Role of the Rice HexokinasesOsHXK5andOsHXK6as Glucose Sensors    

Abstract

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, 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.In higher plants, sugars are known to function as signaling molecules in addition to being a fundamental source of fuel for carbon and energy metabolism. Indeed, sugars have been shown to regulate physiological processes during the entire plant life cycle, from germination to flowering and senescence, and to function during defense responses to biotic and abiotic

Stress-Induced Cytokinin Synthesis Increases Drought Tolerance through the Coordinated Regulation of Carbon and Nitrogen Assimilation in Rice

“…These results appear to be consistent with the role of Glc-6-P as a substrate for Suc synthesis via SPS, in contrast to the wild-type plants, where Suc was degraded. Our results show that increased HK activity in P SARK ::IPT plants is not associated with detrimental effects on photosynthesis and growth or in accelerating senescence, indicating that the differences between the regulation driven by changes in gene expression and protein activity might involve different signaling pathways, as suggested previously (Cho et al, 2009). …”

“…It has been shown that HK regulates photosynthesis, growth, and senescence. Increased levels of HK expression reduced growth and photosynthesis and induced senescence in tomato (Solanum lycopersicum) and rice plants (Dai et al, 1999;Cho et al, 2009) Supplemental Fig. S1): 1 (LOC_Os02g14110, aminotransferase), 1.2 (LOC_Os01g08270, aminotransferase), 1.3 (LOC_Os09g28050, aminotransferase), 1.4 (LOC_Os03g18810, aminotransferase), 1.5 (LOC_Os01g55540, aminotransferase), 2 (LOC_Os07g46460, Fd-GOGAT), 3 (LOC_Os02g20360, Tyr aminotransferase), 4 (LOC_Os04g52450, aminotransferase), 5 (LOC_Os08g01410, phosphate/phosphoenolpyruvate translocator), 6 (LOC_Os01g02020, acetyl-CoA acetyltransferase), 7 (LOC_Os07g42600, Ala aminotransferase), 8 (LOC_Os02g07160, glyoxalase family protein), 9 (LOC_Os04g14790, glycerol-3-phosphate dehydrogenase), 10 (LOC_Os02g41680, Phe ammonia-lyase), 11 (LOC_Os04g33300, amino acid kinase), 12 (LOC_Os02g37040, NTP3 [nitrate transporter]), 13 (LOC_Os04g45970, GDH), 14 (LOC_Os01g09000, glutaminyl-tRNA synthetase), 14.2 (LOC_Os07g07260, Gln-dependent NAD), 15 (LOC_Os02g38200, isocitrate dehydrogenase), 15.2 (LOC_Os01g46610, isocitrate dehydrogenase), 16 (LOC_Os02g10070, citrate synthase), 17 (LOC_Os03g05390, citrate transporter), 18 (LOC_Os04g31700, methylisocitrate lyase 2), 19 (LOC_Os10g08550, enolase), 20 (LOC_Os08g09200, aconitate hydratase), 20.2 (LOC_Os03g04410, aconitate hydratase), 21 (LOC_Os04g56400, Gln synthetase), 22.1 (LOC_Os06g16420, amino acid transporter), 22.2 (LOC_Os04g12499, amino acid transporter), 22.3 (LOC_Os06g36210, amino acid transporter), 22.4 (LOC_Os06g43700, amino acid transporter), 22.4 (LOC_Os11g09020, amino acid transporter), 22.5 (LOC_Os12g42850, amino acid permease), 22.6 (LOC_Os03g25869, amino acid permease), 22.7 (LOC_Os08g23440, amino acid permease), 22.8 (LOC_Os10g30090, amino acid permease), 22.9 (LOC_Os11g05690, amino acid permease), 22.10 (LOC_Os04g35540, amino acid transporter), 22.11 (LOC_Os08g03350, amino acid transporter), 22.12 (LOC_Os01g40410, amino acid transporter), 22.13 (LOC_Os01g63854, amino acid transporter), 22.14 (LOC_Os02g44980, amino acid transporter), 22.15 (LOC_Os02g09810, amino acid transporter), 22.16 (LOC_Os04g38680, amino acid transporter), 22.17 (LOC_Os05g50920, amino acid transporter), 22.18 (LOC_Os01g65000, ammonium transporter protein), 23.1 (LOC_Os02g34580, ammonium transporter protein), 23.2 (LOC_Os03g62200, ammonium transporter protein), 23.3 (LOC_Os04g43070, ammonium transporter protein), 24.1 (LOC_Os03g13240, peptide transporter PTR2), 24.2 (LOC_Os06g49250, peptide transporter PTR2), 24.3 (LOC_Os10g02240, peptide transporter PTR2), 24.4 (LOC_Os10g33170, peptide transporter PTR2), 24.5 (LOC_Os03g51050, peptide transporter PTR2), 25.1 (LOC_Os03g13274, NO 3 peptide transporter PTR2), 25.2 (LOC_Os10g42900, NO 3 peptide transporter PTR3), 26 (LOC_Os11g24450, 2-oxoglutarate/malate translocator), 27 (LOC_Os08g43170, hydroxymethylglutaryl-CoA synthase), and 28 (LOC_Os02g32490, acetyl-CoA synthetase).…”

“…Interestingly, HK roles in sugar sensing and signaling seem to be independent of the enzyme activity, as demonstrated using catalytically inactive OsHXK alleles of OsHXK5 and OsHXK6 in rice plants (Cho et al, 2009). This notion was further supported by the higher HK activity found in P SARK ::IPT plants that correlated well with an increase in Glc-6-P without detrimental effects on photosynthesis, growth, or senescence.…”

Differential Anoxic Expression of Sugar-Regulated Genes Reveals Diverse Interactions between Sugar and Anaerobic Signaling Systems in Rice

“…Especially, two hexokinases of rice (OsHXK5 and 6) also have been demonstrated to serve as glucose sensor and also their catalytically inactive forms still successfully delivered the sugar regulation, too [Cho et al (2009a), see Cho (2009b) for review]. However, the finding of the uncoupling of the metabolic and signaling activities of hexokinase unwittingly led to underscore the possible interaction of sugar signaling with the cellular metabolic status.…”

Impaired Function of the Tonoplast-Localized Sucrose Transporter in Rice, OsSUT2, Limits the Transport of Vacuolar Reserve Sucrose and Affects Plant Growth

“…The amplified product was digested with XbaI and XhoI and was cloned into the region between the CaMV35S promoter and sGFP of the JJ461 binary vector (Cho et al, 2009). The primers used were 5#-GCTCTAGACTCTTCTGAACTAACCCAAA-GAT-3# and 5#-CCCTCGAGATCGGTGACCTCTCCTCCTTGAT-3# (the underlined sequences indicate XbaI and XhoI sites, respectively).…”