Plants have developed mechanisms for regulating the expressions of specialized genes to overcome potentially lethal environmental stresses. For example, plants respond to anaerobic conditions by switching carbohydrate metabolism from an oxidative pathway to a fermentative pathway. Anaerobiosis rapidly represses the synthesis of pre-existing proteins and induces the synthesis of new anaerobic proteins in plants. Most of the anaerobic proteins are enzymes involved in alcoholic fermentation [pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH)] and glycolysis (e.g. glycelaldehyde-3-phosphate dehydrogenase and aldolase) (Xie and Wu 1989, Sachs et al. 1996). The release of Ca²⁺ from mitochondria may play an important role in the elevation of cytosolic Ca²⁺, [Ca²⁺]_c under anaerobic conditions and may participate oxygen-deprivation signaling. For example, in maize seedlings, oxygen deprivation results in an elevation of the [Ca²⁺]_c level through intracellular Ca²⁺ flux, and ruthenium red (RR), which is thought to be a blocker of Ca²⁺ fluxes from organelles (Knight et al. 1992, Subbaiah et al. 1998), represses induction of expression of anaerobiosis-inducible genes such as ADH (Subbaiah et al. 1994).

In rice, however, it is unclear whether the level of cytosolic calcium affects the expressions of genes for anaerobic proteins. In this study, we investigated the role of [Ca²⁺]_c in the induction of the alcoholic fermentation genes, ADH1 and PDC1, under anaerobic conditions.

Seven-day-old aerobically-grown rice seedlings were submerged in water for 24 hours. Total RNA was isolated from seedlings submerged for 0 and 24 hours and Northern hybridization was performed using probes for ADH1 and PDC1. In response to the hypoxic treatment, the ADH1 and PDC1 levels dramatically increased, confirming that the submerged seedlings were suffering from oxygen deprivation (Fig. 1). Treatment of rice seedlings with 100 microM RR under hypoxic conditions decreased the steady-state level of ADH1 and PDC1 mRNAs to 40-50% of the level of untreated hypoxic seedlings (Fig. 1).

Treatment of hypoxic seedlings with 5 mM CaCl₂ in additon to 100 microM RR for 24 hours blocked the effect of RR on the expressions of ADH1 and PDC1 (Fig. 1, RRCa). CaCl₂ itself did not affect gene expression under hypoxic conditions (Fig. 1, Ca). These results suggested that the release of Ca²⁺ from intracellular Ca²⁺ stores is a physiological transducer of oxygen deprivation-signaling in rice, subsequently leading to increases of ADH1 and PDC1 mRNAs.

The results presented here provide several new insights concerning regulation of genes involved in alcoholic fermentation in rice. Transcripts of ADH1 and PDC1 were markedly induced in rice seedlings during the 24 hours of hypoxia, and this response was preceded by [Ca²⁺]_c elevation. This is the first demonstration that induction of PDC1 may be regulated by cytosolic calcium in plants. We suggest that hypoxia-induced elevation of [Ca²⁺]_c, which might be preceded by a release of Ca²⁺ from intracellular stores, is involved in an early step in the oxygen-deprivation-signaling pathway for ADH1 and PDC1.

References

Knight M.R., S.M. Smith and A.J. Trewavas, 1992. Wind-induced plant motion immediately increases cytosolic calcium. Proc. Natl. Acad. Sci. USA 89: 4967-4971.

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.

Subbaiah C.C., D.S. Bush and M.M. Sachs, 1994. Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells. Plant Cell 6: 1747-1762.

Subbaiah C.C., D.S. Bush and M.M. Sachs, 1998. Mitochondrial contribution to the anoxic Ca²⁺ signal in maize suspension-cultured cells. Plant Physiol. 118: 759-771.

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.