VII. Gene Expression and Function

49. Transcription of alternative oxidase genes in rice is increased by low temperature

M. NAKAZONO. Y. ITO, D. SAISHOU and A. HIRAI

Laboratory of Radiation Genetics, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113 Japan

There are two respiratory pathways, the cytochrome pathway and the alternative pathway in the mitochondria of higher plants. The alternative pathway branches from the cytochrome pathway at ubiquinone and donates electrons to oxygen directly by alternative oxidase (AOX). It is known that this pathway produces heat instead of ATP in appendix tissue during flowering of Suaromatum guttatum, a thermogenic plant (see McIntosh 1994 for review). When plant species are placed under lower temperature, the capacities of the alternative pathway are enhanced (McCaig and Hill 1977; Stewart et al. 1990: Vanlerberghe and McIntosh 1992) and their heat production is increased (Moynihan et al. 1995). In maize and tobacco, the amount of AOX protein is increased under lower temperature (Stewart et al. 1990: Vanlerberghe and McIntosh 1992). Therefore, it has been proposed that the alternative pathway reduces chilling injury (see Purvis and Shewfelt 1993 for review).

Rice (Oryza sativa) is cultivated mainly in tropical and subtropical regions and its distribution is primarily governed by temperature. Thus, an improvement in the tolerance of rice to lower temperature has long been desired. In this respect, it is of interest to study the expression of rice AOX genes under low temperature.

A database search has shown that the deduced amino acid sequence of exon 1 of the Arabidopsis thaliana AOX1a gene (Saisho et al., in press) has significant similarity with the putative protein encoded by the EST clone from rice (cv. Nipponbare) calli. The partially sequenced EST clone was provided by the National Institute of Agrobiological Resources, Rice Genome Research Program. The full sequence of the cDNA clone was determined. There was a 1474-bp insert that encoded a complete 996-bp open reading frame (ORF) in the clone. The amino acid sequence of the ORF indicated high homology with the predicted AOX proteins of other plants. This AOX gene was designated as the AOX1a (lto et al. in press).

A genomic clone containing the AOX1a gene was also isolated. Southern blot analysis of the clone revealed that there is an additional AOX gene in the flanking region of the AOX1a gene. Therefore, we determined the complete nucleotide sequence of a genomic clone containing the two AOX genes. Interestingly, the additional AOX gene, designated as the AOX1b gene, was located about 1.9 kb upstream of the AOX1a gene.

To examine the specific expression of the AOX1a and AOX1b genes, we constructed a probe specific to AOX1a or AOX1b, which corresponds to the 3'-UTR of the gene.

Total RNA was extracted from etiolated seedlings of rice which had been grown at 28°C for six days, then transferred to 4°C and harvested on every day to day 7. Northern hybridization was performed using the probe specific to AOX1a or AOX1b (Fig. 1 ). We did not detect any transcripts of AOX1a or AOX1b in etiolated seedlings grown at 28°C (Fig. 1, lanes 1 and 7). However, treatment with low temperature increased the amounts of transcripts of both of the AOX genes starting on the second day of treatment and the levels of each transcript reached a maximum after four days (Fig. 1, lanes 2-6 and lanes 8-12). We can rule out the possibility that these inductions were caused by senescence because increases in both of the AOX mRNAs were not observed when the seedlings were kept at 28°C for more than 6 days in the dark.

Although it is uncertain whether heat produced by the alternative pathway under low temperature directly protect plants (Moynihan et al. 1995), to our knowledge, this is the first evidence of AOX genes whose expressions at the mRNA level are induced by treatment with low temperature.

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Fig. 1. Northern hybridization analysis of transcripts of AOX genes produced by rice seedlings grown under different temperature regimes. Lanes 1-6 and lanes 7-12 show hybridizations with probe specific for the AOX1a and AOX1b genes, respectively. Total RNA was extracted from seedlings grown for the indicated number of days at 28°C and/or 4°C (e.g., 6/3 indicates 6 days at 28°C followed by 3 days at 4°C). Lanes 1-6 or lanes 7-12 indicate 6/0, 6/1, 6/2, 6/3, 6/4 and 6/7, respectively.

References

Ito, Y., D. Saisho, M. Nakazono, N. Tsutsumi and A. Hirai, Transcript levels of tandem-arranged alternative oxidase genes in rice are increased by low temperature. Gene. (in press)

McCaig, T.N, and R.D. Hill, 1977. Cyanide-insensitive respiration in wheat: cultivar differences and effects of temperature, carbon dioxide, and oxygen. Can. J. Bot. 55: 549-555.

McIntosh, L., 1994. Molecular biology of the alternative oxidase. Plant Physiol. 105: 781 -786.

Moynihan, M.R., A. Ordentlich and I. Raskin, 1995. Chilling-induced heat evolution in plants. Plant Physiol. 108: 995-999.

Purvis, A.C. and R.L. Shewfelt, 1993. Does the alternative pathway ameliorate chilling injury in sensitive plant tissues? Physiol. Plant. 88: 712-718.

Saisho, D., E. Nambara, S. Naito, N. Tsutsumi, A. Hirai and M. Nakazono. Characterization of the gene family for alternative oxidase from Arabidopsis thaliana. Plant Mol. Biol. (in press)

Stewart, C.R., B.A. Martin, L. Reding and S. Cerwick, 1990. Respiration and alternative oxidase in corn seedling tissues during germination at different temperatures. Plant Physiol. 92: 755-760.

Vanlerberghe, G.C. and L. Mcintosh, 1992. Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco. Plant Physiol. 100: 115-119.