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The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Gic) sensor. However, the function of hexokinases as Gic sensors has not been clearly demonstrated in other plant species, including rice (Oryzn sativa). To investigate the functions of rice hexokinase isoforms, we characterized Osl-IXK5 and OsHXK6, which are evolutionarily related to AtHXKI. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHK5NNLS-GFP and OsHXK6NLS-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter:: luciferase fusion constructs, these two Osl-IXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zen niays) Rubisco small subunit (RbcS) and rice a-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to GIc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Gic sensors. [ABSTRACT FROM AUTHOR]