中村 郁郎

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis. © 2014 Springer Science+Business Media Dordrecht.

PolA1, a single-copy nuclear gene encoding the largest subunit of RNA polymerase I, comprises highly polymorphic intron 19 and nucleotide tag (Ntag) sequences. We analyzed these sequences in 42 accessions, which differed in ploidy, of Triticum-Aegilops and Hordeum species. The lengths of the intron 19 sequences were ca. 110 bp long in Triticum-Aegilops species, except in four Sitopsis species, Ae. longissima, Ae. searsii, Ae. sharonensis, Ae. speltoides, which had introns similar in length to those of Hordeum species, i. e., ca. 240 bp long. Phylogenetic analyses of the Ntag sequences showed that the four Sitopsis and remaining Triticum-Aegilops species were classified into two discrete Hordeum and Triticum clades, respectively. The A and D genome-specific Ntag sequences of polyploid wheats were highly homologous with those of T. urartu and Ae. tauschii, respectively. In Ae. bicornis, another Sitopsis species, two accessions had the short intron 19 and Triticum-type Ntag sequence, which were highly homologous with those of the B genome in polyploid wheats, whereas one accession contained the long intron 19 and Hordeum-type Ntag sequences. In contrast, partial sequence analyses revealed that the three accessions of Ae. bicornis shared highly homology to single-copy DMC1 and EF-G genes. The discrepancy between these results indicates that the Sitopsis species were probably established by hybrid speciation including ancient introgressive hybridization between progenitors of Triticum-Aegilops and Hordeum. Although many researchers have proposed Ae. speltoides as a candidate for the B genome donor, our data suggest the existence of diploid B genome species in the past that were responsible for the origin of both polyploid wheats and Sitopsis species, including Ae. speltoides. © 2012 Springer Science+Business Media B.V.

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241-243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively. © 2009 Springer Science+Business Media B.V.

The section Sitopsis in the genus Aegilops includes five species, Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, which share the SS genome. Although extensive molecular studies have indi- cated Ae. speltoides as a donor of BB or GG genome to polyploid wheat species, the precise relationships among SS, BB, and GG genomes remain unclear. PolA1 is a single-copy nuclear gene encoding the largest subunit of RNA polymerase I. Highly polymorphic PolA1 exon 20 sequences were analyzed for 11 Triticum- Aegilops, 13 Hordeum and three related species. Phylogenetic analyses of the PolA1 gene showed that Triticum-Aegilops and Hordeum species were distinctly separated into two clades. Two related species, Secale cereale and Dasypyrum villosum, were grouped into Triticum and Hordeum clades, respectively. Interestingly, seven accessions of the Sitopsis species were clustered into the Hordeum clade whereas two accessions belonged to the Triticum clade. In contrast, all accessions of Sitopsis species shared the same haplotype of plastid PSID sequences with Triticum-Aegilops species. This inconsistency in phylogeny between nuclear and cytoplasmic sequences suggested that the Sitopsis species probably originated through introgres- sive hybridization between ancestral species of Triticum-Aegilops and Hordeum.

To develop a rice cultivar that would be suitable for direct-seeding cultivation in cooler temperate regions, we generated transgenic rice plants in which a rice encoding a ζ-class glutathione S-transferase (GST) under the control of a maize ubiquitin promoter. GSTs have been suggested to be responsible for tolerance to various stresses such as cold, salt and drought by detoxification of xenobiotic compounds and reactive oxygen species. A total of 87 R0 transgenic rice plants harboring a chimeric GST gene were generated using Agrobacterium mediated transformation. Three R2 lines homozygous for the transgene were assayed for GST activity and had higher GST and glutathione peroxidase activities than non-transformants. Seedlings of the transgenic lines demonstrated greatly enhanced germination and growth rates at low temperature grown under submergence. The GST transgenic lines should be useful for breeding rice cultivars suitable for direct-seeding cultivation in cooler temperate regions.

Transgenic rice (Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.

Recent complete sequence data of the cyanobacteria genome and the plastid genome of algae and land plants clearly show that plastids originated from the endosymbiotic integration of a photosynthetic prokaryote into eukaryotic host cells. One indication of this event is that the organization of ribosomal protein genes has been conserved from bacteria to plastids of land plants. We are interested in the rpl16 and rpl14 genes, which are interrupted by the rpl29 and rps17 genes in both the cyanobacteria genome and the plastid genome of red alga, but are adjacent in liverwort to higher plants. This suggests that a linker between rpl16 and rpl14 was made by deletion of the rpl29 and rps17 genes at the appearance of land plants, and therefore the rpl16-rpl14 linkers of land plants have diverged from a common ancestral sequence. The rpl16 and rpl14 genes are highly conserved (more than 84 % homology) among rice, spinach and tobacco, whereas their linkers show low homology (28 %). The plastid rpl16-rp114 linker sequences of higher plants, such as barnyard grass, gentian and oak tree could be amplified and sequenced using a pair of common primers, even if their sequences are unknown. We propose short DNA sequence in the 3' noncoding region from the stop codon of the rpl16 gene as plastid subtype ID sequence (PS-ID). PS-IDs of ca. 50 bp are thought to be long enough to address plastid subtypes of higher plants and the establishment of a PS-ID database could contribute to plant phylogeny.