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First published online June 12, 2003; 10.1104/pp.102.017376 Plant Physiology 132:1196-1206 (2003) © 2003 American Society of Plant Biologists Energy Status and Its Control on Embryogenesis of Legumes. Embryo Photosynthesis Contributes to Oxygen Supply and Is Coupled to Biosynthetic Fluxes1Institut für Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany
Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O2 levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O2 concentration (1.1 µM). Embryo respiration was more strongly inhibited by low O2 during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O2 limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O2 and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O2 production elevated the internal level up to approximately 50% of atmospheric O2 concentration (135 µM). Upon light conditions, embryos partitioned approximately 3-fold more [14C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O2 supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O2 and energy that is readily used for biosynthesis and respiration.
Growing seeds are sink organs that import assimilates from the phloem, mainly as Suc and amino acids. Their photosynthetic potential to fix carbon is rather low compared with leaves and pods (Harvey et al., 1976  ;
Atkins and Flinn, 1978;
Flinn, 1985;
Eastmond et al., 1996;
Asokanthan et al., 1997).
Embryos are therefore predominantly heterotrophic. Accordingly, their ATP
supply is mainly covered by mitochondrial respiration, the regulation of which
is therefore important for storage activity and maturation. Mitochondrial
respiration is regulated at different levels like carbohydrate status, light,
and temperature. Especially the availability of respiratory substrates is
often regarded as a limiting factor. Because the overall sugar levels within
embryo tissues are quite high at all stages, its availability should not limit
mitochondrial respiration. During early seed growth (prestorage phase),
embryos contain high levels of hexoses due to high invertase activity
(Weber et al., 1995a). Later
on, invertase decreases and mainly Suc is imported. The switch from hexoses to
Suc is accompanied by cell differentiation and storage product synthesis
(Borisjuk et al., 1995;
Weber et al., 1998). The
analysis of the spatial distribution of both Glc and Suc concentrations in
growing broad bean (Vicia faba) cotyledons detected specific patterns
that change during development. High Glc concentrations were found in
non-differentiated premature regions, whereas mature starch-accumulating
regions contain particularly low concentrations
(Borisjuk et al., 1998). In
contrast, actively elongating and starch-accumulating cells contain highest
Suc concentrations, suggesting a role for Suc in differentiation and storage
processes (Borisjuk et al.,
2002).
Besides sugars, oxygen is a substrate for mitochondrial respiration as well
and can be rate limiting. This aspect, however, was almost neglected in
previous studies on seed physiology, although there was some indirect evidence
for oxygen shortage within seeds (Wager,
1974
Recently, we measured O2 levels within seeds of pea (Pisum
sativum) and faba bean using O2-sensitive microsensors with
high spatial resolution (Rolletschek et
al., 2002a Using a bioluminescence-based metabolite-imaging method (L. Borisjuk, unpublished data), we measured the spatial and temporal distribution pattern of ATP within growing embryos. The local ATP distribution temporally and spatially corresponded to greening pattern, chlorophyll distribution, and photosynthetic O2 production, indicating that the overall increase of the energy state is associated to the greening process. Thus, embryogenic photosynthesis may be particularly important under low O2 conditions delivering both oxygen and ATP. In this study, we provide additional experimental evidence for this model by analyzing the effects of day/night conditions on O2, metabolites, and biosynthetic activity. We found that during the early stages, embryos are ATP limited and responded by fermentation. During further differentiation, the ATP levels increased along with increasing photosynthetic capacity. Light supply and corresponding photosynthetic activity increased respiratory and biosynthetic fluxes.
Oxygen Levels in Embryos Are Low during Early Development and at Night Using oxygen-sensitive microsensors, we measured the spatial O2 distribution within seeds. Figure 1 shows an O2 profile across a transect through a pea seed of 300 mg fresh weight. O2 levels dropped strongly within the seed coat with a much stronger gradient at dark compared with light conditions. O2 fell to very low levels of about 0.5% to 1.5% of atmospheric O2 concentration in darkness (1.3-4 µM). In the light, the mean level was approximately 48% (130 µM). During both light and dark conditions, the O2 level was strikingly stable without detectable gradients within the embryo. The O2 profiles measured in seeds of faba bean were similar to those of pea (data not shown). Within broad bean seeds, O2 levels were dependent on both seed age and light supply (Fig. 2A). When O2 was measured at light, the mean levels inside the embryo were low (3%-20% of saturation, which corresponds to 8.1-54 µM) at early stage IV. Later on, levels increased steadily to approximately 50% (135 µM) at the end of stage VI (300 mg seed fresh weight), and remained stable thereafter. In darkness, concentrations were very low (0.4% of atmospheric O2 concentration, which corresponds to 1.1 µM), but anoxia could never be detected. During stage VII, O2 increased to values observed also under light conditions. In summary, the O2 levels in growing embryos changed during both development and upon dark-light conditions with lowest values at early stages and in darkness.
To analyze whether embryo metabolism is restricted by low oxygen, we measured fermentative activity and nucleotide levels in growing broad bean embryos. Both ethanol emission and alcohol dehydrogenase (ADH) activity were highest at stage IV with maximum rates of 80 and 300 nmol g–1 min–1, respectively (Fig. 2B). During stage V, ADH activity and ethanol emission decreased and remained nearly constant in seeds of >200 mg fresh weight. Ethanol emission was not detectable during stages VI and VII. Together with the low O2 levels and fermentative metabolism, the ATP concentrations were lowest at early stages, but increased to values of approximately 250 nmol g–1 in seeds of stage V to VI (Fig. 2C). From this stage onwards, ATP levels remained relatively constant. AMP decreased initially together with increasing ATP and remained constant thereafter. ADP levels were between 40 to 60 nmol g–1 throughout growth without major changes (data not shown). The profile of the adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was similar to that of ATP with minimum values of 0.38 at early stages and maximum levels of 0.76 at later stages. In summary, at stage IV when oxygen levels are lowest, embryos also have a low energy state and high fermentative activity, indicating hypoxic metabolism. During stage V, energy levels increase and fermentative metabolism disappears.
Low O2 levels within the embryo might affect respiratory activity. We therefore measured respiration related to external O2 levels. The respiration rate, given as percentage of the rate at O2-saturating condition, decreased almost linearly in response to falling O2 levels in the medium (Fig. 3). The decrease occurred already when external O2 levels were still high. This might be attributed to the high diffusional impedance of embryo tissue. With the decrease of the concentration gradient at lower external O2 levels, the driving force for diffusive influx also decreases. Therefore, from the fitted lines in Figure 3 we cannot estimate the extent to which in vivo respiration is inhibited by low O2. However, due to the very low in vivo O2 levels (Fig. 2A), it can be assumed that embryo respiration is inhibited. Remarkably, respiration of mitotically active embryos (stages IV to V) is more strongly inhibited by low O2 than at stage VII after onset of storage. This indicates that embryos of early stages are more susceptible to O2 limitation and that during maturation respiration becomes adapted to low O2.
To further characterize the metabolic adaptations to O2
limitation, we changed experimentally the oxygen supply to isolated pea
embryos of approximately 100 mg seed fresh weight from 100% to 10% and 1% of
the atmospheric O2 concentration (270, 27, and 2.7
µM). Characteristic changes in the metabolite pattern are shown
in Figure 4. The t0
values represent levels in freshly harvested embryos. Decreasing O2
from 100% to 1% of atmospheric O2 concentration did not change the
total free amino acid pool. Ala and
Metabolite levels in immediately frozen embryos represent the in vivo values, assigned as t0. Comparing these to the values of isolated embryos, treated with 100% of atmospheric O2 concentration, reveals the response of the metabolite pools to elevated O2. This is because the embryo is normally surrounded by the seed coat, which is a strong diffusional barrier and will never come in contact to 100% of atmospheric O2 concentration. Upon increased O2 supply, ADP-Glc, ATP, ATP to ADP ratio, UTP to UDP ratio, GTP to GDP ratio, and PEP increased, whereas ADP, hexose phosphates, and pyruvate decreased (Fig. 4). This indicates that increasing O2 supply leads to a higher overall energy state. The in vitro experiments are potentially affected by artifacts. Therefore, we performed near in vivo experiments where intact pods, still attached to the plant, were treated with an air stream containing 100%, 10%, and 1% of atmospheric O2 concentration, respectively. Thereby, the ambient O2 concentration was lowered followed by reduced diffusive O2 uptake. In principle, the results were very similar to the in vitro experiments (Fig. 5). Upon lowering O2 levels, ADP and lactate increased (Fig. 5, A and B), whereas ATP and the ATP to ADP ratio decreased (Fig. 5, C and D). Levels of ADP- and UDP-Glc and the GTP to GDP ratio decreased (data not shown). Lactate accumulated strongly already at 10% of atmospheric O2 concentration. However, within freshly harvested embryos accumulation of lactate had never been detected. Obviously, the treatment with 1% (and 10% in vivo) led to unphysiological low O2 conditions or anoxia.
The results show that lowering the oxygen supply decreases the energy state of the embryo. On the other hand, an experimentally increased O2 supply elevates the energy state. This indicates that in vivo the embryo metabolism is O2 limited.
To estimate the photosynthetic capacity, we measured the chlorophyll content in developing embryos. Chlorophyll a content was constant in embryos up to 120 mg fresh weight, but increased significantly thereafter (Fig. 6). Chlorophyll b showed a similar time course, but the chlorophyll a to b ratio increased from approximately 0.5 in 30 mg of seed to 2 in 400 mg of seed (data not shown). The increase of chlorophyll was significantly correlated to that of O2 levels measured under light but not dark conditions. It indicates that photosynthetic capacity increased during differentiation leading to elevated O2 levels. The relationship between chlorophyll content, photosynthetic production of oxygen, and ATP concentration are also in accordance with their spatial distribution pattern (L. Borisjuk, unpublished data). The increase of chlorophyll as well as of oxygen was accompanied by increasing ATP and AEC levels (Fig. 2C), indicating that embryo photosynthesis improves the energy state.
Embryogenic photosynthesis contributes to O2 and ATP production, which should increase metabolic fluxes. We therefore measured partitioning of [14C]Suc during day and night for embryos of different stages under near in vivo conditions. [14C]Suc was injected into the liquid endosperm surrounding the embryo, and label uptake as well as partitioning was monitored. The overall uptake of [14C] label per embryo fresh weight was not different (data not shown). However, at light conditions, embryos from 200 mg of seed fresh weight onward partitioned less label into the soluble fraction (Fig. 7A), but about 3-fold more label into starch (Fig. 7B), and slightly more into the cell wall fraction (Fig. 7C) compared with darkness. The flux of label into the protein fraction was unaffected by day or night conditions (Fig. 7D). Different fluxes might be caused by changing Suc levels within the liquid endosperm (isotope dilution effects). However, Suc was constant during the day/night cycle (50 ± 15 and 67 ± 14 mM, respectively).
These results indicate that the carbon flux into starch is increased upon light versus dark conditions possibly due to the higher O2/energy supply by photosynthetic activity of the embryo.
Changing O2 levels and respiratory activities as well as light-dark regulation of biosynthetic fluxes may affect steady-state levels of metabolites. Therefore, we measured nucleotides, nucleotide sugars, glycolytic intermediates, lactate, soluble sugars, and free amino acids upon dark or light conditions. Samples were taken at the mid and end of the night phase, the following day and the following night (total of six sampling times). Because samples derived from distinct time points during night and day were not significantly different, we pooled them to one night (n = 16) and day (n = 10) value. Data are summarized in Table I. Nucleotides, hexosephosphates, nucleotide sugars, PEP, pyruvate, lactate, and free sugars and amino acids were not different. The 3-P-glycerate pool increased significantly upon light conditions, most likely caused by photosynthetic CO2 fixation. Thus, light versus dark conditions did not change steady-state levels of metabolites but altered metabolic fluxes as shown by [14C]Suc-partitioning experiments.
The aim the work reported in this paper is to analyze the energy state and its control on legume embryogenesis. Growing embryos are predominantly heterotrophic and require not only carbon and nitrogen precursors but also ATP from mitochondrial respiration. Due to the limited diffusional O2 influx across the seed coat, embryos grow in an environment of low O2 availability. We show here that young embryos respond with hypoxic growth including fermentative metabolism. During maturation, embryos become adapted to low O2 supply and able to increase their energy state. The adaptation process is evident at different levels and part of the differentiation program. It may therefore be regarded as a prerequisition to perform storage product biosynthesis. Greening and the gain of photosynthetic activity in maturing embryos contribute significantly to O2 supply thereby improving the embryo's energy state and metabolic fluxes.
Growing embryos are mainly heterotrophic and dependent on mitochondrial
respiration. However, oxygen concentrations within growing seeds of broad bean
and pea are very low. This seems to be a general phenomenon in seeds and has
also been suggested for Brassica sp., soybean (Glycine max),
and Arabidopsis (Quebedeaux and Hardy,
1975 We show that ethanol emission and ADH activity within embryos are high during the early growth stages, whereas at later stages, levels are constantly low (Fig. 2B). We conclude therefore that only the young embryo responds to hypoxic conditions by inducing fermentative metabolism. Accordingly, ATP concentrations are lowest at stage IV but increase to higher values in seeds of 150 mg onward (Fig. 2C). Thus, metabolic responses upon hypoxia and low ATP levels are characteristic only for the young undifferentiated stage and disappear in the course of further differentiation. In embryos from 150 to 200 mg fresh weight onward, fermentative metabolism is no more evident. An increased diffusional uptake of O2 is unlikely the reason for overcoming hypoxic metabolism because O2 concentrations remain low in embryos up to 400 mg fresh weight in darkness (Fig. 2A).
Low O2 could be sensed and the signal translated into hypoxic
responses (López-Barneo et al.,
2001
Embryo respiration rates gradually decrease in response to falling
O2 levels when oxygen is already relatively high
(Fig. 3). This suggests that
embryonic respiration is apparently O2 limited at all stages.
Furthermore, after experimentally increasing oxygen supply, the ATP and AEC
levels rise (Fig. 5),
indicating that O2 is rate limiting for ATP production. Although
the O2 supply during early and later stages is suboptimal and thus
embryonic respiration is assumed to be restricted, there is an apparent lack
of hypoxic responses or fermentative metabolism at later stages. We
hypothesize therefore that embryos become adapted to the low O2
environment during differentiation mainly due to a drastic decrease of the
overall respiration. As a consequence, they can regulate their metabolism and
maintain their energy state at a constant high level. The early embryo has a
high respiration and consequently becomes fermentatively active. However,
fermentation is very inefficient with respect to ATP synthesis and produces
potentially toxic metabolites. The metabolic and physiological adaptation may
therefore be regarded as a precondition to perform storage product
biosynthesis. A similar decrease of respiration from the early cell division
to the maturation phase was reported for wheat (Triticum aestivum)
endosperm (Emes et al.,
2003
We here show that the metabolic and physiological adaptations to the low
O2 conditions are embedded in differentiation program of the embryo
and are evident on different levels: First, respiration is more strongly
inhibited by low O2 during earlier than later stages
(Fig. 3), and overall rates
decrease during embryo growth (L. Borisjuk, unpublished data). This indicates
that during maturation, embryonic respiration becomes tightly controlled and
adapted to the low O2 conditions. Second, ATP concentrations and
AEC are lowest in embryos of early stages but increase later on
(Fig. 2C). This indicates that
embryos acquire the ability to elevate and stabilize ATP and AEC levels.
Third, during maturation, embryos switch from an invertase and hexose-based
metabolism to one that is Suc-based and controlled by a Suc synthase pathway
(Weber et al., 1995a
Broad bean and pea embryos become green and photosynthetically active
during differentiation. Spatial distribution of photosynthetic activity
corresponds well to the chlorophyll pattern (L. Borisjuk, unpublished data).
We could show that the increase of chlorophyll within the embryo is correlated
to the O2 concentration under light conditions
(Fig. 3). Accordingly, in
oilseed rape (Brassica napus) seeds, chlorophyll content is
correlated to the photosynthesis-dependent O2 evolution
(Eastmond et al., 1996 The primary effect of embryonic photosynthesis is to increase internal O2 contents. A rate of oxygen production of 25 to 40 nmol g–1 min–1 should be sufficient for a 50% of atmospheric O2 concentration (135 µM) within the embryo within approximately 5 min. Changing O2 concentrations upon light/dark transitions are neither reflected at the level of ATP and AEC (Borisjuk et al., unpublished results) nor on the level of several other metabolites—both remain remarkably stable (Table I). Therefore, the increase of the overall energy state during maturation is not directly related to the embryonic photosynthetic activity or the corresponding O2 production. Instead, the adjustment of energy pools is developmentally regulated and coupled to the photosynthetic capacity of the embryo. However, we cannot exclude a change of nucleotide pools or AEC within the plastidial compartment upon light/dark conditions.
Chloroplasts in seeds are characterized by high rates of uncoupled electron
transport, a high chlorophyll a to b ratio, and abundant proteins associated
with photosystem II (Banerji and Rauf,
1979
An important question is what can be the contribution of embryo
photosynthesis to biosynthetic activity and metabolic fluxes? Photosynthetic
activity obviously does not modulate energy and metabolite levels but could
contribute to O2 and ATP supplies. If there is a significant
contribution, metabolic fluxes should change upon light/dark transitions.
Using [14C]Suc-partitioning experiments under near in vivo
conditions, we could show that upon light, embryos partitioned approximately
3-fold more label into starch (Fig.
7). This light-dependent increase of flux into starch synthesis is
developmentally dependent. From these results two important conclusions can be
drawn: First, embryogenic photosynthesis increases biosynthetic fluxes
probably by providing O2 and ATP which is readily used for
respiration and biosynthesis, although it is difficult to estimate the in vivo
contribution in absolute terms. Second, the strategy of maturing embryos to
respond to low O2 supply is to adjust metabolic fluxes rather than
the steady-state levels of metabolites. Our results show that young
undifferentiated embryos are not able to perform this mode of regulation which
has to be acquired in the course of the differentiation process. The
light-dependent increase in the respiratory and biosynthetic flux might also
be related to redox signals coming from photosynthesis. These are known to
alter both gene expression in leaves
(Pfannschmidt et al., 2001
Plant Material Broad bean (Vicia faba L. var minor cv Fribo) and pea (Pisum sativum cv Erbi) were grown in growth chambers under a light/dark regime of 16 h of light and 8 h of dark at 20°C/18°C. For the isolation of embryos, pods were tagged according to days after pollination, collected, and processed further. For metabolite measurements and enzyme assays, seeds were harvested, and embryos were immediately isolated and frozen in liquid nitrogen.
Oxygen concentration inside broad bean and pea seeds was determined using
microsensors (Presens, Neuburg, Germany) as described in detail by Rolletschek
et al. (2002a
To measure respiration, freshly isolated, intact broad bean embryos were
transferred into a 10 mL-measuring chamber equipped with a magnetic stirrer
and the inserted microsensor. Incubation buffer was prepared according to
Millerd et al. (1975
For the preparation of crude extracts, broad bean embryos were homogenized
on ice by precooled pestle and mortar in six volumes of cold extraction buffer
(125 mM MES, 100 mM NaCl, 2.5 mM
MgCl2, 0.5 mM thiamine pyrophosphate, 1 mM
EDTA, and 2 mM dithiothreitol, pH 6.8). Homogenates were kept at
4°C and centrifuged for 10 min at 4°C and 10,000g. ADH (EC
1.1.1.1) was measured spectrophotometrically as given by Waters et al.
(1991
Frozen plant material was extracted with trichloroacetic acid according to
Rolletschek et al. (2002b
To estimate in vivo biosynthetic fluxes of broad bean embryos, we measured the incorporation of [14C] label. Intact plants with attached pods were illuminated (approximately 400 µmol m–2 s–1) or kept in darkness. A small window was cut into the pod wall to get access to the seed. Using a 10-µL syringe, 3 µL of [U-14C]Suc (7.4 MBq mL–1, Amersham-Buchler, Braunschweig, Germany) was injected carefully through the seed coat into the endospermal cavity of the seed surrounding the embryo without injuring the latter. The injection site of the seed coat and the incision site of the pod wall was covered with silicone grease to avoid both water loss and gas exchange. After a 4-h incubation period, the embryo was removed from the seed, rinsed two times in buffer (250 mM Suc and 10 mM MES/KOH, pH 5.6), weighed, and frozen in liquid nitrogen. Only embryos without obvious damage of their surface were used for subsequent analyses. Extraction was carried out once with 80% (v/v) ethanol at 60°C, and in two subsequent steps with 60% (v/v) ethanol (combined supernatants were assigned as soluble fraction). Insoluble material was washed twice in 1 mL of water and homogenized. To hydrolyze starch, an aliquot was incubated with 14 units of amyloglucosidase in 50 mM sodium acetate (pH 4.8) for 24 h at 55°C. After centrifugation (15 min, 14,000g), the pellet was washed once in 1 mL of water, and the combined supernatants were counted. To dissolve protein, the residue was incubated for 24 h at 30°C in 1 mL of 50 mM Tris-HCl (pH 7.4) containing 0.08% (w/v) pronase. The homogenate was centrifuged and washed once in 1 mL of water, and the combined supernatants were counted. The remaining pellet (assigned as cell wall) was resuspended in 1 mL of water and counted. Radioactivity was determined by liquid scintillation counting (Rotiszint, Roth, Germany). Counts were corrected for background and quenching by external standards.
Intact broad bean seeds were removed from the pod and enclosed in 12-mL headspace vials containing a small portion of wet cotton wool (to conserve high humidity). After a 4-h incubation period, the amount of ethanol (released by the seed) within the headspace was measured gas chromatographically (6890 Series, Agilent Technologies, Palo Alto, CA). Chromatographic conditions were as follows: column DB23 (J&W Scientific, Folsom, CA) 30-m x 0.25-mm (i.d.) x 0.25-µm film; injector, 5-µL injection volume; split ratio 5:1, 250°C; oven, 50°C for 5 min; detector, flame ionization detector, 275°C, He makeup gas 25 mL min–1; carrier gas, He with 1 mL min–1. Emission rate was calculated from the amount of ethanol released during the incubation time related to the fresh weight of the incubated embryo.
To study the effect of diminished oxygen supply, pea seeds were aerated with premixed gases (Messer-Griesheim, Germany). For the in vitro experiment, embryos (about 100 mg fresh weight) were harvested and incubated in a nutrient solution (as used for respiration studies) in 50-mL conical flasks in darkness. The medium was aerated with gases containing the atmospheric oxygen level (about 21 vol%, referred to as to 100%), 2 vol% O2 (= 10%, balanced by N2), and 0.2 vol% O2 (= 1%, balanced by N2), respectively. After 5 h of incubation, embryos were rinsed shortly two times in distilled water, frozen in liquid nitrogen, weighed, and stored at –80°C until used. For the in vivo experiment, pea pods attached to the plant were coated with aluminum foil and aerated with premixed gases (100%, 10%, and 1% O2 of atmospheric O2 concentration, respectively) for 5 h. Thereafter, embryos were removed, frozen in liquid nitrogen, weighed, and stored at –80°C.
We are grateful to Ulrich Wobus for continuous support and discussions. Many thanks to Katrin Blaschek for excellent technical assistance and Heiko Weichert for help with ethanol measurements. Received November 7, 2002; returned for revision December 19, 2002; accepted February 25, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.017376.
1 This work was supported by the Deutsche Forschungsgemeinschaft. * Corresponding author; e-mail weber{at}ipk-gatersleben.de; fax 49 -39482-5138.
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ABSTRACT
RESULTS
-aminobutyric acid increased
gradually, whereas Asp decreased. Nucleotide sugars ADP-Glc and UDP-Glc
decreased. The ratio of ATP to ADP decreased significantly due to both
increasing ADP and decreasing ATP contents. Similar patterns were obvious for
the UTP to UDP and the GTP to GDP ratios. Lowering the O2 to 1% of
atmospheric O2 concentration increased lactate by 5-fold. The PEP
to pyruvate ratio decreased due to the 4-fold elevated pyruvate levels. These
changes indicate a partial inhibition of respiration and glycolysis. Hexose
phosphates and the soluble sugars Glc, Fru, and Suc (data not shown) did not
respond to varying O2 supply. 
