38. The expression of a gene encoding Ca2+-binding motif, EF-hand, is induced by sugars in rice embryos

Bioscience Center and Graduate School of Bioagriculture Sciences, Nagoya University, Nagoya, 464-8601 Japan

Sugars act not only as an important source of energy and carbon skeletons, but also as signaling molecules involved in developmental processes of higher plants. Both abundance and depletion of carbohydrates in cells can trigger the expression of "sugar-regulated" genes. In general, carbohydrate depletion enhances the expression of genes for photosynthesis, reserve mobilization, and export processes, whereas abundant carbon resources favor genes for storage and utilization (Koch 1996). Expression of the rice alpha-amylase RAmy3D gene is markedly up-regulated by sugar depletion and would be a lowcarbohydrate-level marker in rice embryos (Toyofuku et al.1998). To understand the role of sugars in developmental processes, gene markers to estimate cellular sugar status as well as RAmy3D are needed.

Using DNA microarray technology, it becomes possible to measure the levels of thousands of different mRNAs in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions (Howell 1999). To detect genes whose expression is under sugar-regulation, we performed this technique using rice embryos under various sugar conditions.

After mature dry seeds of rice (Oryza sativa cv. Notohikari) were incubated for six days in liquid Murashige-Skoog salt mixture containing 2 mg/l 2,4-D, embryos were treated under four conditions. The sample were: 1. immediately sampled for RNA extraction, 2. transferred to sugar-free medium for one day and sampled, 3. transferred to sugar-free medium for three days and sampled, and 4. transferred to sugar-free medium for one day, then incubated in a medium containing 90 mM glucose for two days and sampled. Poly A+ RNAs isolated from the embryos under all conditions were labeled with fluorescence Cy5 dye and hybridized to cDNAs containing 1265 rice EST clones. We obtained several clones whose expressions are regulated by high- or low-sugar levels. Two clones whose mRNAs accumulated in high-sugar condition were named OsSUR1 and OsSUR 2 (sugar up-regulated), and five clones whose mRNAs were reduced were named OsSDR1 - OsSDR5 (sugar down-regulated). Northern blot analysis of these clones showed that OsSUR1 was markedly up-regulated by high-sugar content (Fig. 1, lanes 1, 4). The expression patterns of OsSUR1 contrasts sharply with RAmy3D. This clone could be a useful marker for high carbohydrate level in rice embryos.

OsSUR1 cDNA contains a 414-bp open reading frame, that encodes a putative protein consisting of 137 amino acid residues with a predicted mol wt of 14,973. The deduced amino acid sequence was compared with protein sequences in the public databases with the Blastx algorithm. Although the deduced amino acid sequence did not show high homology to proteins with known functions, it contains two domains that have high homology to a Ca2+-binding domain named EF-hand motif.

The EF-hand motif consists of a Ca2+ chelation loop that bridges two alpha-helices, one at each end, yielding a helix-loop-helix structure. In the Ca2+-binding loop, six residues form an octahedral Ca2+-coordinating structure with oxygen. The oxygens at positions x, y, z, and -z (Fig. 2) are supplied by side chains of amino acid residues or by water of hydration. The oxygen at position -y comes from a carbonyl group in the main chain (Kretsinger 1996).

In plant cells, various physiological stimuli induce the elevation of cytosolic Ca2+ concentration. The protein product of pOsSUR1 may be involved in the regulation and/or

sensing of cellular sugar states mediated by Ca2+ signal-transduction processes. Further characterization of this protein for Ca2+-binding ability and detailed expression pattern will be needed.

This work was supported by MAFF grant (Rice Genome Project, GS-2218)


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