Glycolysis and alcoholic fermentation are important for energy production during seed germination, especially in anaerobic environments (Perata and Alpi 1993). Alcoholic fermentation consists of two steps: the decarboxylation of pyruvate to acetaldehyde catalyzed by pyruvate decarboxylase (PDC) followed by the reduction of acetaldehyde to ethanol with the concomitant oxidation of NADH to NAD⁺ catalyzed by alcohol dehydrogenase (ADH) (Perata and Alpi 1993). This metabolic pathway is recognized as the principal catalytic pathway for recycling NAD⁺ to maintain glycolysis and ATP levels in the absence of oxygen. It is known that expression of the genes involved in glycolysis and alcoholic fermentation (e.g. ADH, PDC, glyceraldehyde-3-phosphate dehydrogenase, and enolase) are dramatically induced by anaerobiosis (Sachs et al. 1996).

Aldehyde dehydrogenases [aldehyde: NAD(P)⁺ oxidoreductases] (ALDHs) are a group of enzymes catalyzing the conversion of aldehydes to the corresponding acids. Mitochondrial ALDH protein (ALDH2) exhibits a high activity for the oxidation of acetaldehyde, an intermediate of alcoholic fermentation, and is thought to play an important role in the detoxification of acetaldehyde. In 1996, the first gene encoding a plant mitochondrial ALDH, the restorer of fertility 2 gene (rf2), was identified in maize (Cui et al. 1996). The rf2 gene was found to be a nuclear restorer gene of Texas-type cytoplasmic male sterility (cms-T). Subsequently, in tobacco, two Aldh genes (Aldh2a and Aldh2b) were identified, and the Aldh2a transcript and the ALDH2a protein were found to be present at high levels in floral tissues, especially stamens, pistils and pollen (op den Camp and Kuhlemeier 1997). Under anaerobic conditions, the expressions of Adh and Pdc are induced in tobacco leaves, but the expression of Aldh2a is not. Therefore, tobacco ALDH2a does not seem to function in anaerobic environments (op den Camp and Kuhlemeier 1997).

Rice has a higher tolerance for anaerobic conditions than does tobacco. The expressions of rice Adh1 (Xie and Wu 1989) and rice Pdc1 (Hossin et al. 1996) are induced in an anaerobic environment. To determine whether the expression of the rice mitochondrial ALDH gene (Aldh2a) is also induced under such conditions, we submerged seven-day-old seedlings grown under aerobic conditions for 12, 24 and 36 hours in the light or in the dark and then carried out Northern hybridization analysis using probes specific to Aldh2a. We also investigated the expressions of Adh1 and Pdc1. In the dark, the steady-state levels of the Aldh2a, Adh1 and Pdc1 mRNAs were dramatically increased by the submergence treatment (Fig. 1). When the submerged seedlings were transferred to an aerobic environment, the amounts of these transcripts decreased. This indicated that ALDH2a, like ADH1 and PDC1, is involved in anaerobic metabolism. In contrast, we observed no increase of transcripts of Aldh2a, Adh1 and Pdc1 under anaerobic conditions in the light. Yamada (1959) reported that rice plants photosynthesize in the light even when submerged. It seems likely that rice plants under submergence in the light are not in an

anaerobic state because of the presence of oxygen generated by photosynthesis. In fact, rice plants in the light are more tolerant to submergence than those in the dark (Yamada 1959).

op den Camp and Kuhlemeier (1997) reported that tobacco Aldh2a transcript levels did not increase during anaerobiosis in leaf tissue, and proposed that a pathway involving ALDH is not important for normal metabolism in tobacco leaves, even in anaerobiosis. In preliminary studies, we found that the expression of the Arabidopsis ALDH2a gene was also not enhanced under submerged conditions (data not shown). Thus, rice may have a greater ability than tobacco and Arabidopsis to detoxify the acetaldehyde that is produced during alcoholic fermentation. This may be due to higher levels of both ALDH and ADH in rice. To date, it is unclear whether the expression of the Aldh2 gene is enhanced under anaerobic conditions in anaerobiosis-intolerant graminaceous plants such as maize and wheat. It will be of interest to examine the expressions of these Aldh2 genes during submergence.

References

Cui X., R.P. Wise and P.S. Schnable, 1996. The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science 272: 1334-1336.

Hossin M.A., E. Huq, A. Grover, E.S. Dennis, W.J. Peacock and T.K. Hodges, 1996. Characterization of pyruvate decarboxylase genes from rice. Plant Mol. Biol. 31: 761-770.

op den Camp R.G.L. and C. Kuhlemeier, 1997. Aldehyde dehydrogenase in tobacco pollen. Plant Mol. Biol. 35: 355-365.

Perata P. and A. Alpi, 1993. Plant responses to anaerobiosis. Plant Sci. 93: 1-17.

Sachs M.M., C.C. Subbaiah and I.N. Saab, 1996. Anaerobic gene expression and flooding tolerance in maize. J. Exp. Bot. 47: 1-15.

Xie Y. and R. Wu, 1989. Rice alcohol dehydrogenase genes: anaerobic induction, organ specific expression and characterization of cDNA clones. Plant Mol. Biol. 13: 53-68.

Yamada N., 1959. Physiological basis of resistance of rice plantt against overhead flooding. Bulletin of the National Institute of Agricultural Sciences D (Japan). 8: 1-110. (in Japanese with abstract in English)