54. COX6b in rice has a long N-terminal sequence compared with those in other species

K. Ohtsu, S. HAMANAKA, M. NAKAZONO and A. Hirai

Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Science, The University ofTokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-3657 Japan

     Mitochondria of higher plants contain two respiratory pathways: the cyanide-sensitive cytochrome pathway, and the cyanide-resistant alternative pathway. Cytochrome c oxidase (COX) is a terminal oxidase in the cytochrome pathway that converts oxygen to H20. The COX of higher plants is composed of at least 10 subunits. The three largest subunits (COX1, COX2 and COX3) are encoded by the mitochondrial genome, and the remaining subunits are encoded by the nuclear genome. The mitochondnal-encoded COX genes have been extensively characterized in higher plants including rice (Schuster and Brennicke 1994). However, only a limited number of studies have been carried out on the nuclear-encoded CUX genes of plants. It is required to accumulate more information about them.

     The gene for the human COX6b subunit has been described by Taanman eta!. (1990) and Carrero-Valenzuela et al.. (1991). The COX12 gene, which encodes the COX6b subunit of Saccharomyces cerevisiae has been described by LaMarche et a!. (1992). The protein has a molecular size of approximately 10 kDa, and analysis of the CUXJ2-disrupted strain indicated that the COX6b subunit is essential during assembly for full cytochrome c oxidase activity. So far, information about the COX6b subunit in plants has not been available. In this report, we characterized a novel gene encoding COX6b subunit in rice.

     As a first step in detecting novel CUX genes in higher plants, we searched the rice EST clone database for genes that are homologous to human nuclear-encoded CUX genes. As a result, the amino acid sequence of human COX6b protein was found to show a significant homology to the putative protein encoded by the EST clone S 12878 from rice (Uryza sativa cv. Nipponbare) green seedlings. The 600 bp insert of the cDNA clone S 12878 was completely sequenced. However, no putative initiation codon was included in the S 12878 clone. Therefore the 5’RACE experiment was carried out using the Cap site cDNA derived from green leaves. We obtained PCR fragments and determined their complete nucleotide sequences. With these sequences, an open reading frame (ORF) could be identified. The ORF, which was designated as COX6b, encodes 169 amino acids with a calculated molecular mass of 19 kDa. A comparison of the deduced amino acid sequences of the COX6b precursor proteins from rice, human, cow and Saccharomyces showed that, surprisingly, the N-terminal portion of the rice COX6b precursor protein was extended (Fig. 1). We also sequenced a cDNA clone of an Arabidopsis gene that is homologous to human CUX6b and yeast COX12 (data not shown). The deduced amino acid sequence of the Arabidopsis COX6b gene was also extended toward the N-terminus. Thus, it is likely that this feature may be common among higher plants. Although the structure of rice COX6b protein is unusual, the result of Northern blot analysis showed that in rice, this gene was constitutively expressed in various organs in several growth stages (Ohtsu eta!. 1999). Therefore the rice COX6b gene we characterized here may produce functional COX6b protein in rice.