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
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
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)
Howell S.B., 1999. DNA microarrays for analysis of gene expression. Mol.
Urol. 3: 295-300.
Koch K.E., 1996. Carbohydrate-modulated gene expression in plants. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 47: 509-540.
Kretsinger R.H., 1996. EF-hands reach out. Nat. Struct. Biol. 3:
Toyofuku K., T. Umemura and J. Yamaguchi, 1998. Promoter elements required
for sugar-repression of RAmy3D gene for alpha-amylase in rice.
FEBS Letters. 428: 275-280.