51. Subcellular localization of rice hexose kinase, OsHXK1

T. SUNAKO, A. MORITA and J. YAMAGUCHI

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

     Hexose kinases, which catalyze the production of hexose-6-phosphate from hexoses such as glucose or fructose, are involved in the initial metabolic step of glycolysis in cells, but experimental evidence suggests that they may also act as sugar sensors. Transient expression experiments using the RAmy3D gene promoter indicate a possible role of hexose kinases in the sugar-sensing mechanisms triggering repression of the RAmy3D gene in rice embryos (Umemura et a!. 1998). cDNAs encoding two hexokinase (OsHXKI, 2) and two fructokinase (OsFRKJ, 2) were cloned and characterized in rice. Coexpression of antisense OsHXKJ cDNA largely abolished sugar-repression of the RAmy3D gene. These results indicate that OsHXK1 is an intermediate of the sugar-signaling pathway that allows sugar-repression of the cz-amylase gene to occur.

     To determine the subcellular localization of OsHXK1, the chimeric gene of OsHXK1 fused to the coding sequence of a green fluorescent protein (GFP) was prepared. This construct (OsHXKI-GFP) is shown in Fig. 1. The fused protein was transiently expressed in onion epidermal cells using particle gun bombardment. After bombardment at pressure 1100 psi with PDS-1000/He (Bio-Rad), the onion was incubated at 26°C in the dark, and then imaged using a confocal laser scanning microscope (MRC 1024 Bio-Rad) with mitochondrion stained by the MitoTracker dye. GFP protein, as a control, was detectable in the nucleus and cytoplasm (Fig. 2D). Taken together, OsHXK1 appeared to be localized in the mitochondria.