Sugars are an important source of energy and carbon skeletons for plant growth and development, but they also act as signaling molecules affecting developmental and metabolic processes. Hexokinase, catalyzing the production of hexose-6-phosphate from hexose such as glucose and fructose, is involved in the initial metabolic step of glycolysis in cells growing on free sugars, but experimental evidence suggests that they may also act as sugar sensor (Leon and Sheen, 2003). Indeed, Transient expression experiments using promoter of the RAmy3D, a rice Alpha-amylase gene, indicate a possible role of hexokinase in the sugar-sensing mechanisms triggering repression of the RAmy3D in rice embryos (Umemura et al., 1998). The database searching o rice genomic sequences by using the predicted amino acid alignment encoded in the rice hexokinase showed that at least 7 homologous genes designated OsHXKs exist in the genome. Among them, OsHXK1 (GenBank accession number D46661) is most likely to be a candidate of sugar sensor (Guglielminetti et al., 2000). In order to investigate molecular function of rice hexokinase, we have characterized loss-of-function mutant for OsHXK1 gene in terms of sugar regulation of the RAmy3D.

We isolated a loss-of-function mutant from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. The insertions of the transposable element into the OsHXK1 gene can be detected by PCR-based screening. For efficient screening, we adopted a pool sampling system in which plants were arranged in a three-dimensional matrix as described previously (Kumar and Hirochika, 2001). Finally, a candidate line, ND0370, designated hxk1-1, was isolated. Tos17 was inserted into the exon 4, which corresponds to the protein-coding region (Fig. 1A). We directly studied the presence of the transcript for the OsHXK1 by RNA gel blot and immunoblot analyses, indicating that no transcript (Fig. 1B) and protein (data not shown) were detectable. These results indicate that the Tos17 insertion leads to loss-of-function mutation of the OsHXK1.

Sugar response in the hxk1-1 mutant was examined. Isolated embryos from wild-type, Nipponbare were treated with up to 90 mM glucose for 4 days and then harvested for RNA preparation. RT-PCR was performed to quantify RAmy3D transcript (Fig. 2A). The tran-

script gradually reduced and disappeared at the application of 60 mM glucose, indicating that the RAmy3D transcription is strictly under glucose control. Interestingly, Actin used as a control for normalization of RNA amount was also slightly under glucose regulation. Similar glucose repression to the wild-type plant was also observed in the hxk1-1 mutant (Fig. 2B), suggesting that the knock-out mutation of OsHXK1 does not affect sugar response in terms of the RAmy3D transcription. This result is inconsistent with the present working hypothesis that the OsHXK1 protein is responsible as a sugar sensor. Since the OsHXK genes consist as a multigene family, it is likely that the other family gene(s) complements functions of the OsHXK1. Further studies will be required to clarify the molecular functions of OsHXK protein family in rice.

Acknowledgement

We gratefully thank the NIAS for providing the mutant line ND0370.

References

Leon, P. and J. Sheen, 2003. Sugar and hormone connections. Trends Plant Sci. 8: 110-116.

Guglielminetti, L., P. Perata, A. Morita, E. Loreti, J. Yamaguchi and A. Alpi, 2000. Characterization of isoforms of hexose kinases in rice embryo. Phytochem. 53: 195-200.

Kumar, A. and H. Hirochika, 2001. Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci. 6: 127-134.

Umemura, T., P. Perata, Y. Futsuhara and J. Yamaguchi, 1998. Sugar sensing and Alpha-amylase gene repression in rice embryos. Planta 204: 420-428.