperm organellar genomes. During the C-to-U RNA editing event, incomplete edited transcripts have been observed at many sites in the steady-state mRNA population (partial editing). Here, by using coexpression analysis and the surveillance of whole editing status on the mitochondrial genome, we have revealed that a pentatricopeptide repeat (PPR) protein classified into the P subfamily (PPR596) has site-specific influence on the efficiency of C-to-U RNA editing events at partial editing sites on the Arabidopsis thaliana mitochondrial genome. Previous works have revealed that PPR proteins classified into the PLS subfamily containing the E or E and DYW motif are involved in RNA editing as trans-factors; they are believed to recruit deaminase at editing sites. In contrast with the mutant analyses of PLS-subfamily PPR proteins, the editing efficiency at rps3eU1344SS was revealed to be significantly increased in ppr596 mutants. Our study implies P-subfamily PPR protein is involved in the control of the degree of partial editing.

Transfer of the mitochondrial rps10 gene to the nucleus in rice: acquisition of the 5′ untranslated region followed by gene duplication

Abstract Mitochondrial ribosomal protein S10 (rps10) is encoded by the mitochondrial genome in potato and pea. Here we show that the rps10 gene is absent from the mitochondrial genome of rice and has been transferred to the nucleus. Cloning and transcriptional analysis show that there are two rps10 genes in the rice nuclear genome and that their transcripts differ in abundance. Western analysis detected the RPS10 protein in the soluble fraction of rice mitochondria, although neither RPS10 has any obvious N-terminal presequence for targeting to mitochondria. This result suggests that targeting information is present in the internal region of rice RPS10. Genomic sequence analysis indicated that each rps10 gene has an intron in the 5′ untranslated region (5′ UTR) and that these intron sequences are homologous to each other. This result strongly suggests that a duplication event occurred after transfer of the rps10 gene to the nucleus. The duplicated rps10 genes have since been translocated to different chromosomes, because the two rps10 genes were mapped on chromosomes 6 and 12 by RFLP analysis. Interestingly, the 5′ UTR and the intron of the rice rps10 genes are homologous to sequences found in several rice genes with various functions, such as osk4, EF-1β2 and RAG1, suggesting a common origin and a functional role for the 5′ UTR. Acquisition of the 5′ flanking region might have accelerated the activation of the mitochondrial rps10 gene which was transferred to the nuclear genome.

Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid‐soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.

A ribosomal protein L2 gene is transcribed, spliced, and edited at one site in rice mitochondria

The mitochondrial ribosomal protein L2 gene (rpl2) is coded by two exons of 840 and 669 bp separated by an intron sequence of 1481 bp in the rice mitochondrial genome. The rpl2 gene is located three nucleotides upstream of the ribosomal protein S19 gene (rps19) and both genes are co-transcribed. cDNA sequence analysis indentified splicing of the intron sequence from the rpl2 mRNA as well as RNA editing events. The deduced secondary structure of the rpl2 intron sequence shows the characteristic features of a group-II intron. A single RNA editing site is identified in rpl2 and six editing sites in rps19 transcripts. In addition, one editing site is observed in the 3 nucleotide intergenic region. Analysis of individual cDNA clones showed a different extent of RNA editing. The rice rpl2 intron is located at a different site and shows no significant nucleotide sequence similarity with the rpl2 intron of liverwort. However, 60% nucleotide sequence identity is observed between the rice rpl2 intron and the Oenothera nad5 intron in a 234 nucleotide region. The mitochondrial rpl2 sequence is absent from the pea mitochondrial genome and we consequently propose that the mitochondrial RPL2 protein is encoded by a nuclear gene in pea.

Induction of two alcohol dehydrogenase polypeptides in rice roots during anaerobiosis

Abstract In vivo pulse labeling of rice roots exposed to anaerobiosis shows that new proteins are synthesized while the synthesis of other proteins ceases as a result of the stress. Alcohol dehydrogenase (ADH) activity in the rice roots gradually increased over a 24-h period, leveling off subsequently. An antiserum against ADH was used to show that ADH is present at low levels in aerobically grown roots, and that two molecular forms of ADH, differing in molecular weight, are synthesized de novo when the roots are stressed. The rise in ADH activity under a lack of oxygen does not involve the conversion of an inactive protein into active enzyme.