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Biol. Chem., Vol. 381, pp. 723 – 740, August 2000 · Copyright © by Walter de Gruyter · Berlin · New York
Metabolic Activity Decreases as an Adaptive Response to
Low Internal Oxygen in Growing Potato Tubers
Peter Geigenberger1, Alisdair R. Fernie2,
Yves Gibon1, Maja Christ1 and Mark Stitt1, *
1
Botanisches Institut, Universität Heidelberg,
Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany
2
Max-Planck-Institut für Molekulare Pflanzenphysiologie,
Am Mühlenberg 1, D-14476 Golm, Germany
* Corresponding author
Plants lack specialised organs and circulatory systems, and oxygen can fall to low concentrations in
metabolically active, dense or bulky tissues. In animals that tolerate hypoxia or anoxia, low oxygen triggers an adaptive inhibition of respiration and metabolic activity. Growing potato tubers were used to investigate whether an analogous response exists in plants.
Oxygen concentrations fall below 5% in the centre of
growing potato tubers. This is accompanied by a decrease of the adenylate energy status, and alterations
of metabolites that are indicative of a decreased rate
of glycolysis. The response to low oxygen was investigated in more detail by incubating tissue discs from
growing tubers for 2 hours at a range of oxygen concentrations. When oxygen was decreased in the range
between 21% and 4% there was a partial inhibition of
sucrose breakdown, glycolysis and respiration. The
energy status of the adenine, guanine and uridine nucleotides decreased, but pyrophosphate levels remained high. The inhibition of sucrose breakdown
and glycolysis was accompanied by a small increase
of sucrose, fructose, glycerate-3-phosphate, phosphenolpyruvate, and pyruvate, a decrease of the acetyl-coenymeA:coenzymeA ratio, and a small increase
of isocitrate and 2-oxoglutarate. These results indicate that carbon fluxes are inhibited at several sites,
but the primary site of action of low oxygen is probably
in mitochondrial electron transport. Decreasing the
oxygen concentration from 21% to 4% also resulted in
a partial inhibition of sucrose uptake, a strong inhibition of amino acid synthesis, a decrease of the levels
of cofactors including the adenine, guanine and uridine nucleotides and coenzymeA, and attenuated the
wounding-induced increase of respiration and invertase and phenylalanine lyase activity in tissue discs.
Starch synthesis was maintained at high rates in low
oxygen. Anoxia led to a diametrically opposed response, in which glycolysis rose 2-fold to support fermentation, starch synthesis was strongly inhibited,
and the level of lactate and the lactate: pyruvate ratio
and the triose-phosphate:glycerate-3-phosphate ra-
tio increased dramatically. It is concluded that low
oxygen triggers (i) a partial inhibition of respiration
leading to a decrease of the cellular energy status and
(ii) a parallel inhibition of a wide range of energy-consuming metabolic processes. These results have general implications for understanding the regulation of
glycolysis, starch synthesis and other biosynthetic
pathways in plants, and reveal a potential role for pyrophosphate in conserving energy and decreasing oxygen consumption.
Key words: Adenylates / Glycolysis / Hypoxia / Oxygen /
Potato tubers / Respiration.
Introduction
Animals have evolved specialised respiratory organs that
absorb oxygen from the environment, and circulatory systems that pump fluids containing high concentrations of
oxygen-binding proteins round the organism. These allow
large amounts of oxygen to be delivered, and facilitate
rapid changes in oxygen delivery in response to changes
in metabolic activity. In contrast, plants lack specialised
systems for oxygen delivery. Oxygen moves by diffusion
from the surrounding air (21% oxygen, equivalent to 21
KPa at sea level) through regulated apertures in the epidermis and intercellular air spaces within the tissue. Although plants can adapt to low oxygen by producing larger intercellular air spaces (Laan et al., 1990; Drew et al.
1994, 1995), this response is slow and inefficient compared to the physiological adaptations in animals. The absence of specialised systems for oxygen delivery is not
generally considered to be a problem for plant growth and
metabolism because most plant organs have a relatively
high surface-to-volume ratio, and their respiration rates
per unit volume of tissue are usually lower than in animals
because plant cells usually have a large vacuole. However, tissues with high rates of metabolism can become hypoxic (a severe decrease of the oxygen concentration) or
anoxic (zero oxygen), especially when they lack large intercellular air spaces, contain cells that are not highly vacuolated, or are located in the centre of organs or at other
sites remote from the sites at which oxygen enters the
plant.
Low internal oxygen has been implicated in the development of seed dormancy in some species (Honek and
Matinkova, 1992; Jauzein and Mansour, 1992; de Meillon
et al., 1994). Hypoxia can occur in germinating seeds
(Bewley and Black, 1994; Edelstein et al., 1995) especially
when external oxygen is low due to water logging or microbial activity. In vegetatively growing plants, low oxygen
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concentrations have been reported in meristems (Armstrong and Beckett, 1985; Gibbs et al., 1995) and vascular
tissues (Kimmers and Stringer, 1988). Vascular tissues are
especially prone to hypoxia in roots (Thomson and Greenway, 1991; Sorrel, 1994; Armstrong et al., 1994; Ober and
Sharpe, 1996), where the external oxygen concentration is
anyway often low and the phloem and xylem are located in
the stele in the centre of the root. Low oxygen tensions in
the stele interfere with sugar retention in the phloem and
ion loading into the xylem (Drew, 1997; Gibbs et al., 1998).
The potential importance of oxygen delivery for reproductive growth is revealed by the observation that seed production is totally inhibited at 2% external oxygen in rice
(Akita and Tanaka, 1973), soybean, wheat, sorghum (Quebedeaux and Hardy, 1975, 1976), rape and Arabidopsis
(Porterfield et al., 1999). When the external oxygen concentration is decreased in the range below 15% there
is a linear reduction of Arabidopsis seed size (Porterfield
et al., 1999). This implies that substantial concentration
gradients are required to drive oxygen diffusion into these
tissues. In agreement, when Arabidopsis and rape were
grown in air the oxygen concentration in the airspace inside the silique in the dark was already decreased to 12%
and 6%, respectively, (Porterfield et al., 1999). Several indirect lines of evidence, including induction of enzymes involved in fermentation, accumulation of lactate or ethanol,
and declining adenylate energy change indicate that oxygen falls to low levels in developing seeds of Phaseolus
(Boyle and Yueng, 1983), barley (Macnicol and Jacobson,
1992), rape (Ching et al., 1974) and soybean (Quebedeaux,
1981; Shelp et al., 1995). Decreased internal oxygen concentrations have also been measured in bulky storage organs, including apples (Magness 1920), bananas (Banks,
1983), advocado (Ke et al., 1995), carrots (Lushuk and
Salveit, 1991) and potato tubers (Magness 1920; Steward
et al., 1932; Stiles, 1960; Burton, 1950, 1989). As these
studies (see Stiles, 1960 for a review) focused on stored
fruits and tubers at low temperatures, they may underestimate the severity of hypoxia in growing tubers and fruits at
ambient growth temperatures.
Cytochrome oxidase catalyses the terminal reaction of
the mitochondrial electron transport chain, which transfers electrons from NADH to oxygen. Cytochrome oxidase
has a very high affinity for oxygen with a Km(oxygen) of
about 14 M (equivalent to the oxygen concentration that
is in equilibrium with a gas phase containing 0.013% oxygen at 20 °C; see Drew, 1997). Unregulated operation of
cytochrome oxidase would decrease internal oxygen to
very low levels when the oxygen supply is restricted. Although this would promote oxygen delivery by diffusion, it
carries the concomitant risk that sectors of the tissue will
be driven into anoxia by small fluctuations of the external
oxygen concentration, the resistance to oxygen entry, or
oxygen consumption in other parts of the plant. Anoxia
represents a stress because the efficiency of ATP formation is sharply reduced, cytosolic pH decreases, and lactate and ethanol accumulate. Furthermore, harmful oxygen radicals are formed when oxygen re-enters highly re-
duced anoxic tissues (Drew, 1997; Biemelt et al., 1998).
Oscillation between anoxia and hypoxia would be particularly harmful, leading to fluctuations in the ATP supply and
cellular pH, and repeated exposure to oxygen radicals
(Drew, 1997). The importance of mechanisms to avoid, or
at least delay, the depletion of oxygen to very low levels is
also emphasised by the finding that a period of pre-adaptation in low oxygen is important for the development of
tolerance to anoxia (Drew et al., 1997; Chang et al., 2000).
Animals that tolerate low oxygen tensions are able
to decrease their oxygen consumption at low oxygen
concentrations (Storey, 1996: Kwast and Hand, 1996;
Hochachka et al., 1996; Hochachka, 1997). This adaptive
response includes a partial inhibition of respiration, and a
concomitant decrease of ATP consumption due to decreased protein synthesis and ion pumping activity. The
inhibition of respiration is rapid, and occurs at oxygen concentrations well above the Km(oxygen) of cytochrome oxidase. Characteristic changes in gene expression that are
induced by hypoxia are not simulated when respiratory inhibitors are added to animal tissues (Land and Hochachka, 1995), indicating that an oxygen sensing system
triggers a co-ordinated inhibition of ATP formation and
consumption.
Biochemical adaptation to low oxygen might be especially important in plants because (see above) they lack
sophisticated physiological mechanisms for oxygen transport. Although the response of plants to external hypoxia
has been intensively studied (Crawford and Brandle,
1996; Drew et al., 1997), this research has been driven by
questions related to adaptation to anoxia. Low external
oxygen induces a characteristic set of anaerobic response
(ANR) genes including lactate dehydrogenase (LDH),
pyruvate decarboxylase, alcohol dehydrogenase (ADH)
and several glycolytic enzymes (Andrews et al., 1993,
1994; Fennoy and Bailey-Serres, 1995; Dolferus et al.,
1997; Germain et al., 1997b). On the other hand, it has
also been reported that low oxygen inhibits respiration of
tubers (Stiles, 1960; Burton, 1989), seeds (Raymond and
Pradet, 1980; Al Ani et al., 1985) and roots (Saglio et al.,
1983). In seeds, this is accompanied by a decrease of the
adenylate energy charge (Raymond and Pradet, 1980;
Saglio et al., 1985). In maize root tips, low oxygen inhibits
protein synthesis (Chang et al., 2000). Low external oxygen has opposing effects on enzymes for sucrose breakdown, repressing invertase and inducing sucrose synthase (SuSy) in maize root tips (Germain et al., 1997a; Zeng
et al., 1999). In ripening banana fruits low oxygen inhibits
the synthesis and breakdown of starch, and activates cycles in sucrose metabolism and glycolysis (Hill and ap
Rees, 1995). Some of the metabolic responses to low
external oxygen, for example the induction of enzymes
required for fermentation, represent a pre-adaptation to
allow better survival under anoxia, (Drew, 1994; 1997;
Chang et al., 2000). It is unclear whether the other changes
represent an adaptation to decrease oxygen consumption
and avoid anoxia. Although there are differences between
the response to low and zero external oxygen (Andrews
Metabolic Adaptation to Low O2 in Potato Tubers
725
et al., 1994; Dolferus et al., 1997; Germain et al., 1997b;
Ying et al., 1998; Zeng et al., 1999), this may be because
sudden anoxia is interferes with adaptive responses
(Chang et al., 2000). Interpretation is further complicated
because some cells or tissues may become anaerobic
when plants are exposed to low external oxygen concentrations (Armstrong and Beckett, 1987; Drew, 1997). Most
previous studies have used rather low external oxygen
concentrations, and did not investigate the effect on internal oxygen concentrations.
The following experiments ask whether the oxygen
concentration is significantly decreased in growing potato
tubers, and investigate the accompanying changes in
energy status. In a complementary approach, tissue discs
from growing tubers were exposed to a range of oxygen
concentrations to explore the consequences for cellular
energy status, metabolite levels, fluxes in sucrose, starch
and respiratory metabolism, and the response to wounding.
Results
Oxygen Tension inside Growing Potato Tubers
Oxygen concentrations inside Solanum tuberosum cv.
Desiree tubers were measured using an oxygen mini-electrode. The oxygen concentration in the centre of tubers
which had been harvested and stored for 4 weeks at 20 °C
was 12 – 14% (Figure 1A), which is similar to values reported for tubers of other cultivars stored at this temperature
(see e. g. Magness, 1920; Burton, 1950, 1989). The oxygen concentration in the centre of growing tubers at ambient growth temperature (20 °C) was below 5% (Figure 1A).
The tubers were growing in well-aerated soil and the oxygen concentration surrounding the tubers in situ was
> 18% (data not shown). Figure 1B shows a typical oxygen concentration profile through a growing tuber (fresh
weight approx. 20 g). The oxygen concentration was 11 –
15% immediately under the periderm, falling to 5% in the
centre. Figure 1C illustrates the interaction between tuber
size and the internal oxygen concentration. As tuber size
increased from 8 g to 32 g, the oxygen concentration immediately under the periderm decreased from 15% to 5%
and the concentration in the centre decreased from 8% to
2%.
Adenine Nucleotide Levels along a Transect across
Growing Tubers
To investigate gradients of metabolites, a cork borer was
forced through the tuber, the tissue plug was immediately
forced out and simultaneously sliced into discs that fell
into successive containers filled with liquid nitrogen. In
two tubers of ca. 20 g harvested from separate plants
there was a decrease of ATP (Figure 2A), no clear change
of ADP (Figure 2B), an increase of AMP (Figure 2C) and a
decrease of the ATP/ADP ratio (Figure 2D) and the adenylate energy change (Figure 2E) in the centre compared to
Fig. 1 Oxygen Tensions in Potato Tubers Analysed with a Microelectrode.
(A) Oxygen tensions in the centre of growing tubers and of tubers
which had been harvested and stored for 4 weeks at 20 °C.
(B) Oxygen concentration along a ca. 3.8 cm transect across a
growing tuber (ca. 20 g). (C) Interaction of tuber size and internal
oxygen concentration, measured immediately under the periderm (open bars) and in the centre of the tuber (filled bars). Data are
mean ± SE (n = 3 – 4 separate tubers) for (A). In (B) and (C) data of
a typical experiment are shown.
the peripheral zones. The total adenine nucleotide pool
decreased slightly in the centre of the tubers (Figure 2F).
Whereas the adenylate energy charge in the outer zones
(0.75 – 0.85) was similar to that expected in aerobic tissues
(see Raymond and Pradet, 1980), the energy charge in the
centre (0.45 – 0.6) was much lower. This decrease occurred at internal oxygen concentrations (ca. 5%, see Figure 1B) two orders of magnitude above those needed to
saturate cytochrome oxidase (ca. 0.04% oxygen, see Introduction).
In numerous experiments over a period of two years,
we reproducibly found low ATP/ADP ratios (1.5 – 3) when
discs from growing tubers were harvested immediately
into liquid nitrogen, and much higher values (6 – 12) when
the discs were allowed to equilibrate with air (data not
shown). The tubers investigated in Figure 2 showed similar trends, even though there are differences in the exact
magnitude from tuber to tuber. This may be due to differences in tuber size, position or (Geigenberger et al., 2000)
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supply. During this period the tuber remained attached to
the mother plant. Submergence led to a decrease of ATP,
whereas ADP remained unaltered and AMP increased
(Figure 2A – C). The ATP/ADP ratio (Figure 2D) and the
adenylate energy change (Figure 2E) were far lower and
the total adenine nucleotide pool (Figure 2F) was slightly
lower than in tubers in air. There was still a marked gradient, with a dramatic decrease of ATP and increase of AMP
in the centre of the tuber, where the adenylate energy
charge decreased to under 0.3. A similarly low adenylate
status was found when tubers were preequilibrated in
nitrogen gas (data not shown).
Levels of Glycolytic Metabolites and Organic Acids
along a Transect across Growing Tubers
Fig. 2 Levels of Adenine Nucleotides in Transects of Growing
Potato Tubers.
To investigate adenine nucleotide levels inside the tuber, a cork
borer was forced through the middle, removed, and the tissue
plug rapidly forced out and simultaneously sliced into ca. 1 mm
thick discs which fell directly into liquid nitrogen. For each sample, three subsequent discs were pooled, extracted and adenine
nucleotide levels analysed. (A) ATP, (B) ADP, (C) AMP, (D) ATP/ADP,
(E) adenylate energy charge, (F) total adenine nucleotide pool.
The data show typical transects of 2 aerated (closed symbols)
and 1 submerged tuber (open symbol). The latter was excavated
and submerged under water for 24 h to restrict the external oxygen supply. During this period the tuber remained attached via the
stolon to the mother plant. One of the aerated tubers (solid
squares) corresponds to the tuber used in Figure 1C, the other
aerated tuber and the submerged tuber were a similar size).
metabolic status. The internal oxygen gradient in one of
the tubers was measured along a transect about 2 cm removed from that used to harvest discs for metabolite
analysis (compare Figure 1B with the data set shown as
solid squares in Figure 2). There was good agreement between the internal changes of oxygen and the changes in
the adenylate energy status.
For comparison, a growing tuber was excavated and
submerged for 24 h to severely restrict the external oxygen
Sugars and glycolytic metabolites were investigated
along the transects through the aerated tubers (Figure 3,
solid symbols). Both tubers contained slightly elevated
sucrose levels (Figure 3A) in the central zone. Fructose
was 3-fold higher in the central zone of one tuber, but only
slightly increased in the other (Figure 3B). The central region contained unaltered or slightly increased glucose-1phosphate (Glc1P, data not shown), glucose-6-phosphate
(Glc6P, Figure 3C) and fructose-6-phosphate (Fru6P, data
not shown). Fructose-1,6-bisphosphate (Fru1,6P2) (Figure 3D) and triose phosphates (Figure 3E) were below the
limit of detection (ca. 1 nmol/gFW). The centre contained
higher glycerate-3-P (3PGA, Figure 3F) and phosphoenolpyruvate (PEP, Figure 3G) than the periphery, with a
large increase in one and a small increase in the other
tuber. Increased 3PGA and PEP are typically seen when
glycolysis is inhibited in plants (Dennis and Greyson, 1987;
Hatzfeld and Stitt, 1991; Dennis et al., 1997). Pyruvate was
unaltered or increased slightly in the centre (Figure 3H).
Lactate (Figure 3I) remained very low in the centre of one
tuber and rose slightly in the centre of the other. The lactate:pyruvate (Figure 3J) ratio remained very low. Although
similar trends were found in both tubers, the extent of the
changes varied, with one containing slightly more lactate
and showing a much more marked increase of fructose,
3PGA and PEP in the centre. This may reflect a slightly
lower internal oxygen concentration.
Submergence of tubers for 24 h led to a slight decrease
of Glc1P (data not shown), Glc6P (Figure 3C) and Fru6P
(data not shown), a dramatic increase of Fru1,6P2 (Figure
3D) and triose phosphates (Figure 3E), no change or a
slight decrease of 3PGA and PEP (Figure 3F – G), a decrease of pyruvate (Figure 3H), and a 30-fold increase of
lactate (Figure 3I). The lactate :pyruvate ratio (Figure 3J)
and the triose phosphate:3PGA ratio (compare Figures 3E
and 3F) rose dramatically. Inspection of the transect
across the submerged tuber reveals a marked increase of
triose phosphate (Figure 3E), no unaltered or inconsistent
changes of 3PGA and PEP (Figure 3F – G), and a marked
decrease of pyruvate (Figure 3H) and dramatic increase of
lactate (Figure 3I) in the central zone.
The low lactate content of aerated tubers was not due to
absence of lactate dehydrogenase (LDH). Aerated and
Metabolic Adaptation to Low O2 in Potato Tubers
727
Fig. 3 Metabolite Levels in Transects of Growing Potato Tubers.
In the same samples as in Figure 2, metabolite levels were analysed. (A) sucrose, (B) fructose, (C) Glc6P, (D) Fru-1,6-P2 (E) Triose-P, (E)
3-PGA, (F) PEP, (G) pyruvate, (H) lactate, (I) lactate/pyruvate ratio. For details, see legend of Figure 2.
submerged tubers contained similar activities of LDH (Figure 4A) and alcohol dehydrogenase (ADH, Figure 4B).
Phosphofructokinase (PFK) activity was identical in aerated and submerged tubers (Figure 4C). As the reactions
catalysed by lactate dehydrogenase and glyceraldehyde3-phosphate dehydrogenase are close to equilibrium in
vivo, the low lactate:pyruvate ratio (Figure 3J) and very low
triose phosphate:3PGA ratio (compare Figures 3E and 3F)
in the centre of tubers in air shows that the NAD+ system is
highly oxidised. Due to uncertainties about the subcellular
location of metabolites in tubers, the precise cytosolic
(NADH)/(NAD+NADH) ratio could not be calculated.
Influence of the Oxygen Concentration on Adenine
Nucleotide Levels in Discs
A complementary approach was taken to allow a more
precise analysis of the effect of different oxygen concentrations on metabolism. Tissue discs (1 mm thick, 8 mm
diameter) were prepared from small growing tubers (ca.
15 g FW) and incubated for 2 h with 20 mM sucrose in the
presence of 0, 4%, 8%, 12%, 21% or 40% oxygen. For
comparison, metabolites were investigated in discs that
Fig. 4 Enzyme Activities in Transects of Growing Potato Tubers.
In samples taken in parallel to those in Figure 2, enzyme activities
were analysed. (A) lactate dehydrogenase, (B) alcohol dehydrogenase, and (C) PFK. For details, see legend of Figure 2.
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were quenched immediately after removing them from the
centre of the tubers used for these experiments (shown on
the right hand side of the following Figures).
Decreasing oxygen concentrations led to a progressive
decrease of ATP (Figure 5A), an increase of ADP (Figure 5B) and AMP (Figure 5C), and a marked decrease of
the ATP/ADP ratio (Figure 5D) and the adenylate energy
charge (Figure 5E). Marked changes of the adenine nucleotide levels occurred when oxygen was decreased
from 40% to 12%, and dramatic changes occurred when
oxygen was decreased below 12%. The overall adenylate
content declined (Figure 5F), even though the incubation
lasted only 2 h. GTP, GDP and the overall guanine nucleotide content were 90% lower than the corresponding
adenine nucleotide pool, and showed a similar response
to oxygen (data not shown).
Adenine nucleotide levels respond in a similar manner
to a low oxygen concentration in discs, and low internal
oxygen in intact tubers. The absolute levels of ATP, ADP
and AMP and the values of the ATP/ADP ratio and adenylate energy charge in tissue harvested immediately from
the centre of tubers (right hand data point, Figure 5A – F)
resemble those found in discs incubated with 4 – 8% oxygen (Figure 5A – F). Based on our previous measurements
(see Figure 1), the oxygen concentration in the centre of
these tubers would have been in this range. The value of
the ATP/ADP ratio and the adenylate energy change also
resemble those found in the centre of the two tubers
shown in Figure 2, although the absolute levels of adeny-
lates varied by a factor of two. This may reflect differences
between separate batches of tubers or plants.
Influence of the Oxygen Concentration on
Metabolites Involved in Sucrose Breakdown
Sucrose breakdown in growing potato tubers occurs
via sucrose synthase (SuSy), fructokinase (FK) and UDP
glucose pyrophosphorylase (UGPase). SuSy catalyses
the reversible conversion of sucrose and UDP to UDPglucose (UDPGlc) and fructose, FK phosphorylates fructose to Fru6P, and UGPase catalyses the reversible
conversion of pyrophosphate (PPi) and UDPGlc to Glc1P
and UTP. Plants contain a significant pool of PPi that, at
least in leaves, is located in the cytosol (Weiner et al.,
1987).
Sucrose was not measured because it was supplied in
the medium, and repeated washing to remove extracellular sucrose would have introduced the risk of changing the
oxygen concentration. Fructose (Figure 6A) increased by
about 50% when the oxygen concentration was decreased to 8% and by 2- to 3-fold when oxygen was
decreased to 4 and 1%, and decreased again by 50% in
zero oxygen. For comparison, fructose increased slightly
(Figure 5A, Figure 3B) or markedly (Figure 3B) in the centre
of intact tubers.
The overall uridine nucleotide pool declined in parallel
with the total adenine nucleotide pool (Figure 5K). UDPglucose (UDPGlc) accounted for most of the uridine nu-
Fig. 5 Levels of Adenine and Uridine Nucleotides in Tuber Slices Incubated at Different Oxygen Tensions.
Freshly cut slices of growing potato tubers were either immediately quenched in liquid nitrogen (t0), or incubated in buffer including 20 mM
sucrose under continuous aeration using premixed gases containing 0, 4, 8, 12, 21, or 40% oxygen. After 2 h incubation, slices were harvested by immediately quenching in liquid nitrogen, and then extracted to measure nucleotide levels (nmol gFW–1). (A) ATP, (B) ADP, (C)
AMP, (D) ATP/ADP ratio, (E) adenylate energy charge, (F) total adenylates, (G) UTP, (H) UDP, (I) UTP/UDP ratio, (J) UDPGlc, and (K) total
uridinylates. (L) ADPGlc. Data are mean ± SE (n = 3).
Metabolic Adaptation to Low O2 in Potato Tubers
729
Fig. 6 Metabolite Levels in Tuber Slices Incubated at Different Oxygen Tensions.
Freshly cut slices of growing potato tubers were either immediately quenched in liquid nitrogen (t0), or incubated in buffer including 20 mM
sucrose under continuous aeration using premixed gases containing 0, 1, 4, 8, 12, 21, or 40% oxygen. After 2 h incubation, slices were
harvested by immediately quenching in liquid nitrogen, and then extracted to measure metabolite levels. (A) Fructose, (B) PPi, (C) Glc1P,
(D) Glc6P), (E) Fru6P, (F) Fru-1,6-P2, (G) DHAP, (H) 3-PGA, (I) PEP, (J) pyruvate, (K) lactate, (L) lactate/pyruvate, (M) malate, (N) isocitrate, (O)
2-ketoglutarate, (P) citrate, (Q) acetyl-CoA, and (R) CoA-SH. Data are expressed as nmol gFW-1, except for fructose, lactate, malate and
citrate, which are expressed as µmol gFW–1. Data are mean ± SE (n = 3).
cleotide pool (see also Loef et al., 1999). UDPGlc declined
slightly as the oxygen concentration decreased (Figure 5J). UTP decreased progressively as the oxygen concentration decreased (Figure 5G). UDP rose slightly as
oxygen was decreased from 40% to 21%, remained unaltered as oxygen was decreased from 12% to 4%, and rose
markedly in zero oxygen (Figure 5H). The UTP/UDP ratio
fell progressively as the oxygen concentration was decreased (Figure 5I). Uridine nucleotide levels in discs incubated with 4 – 8% oxygen differed from those found in the
centre of intact tubers (Figure 5G – K). The overall pool was
smaller, UDPGlc was lower, UDP was higher and the
UTP/UDP ratio was lower. This may be due to secondary
changes triggered by disc preparation, and because the
sucrose supplied to the discs does not fully replace the
inflow of sucrose from the phloem in intact tubers. There is
a rapid decrease of the overall uridine nucleotide pool in
discs even when they are incubated in air (see also Loef
et al., 1999), and UTP and UDP levels are sensitive to the
sucrose concentration supplied to the discs. When discs
were supplied with zero, 25 and 100 mM sucrose for 20 min
at 21% oxygen, the UDP level decreased from 3.25 ±
0.024 to 3.08 ± 0.5 and 2.37 ± 0.01 nmol/g FW, UTP increased from 29.7 ± 0.2 to 29.9 ± 1.7 and 31.3 ± 0.3 nmol/g
FW, and the UTP/UDP ratio increased from 8.6 ± 0.7 to
11.0 ± 1.7 and 13.3 ± 0.1, respectively (mean ± SE, data
not shown).
PPi was very low in discs incubated in 40% oxygen, increased markedly in 21% oxygen, rose further to a maximum at 4 – 8% oxygen, and fell to low levels in zero oxygen
(Figure 6B). The PPi level in discs incubated with 4 – 8%
oxygen resembled those in the centre of intact tubers (Figure 6B). PPi levels in plant tissues usually do not change
greatly (Stitt, 1998). It is striking that PPi remains high or
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P. Geigenberger et al.
even increases slightly at low oxygen concentrations even
though the energy status of the adenine, guanine and uridine nucleotides (see Figure 5) decreases. Independent
changes of PPi and adenine nucleotides have also been
seen in response to uncouplers and anoxia in pea roots
and Arum maculatum spadices (Dancer and ap Rees,
1989), where PPi remained high even under anoxia.
Influence of the Oxygen Concentration on the Levels
of Glycolytic Metabolites and Organic Acids in Discs
Glc1P (Figure 6C), Glc6P (Figure 6D) and Fru6P (Figure 6E)
showed a slight decrease at 8% and 4% oxygen, and rose
again slightly in 1% and zero oxygen. Fru1,6P2 (Figure 6F)
and triose phosphates (Figure 6G) remained low until 1%
oxygen, and rose 2-fold in zero oxygen. 3PGA (Figure 6H)
and PEP (Figure 6I) increased progressively as oxygen
was decreased, and decreased in zero oxygen. The triose
phosphate:3PGA ratio was very low, except in zero oxygen (compare Figures 6G and 6H). Pyruvate decreased
slightly between 21% and 8% oxygen, increased at 4%
and 1% oxygen, and decreased in zero oxygen (Figure 6J).
Lactate was low except in zero oxygen, where it rose dramatically (Figure 6I). The lactate:pyruvate ratio (Figure 6J)
was very low in high oxygen, rose very slightly as oxygen
was decreased below 12%, rose slightly in 1% oxygen,
and increased dramatically in zero oxygen.
Potato tubers contain large pools of malate (Figure 6M)
and citrate (Figure 6P), and much smaller pools of isocitrate (Figure 6N) and 2-oxoglutarate (Figure 6O). Most of
the citrate and malate are in the vacuole (Martinoia and
Rentsch, 1994). Decreasing concentrations of oxygen led
to a progressive decrease of malate, whereas citrate remained unaltered. Isocitrate and especially 2-oxoglutarate increased slightly in 8% oxygen, and decreased in
lower oxygen and zero oxygen. Acetyl-CoA decreased
slightly as oxygen was reduced from 40% to 12%, decreased 50% between 12% and 4% oxygen, and remained low in 1% and zero oxygen (Figure 6Q). This was
accompanied by a slight increase of CoA-SH (Figure 6R).
As a result, there was a 75% decrease of the acetylCoA/CoA-SH ratio as oxygen decreased (compare Figures 6Q and 6R). The total CoA pool (the sum of acetylCoA plus CoA-SH) decreased by 40% at low oxygen (values calculated from Figures 6Q and R). The transient
increase of isocitrate and 2-oxoglutarate in 8% oxygen
(Figures 6N – O) occurred when acetyl-CoA was still relatively high and pyruvate (Figure 6J) was relatively low.
When oxygen was decreased further there was a larger
decrease of acetyl-CoA, 2-oxoglutarate and isocitrate
decreased, and pyruvate rose (compare Figures 6N – O
with Figure 6J).
Most glycolytic metabolites and organic acids respond
in a similar manner to low oxygen in discs (Figure 6) and to
depletion of oxygen in the centre of intact tubers (Figures 3, 6). In particular, 3PGA and PEP increase, lactate
remains low, and the lactate:pyruvate and triose phosphate:3PGA ratios remain very low in both experimental
systems. This contrasts with the response of discs to zero
oxygen (Figure 6) and the changes in the centre of submerged tubers (see Figure 3), when 3PGA and PEP remain
unaltered or fall, lactate increases, and the lactate:pyruvate and triose phosphate:3PGA ratios rise dramatically.
Although a precise comparison is complicated by differences between tubers and by the dynamic changes in
discs in the range between 12 and 4% oxygen, most
metabolites including the hexose phosphates, 3PGA,
pyruvate, lactate, malate and isocitrate were present at
similar concentrations in discs incubated at 4 – 8% oxygen (Figure 6) and in the middle of intact aerated tubers
(Figures 3 and 6). The major exceptions were Fru1,6P2 and
triose phosphates which were 2- and 3-fold higher, and
acetyl-CoA and 2-oxoglutarate which were slightly lower
in discs than in the centre of tubers. Elevated levels of
Fru1,6P2 and triose phosphates are typically seen in discs
(data not shown), possibly due to a wounding-related
stimulation of respiration.
Influence of the Oxygen Concentration
on the Level of ADPGlc in Discs
The first committed reaction in starch biosynthesis is the
conversion of ATP and Glc1P to ADPGlc and PPi, which is
catalysed by ADPglucose pyrophosphorylase (AGPase).
ADPGlc decreased slightly at 12% oxygen, fell further at
4 – 8% oxygen, and decreased markedly at zero oxygen
(Figure 5L).
Influence of the Oxygen Concentration on the
Metabolism of [U-14C]Sucrose by Discs
To investigate the effect of the oxygen concentration on
fluxes, discs were incubated with 20 mM [U-14C] sucrose
for 2 h at 40%, 21%, 12%, 8%, 4%, 1% or zero oxygen
(Figure 7). Sucrose uptake was partially inhibited when
oxygen was decreased from 12 to 8% (Figure 7A), but did
not decrease further at 4%, 1% or zero oxygen. High oxygen (40%) also inhibited sucrose uptake. Figure 7B shows
the proportion of the absorbed [U-14C] sucrose that was
metabolised to other compounds. Only a small proportion
of the absorbed sucrose is metabolised during the 2 h incubation (Figure 7B, see also Geigenberger et al., 1997;
Geiger et al., 1998). The proportion that was metabolised
showed a slight decrease as oxygen was decreased from
21% down to 4%, and decreased markedly at 1% and
zero oxygen.
Figures 7C – F depict the fate of the metabolised sucrose. The label recovered in each fraction is shown as a
percentage of the metabolised [U-14C] sucrose. Part of the
label is retained as phosphorylated intermediates (Figure 7D). This pool is dominated by the hexose phosphates
(see Figure 5). As seen previously (Geigenberger and
Stitt, 1993; Geigenberger et al., 1997; Geiger et al., 1998),
starch (Figure 7C) represents the single largest product. A
considerable proportion of the metabolised label is also
converted to organic acids (Figure 7E) and amino acids
(Figure 7F), representing the flux through glycolysis. Very
Metabolic Adaptation to Low O2 in Potato Tubers
731
Fig. 7 Metabolism of [14C]Sucrose in Tuber Slices Incubated at Different Oxygen Tensions.
Freshly cut slices of growing potato tubers were incubated in a medium containing 20 mM sucrose under continuous aeration using premixed gases containing 0, 1, 4, 8, 12, 21, or 40% oxygen as in Figure 6. After 20 min, [U-14C]sucrose was added and the incubation was
continued for 2 h before slices were washed and extracted to determine label distribution. (A) [14C]sucrose absorbed by the tissue, (B) percentage of the absorbed label that is metabolised to other compounds. Incorporation of [14C] into (C) starch, (D) phosphate ester, (E) organic acids and (F) amino acids is expressed as a percentage of the label metabolised. The specific activity of the hexose phosphate pool
(G) was estimated by dividing the label retained in the phosphate ester pool by the summed carbon of the hexose phosphates (see Figure 6C – E). The values were divided by 2 to give the mean specific activity during the course of the 2 h. The specific activity of the hexose
phosphate pool and label incorporation into the relevant fractions was used to calculate (H) the absolute rate of starch synthesis and (I)
glycolytic flux (the sum of the flux to the organic acids and amino acids). Panel (J) shows the relative rates of starch synthesis and glycolysis and (K) the summed fluxes to starch and glycolysis. The results are given as mean ± SE (n = 4).
little [14C]CO2 was released during the 2 h incubation (data
not shown) because most of the label is initially trapped in
the large pools of organic acids. Less than 2% of the label
is converted to protein or cell wall components (data not
shown, see Geigenberger et al., 1997).
Label allocation to starch increased slightly as the oxygen concentration was decreased from 40% to 12%, and
decreased as the oxygen concentration was decreased to
8, 4 and 1% (Figure 7C). Labelling of starch was nevertheless high, even in 1% oxygen. Zero oxygen led to a strong
inhibition of label incorporation into starch (Figure 7C). The
residual level of label incorporation into starch in zero oxygen (< 10%) is similar to that seen in stored tubers (see
e. g. Hajirezaei et al., 1994). Low oxygen slightly increased
the proportion of the label that was retained in phosphorylated intermediates (Figure 7D). The proportion recovered
in organic acids (Figure 7E) decreased slightly as oxygen
was decreased down to 1%, and rose dramatically in zero
oxygen. The marked increase of label in organic acids in
discs at zero oxygen coincided with the accumulation of
lactate (Figure 5K). The proportion of the metabolised label recovered in amino acids (Figure 7F) decreased continuously as the oxygen concentration was decreased.
Absorbed [U-14C]sucrose will mix with internal unlabelled pools, so movement of the label will not necessarily reflect fluxes into the various pools (Geigenberger
and Stitt, 1993; Geigenberger et al., 1997). Interpretation
of labelling experiments is especially complicated when
732
P. Geigenberger et al.
treatments are compared that modify label uptake or the
turnover of internal pools, as is the case in the experiment
of Figure 7. The label in the phosphorylated intermediate
fraction (see Figure 7E) was divided by the total carbon
found in Glc1P, Glc6P and Fru6P (Figures 6C – E) to calculate the specific activity of the hexose phosphate pool
(Figure 7G) (see Geigenberger et al., 1997, for a discussion
of the assumptions involved in these calculations). To estimate the absolute rate of starch synthesis (Figure 7H),
the label in starch was divided by the specific activity of the
phosphorylated intermediate pool. Starch synthesis increases slightly as the oxygen concentration is decreased
from 40% to 12%, decreases slightly as the oxygen concentration decreases from 12% down to 8%, 4% and 1%,
and falls sharply in zero oxygen. To estimate glycolytic flux
(Figure 7I), label in organic acids and amino acids was
summed and divided by the specific activity of the phosphorylated intermediate pool. The estimated rate of glycolysis fell almost linearly as the oxygen concentration
was decreased from 40% to 1%, and then increased
2-fold in zero oxygen. In addition to lactate, some ethanol
is produced during fermentation in potato tubers. It was
not possible to accurately measure ethanol formation in
our experimental system because it was volatilised by the
gas passed though the solution. Our results may therefore
underestimate the stimulation of glycolysis during fermentation. The ratio between the rate of starch synthesis and
the rate of glycolysis is shown in Figure 7I. Starch synthesis rose relative to glycolysis as the oxygen concentration
was decreased from 40% to 12%, remained high as the
oxygen concentration was decreased from 12% down to
1%, and fell dramatically in zero oxygen.
The estimated fluxes to starch and through glycolysis
are summed in Figure 7K. As these are the two major
fluxes in potato tubers, their sum provides an estimate
of the net rate of sucrose breakdown. The combined
flux is inhibited when oxygen is decreased below 12%.
This shows that the inhibition of [U-14C] sucrose breakdown observed when oxygen falls below 12% (see Figures 7A – B) is not an artefact due to increased equilibration of the incoming labelled sucrose with unlabelled endogenous sucrose pool. Indeed, comparison of Figure 7G
with Figures 7A – B reveals that the specific activity of
the phosphorylated intermediate pool decreases less
than the rate of [U-14C] sucrose metabolisation, showing
that low oxygen also restricts mobilisation of endogenous
carbohydrates. Zero oxygen did not lead to a further inhibition of sucrose breakdown (Figure 7K). Carbon is available for the 2-fold stimulation of glycolysis in zero oxygen
(Figure 7I) because starch synthesis (Figure 7H) is strongly inhibited.
Influence of the Oxygen Concentration on the Rate
of Respiration of Tuber Discs
To measure respiration, discs (1 mm thick, 8 mm diameter)
were prepared from growing tubers, placed in an oxygen
electrode at pre-adjusted oxygen concentrations, and
Fig. 8 Respiration by Tuber Slices Incubated at Different Oxygen Tensions.
Freshly prepared potato tuber slices were transferred into the
temperature controlled measuring chamber of an oxygen electrode pre-equilibrated to 4, 8 or 21% oxygen by using premixed
gases (see Figure 5) to measure O2 consumption. Data are given
as mean ± SE (n = 3).
oxygen uptake was measured during the next 20 min (Figure 8). Compared to 21% oxygen, respiration was inhibited by 22 and 53% when the oxygen concentration was
decreased to 8 and 4% oxygen, respectively. In a separate
experiment, 20 mM sucrose was included in the suspending medium. Decreasing the oxygen concentration from
21% to 8% resulted in a 40% inhibition of respiration (data
not shown). The inhibition of respiration in low oxygen is of
a similar order to the inhibition of glycolytic flux estimated
from the labelling experiment (Figure 7H). This inhibition
occurs within minutes. When oxygen uptake was monitored over a longer period in the oxygen electrode, respiration decreased as oxygen was depleted and the inhibition was rapidly reversed when oxygen was reintroduced
(data not shown).
Influence of the Oxygen Concentration on the
Wounding Response
Slicing of tubers leads to a wound response in which respiration is stimulated, the activities of glycolytic enzymes
increase, and secondary metabolism is induced (Kahl,
1974; Burton, 1989). Anaerobiosis inhibits these wounding-induced changes in gene expression (Butler et al.,
1990), including the induction of PAL (Rumeau et al.,
1990). We investigated whether the wounding response
is attenuated when discs are incubated at oxygen concentrations similar to those found inside growing tubers
(Figure 9).
When control discs were incubated for 8 and 24 h at
21% oxygen, there was a marked increase of respiration
(Figure 9A), invertase activity (Figure 9B) and phenylalanine lyase (PAL) activity (Figure 9C). As expected (see
above), these changes were completely suppressed in
zero oxygen. They were also attenuated at 4% and 8%
oxygen. Incubation at low oxygen attenuated the rise of
respiration during the first 8 h, and led to a decline of respiration between 8 and 24 h. When tuber discs that had been
incubated for 24 h at 4% or 8% oxygen were transferred
Metabolic Adaptation to Low O2 in Potato Tubers
Fig. 9 Wounding Induced-Changes in Tuber Slices Incubated at
Different Oxygen Tensions.
Tuber slices were cut and pre-incubated for 20 min in 0, 4, 8, or
21% oxygen using pre-mixed gases as in Figure 5. At this time
point (t0) or after further incubation for 8 and 24 h, slices were
(i) transferred to an oxygen electrode to analyse (A) respiration
rates at the respective oxygen concentrations, or (ii) frozen in liquid nitrogen and extracted to analyse (B) invertase activity, and (C)
phenylalanine lyase (PAL) activity. The results are mean ± SE
(n = 3 – 4).
back to 21% oxygen, respiration was immediately stimulated but was much lower than in discs that had been incubated for 24 h in 21% oxygen (data not shown). The increase of invertase activity (Figure 9B) was decreased by
about 50%, and the increase of PAL activity (Figure 9C)
was inhibited by 80% in 4 – 8% oxygen.
Discussion
Respiration and Metabolic Activity Are Decreased in
Response to Low Oxygen in Potato Tuber Tissue
Growing potato tubers are bulky organs with a fairly homogenous morphology and a moderately high rate of metabolism, providing a good system to explore the conse-
733
quences of internal oxygen gradients in plants. The oxygen concentration is significantly decreased in the outer
zones of growing tubers, and there is a further decline between the outer and inner zones where the oxygen concentration drops below 5%. These low oxygen concentrations are accompanied by a decrease of the adenylate
energy status and changes of metabolites, that are indicative of a partial inhibition of respiration. The magnitude of
the changes varied between tubers, presumably depending on their size and metabolic activity. To allow more precise analysis of the effect of decreased oxygen on metabolism, freshly cut tuber slices were incubated for a short
time at a range of oxygen concentrations. This model system provided three lines of evidence that low oxygen restricts respiration. First, respiration was inhibited when the
oxygen concentration was reduced from 21% to 4 – 8%.
Second, labelling experiments revealed a progressive inhibition of glycolysis across a wide range from 40% down
to 1% oxygen. Third, decreasing oxygen led to a progressive and marked decrease of the ATP/ADP ratio and the
adenylate energy charge.
The inhibition of respiration in low oxygen is not due to
direct oxygen limitation of cytochrome oxidase. The inhibition of respiration and the decrease of the adenylate status is already evident in discs incubated with 8% oxygen,
which is almost three orders of magnitude above the
Km(oxygen) of cytochrome oxidase. Further, it occurs in
the absence of lactate accumulation, and in the presence
of low lactate:pyruvate and triose-P:3PGA ratios which
are indicative of a low (NADH)/(NAD + NADH) ratio. This
rules out the possibility that oxygen falls to very low levels
inside the tissue discs. Our results provide indirect evidence that respiration is also inhibited by falling oxygen
concentrations inside intact tubers, where there is a similar decrease of the adenylate energy status even though
the oxygen concentration is still in the range of 5%, and
low lactate levels and low lactate:pyruvate and trioseP:3-PGA ratios show that the tissue has not become
anaerobic. Earlier studies, which found that low external
oxygen reduces the rate of respiration of intact potato tubers but assumed this occurred because zones inside the
tuber were becoming anoxic (Stiles, 1960; Burton, 1989),
may require reinterpretation in the light of our results.
The responses to low oxygen and anoxia are actually
diametrically opposed. Low oxygen leads a progressive
inhibition of glycolysis and an increase of the terminal
glycolytic intermediates (3PGA, PEP) as is typically seen
when glycolysis is inhibited in plants (Dennis and Greyson,
1987; Hatzfeld and Stitt, 1991; Dennis et al., 1997). In contrast, zero oxygen leads to a dramatic increase of the lactate:pyruvate and triose phosphate:3PGA ratios, a further
decrease of ATP, a stimulation of glycolysis, a decrease of
3PGA and PEP, and accumulation of lactate. Glycolysis is
presumably stimulated in zero oxygen because ATP formation is less efficient during fermentation. There is an interesting parallel to the differing effect of low oxygen and
anoxia on the levels of the transcripts for two glycolytic enzymes (enolase, aldolase), LDH and ADH in maize roots
734
P. Geigenberger et al.
(Andrews et al., 1994). Whereas anoxia led to an increase
of all four transcripts, low oxygen led to a marked increase
of the transcripts for LDH and ADH as a pre-adaptation to
anoxia (see also Chang et al., 2000), whereas transcript for
enolase increased only slightly and transcript for aldolase
decreased.
Low oxygen also inhibits a range of metabolic processes that consume ATP in potato tuber discs. When oxygen
was decreased below 12% there was a partial inhibition of
sucrose uptake (Figure 7A). Amino acid synthesis was
progressively inhibited as oxygen was decreased below
21% (Figure 7F). The overall adenine, uridine and guanine
nucleotide pools, and the CoA pool decreased within 2 h
(Figures 5 – 6), revealing that synthesis or turnover of these
cofactors responds rapidly to low oxygen. A similar decrease of CoA was observed during anaerobic incubation
of bacteria cells (Chohnan et al., 1997). Biosynthesis of nucleotides and CoA requires energy (Brown, 1959; Stryer,
1990), and decreased levels of these cofactors may also
represent a mechanism to decrease fluxes through the
biosynthetic pathways in which they participate (Loef
et al., 1999, 2000). Further experiments are needed to investigate the impact of low oxygen on cell wall synthesis,
RNA synthesis and the overall rate of protein synthesis.
Chang et al. (2000) recently showed that hypoxia leads to
an approximately 50% inhibition of protein synthesis in
maize roots, due to inhibition of translation of a wide range
of proteins. We investigated the effect of low oxygen on
the wounding induced stimulation of respiration and activation of phenylpropanoid metabolism (Kahl et al., 1974;
Burton et al., 1989), an example for a cellular response that
requires a large increase of metabolic activity. Low oxygen
decreased the wounding-induced increase of respiration
and invertase activity by 50%, and strongly inhibited the
wounding-induced increase of PAL activity.
Mechanisms Contributing to the Inhibition of
Respiration and Metabolism at Low Oxygen
Our results indicate that low oxygen concentrations are
sensed in plants, leading to an inhibition of respiration and
a lowering of the adenylate energy status. Although accumulation of carbon dioxide, ethylene or other gases might
play a role in triggering morphological adaptation to low
oxygen (Drew et al., 1994, 1997), this cannot be responsible for the changes of metabolism in tuber discs because
the oxygen concentration was decreased without decreasing the rate at which other gases exit the tissue.
Studies with protoplasts and cell suspension cultures
(Paul and Ferl, 1991; Bailey-Serres and Dawe, 1996) indicate that LDH expression is regulated in response to low
oxygen, because low oxygen cannot be mimicked by respiratory inhibitors. LDH and ADH activity were already
high in tubers in air, indicating that they are induced at
the oxygen concentrations found inside growing tubers.
Further studies are needed to characterise the oxygen
sensing mechanism in plants. E. coli senses oxygen via a
two component sensor-response system (Guest, 1992),
heme-containing proteins are implicated in the oxygenregulation of gene expression in Rhizobium meliloti (Monson et al., 1992; Lois et al., 1993) and yeast (Zitomer and
Lowry, 1992; Sabova et al., 1993) and in the depression of
metabolic activity in reptiles (Hand and Hochachka, 1995),
and oxygen-sensitive ion channels have been implicated
in oxygen sensing in mammals (Lopez-Barneo et al.,
1994).
Some of the changes in low oxygen will be secondary
responses that are triggered by the decreased adenylate
energy status or other consequences of a decreased rate
of respiration. For example, changes in adenylate status
explain the inhibition of sucrose breakdown. SuSy and
UGPase catalyse reversible reactions that are close to
equilibrium in vivo (Geigenberger and Stitt, 1993; Geigenberger et al., 1994), so sucrose breakdown will depend
strongly on the removal of fructose by FK. ADP inhibits
potato tuber FK, acting competitively to ATP (Renz et al.,
1993). The physiological significance of these properties
was previously unclear, because inhibition is negligible
until the ATP/ADP ratio falls to low values. It is now evident
that they facilitate inhibition of FK in hypoxic conditions.
The observed increase of fructose in discs incubated with
less than 12% oxygen provides strong evidence that FK
is inhibited in vivo at low oxygen concentrations. Fructose
is, in turn, a potent feedback inhibitor of SuSy (Doehlert
1987, Dancer et al., 1990). Comparison of the Ki (fructose)
(1 – 5 mM) with the Km (sucrose) (50 – 100 mM; Avigad,
1982) of SuSy and the overall level of sucrose in tubers
(ca. 40 mM, Figure 2) indicates that the increase of fructose
from 0.8 mM in high oxygen to over 2 mM under hypoxic
conditions will lead to an approx. 50% inhibition of SuSy
activity. This is in good agreement with the inhibition
measured in the labelling experiment of Figure 7. The
changes of sucrose and fructose (Figures 3A – B and 6A) in
the centre of tubers indicate that this mechanism is
switched on with varying intensity in intact growing tubers.
Low oxygen led to a series of small changes in many
intermediates of glycolysis and the Krebs cycle, revealing
that these pathways are being coordinately regulated at
multiple sites and indicating that the primary site is likely to
be in the mitochondrial electron chain. UDPGlucose and
hexose phosphates showed only slight changes, showing
that the rate of carbon consumption for glycolysis and
starch synthesis is regulated in parallel with the rate of sucrose breakdown. There was a 2-fold increase of 3PGA
and PEP, showing that pyruvate kinase and/or PEP carboxylase activity has been decreased. The maintenance
of high pyruvate and the decrease of the acetyl-CoA:CoA
ratio pinpoint pyruvate dehydrogenase as a further important site for regulation. The precise reasons for the inhibition of pyruvate kinase, PEP carboxylase and pyruvate dehydrogenase are not revealed by our results. Nevertheless, organic acids that participate in the tricarboxyllic
acid cycle including 2-oxoglutarate and isocitrate remain
high or even increase slightly, especially in 8% oxygen
when 3PGA and PEP have only increased slightly and
acetyl-CoA has decreased only slightly. This may be be-
Metabolic Adaptation to Low O2 in Potato Tubers
cause glycolysis is inhibited less than respiration, or because consumption of organic acids for biosynthetic
processes including amino acid synthesis is decreased
(see Figure 6F), or because malate is re-mobilised (see
Figure 6M) to compensate for the short fall of carbon from
glycolysis. Increased malate mobilisation via NAD-malic
enzyme has been reported in response to extreme hypoxia (Edwards et al., 1996). The slight differences in the responses of different metabolite pools as the oxygen concentration is decreased indicates that the inhibition of the
various enzymes in carbon metabolism is not totally synchronous. This may contribute to the variable responses
of individual metabolites in intact tubers.
Starch synthesis is well adapted to the hypoxic conditions inside growing potato tubers. The highest rates of
starch synthesis, both in absolute terms and relative to
other fluxes, were found at 12% oxygen. Although a further decrease of the oxygen concentration down to 1%
slightly inhibited the absolute rate, starch synthesis remained high relative to other fluxes. The changes in the
rate of starch synthesis are in agreement with the changes
of the ADPGlc level. Although ADPGlc decreases slightly
under hypoxic conditions, the in vivo level remains above
1.5 nmol/gFW, which is sufficient to support near-maximal
rates of starch synthesis in vivo (Geigenberger et al.,
1997). Two mutually compensating changes explain why
starch synthesis operates effectively down to very low
oxygen concentrations. Although low ATP will restrict
AGPase activity (see Loef et al., 2000), this is compensated because low oxygen leads to a restriction of glycolysis and an increase of 3-PGA, which is a potent allosteric
activator of AGPase (Preiss, 1988). Starch synthesis is not
severely inhibited until tuber tissue becomes anoxic, when
ATP decreases even further and there is also a decrease of
3-PGA because glycolysis is stimulated to support fermentation. Our results differ from those obtained by Hill
and ap Rees (1995) with ripening banana fruit; however,
they did not monitor the internal oxygen concentrations,
and there may be differences between tissues which are
accumulating starch and tissues which are ripening and
re-mobilising starch.
General Implications for the Regulation of
Plant Metabolism
Many important plant tissues become hypoxic in the presence of a high external oxygen concentration (see Introduction). The finding that growing potato tubers respond
by decreasing their rate of respiration and adenylate status has general implications for our understanding of plant
metabolism.
First, it provides a rationale for the rather unusual way in
which glycolysis is regulated in plants. PFK is subject to
exquisite regulation by the ATP/AMP ratio in most microbes and animals (Stryer, 1990), allowing sensitive activation of glycolysis and respiration in response to falling
ATP. In plants, however, cytosolic and plastid PFK are only
weakly regulated by adenylates (Dennis and Greyson, 1987),
and our results demonstrate in vivo that a declining adeny-
735
late energy status does not override the partial inhibition of
glycolysis in low oxygen. When the oxygen supply is restricted, activation of PFK by falling ATP and rising ADP
and AMP would be counterproductive because the resulting stimulation of glycolysis would either increase oxygen
consumption or lead to accumulation of NADH and require fermentation. Low sensitivity of PFK and other glycolytic enzymes to regulation by adenylates may be an important component of the regulatory networks that allow
respiration to be decreased in response to low oxygen.
Second, plants will require regulatory systems that inhibit energy-consuming processes (see above for examples) when oxygen is low. The plant SNF1 homolog resembles the mammalian counterpart in being activated by
AMP (Sudgen et al., 2000). SNF1-related kinases have
been shown in vitro to phosphorylate and inactivate key
enzymes in the pathways of sucrose synthesis, nitrate
assimilation and isoprenoid synthesis in plants (Sugden
et al., 1999). Based on the role of the AMP-kinase in animals (Hardie et al., 1998), it will be important to investigate
whether plant SNF1 kinase homologs facilitate a global
regulation of biosynthetic processes to allow ATP consumption to be reduced when respiration falls and AMP
rises.
Thirdly, there will be a premium on biochemical adaptations that conserve energy and allow oxygen consumption
to be decreased. This may explain the unusual use of PPi
as an alternative energy donor in plants (Stitt, 1998). PPi
is a side product of numerous biosynthetic reactions and,
in most organisms, is hydrolysed to Pi (Stryer, 1990). The
cytosol of plant cells contains significant levels of PPi,
which is utilised as an energy donor for sucrose mobilisation via SuSy and UGPase (see above), for glycolysis via
pyrophosphate:fructose-6-phosphate phosphotransferase (PFP), and for tonoplast energisation via a PPi-dependent proton pump (Stitt, 1998). Each of these PPi-dependent reactions duplicates an ATP-consuming reaction
(Stitt, 1998). Substitution of part of the ATP consumption
by PPi will recycle ‘waste’ energy to support important
central metabolic and cellular functions, and allow ATP
and oxygen consumption to be decreased. PPi is maintained at high levels in hypoxic tissues (Figure 6B), in contrast with the progressive decrease of the energy status of
the adenine, uridine and guananine nucleotide systems
(Figure 5). The stimulation of substrate cycles in sucrose
metabolism and glycolysis in hypoxic banana fruit (Hill
and ap Rees, 1995) is also consistent with our proposal.
The cycle between sucrose and hexose phosphates is
mainly due to label equilibration via the PPi-dependent
sequence catalysed by the reversible SuSy and UGPase
reactions (Geigenberger and Stitt, 1993), and the substrate cycle between hexose phosphates and triose phosphates is due to the reversible PPi-dependent reaction
catalysed by PFP (Hajirazaei et al., 1994).
Independent evidence that PPi may play a role in conserving oxygen was recently provided by Zeng et al.,
(1999), who showed that hypoxia and anoxia repress invertase and induce a specific SuSy gene in maize roots.
736
P. Geigenberger et al.
Whereas breakdown of a molecule of sucrose via invertase requires 2 molecules of ATP, breakdown via SuSy and
UGPase requires only one molecule of PPi (Stitt, 1998).
The development- and cell-specific expression patterns
of these two enzymes also takes on a new significance
when they are related to oxygen delivery. In seeds and
tubers, invertase is typically expressed during the early
stages and SuSy during the later stages of development
(Appeldoorn et al., 1997; Weber et al., 1997). This switch
correlates with a transition from cell division to cell expansion and storage, and to date has been interpreted in
terms of the regulation of seed development by sugar-related signals. It will be interesting to learn whether the
switch from invertase to SuSy is also related to problems
that develop with respect to oxygen delivery as seeds
(Ching et al., 1974; Quebedeaux, 1981; Macnicol and
Jacobson, 1992; Shelp et al., 1995; Porterfield et al., 1999)
and tubers (see Figure 1) grow. Intriguingly, ectopic overexpression of invertase in tubers failed to improve starch
synthesis despite a massive increase in the levels of glycolytic intermediates, but instead led to a large stimulation
of respiration (Trethewey et al., 1998, 1999). The oxygen
concentration in the centre of these transgenic tubers is
almost zero (unpublished data). Sucrose metabolism in
the phloem complex occurs exclusively via SuSy (Geigenberger et al., 1995) and is disrupted when PPi levels are reduced by phloem-specific overexpression of pyrophosphatase (Lerchl et al., 1995; Geigenberger et al., 1996).
This may represent an adaptation to low oxygen tensions
in the phloem (see Introduction). In growing leaves, expression of SuSy and other PPi-utilising proteins is high in
the immature sector at the base of the leaf, and decreases
dramatically as the leaf matures (Stitt, 1998). As a leaf matures, the energy consumption for growth decreases, cell
extension and the accompanying increase of the vacuole
leads to a further decrease of the protein concentration
and the rate of respiration per unit volume, and intercellular air spaces are formed.
Finally, the finding that oxygen falls to low levels in
growing potato tubers provides a physiological explanation for two important agronomic factors that affect yield.
Potatoes require well-aerated soil, and yield is inhibited at
high temperatures (Burton, 1989). Partial water logging of
soil will decrease oxygen availability in the soil. High temperatures will lead to a dramatic decrease of the internal
oxygen concentration (Burton, 1950) because respiration
rises markedly as the temperature is increased but oxygen
diffusion only increases in proportion to the absolute temperature, and oxygen solubility in the liquid phase actually
declines. The resulting accentuation of problems related
to oxygen delivery will reduce tuber growth and, by suppressing the wound response, may also increase susceptibility to fungal or bacterial attack.
Materials and Methods
Plant Material
Potato plants (Solanum tuberosum L. cv. Desirée, Saatzucht Fritz
Lange, Bad Schwartau, Germany) were grown in well-aerated soil
(3 liter pots) supplemented with Hakaphos grün (100 g per 230
liter soil; BASF, Ludwigshafen, Germany) in a growth chamber
(350 mol photons m–2 s–1 light intensity, 14 h/10 h day/night
regime, 20 °C, 50% relative humidity). Growing tubers from
9 week-old daily-watered plants with high activities of sucrose
synthase, which is taken as an indicator for rapidly growing tubers
(Merlo et al., 1993) were used for the experiments.
Analysis of Oxygen Tensions in Potato Tubers
Intact tubers growing near the surface of the pot (where the oxygen concentration of the soil was above 18%, data not shown)
were excavated. The internal oxygen tension was measured 1 – 2
min later by introducing an O2 microelectrode (diameter of the tip
< 1 mm; Toepffer Lab Systems, Germany) into the tuber tissue.
When gradients through the tuber were investigated, the electrode was pushed gradually through the tuber, pausing for ca.
3 – 4 minutes at each position to obtain a measurement.
Sampling of Tissues from Intact Tubers
To investigate adenine nucleotide levels inside the tuber, a tuber
growing at the surface of the pot and that was still attached via the
stolon to the plant was excavated and exposed to air for ca. 2 min.
A cork borer (diameter 8 mm) was forced through the middle, removed, and the tissue plug rapidly forced out and simultaneously sliced into ca. 1 mm thick discs which fell directly into liquid
nitrogen. For each sample, three subsequent discs were pooled
and stored in liquid nitrogen until extraction.
Labelling Experiments with Tuber Slices
Tuber discs (diameter 8 mm, thickness 1 mm) were cut directly
from a core removed in a cork borer from the centre of growing
tubers (ca. 15 g FW) attached to the fully photosynthesising mother plant, washed quickly with 10 mM 2-(N-morpholino) ethane sulfonic acid (MES) (pH 6.5; KOH), pre-incubated for 20 min in buffer
containing 20 mM sucrose using 50 ml Falcon tubes in a water
bath at 25 °C (ca. 8 discs in 20 ml), and U-[14C]sucrose (final specific activity 1.4 KBq/mol) (Amersham-Buchler, Freiburg, Germany) was added and incubation continued for another 2 h. During the whole incubation and pre-incubation time, discs were
aerated by a continuous stream of pre-mixed gases containing 0,
1, 4, 8, 12, 21 or 40% oxygen. The oxygen concentration in the
solution was routinely checked using an oxygen electrode. After
2 h, discs were immediately washed 3 times with buffer to remove
external radioactivity, and frozen in liquid nitrogen to analyse label
distribution.
Tissue slices were prepared and incubated in a similar manner
to measure metabolite levels in dependence on oxygen, except
that no radioactivity was added and a subset of discs from the
centre of the tubers were allowed to fall directly after cutting into
liquid nitrogen. Tissue slices (30 discs in ca. 80 ml medium) were
incubated using glass vessels allowing continuous aeration with
premixed gases (see above). The oxygen concentration was
checked using an oxygen electrode. Slices were harvested by
pouring the medium immediately through a strainer and throwing
the slices into liquid nitrogen within 1 second. The slices were
stored in liquid nitrogen until extraction.
Metabolic Adaptation to Low O2 in Potato Tubers
Fractionation of 14C-Labelled Tissue Extracts
Discs were extracted with 80% (v/v) ethanol at 80 °C (1 ml per
2 disc), re-extracted in two subsequent steps with 50% (v/v)
ethanol (1 ml per 2 discs for each step), the combined supernatants dried under an air stream at 40 °C, taken up in 1 ml H2O
(‘soluble fraction’), and separated into neutral, anionic, and basic
fractions by ion-exchange chromatography; the neutral fraction
(3.5 ml) was freeze dried, taken up in 100 l water, and further
analysed by thin-layer chromatography (Geigenberger et al.,
1997). To measure phosphate esters, samples (150 l ) of the
soluble fraction were incubated in 50 l buffer (10 mM Mes-KOH,
pH 6.0) with or without 1 unit potato acid phosphatase (Grade II,
Boehringer Mannheim) for 3 h at 37 °C, boiled for 2 min, and
analysed by ion-exchange chromatography (Geigenberger et al.,
1997). The insoluble material left after ethanol extraction was
homogenised, taken up in 1 ml water and counted for starch. In
discs from growing tubers, starch accounts to over 90% of the
label in the insoluble fraction (Geigenberger et al., 1994).
Metabolite and Nucleotide Analysis
Tissue slices were extracted with trichloroacetic acid, and
metabolites and nucleotides measured as given in Geigenberger
et al. (1998). The recovery of small, representative amounts of
each metabolite through the extraction, storage, and assay
procedures has been documented previously (see Jelitto et al.,
1992; Merlo et al., 1994; Hajirezaei et al., 1994; Geigenberger
et al., 1994; Farré et al., 2000). Acetyl-CoA and CoA-SH were
measured according to Bergmeyer (1987), using the same extracts.
Enzyme Analysis
Acid invertase and PFK activities were analysed in tuber extracts
as in Geigenberger et al. (1998). PAL activity was analysed according to Lamb et al. (1979), lactate dehydrogenase and alcohol
dehydrogenase according to Bergmeyer (1987).
Respiration Measurements
Two freshly prepared potato tuber slices (1 mm thick, 8 mm diameter) were transferred into the temperature controlled measuring chamber of an oxygen electrode (Hansatech, Kings Lynn,
Norfolk, UK) containing 1 ml buffer solution (10 mM MES-KOH,
pH 6.5 and different sugars) preequilibrated to 4, 8 and 21% oxygen by using premixed gases, to measure O2 consumption of the
discs. Each measurement took ca. 5 min, during which time oxygen depletion in the buffer solution was less than 20% of the initial value.
Incubation for Inducing the Wounding Response
Discs from the middle of intact tubers (1 mm thick, 8 mm diameter) were sliced into 80 ml incubation medium (10 mM MES-KOH,
pH 6.5). The whole procedure took place inside a plastic bag, and
both the atmosphere inside the bag and the incubation medium
was equilibrated to 21, 8, 4 or zero oxygen by using premixed gases passing through the bag and the medium. The temperature
was 20 °C. Modified glass vials were used allowing complete aeration of the medium via a continuous air stream. After ca. 20 min
(= t0), one set of discs was harvested and frozen immediately in
liquid nitrogen to analyse enzyme activities, whereas a second set
of discs was directly transferred into the measuring chambers of
oxygen electrodes (pre- equilibrated at the different oxygen concentrations, respectively) to measure O2 consumption at 21, 8
and 4 % oxygen, respectively. The remaining discs were incubated for another 8 or 24 h, before they were sampled in the same
way as above.
737
Acknowledgements
This research was supported by the Deutsche Forschungsgemeinschaft (Sti78/1-2 and SFB 199 TP-C4). We are grateful to
Chris Somerville for alerting us to the publications of Quebedeaux
and Hardy.
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Received May 4, 2000; accepted July 5, 2000