Os05g0522500

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Annotated Information

Function

the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5DmTP-GFP and OsHXK6DmTP-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 OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) 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 Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.

Expression

OsHXK5 cDNA full length 2058bp, contains nine exons encoding a 507 amino acid composition of hexokinase, OsHXK5 contain nuclear localization signal peptide, mitochondrial targeting signal peptide and sugar binding domain.These analyses revealed that of the 10 OsHXKs, OsHXK5 and OsHXK6 had a predicted N-terminal mTP, 1MGKAAAVGTAVVVAAAVGVAVVLA24 for OsHXK5 and 1MGKGTVVGTAVVVCAAAAAAVGVAVVVS28 for OsHXK6. These analyses also indicated that both proteins contained a predicted NLS, 25RRRRRRDLELVEGAAAERKRK45 for OsHXK5 and 29RRRRSKRCho.EAEEERRRR44 for OsHXK6, within their N-terminal domains. Together with our previous phylogenetic analyses of rice HXKs (Cho et al., 2006a), these data suggest that OsHXK5 and OsHXK6 are evolutionarily closely related to the Arabidopsis Glc sensor AtHXK1.Results of subcellular localization experiments showed that signals of OsHXK5-GFP and OsHXK6-GFP fusion proteins were primarily colocalized with the mitochondrial dye MitoTracker in maize protoplasts.We confirmed that OsHXK5DmTP-GFP (79.0 kD) and OsHXK6DmTPGFP(80.1 kD) fusion proteins were effectively produced in vivo using protein-gel blot analysis with an anti-GFP antibody (Fig. 2G). In control experiments,signals in maize protoplasts expressing only GFP were observed strongly both in the cytosol and in the nucleus.

Evolution

OsHXK5 is inactive mutants of the candidate rice Glc sensors.In the mutant alleles, ATP binding was eliminated by mutating the conserved Gly (G) in the phosphate 1 domain of the ATP-binding site to Asp (D) and phosphoryl transfer was prevented by mutating the conserved Ser (S) in the sugar-binding domain to Ala (A; Kraakman et al., 1999; Moore et al., 2003; Cho et al.,2006a). These mutant alleles were referred to as OsHXK5-G113D, OsHXK5-S186A, OsHXK6-G112D, and OsHXK6-S185A, according to their mutation sitesTo determine whether enzyme catalytic activity was abolished in the mutant alleles, the individual cDNA clones were tested to complement the yeast triple mutant YSH7.4-3C (hxk1, hxk2, glk1), which lacks endogenous hexokinase activity. While yeast cells transformed with wild-type cDNAs of OsHXK5and OsHXK6 were able to grow on selection mediumcontaining Glc as the sole carbon source (Cho et al.,2006a), yeast cells transformed with the OsHXK mutant alleles or the empty pDR196 vector did not grow on the selection medium (Fig. 4B, top). In the control experiment, all transformed yeast cells grew on the Gal-containing medium

Reference

1.Jung-Il Cho;Nayeon Ryoo;Joon-Seob Eom;Dae-Woo Lee;Hyun-Bi Kim;Seok-Won Jeong;Youn-Hyung Lee;Yong-Kook Kwon;Man-Ho Cho;Seong Hee Bhoo;Tae-Ryong Hahn;Youn-Il Park;Ildoo Hwang;Jen Sheen;Jong-Seong Jeon.Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors.Plant Physiology.2009, 149(2): 745-759 2. Jung-Il Cho;Nayeon Ryoo;Seho Ko;Sang-Kyu Lee;Junok Lee;Ki-Hong Jung;Youn-Hyung Lee;Seong Hee Bhoo;Joris Winderickx;Gynheung An;Tae-Ryong Hahn;Jong-Seong Jeon.Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.).Planta, 2006, 224(3): 598-611

Labs working on this gene

1.Plant Metabolism Research Center and Graduate School of Biotechnology, Kyung Hee University, Yongin 446–701, Korea (J.-I.C., N.R., J.-S.E., D.-W.L., H.-B.K., Y.-K.K., M.-H.C., S.H.B., T.-R.H., J.-S.J.) 2.Department of Biology, Chungnam National University, Daejeon 305–764, Korea (S.-W.J., Y.-I.P.) 3.Department of Horticultural Biotechnology, Kyung Hee University, Yongin 446–701, Korea (Y.-H.L.) 4.Department of Life Sciences, Pohang University of Science and Technology, Pohang 790–784, Korea (I.H.) 5. Department of Molecular Biology,Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114 (J.S.)