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Abstract. The entire mitochondrial genome of rice (Oryza sativa L.), a monocot plant, has been sequenced. It was found to comprise 490,520 bp, with an average G+C content of 43.8%. Three rRNA genes, 17 tRNA genes and five pseudo tRNA sequences were identified. In addition, eleven ribosomal protein genes and two pseudo ribosomal protein genes were found, which are homologous to 13 of the 16 genes for ribosomal proteins in the mitochondrial genome of the liverwort (Marchantia polymorpha). A greater degree of variation in terms of presence/absence and integrity of genes was observed among the ribosomal protein genes and tRNA genes of rice, Arabidopsis and sugar beet. Transcription and post-transcriptional modification (RNA editing) in the rice mitochondrial sequence were also examined. In all, 491 Cs in the genomic DNA were converted to Ts in cDNA. The frequency of RNA editing differed markedly depending upon the ORF considered. Sequences derived from plastid and nuclear genomes make up 6.3% and 13.4% of the mitochondrial genome, respectively. The degree of conservation of plastid sequences in the mitochondrial genome ranged from 61% to 100%, suggesting that sequence migration has occurred very frequently. Three plastid DNA fragments that were incorporated into the mitochondrial genome were subsequently transferred to the nuclear genome. Nineteen fragments that were similar to transposon or retrotransposon sequences, but different from those found in the mitochondrial genomes of dicots, were identified. The results indicate frequent and independent DNA sequence flow to and from the mitochondrial genome during the evolution of flowering plants, and this may account for the range of genetic variation observed between the mitochondrial genomes of higher plants.

It is known that alcoholic fermentation is important for survival of plants under anaerobic conditions. Acetaldehyde, one of the intermediates of alcoholic fermentation, is not only reduced by alcohol dehydrogenase but also can be oxidized by aldehyde dehydrogenase (ALDH). To determine whether ALDH plays a role in anaerobic metabolism in rice (Oryza sativa L. cv Nipponbare), we characterized a cDNA clone encoding mitochondrial ALDH from rice (Aldh2a). Analysis of sub-cellular localization of ALDH2a protein using green fluorescent protein and an in vitro ALDH assay using protein extracts from Escherichia coli cells that overexpressed ALDH2a indicated that ALDH2a functions in the oxidation of acetaldehyde in mitochondria. A Southern-blot analysis indicated that mitochondrial ALDH is encoded by at least two genes in rice. We found that the Aldh2a mRNA was present at high levels in leaves of dark-grown seedlings, mature leaf sheaths, and panicles. It is interesting that expression of the rice Aldh2a gene, unlike the expression of the tobacco (Nicotiana tabacum) Aldh2a gene, was induced in rice seedlings by submergence. Experiments with ruthenium red, which is a blocker of Ca(2+) fluxes in rice as well as maize (Zea mays), suggest that the induction of expression of Adh1 and Pdc1 by low oxygen stress is regulated by elevation of the cytosolic Ca(2+) level. However, the induction of Aldh2a gene expression may not be controlled by the cytosolic Ca(2+) level elevation. A possible involvement of ALDH2a in the submergence tolerance of rice is discussed.

We identified a gene for subunit 9 of NADH dehydrogenase (nad9) in rice mitochondrial DNA. Southern and Northern hybridizations demonstrated that rice nad9 is present in a unique region in mtDNA and is transcribed at a high level. The transcript of rice nad9 is edited at twelve positions.

The nucleotide sequence of a 27,588-bp region of rice mitochondrial DNA was determined. This sequence contains putative genes that encode initiator methionine tRNA (trnfM), subunits III (nad3) and IV (nad4) of the NADH dehydrogenase complex, and ribosomal proteins S3 (rps3), S12 (rps12) and L16 (rpl16). An open reading frame that contains sequences homologous to parts of rps2 and atpA is also present. In addition to these regions, there are many short sequences with homology to fragments of mitochondrial DNAs from rice or other plants. These sequences may be remnants of multiple rearrangements of the genome and their presence seems to explain, in part, the large sizes of the mitochondrial genomes of higher plants.

An unusual case of coexisting acromegaly and Cushing’s syndrome is reported in a 34-yr-old female. There was no biochemical or morphological evidence to suggest the presence of other endocrinopathies. She did not have any family history to suggest a hereditary tendency to endocrine disorders. Her acromegaly and Cushing’s syndrome were proven to be due to a pituitary somatotroph adenoma and a cortisol-producing adenoma in the right adrenocortex, respectively. Surgical removal of both tumors led to a marked biochemical improvement of the two endocrinopathies. To account for the simultaneous occurrence of the two endocrine tumors, at least two endocrine syndromes may be considered. One of them is Carney’s complex. However, Cushing’s syndrome in this complex is unexceptionally due to primary pigmented nodular adrenocortical disease, differing from the adrenal pathology of our patient. In addition, a lack in this case of any other characteristic suggestive of this syndrome appears to speak against this possibility. A second possibility is multiple endocrine neoplasia type 1. The absence of a parathyroid or pancreatic islet cell tumor does not strongly support this possibility, but adrenocortical lesions are not rare in this syndrome although they are only rarely functional. However, existence of similar case reports, although very few, in the literature leaves the possibility that she represents another rare variant of sporadic multiple endocrine neoplasia type I syndrome.

Mitochondrial DNA (mtDNA) was isolated from young green leaves of rice plants. DNA fragments were cloned into lambda DNA, and clones that hybridized to mitochondrial genes from other plants were selected. Distal restriction fragments of these clones were used as probes for the selection of overlapping clones. A genetic map was finally created from the library by “walking” along the genome. The mitochondrial genome consists of five basic circles, with each circle sharing homologous sequences with one or two other circles. A master circle was constructed from the results of recombination across repeated sequences, and its size was estimated to be 492 kb. A physical map and a bank of overlapping clones were also constructed.

The chloroplast-derived sequencetrnS-rps4/3′trnL-trnF-ndhJ-ndhK (4066 bases in length) is present in a region that starts 355 bases upstream of the gene for subunit 9 of NADH dehydrogenase (nad9) in the mitochondrial genome of rice. Northern blot hybridization revealed that three large transcripts of 3.05, 1.62 and 1.05 kb hybridized to strand-specific probes for both thenad9 gene and the chloroplast-derived sequence, indicating that thenad9 gene was transcribed together with the chloroplast-derived sequence. From the results of in vitro capping and ribonuclease protection experiments, as well as primer extension analysis, we identified at least seven sites for the initiation of transcription ofnad9 in the chloroplast-derived sequence. All of the initiation sites for transcription of thenad9 gene were located in sequences homologous to chloroplast DNA. Two of seven initiation sites were flanked by a sequence homologous to the consensus promoter motif that includes the CRTA motif (where R is A or G) of the rice mitochondrion. However, the sequences surrounding the other five sites showed only limited similarity to the conserved sequence. It is suggested that all the promoters of the ricenad9 gene exist in a sequence that was transferred from the chloroplast during evolution. Thus, the chloroplast-derived sequence has a novel, significant function in the mitochondrial genome of this higher plant.