Purification and Characterization of S-Adenosyl-L-Methionine: Desoxyhemigossypol-6-O-Methyltransferase from Cotton Plants. An Enzyme Capable of Methylating the Defense Terpenoids of Cotton

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.     

In plants, 3-o-methylglucose is phosphorylated by hexokinase but not perceived as a sugar

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man.     

Abscisic acid inhibition of radicle emergence but not seedling growth is suppressed by sugars

Low concentrations of sugars altered the sensitivity of seed germination to inhibition by exogenous abscisic acid (ABA). Germination of wild-type and ABA-insensitive (abi) Arabidopsis seeds was assayed on media containing ABA and a variety of sugars and sugar alcohols. The inhibitory effects of ABA were strongly repressed in the presence of 15 to 90 mM glucose (Glc), sucrose, or fructose, but not by comparable concentrations of sorbitol or mannitol. Several features of the response to Glc are inconsistent with a purely nutritional effect: The optimal sugar concentration is low and differs between the wild type and the abi mutants. Furthermore, Glc suppression of ABA inhibition is light dependent and limited to the process of radicle emergence.     

Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism

We characterized the function of the rice cytosolic hexokinase Os HXK7(Oryza sativa Hexokinase7),which is highly upregulated when seeds germinate under O_2-deficient conditions. According to transient expression assays that used the promoter:luciferase fusion construct,Os HXK7 enhanced the glucose(Glc)-dependent repression of a rice a-amylase gene(RAmy3D) in the mesophyll protoplasts of maize,but its catalytically inactive mutant alleles did not. Consistently,the expression of Os HXK7,but not its catalytically inactive alleles,complemented the Arabidopsis glucose insensitive2-1(gin2-1) mutant,thereby resulting in the wild type characteristics of Glc-dependent repression,seedling development,and plant growth. Interestingly,Os HXK7-mediated Glc-dependent repression was abolished in the O_2-deficient mesophyll protoplasts of maize. This result provides compelling evidence that Os HXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions,but its signaling role is suppressed when O_2 is deficient. The germination of two null Os HXK7 mutants,oshxk7-1 and oshxk7-2,was affected by O_2 deficiency,but overexpression enhanced germination in rice. This result suggests the distinct role that OsH XK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O_2-deficient rice.     

The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.     

A role for F-actin in hexokinase-mediated glucose signaling

HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.     

The role of sugars and hexose phosphorylation in regulating the re-establishment of desiccation tolerance in germinated radicles of Cucumis sativa and Medicago truncatula

This study examined whether sugars and hexose phosphorylation participate in the regulatory mechanisms that induce desiccation tolerance (DT) in seeds. In germinated desiccation-sensitive radicles of Cucumis sativa and Medicago truncatula , DT was re-established by an osmotic treatment using polyethylene glycol (PEG) for several days. In cucumber, Glc kinase activity (EC 2.7.1.1) transiently peaked early during PEG incubation before the induction of DT in protruded radicles, whereas Fru kinase activity (EC 2.7.1.4) increased progressively during the re-establishment of DT. Glucosamine (GAM, a competitive inhibitor of HXK) was able to repress the PEG-induced DT in both species, whereas hexose and poorly metabolizable hexose analogues had no effect. GAM was effective in repressing DT only early during PEG incubation, indicating that this effect is transient. Both Glc and Man fully rescued GAM-inhibited DT. PEG-induced accumulation of Suc was not affected by GAM. Isocitrate lyase (ICL) gene expression, which is known to be regulated by hexoses, responded to the re-establishment of DT and GAM feeding. In cucumber, expression of ICL was repressed after 6 h of PEG incubation whereas GAM feeding led to ICL de-repression. When GAM could no longer inhibit the re-establishment of DT, neither were steady-state levels of ICL influenced. The implication of HXK as a catalytic regulator and sugar-sensor in DT is discussed.     

Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells

We present evidence that the activity of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.     

Glucose delays seed germination in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Here we report that glucose delays germination of Arabidopsis thaliana (L.) Heynh. seeds at concentrations below those known to inhibit early seedling development. This inhibition acts on embryo growth and is independent of hexokinase (HXK) function. Hormones and hormone inhibitors were applied to the germination media and several hormone biosynthesis and signalling mutants were tested on glucose media to investigate a possible role of abscisic acid (ABA), gibberellin and ethylene in the glucose-induced germination delay. Results indicate that the germination inhibition by glucose cannot be antagonized by ethylene or gibberellin and is independent of the HXK1/ABA/ABI4 signalling cascade. These findings suggest that there is a separate regulatory pathway independent of ABI2/ABI4/ABI5. Thus, in a relatively short time frame sugars utilize different signalling cascades to inhibit germination and post-germination growth, underlining the complexity of sugar responses.Abbreviations ABA Abscisic acid - ABI ABA insensitive - ACC 1-Aminocyclopropane-1-carboxylic acid - BR Brassinosteroid - CAB Chlorophyll a/b-binding protein - FUS3 Fusca3 - GA Gibberellin - GA ₃ Gibberellic acid - HXK Hexokinase - LEC1 Leafy cotyledon1 - RBCS Ribulose-1,5-bisphosphate carboxylase small subunit - WT Wild type     

Phytochrome E Controls Light-Induced Germination of Arabidopsis

Germination of Arabidopsis seeds is light dependent and under phytochrome control. Previously, phytochromes A and B and at least one additional, unspecified phytochrome were shown to be involved in this process. Here, we used a set of photoreceptor mutants to test whether phytochrome D and/or phytochrome E can control germination of Arabidopsis. The results show that only phytochromes B and E, but not phytochrome D, participate directly in red/far-red light (FR)-reversible germination. Unlike phytochromes B and D, phytochrome E did not inhibit phytochrome A-mediated germination. Surprisingly, phytochrome E was required for germination of Arabidopsis seeds in continuous FR. However, inhibition of hypocotyl elongation by FR, induction of cotyledon unfolding, and induction of agravitropic growth were not affected by loss of phytochrome E. Therefore, phytochrome E is not required per se for phytochrome A-mediated very low fluence responses and the high irradiance response. Immunoblotting revealed that the need of phytochrome E for germination in FR was not caused by altered phytochrome A levels. These results uncover a novel role of phytochrome E in plant development and demonstrate the considerable functional diversification of the closely related phytochromes B, D, and E.     

Relationship of Endo-[beta]-D-Mannanase Activity and Cell Wall Hydrolysis in Tomato Endosperm to Germination Rates

The endosperm tissue enclosing the radicle tip (endosperm cap) governs radicle emergence in tomato (Lycopersicon esculentum Mill.) seeds. Weakening of the endosperm cap has been attributed to hydrolysis of its mannan-rich cell walls by endo-[beta]-D-mannanase. To test this hypothesis, we measured mannanase activity in tomato endosperm caps from seeds allowed to imbibe under conditions of varying germination rates. Over a range of suboptimal temperatures, mannanase activity prior to radicle emergence increased in accordance with accumulated thermal time. Reduced water potential delayed or prevented radicle emergence but enhanced mannanase activity in the endosperm caps. Abscisic acid did not prevent the initial increase in mannanase activity, although radicle emergence was markedly delayed. Sugar composition and percent mannose (Man) content of endosperm cap cell walls did not change prior to radicle emergence under any condition. Man, glucose, and other sugars were released into the incubation solution by endosperm caps isolated from intact seeds during imbibition. Pregerminative release of Man was suppressed and the release of glucose was enhanced when seeds were incubated in osmoticum or abscisic acid; the opposite occurred in the presence of gibberellin. Thus, whereas sugar release patterns were sensitive to environmental and hormonal factors affecting germination, neither assayable endo-[beta]-D-mannanase activity nor changes in cell wall sugar composition of endosperm caps correlated well with tomato seed germination rates under all conditions.     

Hexokinase as a sugar sensor in higher plants.

The mechanisms by which higher plants recognize and respond to sugars are largely unknown. Here, we present evidence that the first enzyme in the hexose assimilation pathway, hexokinase (HXK), acts as a sensor for plant sugar responses. Transgenic Arabidopsis plants expressing antisense hexokinase (AtHXK) genes are sugar hyposensitive, whereas plants overexpressing AtHXK are sugar hypersensitive. The transgenic plants exhibited a wide spectrum of altered sugar responses in seedling development and in gene activation and repression. Furthermore, overexpressing the yeast sugar sensor YHXK2 caused a dominant negative effect by elevating HXK catalytic activity but reducing sugar sensitivity in transgenic plants. The result suggests that HXK is a dual-function enzyme with a distinct regulatory function not interchangeable between plants and yeast.     

Regulation of carbon and nitrogen metabolisms in rice roots by 2-oxoglutarate at the level of hexokinase

Feeding experiments were designed, to investigate the role of 2-oxoglutarate (2-OG) in regulation of carbon and nitrogen metabolisms in non-photosynthetic tissues of rice ( Oryza sativa L.), and enzyme activities involved in the metabolisms as well as contents of several relating metabolites were determined in the roots. The enhancement of 2-OG level by feeding 2-OG or metabolizable sugars [sucrose (Suc) or glucose (Glc)], rather than by feeding non-metabolizable carbon sources (mannose or mannitol), led to increase in enzyme activities, including hexokinase (HXK, EC 2.7.1.1), nicotinamide adenine dinucleotide phosphate (NADP)⁺-dependent isocitrate dehydrogenase (NADP⁺-ICDH, EC 1.1.1.42), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), glutamine synthetase (GS, EC 6.3.1.2) and the reduced form of nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14). In addition, the increase in ammonium uptake, glutamine and glutamate (Glu) as well as the decrease in soluble carbohydrates were observed. The effects of feeding 2-OG or metabolizable sugars were reversed by feeding of N- acetyl-glucosamine (NAG; a HXK inhibitor). The decreased 2-OG level by the feeding of NAG alone led to increase in soluble carbohydrates and decrease in the enzyme activities, ammonium uptake as well as Glu content. The effects of NAG were reversed by supply of 2-OG, Suc and Glc. These results suggest that nitrogen uptake and assimilation as well as their related carbohydrate metabolism in rice roots were regulated in coordination by 2-OG level, and HXK activity was involved in the regulation of 2-OG.