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Hexokinase(HXK) plays a significant role in Glucose metabolism and Glucose signal transduction. Rice has ten HXK genes including OsHXK1~10. The hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. It was also proved that OsHXK5 and OsHXK6 are evolutionarily related to AtHXK1. To isolate rice hexokinases homologous to AtHXK1, Jung-Il Cho et al. first predicted the subcellular localization of OsHXKs using the TargetP program for determination of the presence of any N-terminal presequences, including putative mitochondrial targeting peptides (mTPs), and the predictNLS program for determination of NLSs. 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 29RRRRSKRChoEAEEERRRR44 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.
Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria.To test whether both OsHXKs could localize to both mitochondria and nuclei, we generated the OsHXK mutants OsHXK5DmTP and OsHXK6DmTP fused to GFP by deleting predicted mTPs . Interestingly, signals of OsHXK5DmTP-GFP and OsHXK6DmTP-GFP were detected strongly in nuclei and weakly in cytosols, as confirmed by colocalization studies with the SYTO nuclear dye, but were not localized to mitochondria. The quantitative analysis of GFP fluorescence intensity supported that GFP signals were mostly present in nuclei of maize protoplasts expressing OsHXK5DmTP-GFP or OsHXK6DmTP-GFP. 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. In control experiments, signals in maize protoplasts expressing only GFP were observed strongly both in the cytosol and in the nucleus .
These OsHXKs including OsHXK6 retain a dual-targeting ability to mitochondria and nuclei. Some results also provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors. 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. To further examine function of the predicted NLSs, The NLSs of OsHXK5 and OsHXK6 to GFP were fused, respectively, thereby generating OsHXK5NLS-GFP and OsHXK6NLS-GFP. In transient expression assay using maize protoplasts, signals of the GFP fusion products were predominantly localized in nuclei, indicating that the NLSs of OsHXK5 and OsHXK6 are functional nuclear targeting sequences in vivo. The quantitative analysis of GFP fluorescence intensity again supported that GFP signals were mostly detected in nuclei of maize protoplasts expressing OsHXK5NLS-GFP or OsHXK6NLSGFP. To confirm this result, OsHXK5DNLS-GFP and OsHXK6DNLS-GFP were constructed by deleting the NLSs of OsHXK5 and OsHXK6. Consistently, transient expression assays revealed that both GFP fusion products were primarily localized to mitochondria. By deleting both mTP and NLS of the two OsHXKs, OsHXK5DmTPDNLS-GFP and OsHXK6DmTPDNLS-GFP were generated. These two GFP fusion products were mainly detected in cytosols. Our results suggest that these OsHXKs are targeted to mitochondria and also possibly to nuclei, raising the possibility that OsHXK5 and OsHXK6 are functional homologues of the Arabidopsis Glc sensor AtHXK1.
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. 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. To examine a possible role for the two rice hexokinase isoforms OsHXK5 and OsHXK6 as Glc sensors, Whether either OsHXK could complement the Arabidopsis gin2-1 was tested. To individually express OsHXK5, OsHXK6, and the catalytically inactive mutant alleles OsHXK5-G113D, OsHXK5-S186A, OsHXK6- G112D, and OsHXK6-S185A, each cDNA was placed under the control of the CaMV35S promoter. The resulting constructs were transformed into the gin2-1 mutant by the floral-dip method. More than 10 independent transgenic lines for each construct were selected on the basis of hygromycin resistance. Expression levels of transgenes in the transformed plants were measured by RNA gel-blot analysis. As a result, homozygous lines of two independent transgenic plants for each OsHXK with relatively high transgene expression were used in subsequent analyses.Both wild-type and all transgenic plants expressing OsHXK5, OsHXK6, or mutant alleles significantly suppressed expression of these photosynthetic genes in response to high Glc treatment.
The expression of OsHXK5, OsHXK6, or their mutant alleles complemented the glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. It has been reported in Arabidopsis that the sugar sensing and signaling functions of AtHXK1 do not depend on its Glc phosphorylation activity. To uncouple the sugar sensing and signaling activities from Glc phosphorylation, we employed a targeted mutagenesis experiment to generate catalytically inactive mutants of the candidate rice Glc sensors OsHXK5 and OsHXK6. 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. These mutant alleles were referred to as OsHXK5-G113D, OsHXK5-S186A, OsHXK6-G112D, and OsHXK6-S185A, according to their mutation sites. To 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 OsHXK5 and OsHXK6 were able to grow on selection medium containing Glc as the sole carbon source , yeast cells transformed with the OsHXK mutant alleles or the empty pDR196 vector did not grow on the selection medium. In the control experiment, all transformed yeast cells grew on the Gal-containing medium. In addition, expressions of HXK5, HXK6, and their catalytically inactive mutant alleles were confirmed by reverse transcription (RT)-PCR analysis. These findings demonstrate that the mutant OsHXKs lacked catalytic activity.
[[Figure 1. Transformation of catalytically inactive mutants for OsHXK5 and OsHXK6 into a yeast hexokinase mutant. A, Schematic representation of OsHXK5 and OsHXK6 and their catalytically inactive mutation sites. Mitochondrial targeting signals and NLSs are indicated as white (M) and black (N) rectangles. 1, 2, and A indicate the conserved phosphate 1, 2, and adenosine interaction regions within the ATP-binding site, respectively. The region S indicates the conserved sugar-binding domain. B, Complementation of the hexokinase-deficient yeast triple mutant YSH7.4-3C (hxk1, hxk2, glk1) with OsHXK5, OsHXK6, and their catalytically inactive mutant alleles. The transformed colonies were streaked on the SD-Ura medium (synthetic defined minimal medium lacking uracil) containing 2% D-Glc as a sole carbon source and grown for 3 d at 30�C (top). The YSH7.4-3C mutant strain transformed with the pDR196 vector was used as a control. As control experiment, YSH7.4-3C mutant strains transformed with pDR196, OsHXK5, OsHXK6, and their catalytically inactive mutant alleles were streaked on the SD-Ura medium containing 2% D-Gal (middle). Expression levels of HXK5, HXK6, and their mutant alleles in these strains were measured by RT-PCR analysis (bottom).]]
1. Jung-Il Cho2, Nayeon Ryoo2, Joon-Seob Eom, et al (2009) Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors1[C][W]. Plant Physiology 149:745-759 2. Jung-Il Cho, Nayeon Ryoo, Seho Ko Sang-Kyu Lee, et al (2006) Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.). Planta 224: 598–611