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First published online January 23, 2003; 10.1104/pp.010538 Plant Physiol, February 2003, Vol. 131, pp. 824-837 In Plants, 3-O-Methylglucose Is Phosphorylated by Hexokinase But Not Perceived as a Sugar1Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, and Université Joseph Fourier, Unité Mixte de Recherche 5019 Physiologie Cellulaire Végétale, 17 rue des Martyrs, 38054 Grenoble cedex 9, France (S.C., M.G., A.E., C.R.); Centre National de la Recherche Scientifique, Unité Propre de Recherche 5301 Centre de Recherches sur les Macromolécules Végétales, Boîte Postale 53, 38041 Grenoble cedex 9, France (A.H.); and Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Unité Mixte de Recherche 619 Biotechnologie et Physiologie Végétales, Boîte Postale 81, 33883 Villenave d'Ornon cedex, France (D.B.R., P.R., R.M.B.)
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.
In plants, the supply of
carbohydrates to growing organs can vary greatly with plant development
and external conditions. Changing the sugar supply modulates cell
metabolism and gene expression (Koch, 1996 The mechanisms used by cells to sense sugars have been
extensively studied in yeast, animal, and plants, and there is
increasing evidence that sugar-sensing mechanisms are partly conserved
in eukaryotes (Jang and Sheen, 1997 These somewhat conflicting data prompted us to reinvestigate the
effects and fate of 3-OMG in heterotrophic plant cells, asking the
following questions: Is 3-OMG phosphorylated by HXK, is it a
respiratory substrate, and to what extent can it be used to study sugar
signaling? To answer these questions, we characterized the biochemical
and physiological responses of excised maize root tips and isolated
plant cells when 3-OMG was substituted for Glc in the culture medium.
For root tips, the time courses of metabolite concentrations (sugars,
ester-phosphates, adenine nucleotides [AdNs], and amino acids) were
monitored in vivo using 13C and
31P NMR along with respiration rate measurements.
The effects of 3-OMG on protein contents, proteolytic activities, and
RSIP amounts were observed after a 48-h incubation period, after which
time the changes in enzymatic activities and metabolic contents are clearly marked and yet fully reversible (Brouquisse et al.,
1991
Effects of 3-OMG on Protein Content, Proteolytic Activities, and RSIP Expression Figure 1, A and B, report the changes in total protein content and endopeptidase activity (measured against azocasein) that occurred in maize root tips incubated for 48 h in the presence of either 200 mM Glc or increasing concentrations of 3-OMG, or in the absence of any sugar. In comparison with the control (freshly excised root tips incubated for 4 h in 200 mM Glc), sugar starvation triggered a 50% decrease in protein content and a 5-fold increase in endopeptidase activity, whereas small changes occurred in Glc-fed root tips. In the presence of 3-OMG, protein content and endopeptidase activity changed nearly as much as in sugar-starved root tips. However, high concentrations of 3-OMG in the incubation medium (100 and 200 mM) led to slightly higher protein content and lower endopeptidase activity than lower concentrations (10 and 50 mM). Because of the similar effect of 100 and 200 mM 3-OMG on proteolysis and protease induction, 100 mM was routinely used. The expression of the RSIP and its contribution to endopeptidase activity were estimated through western-blot and immunoprecipitation experiments, using polyclonal antibodies raised against RSIP (Fig. 1C). RSIP was barely present in control root tips or root tips incubated with 200 mM Glc, where it represented 18% and 28%, respectively, of the total endopeptidase activity in vitro. In root tips, either starved or incubated with 100 mM 3-OMG, the increase in total endopeptidase activity was related to an increase in the amount of RSIP, the activity of which represents respectively 82% and 76% of the endopeptidase activity. From these data, the induction factors for RSIP were calculated to be 1.7, 20, and 17, respectively, in Glc-fed, -starved, and 3-OMG-fed root tips. Incubation of root tips for 48 h with 100 mM 6-DOG triggered a decrease in protein, and an increase in endopeptidase activity and RSIP expression, similar to that observed for starved root tips (data not shown). Therefore, at this stage, the biochemical response of the plant cells to 3-OMG is similar to the responses observed for starved or 6-DOG-fed cells. Our results are consistent with the concept that 3-OMG does not repress the sugar starvation response because, although transported into eukaryotic cells, it is not metabolized. To check this point, we investigated the effects of 3-OMG on respiratory metabolism in maize root tips.
Effects of 3-OMG on Growth and The growth of excised maize root tips incubated with 3-OMG was
very limited compared with that of Glc-fed tips and similar to the
growth observed in sugar starvation (Fig.
2). The slight increase of mass and the
related increase in length, observed during the first 15 to 20 h
of incubation, are attributed to the consumption of endogenous sugars
and to water uptake resulting from the increase in cell osmolarity
(Brouquisse et al., 1991
After a 48-h incubation period, the respiration rate of 3-OMG-fed root tips dropped by 75% and was close to that of carbon-starved root tips (Table I). Thus, 3-OMG was not able to sustain cell metabolism, and 3-OMG-fed root tips became rapidly carbon starved. AdN contents of maize root tips incubated for 48 h in media of different composition were measured (Table I). The total AdN content decreased in 3-OMG-treated root tips as in starved root tips, but the ATP to ADP ratio and AEC remained high (around 5 and 0.90, respectively), as in the control. The addition of 10 mM Pi in the incubation medium did not change either the total AdN content or the AEC value significantly. Together, these data show that 3-OMG triggers the same effects as starvation on common indicators of growth and respiratory metabolism.
Effects of 3-OMG on the Metabolite Contents 13C and 31P in Vivo NMR Study 13C and 31P NMR spectroscopy were used to monitor the time course changes in major pools of metabolites in excised root tips. Figures 3 and 4 display typical 13C and 31P spectra obtained with root tips successively fed with 200 mM Glc, 100 mM 3-OMG, and 200 mM Glc again. 13C NMR (Fig. 3) was used to measure the amounts of Suc, 3-OMG, and Asn, a transient marker of protein degradation (Brouquisse et al., 1992 ).
31P NMR (Fig. 4) was used to analyze the most
abundant phosphorylated compounds, such as Glc-6-phosphate (G6P);
phosphorylcholine (P-chol), which is a marker of membrane degradation
(Roby et al., 1987); cytoplasmic phosphate (cyt-Pi) and
vacuolar phosphate (vac-Pi); UDP-Glc (UDPG); and nucleoside
triphosphates (NTPs). The growth of root tips was taken into account
for the quantification of metabolites throughout the
experiment.
 1 (about 120 mM) and then
stabilized (Fig. 5A). As previously
observed in sugar-starved root tips (Saglio and Pradet,
1980; Brouquisse et al., 1991), endogenous Suc
and Fru were totally consumed within 15 h. Unexpectedly, Glc was
also totally consumed within 15 h after the transition to 3-OMG
(data not shown) instead of stabilizing at 30% of its initial value
(Brouquisse et al., 1991). Such a decrease in Glc
content may be explained by the stimulation of Glc efflux in response
to 3-OMG uptake (Gogarten and Bentrup, 1983).
Respiration rate dropped quickly by 50% after the transition to 3-OMG,
but decreased at a lower rate beyond 10 h (Fig. 5B). After a
transient increase, the concentrations of NTP (Fig. 5B) and UDPG (Fig.
5D) continuously decreased to reach about 60% of their initial values
after 48 h of 3-OMG treatment. Cyt-Pi concentration evolved with
kinetics similar to NTP (Fig. 5D), whereas the Cyt-Pi resonance
chemical shift indicated cytoplasmic acidification (data not shown). In
the same time, the concentrations of P-chol, vac-Pi, and Asn, three
common markers of intracellular autophagy (Roby et al.,
1987; Genix et al., 1990), increased during the
incubation with 3-OMG (Fig. 5C). To summarize, in 3-OMG-fed root tips,
the decrease in respiration rate and in NTP and UDPG concentrations, and the steady accumulation of P-chol, vac-Pi, and Asn (Fig. 5), added
to the protein decrease and the induction of proteolytic activities
(Fig. 1), characterize carbon starvation (Journet et al.,
1986; Brouquisse et al., 1991; Yu,
1999).
1 (115 mM) before returning to
standard levels. In the same time, NTP, cyt-Pi, and UDPG pools (Fig. 5,
B and D) gradually returned to levels close to the control. The
decrease in Asn and P-chol contents (Fig. 5C) matches the cessation of
protein and lipid remobilization.
Accumulation of an Unusual Phosphomonoester In control root tips, the chemical shift of the G6P resonance is 4.3 ppm (Fig. 4; Saint-Ges et al., 1991;
Roscher et al., 1998; Brouquisse et al.,
2001). Upon replacement of Glc by 3-OMG in the external medium,
the 4.3-ppm resonance first decreased by 15% (Fig. 5D). Then, the
intensity of a resonance progressively centered at 4.2 ppm increased
dramatically (Figs. 4 and 5D). This resonance, which arises from at
least one phosphomonoester, might have been attributed to G6P on a
chemical shift basis because this parameter is very sensitive to pH
(Robitaille et al., 1991). However, the intensity of
this 4.2-ppm resonance evolved quasi-linearly as a function of time
(Fig. 5D), revealing a quite unusual rate of accumulation for G6P. When
Glc was substituted for 3-OMG in the external medium, not only did the
accumulation of this molecule cease, but the intensity of the 4.2-ppm
resonance decreased quickly down to a point where a resonance centered
at 4.3 ppm could be easily distinguished (Fig. 4).
Such an increase in the G6P resonance was unexpected for several
reasons. First, it is known that G6P content decreases in sugar-starved
cells (Roby et al., 1987; Brouquisse et al.,
2001). Second, variations in UDPG and G6P concentrations are
usually correlated. The phosphoglucomutase and UDPG pyrophosphorylase enzymes generally function near equilibrium and stabilize the G6P to
UDPG ratio (ap Rees, 1988; Roscher et al.,
1998). This is clearly not the case in 3-OMG-fed root tips
(Fig. 5D). Third, demethylation of 3-OMG, which could provide Glc to
HXK to generate G6P, is very limited in vivo (Csaky and Wilson,
1956; see 14C observations below). These
considerations led us to hypothesize that, during 3-OMG treatment, the
accumulated phosphomonoester was not G6P but possibly a phosphorylated
form of 3-OMG.
Identification of 3-OMG-6P in Tissue and Cell Extracts 31P NMR Analysis of Maize Root Tip Acid Extracts To test the hypothesis of 3-OMG phosphorylation, we proceeded toward the formal identification of 3-OMG-phosphate (3-OMG-P) in acid and ethanolic extracts of excised maize root tips and suspension-cultured cells incubated with 3-OMG for 48 h by analyzing 31P and 13C NMR spectra of these extracts. Figure 6 presents the spectral region of the phosphomonoester resonances in a 31P NMR spectra of plant material. In the spectrum of the control extract, G6P is characterized by the two resonances of the - and  -anomers, at 4.38 and 4.34 ppm,
respectively. With sugar-starved root tips, the starvation symptoms
clearly appear through an increase in the P-chol and P-ethanolamine
resonance intensities and a decrease in the G6P one. In the spectrum of
the 3-OMG-fed root tips, an increase is also observed for the P-chol
and P-ethanolamine resonances, but contrary to sugar starvation, there
is a significant increase of the intensities of two unassigned
resonances at 4.36 and 4.30 ppm. These intensities are in the
proportion of 60% to 40%, as usually measured for the - and
-resonances of G6P, respectively. A small amount of G6P was added
directly to the extract (0.4 mM final) and a new spectrum
was recorded. The appearance of a resolved -G6P resonance
demonstrated that the intense resonances of the 3-OMG spectrum do not
arise from G6P (the -G6P resonance 0.4 ppm upfield is hidden). The
chemical shifts of these unassigned resonances sustain the hypothesis
of a phosphorylation of 3-OMG in C6 because the
31P resonance of C6-phosphohexoses always lie in
the 5- to 4-ppm interval at neutral pH (Fan,
1996).
G6P Enzymatic Assay of Maize Root Tip Ethanolic Extracts To confirm this hypothesis, G6P contents were measured in ethanolic extracts using an enzymatic assay. The G6P concentrations were 3.2 ± 0.6, 2.4 ± 0.4, and 1.3 ± 0.4 nmol tip1 in control, 48-h-starved, and 48-h
3-OMG-fed (100 mM) root tips, respectively (mean ± SD of five independent experiments). Thus, G6P
content decreased from control to starvation, as expected (Roby
et al., 1987; Brouquisse et al., 2001), but
decreased even more in root tips incubated with 3-OMG. This proved
definitely that the phosphomonoester accumulated in 3-OMG-fed root tips
was not G6P.
13C NMR Analysis of Maize Root Tip Ethanolic Extracts The 13C spectra of phosphomonoesters being characteristic of their molecular structure, ethanolic extracts of maize root tips were analyzed by 13C NMR spectroscopy. Because the attribution of the resonances is based on the spectra of pure compounds, 3-OMG-P was produced in vitro from 3-OMG using yeast HXK (Gancedo and Gancedo, 1984) and
purified. It was thoroughly characterized with 1H
and 13C NMR and shown to be 3-OMG-6P (M. Gromova,
S. Cortès, A. Heyraud, R. Brouquisse, and C. Roby, unpublished
data). The 13C spectra of 3-OMG (Bock and
Pedersen, 1983) and 3-OMG-6P (Fig. 7, top) were identified among the
different spectra arising from the more abundant components in the
extract of 3-OMG-fed maize root tips (Fig. 7, bottom). Quantification
of the amounts of 3-OMG and 3-OMG-6P from the 13C
NMR spectra of the extracts indicated that 8% ± 1% of the substrate had been phosphorylated in planta after 48 h.
NMR Analysis of Arabidopsis and Tomato Cell Acid Extracts To generalize this finding to other plants, we also looked for 3-OMG phosphorylation in two dicotyledon species, Arabidopsis and tomato, by analyzing acid extracts of heterotrophic cells incubated for 24 h with 50 mM 3-OMG. In analogy to Figure 6, Figure 8A shows that the 31P NMR resonances of the phosphomonoester accumulated in tomato cells have the same chemical shift as 3-OMG-6P. Figure 8B proves that 3-OMG-6P is present in the acid extract of Arabidopsis. Determination of the amounts of 3-OMG and 3-OMG-6P from the 13C NMR spectrum indicated that 12% ± 1% of the substrate had been phosphorylated in Arabidopsis cells.
Activity and Kinetic Parameters of Maize HXK Purification of maize root HXK was undertaken to assess consistently HXK involvement in the phosphorylation of 3-OMG. High neutral phosphatase (measured at pH 7.5) and G6P phosphatase activities were found in clarified homogenates of freshly excised maize root tips (Table II). Moreover, as compared with the control, neutral phosphatase and G6P phosphatase activities doubled in 48-h-starved and 48-h 3-OMG-fed root tips, whereas HXK activity decreased by a factor of 1.7 (Table II). Considering the slow kinetics of 3-OMG phosphorylation in vivo (Fig. 5D), contaminating phosphatase activities are likely to impair the in vitro measurements of 3-OMG phosphorylation kinetics. Thus, the activity and kinetic parameters of maize HXK against 3-OMG, Glc, and Man were investigated with a HXK preparation devoid of phosphatase activity.
The HXK-specific activity was 100-fold higher than that of phosphatase
activity in the HXK-enriched fraction obtained after a five-step
purification procedure (Table II). Remaining phosphatase was inhibited
(>95%) with the use of phosphatase inhibitors (Pi, phosphatase
inhibitor cocktail II). The Km and
Vmax values of purified HXK for Glc, Man,
and ATP are given in Table III. ADP inhibited HXK activity measured against 2 mM ATP
in a partially noncompetitive mode (data not shown). The
Km and
Vmax/Km values found for Glc and ATP, as well as inhibition by ADP, were similar to
those previously found for the organelle-bound maize root HXK (Galina et al., 1995
Overall metabolism of 3-OMG Maize root tips were incubated for 48 h in the
presence of 100 mM
[methyl-14C]3-OMG. During this period, the
production of [14C]CO2
was measured and root tip samples were harvested after 24 and 48 h
for [14C] metabolite analysis. As
reported in Table IV, after 24 and 48 h of incubation, respectively, 88% and 84% of the total
radioactivity were recovered in neutral fractions. Anions represented
8% to 11% of the total radioactivity. After treatment of the anionic fractions with alkaline phosphatase and fractionation on an anionic exchange column, more than 98% of the radioactivity was retrieved in
the neutral fraction obtained. This result and that presented in Figure
5D indicate that the radioactivity of the anionic fraction was
associated with esterphosphates, most being
3-OMG-6P. This matches a previous observation showing that after 6 d, 8% of the radioactivity incorporated as
[U-14C]3-OMG comigrated with sugar phosphates
in Chenopodium rubrum suspension cells (Gogarten and
Bentrup, 1989
3-OMG Phosphorylation in Plant Cells The present study shows that 3-OMG is phosphorylated steadily in
monocotyledon and dicotyledon plant cells (Figs. 4, 6, and 8). It also
demonstrates that in planta 3-OMG is phosphorylated at C6 (Figs. 7 and
8B), and that in vitro it is phosphorylated by maize root HXK, as are
Glc and Man, but at a very slow rate (Table III). Soluble and
membrane-bound HXK activities have been characterized in whole maize
roots (Galina et al., 1995 In animal and yeast systems fed with
14C-labeled 3-OMG, previous studies found that a
phosphorylated molecule in the anionic phase extract was radioactively
labeled, and it was concluded that this molecule was 3-OMG-P
(Gancedo and Gancedo, 1984 3-OMG Metabolism The present work also shows that the product of the
reaction, 3-OMG-6P, accumulates in the cytoplasm (Figs. 4 and 5D), and is practically not used as a respiratory substrate. The drop in respiration rate (Fig. 5B), and the very low amount of
[14C]CO2 produced by
[14C]3-OMG-fed root tips (Table IV) proves this
directly. As a consequence, endogenous proteins and lipids are
mobilized to support respiration and minimal biosynthetic processes
(Fig. 5C), in analogy with sugar-starved plant cells where the amounts
of proteins and lipids decrease strongly (Saglio and Pradet,
1980 Cellular bioenergetics were modified during the 48-h 3-OMG treatment
(decrease in respiration rate, NTP, and Cyt-Pi; Fig. 5, B and D) in a
way similar to starvation (Table I). However, despite the intracellular
accumulation of a large amount of phosphomonoesters (Figs. 4 and 5D),
there was no imbalance in energy metabolism (Table I). Replacement of
3-OMG by Glc triggered a return to the reference metabolic state (Fig.
5). When root tips were fed again with Glc after 48 h of treatment
with 3-OMG, their prompt respiratory and metabolic response (Fig. 5, A
and B) was typical of a healthy organ. Thus, 3-OMG is not toxic to
plant cells, in contrast to other Glc analogs such as Man, 2-DOG, and
glucosamine, which induce Pi starvation, a drop in AdN content and in
the AEC value, and an imbalance of metabolism (Herold and Lewis,
1977 Phosphatase Activities in 3-OMG-Fed and -Starved Root Tips Along with phosphorylation of 3-OMG, a dephosphorylation of
3-OMG-6P was observed in living root tips (Figs. 4 and 5). The two in
vivo activities, estimated from the rates of accumulation and
consumption of the substrate and product, appear to be of the same
order of magnitude. This observation reveals the existence of a
non-negligible ester-phosphate phosphatase activity in maize root tips
that has been estimated in clarified homogenates (Table II). The
presence of phosphatase activities in root tips and their increase
after 3-OMG feeding (Table II) can explain in vivo the slowing of
accumulation of 3-OMG-6P in 3-OMG-fed root tips after 40 h, and
the decrease in the 3-OMG-6P pool after Glc refeeding (Fig. 5D). In
plants, phosphatase activities, mainly acid isoforms, have been shown
to increase in response to Pi starvation, thus remobilizing the Pi
moiety of phosphorylated compounds to maintain Pi turnover and cell
metabolism (Plaxton, 1998 Use of Glc Analogs The question of sugar sensing is only indirectly addressed
when studying the metabolic and physiological consequences of 3-OMG substitution for Glc. It is the biochemical response of the integrated system to the sugar signal that is observed over 48 h, and not the
signal transduction process itself. However, when carefully screening
the metabolic responses to Glc analogs, one can make some
interpretations in terms of signaling from these systemic properties.
In previous work, after making sure that the observed responses were
not the result of a metabolic imbalance caused by Pi starvation, we
have shown that Man could be considered to have the same signaling
effect as Glc (Brouquisse et al., 2001 In yeast, it was concluded recently that HXK sugar sensing was
correlated to the onset of catalysis by the eventual formation of a
stable transition intermediate (Hohmann et al., 1999
The present work shows that 3-OMG is phosphorylated by HXK in heterotrophic plant cells, but not used as a respiratory and growth substrate. Proteolysis and RSIP expression are induced in 3-OMG-fed as in carbon-starved tissues. Thus, despite being phosphorylated, 3-OMG is not sensed as a sugar and behaves as a transported, but not phosphorylated, Glc analog. Considering the low catalytic efficiency of HXK for 3-OMG, it remains valid to use this analog for further study of the potential role of HXK phosphorylation rate in sugar sensing.
Preparation and Incubation of Excised Maize Maize (Pioneer France Maïs, Toulouse, France) seed
germination, root tip preparation, and incubation conditions were
described (Brouquisse et al., 2001 In vivo NMR experiments were performed as described (Brouquisse
et al., 2001 Preparation of Suspension-Cultured Cells Arabidopsis cells (ecotype Columbia), obtained from Pierre
Carol (Unité Mixte de Recherche 5575, Grenoble, France)
originated from the cell suspension culture described by Axelos
et al. (1992) Tomato (Lycopersicon esculentum Mill. var cerasiformae
cv Sweet 100) cells, obtained from Jean-Luc Montillet (DEVM,
Commissariat à l'Energie Atomique, Cadarache, France), were
grown at 23°C in Murashige and Skoog medium containing 166 mM Glc instead of Suc. They were kept in the dark and
continuously agitated on a rotary shaker (HT, Infors AG, Bootmingen,
Switzerland) at 150 rpm. Cells were subcultured every 5 d by
diluting 10 mL of cell solution in 90 mL of fresh medium in 250-mL
flasks. Initial cell density was 60 mg fresh weight
mL Preparation of Extracts Clarified Homogenates of Maize Root Tips Clarified homogenates were prepared as described (Brouquisse et al., 2001). Before HXK and phosphatase
activity measurements and immunoprecipitation experiments, 0.5 to 1 mL
of supernatant was first desalted through an ECONO-PAC 10 DG column
(Bio-Rad Laboratories, Hercules, CA) equilibrated with 50 mM Tris (pH 7.5) and 25 mM NaCl.
Acid Extracts of 3-OMG-Fed Plant Materials Water-soluble metabolites were extracted using perchloric acid (Brouquisse et al., 2001) from plant materials
conditioned in the following way. For maize, 2,000 excised root tips
were incubated for 48 h in medium A supplemented with 100 mM 3-OMG. For cells, 14 g of 5-d-old Arabidopsis cells
and 9 g of 4-d-old tomato cells were incubated for 24 h in
their own nutritive medium plus 100 mM 3-OMG. Plant
materials were harvested by filtration and quickly rinsed with a
3-OMG-free medium before freezing in liquid N2.
13C NMR spectra were recorded first. For 31P
NMR, CDTA was added to chelate divalent cations and the pH was adjusted
to the desired value.
Ethanolic Extracts and Fractionation of ). Neutral, anionic, and
cationic compounds were then fractionated on cationic (AG 50W-X8
Superfine, NH4+ form, Bio-Rad Laboratories),
and anionic (AG 1-X8 Superfine, OH form, Bio-Rad
Laboratories) columns according to the manufacturer's instructions.
14C Experiments Two hundred excised maize root tips were incubated for 2 d
in a 50-mL syringe containing 40 mL of medium A supplemented with antibiotic-antimycotics (A-7292, Sigma) and 100 mM
[methyl-14C]3-OMG (0.37 MBq mmol Synthesis and Purification of 3-OMG-6P The phosphomonoester 3-OMG-6P was synthesized at 22°C from a 2.5-mL solution containing 100 mM 3-OMG, 50 mM KH2PO4, 10 mM MgCl2, 1 mM Mg-ATP, 100 mM phosphoenolpyruvate, 5 units of pyruvate kinase, and 10% (v/v) D2O at pH 7.7. The addition of 45 units of yeast (Saccharomyces cerevisiae) HXK (1426362, Roche Molecular Biochemicals, Grenoble, France) started the reaction, which was stopped after 7 d with boiling ethanol. The sample was evaporated and resuspended in water. Three steps of exclusion diffusion chromatography were used to isolate 3-OMG-6P. The extract was first chromatographed at 60°C on a BIO-GEL P2 column (1.5 × 200 cm, Bio-Rad Laboratories) equilibrated with water. The fractions containing 3-OMG-6P were concentrated and chromatographed again on the same column equilibrated with 50 mM NaNO3. New 3-OMG-6P fractions were desalted, using an HPLC system, through an HW 40-50 F column (50 × 2.1 cm, Interchim, Montluçon, France) equilibrated with water. The enrichment in 3-OMG-6P was assessed using 1H and 13C NMR spectroscopy. Commercial yeast HXK was checked for potential enzymatic contaminations. No phosphotransferase activity was detected. The only relevant contaminant activities were isomerase and phosphatase, each with a ratio (1:30-60) with respect to the formation rate of 3-OMG-6P. Partial Purification of HXK One hundred grams of primary maize roots were homogenized in a
blender (Waring, New Hartford, CT) using 500 mL of a 50 mM Tris buffer at pH 8.0, containing 5 mM
MgCl2, 1 mM Na-EDTA, 5 mM
Enzyme Activity Assays Endopeptidase activity was measured, at pH 6.1, against
azocasein (James et al., 1993 Other Analytical Methods Proteins were quantified (Bradford, 1976 NMR Spectroscopy All the NMR measurements were performed using an AMX400 WB
spectrometer (Bruker S.A., Wissembourg, France): 31P and
13C NMR spectra were acquired at 162 and 100.6 MHz,
respectively. During in vivo experiments, they were recorded
alternatively for 10 and 50 min, respectively, using a dual
electronically switched 25-mm probe. Acid extracts were analyzed using
a 10-mm broadband probe. General acquisition conditions have been
described (Brouquisse et al., 2001 A solution composed of 50 mM methylenediphosphonic acid (MDP; 9508, Sigma) and 8 M ethanol contained in a concentric capillary provided the chemical shifts and intensity references for all spectra. The resonance assignment of metabolites was based upon chemical shifts. The concentration time course of a given metabolite was obtained in vivo in the following way. First, the intensity of its characteristic resonance was measured in each spectrum, eventually corrected to take sample growth out of the detection coil into account (normalization to the initial number of root tips within the detection coil), as well as spectral fluctuations, and plotted as a function of time. Second, the curve obtained was calibrated against concentration as described below for each isotope. Carbon NMR The chemical shifts were referenced to tetramethylsilane using the Suc fructosyl C2 resonance (C2f) fixed at 104.4 ppm as a secondary internal reference. In the absence of Suc resonances in a spectrum, the resonances of ethanol contained in the capillary mixture were used as secondary external reference (their chemical shifts were measured at 58.25 and 18.03 ppm). The Suc amount was derived from the intensity of the C2f resonance, the 3-OMG amount was derived from the sum of the intensities of the C3 (86.2 ppm), and C3 (83.6 ppm) resonances
and Asn amount was derived from the intensity of the CH resonance at
35.6 ppm. These intensities were corrected for the saturation effect
arising from the fast acquisition procedure and for the nuclear
Overhauser enhancement developed during the relaxation delay (the
correction factor was calculated from three interleaved standard and
relaxed spectra). The absolute metabolite amounts were calculated from
the corrected intensities using the ethanol CH2 resonance
intensity calibrated itself in moles.
Phosphorous NMR Chemical shifts were referenced to 85% (w/v) H3PO4 using internal glycerylphosphorylcholine as a secondary reference ( 0.05 ppm) for in vitro spectra and internal
UDPG (12.65 ppm) for in vivo spectra (the chemical shift of MDP
contained in the capillary mixture was measured at 16.72 ppm).
Quantification of acid extract spectra was done by integration of the
resolved resonances and calibration of the intensities was achieved
using the resonance intensity of MDP. The in vivo data were calibrated
using the intensities of a relaxed spectrum of maize root tips fed on
200 mM Glc. The intensities, devoid of nuclear Overhauser
enhancement, were calibrated against concentrations using the MDP
reference. The amounts of G6P and 3-OMG-6P could not be determined
separately from in vivo spectra because there is an overlap of their resonances.
Distribution of Materials Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Marie Hélène Andrieu and Nathalie Pochon who helped us to excise and analyze about 40,000 maize root tips necessary for this study, Monique Gaudillère for her help in AdN extraction and analysis, Pierre Carol and Eliane Charpentier for the Arabidopsis cells and advice, and Jean-Luc Le Bail for his dedicated technical assistance with the NMR spectrometer and perifusion system. Discussions arising from in-depth criticism of the work by the referees added value to our report. We are sincerely grateful to Jan A. Miernyk for his thorough reading of the manuscript, sharp language corrections, and pertinent suggestions to improve the original version.
Received June 26, 2002; returned for revision September 9, 2002; accepted November 21, 2002. 1 This work was supported by the Centre National de la Recherche Scientifique, by the Commissariat à l'Energie Atomique, by the Institut National de la Recherche Agronomique, by the Université Victor Segalen in Bordeaux, by the Université Joseph Fourier in Grenoble, in part by the EC (grant no. BIO4-CT96-0311), and by the Ministère de l'Education Nationale, de la Recherche, et de la Technologie (grants to S.C. and A.E.).
* Corresponding author; e-mail croby{at}cea.fr; fax 33-4-38-78-54-83.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010538.
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TOP
ABSTRACT
INTRODUCTION
AdN and adenylate energy charge (AEC)
were calculated as described in "Materials and Methods." These data
represent the mean of at least two (3-OMG and 3-OMG + Pi), four
(starvation), and six (control) independent experiments.
-globulin was used as the
standard. SDS-PAGE, western-blot, and immunoprecipitation experiments
were performed as described (Brouquisse et al., 2001