Masahiro Sakamoto

The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

SummaryThe entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.

Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions.

SummaryWe have developed a method for reproducibly obtaining transgenic rice at a high frequency (10-4): electroporation with a buffer in which chloride ions are replaced with organic acids. Co-transformation frequencies of the β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes located on two separate plasmids were higher than 50%. Transgenic rice plants contained multiple copies of introduced genes integrated into their genomes in a complex manner. GUS enzyme activity was not proportional to gene copy number. Introduced HPT genes were detected and expressed in the progeny of transformants.

A unique sequence located downstream from the rice mitochondrial atp6 may cause male sterility

Asymmetric cell-fusion of the japonica cultivar ofOryza sativa (rice) with cytoplasmic-male-sterile (CMS) plants bearing cytoplasm derived from Chinsurah Boro II, resulted in two classes of cytoplasmic hybrids (cybrids), fertile and CMS. Southern-blot analysis of the mitochondrial DNA (mtDNA) indicates recombination events around a number of genes; however, the appearance of the CMS character is tightly correlated to reorganization around theatp6 gene, suggesting recombination downstream from theatp6 gene is involved in CMS. The nucleotide sequence downstream fromatp6 contains a pseudogene which was probably created by recombination of the mitochondrial genome. Sense and antisense transcripts of the downstream region ofatp6 were found in CMS-and restored CMS (fertile)-lines, but not in the normal (fertile) line. In the CMS line, several antisense transcripts of theatp6 gene were also found. However, in the restored line which contains a nuclear-encoded gene,Rf-1, the levels of these transcripts were lower than in the CMS line. These results suggest abnormal transcripts of theatp6 gene produced in the antisense direction may be involved in CMS, and that products of the nuclear-encoded restorer gene may reduce abnormal transcription in this region of the mitochondrial genome.

Construction of rice cybrid plants

SummaryThe mitochondrial genomes of rice cells were transferred to a fertile rice variety (N8) from a cytoplasmic male sterile variety (CMS) by asymmetric protoplast fusion based on metabolic complementation. Protoplasts derived from CMS were X-irradiated (125 krad) and electrofused with protoplasts which had been treated with iodoacetamide. Metabolic complementation, presumably between nuclear and cytoplasmic compartments, enabled fused protoplasts to form colonies at high efficiency. Restriction digest analysis of mitochondrial DNA (mtDNA) indicated that hybrid cells carried mtDNA derived from both parents. Of the plants regenerated from hybrid calli, 68% carried a diploid chromosome set (2n=24) and the rest of them carried 48 chromosomes. All of them expressed the aryl acylamidase I deficient phenotype encoded by the recessive allele of the fertile N8 parent. These results indicate that the novel somatic hybrid plants regenerated were cybrids, deriving their nucleus from the iodoacetamide treated parent and their mitochondria from both parents.

Structure and RFLP mapping of a rice sucrose phosphate synthase (SPS) gene that is specifically expressed in the source organ

Abstract A rice sucrose phosphate synthase (SPS) gene was isolated and mapped on chromosome 1, based upon RFLPs between a japonica line ‘FL134’ and an indica cultivar ‘Kasalath’. This gene consisted of 12 exons with 11 introns. In the exon 1, an additional sequence occurred bearing an insertion encoding 24 glycine residues when it was compared with the spinach one. Although the structure of this region possessed the consensus intron borders, its size was only 48 bp and it occurred with neither breakage of open reading frame nor shift of the reading frame. The deduced amino acid sequence of the rice SPS showed a high degree of homology to the known ones. Sites for modification by kinase/phosphatase and for binding with UDP-glucose were highly conserved. No typical promoter sequences were found upstream of the coding region, suggesting that function of the promoter of this gene is weak, although further upstream of the coding region, sequences similar to the promoter and light-responsive elements were found. Expression of this gene was detected only in the leaves, suggesting that this gene is specifically expressed in the source organs. Level of expression was extremely low, reflecting weakness of its promoter activity.