Download Stimulation of glycolysis in anaerobic elongation of pondweed

Journal of Experimental Botany, Vol. 53, No. 376, pp. 1847±1856, September 2002
DOI: 10.1093/jxb/erf036
Stimulation of glycolysis in anaerobic elongation of
pondweed (Potamogeton distinctus) turions
Tatsuhisa Sato, Taro Harada and Kimiharu Ishizawa1
Department of Developmental Biology and Neuroscience, Graduate School of Life Sciences, Tohoku University,
980-8578 Sendai, Japan
Received 28 January 2002; Accepted 9 May 2002
Abstract
Introduction
Stem segments prepared from pondweed (Potamogeton distinctus A. Benn.) turions (overwintering
buds) elongate in anaerobic conditions, whereas
there is almost no elongation in air. The anaerobic
elongation was accompanied by a decrease in dry
weights of stem segments, mainly due to consumption of storage starch in the amyloplasts of stem
cells. On the other hand, total contents of amino
acids increased in stem segments, in which contents
of alanine, valine, leucine, and isoleucine increased,
but contents of asparatic acid decreased. Moreover,
contents of lactate in stem tissues increased at an
early stage of anaerobic incubation. In tracer experiments with 14C-glucose, 14C incorporation into stem
tissues in anoxia was only half of that in normoxia.
However, conversion of 14C to ethanol occurred
exclusively in anoxia. 14C-labelled metabolites were
analysed by two-dimensional cellulose thin-layer
chromatography. 14C incorporation into sucrose and
alanine was signi®cantly increased in anoxia. The
activity of alanine aminotransferase was enhanced by
anoxia, suggesting that pyruvate is a precursor of
alanine synthesis. The results suggest that pondweed turions produce energy necessary for
anaerobic elongation by activating conversion of
storage starch in the amyloplasts to ethanol, alanine
and lactate.
Some aquatic plants have been found to exhibit strong
tolerance to anaerobic conditions. These include rice
coleoptiles (Tsuji, 1972), barnyard grass coleoptiles
(VanderZee and Kennedy, 1981), Scirpus maritimus
rhizomes (Barclay and Crawford, 1982), Trapa natans
seedlings (Menegus et al., 1992), Acorus calamus rhizomes (Monk et al., 1984), Potamogeton pectinatus stems
(Summers and Jackson, 1994), Potamogeton distinctus
stems and Sagittaria pygmaea leaves (Ishizawa et al.,
1999). In the last three species, elongation in the absence
of oxygen has been reported to be greater than that in air.
Strong tolerance to anaerobic conditions is known to be
one of the adaptive characteristics of aquatic plants for
their survival in anaerobic environments. However, the
mechanisms of the tolerance are not clear.
Studies on the regulation of energy metabolism in
anoxia are important to understand how aquatic seed plants
survive when they are exposed to anaerobic conditions.
One of the most important issues for the survival of seed
plants in anoxic conditions is the mechanism of ATP
production, which supports anaerobic growth. Anaerobic
conditions stimulate glycolysis, which is a ubiquitous
pathway for the production of ATP in anaerobic conditions
(Davies, 1980). Crawford (1977, 1978) suggested that
anaerobic-tolerant species exhibit a smaller Pasteur effect
than do intolerant species. A smaller Pasteur effect would
contribute to the preservation of respiratory substrates and
to the prevention of over-production of toxic end-products
of fermentation. In fact, it has been shown that anaerobictolerant plants can produce various metabolites, such as
alanine, succinate, g-aminobutyrate (GABA), and malate,
as fermentation end-products (Crawford, 1978). However,
it is still obscure whether the accumulation of such
metabolites correlates with anaerobic tolerance. In many
Key words: Alanine, amyloplast, anoxia, elongation, ethanol,
fermentation, glycolysis, lactate, Potamogeton distinctus A.
Benn., turion.
1
To whom correspondence should be addressed. Fax: +81 22 217 734. E-mail: [email protected]
ã Society for Experimental Biology 2002
1848 Sato et al.
plants showing extremely strong tolerance to anoxia, such
as Trapa natans (Menegus et al., 1992), Acorus calamus
(Monk et al., 1984), Potamogeton pectinatus (Summers
et al., 2000), P. distinctus and Sagittaria pygmaea
(Ishizawa et al., 1999), alcoholic production is greatly
stimulated in anoxia, suggesting that the stimulation of
alcoholic fermentation is essential for these plants to
survive in anoxia. Kennedy et al. (1992) concluded that the
occurrence of a Pasteur effect per se does not correlate
with tolerance or intolerance to anoxia in plants.
Pondweed (Potamogeton distinctus A.Benn.), a ¯oating
plant, forms yellow banana-shaped overwintering buds
(called turions) underground in autumn. Turions can start
growing in the following spring when they are exposed to
oxygen-de®cient conditions of less than 5 kPa of oxygen
(Ishizawa et al., 1999). The stem elongation of pondweed
turions is maximal in the absence of oxygen. The
anaerobic growth is supported by the maintenance of a
large energy charge between 0.8 and 0.9 during an anoxic
state lasting over 2 weeks, suggesting the operation of
active energy metabolism.
The aims of this study were to determine what
substances are used for an anaerobic-respiratory substrate
and to determine what metabolites are produced when 14Cglucose and 14C-pyruvate are fed to pondweed turions
under anaerobic conditions.
Materials and methods
Plant materials
Pondweed (Potamogeton distinctus A.Benn.) was cultured in a small
pool constructed in a greenhouse belonging to the department.
Banana-shaped turions formed at the top of rhizomes were harvested
and stored in the dark at 4 °C until used.
Incubation and growth experiments
Pondweed turions were placed on moist ®lter paper, pre-incubated
overnight at 25 °C in the dark, and then stripped of their scaly leaves.
These naked turions were used as experimental material. Ten-mmlong stem segments were prepared from naked turions by cutting the
base and the apex with a razor blade. Three segments were stood in
an 8310±7 m3 glass tube (5310±3 m in diameter) with incubation
medium. Distilled water was usually used as the incubation medium.
For 14C-glucose feeding experiments, distilled water containing
carbenicillin 50 g m±3 and vancomycin 500 g m±3 was used as the
incubation medium, whereas for 14C-pyruvate feeding experiments,
5 mol m±3 2-(N-morpholino)ethanesulphonic acid (MES) buffer
(pH 5.5) containing the same antibiotics was used. The glass tubes
were set in a 10±4 m3 glass vessel that was connected to a gas-¯ow
system (Ishizawa et al., 1999) to supply water-saturated air or
nitrogen gas continuously. In the case of the 14C-glucose and
14
C-pyruvate feeding experiments, a vial containing 10±6 m3 of
43103 mol m±3 KOH as an absorbent of carbon dioxide and a bag of
Anaeropackâ (Mitsubishi Gas Chemical Co., Tokyo, Japan) as an
absorbent of oxygen were put together in the 10±4 m3 glass vessel.
Analysis of metabolites in stem segments
Extraction: After the incubation, length, fresh weight and dry weight
of stem segments were measured. For the dry weight measurement,
the segments were heated at 110 °C for 30 min to kill tissues as soon
as possible and further dried at 70 °C for 3 h. To determine the
contents of sugars and amino acids, three dried segments were
serially extracted at 80 °C with 2310±6 m3 of 80% ethanol for 1 h,
with the same volume of 80% ethanol for 30 min and ®nally with
10±6 m3 of 80% ethanol for 30 min. The combined extract was dried
under reduced pressure and redissoloved in deionized water. The
ethanol-insoluble materials were used for analysis of polysaccharides. The water-soluble substances were passed through an ionexchange column packed with Dowex 50W-X8 (NH4+ form) and
then through one packed with Dowex 1-X8 (HCOO± form). The
fraction passing through both columns was evaporated to dryness,
redissolved in deionized water, and used as a neutral fraction for
sugar analysis. Substances bound to the Dowex 50W-X8 column
were eluted with 23103 mol±3 NH4OH, evaporated to dryness with a
rotary evaporator, redissolved in deionized water, and used for
amino acid analysis. Substances bound to the Dowex 1-X8 column
were eluted with 63103 mol m±3 formic acid and used for the
analysis of organic acids. Recoveries of sugars and amino acids
eluted from the ion-exchange columns were more than 80% when
authentic glucose and leucine were separated with them.
Sugar analysis: Total contents of sugars and reducing sugars in the
ethanol-soluble fraction were determined by the method using
phenol±sulphuric acid (Dubois et al., 1956) and by the Somogyi±
Nelson method (Nelson, 1944). To identify sugars in an ethanolsoluble fraction, the neutral substances were applied to a silica gel G
plate (Analthech Imc. Newark, USA) and developed in acetone:chloroform:methanol:water (16:2:2:1, by vol.) and detected by
heating at 100 °C after spraying with an anisaldehyde±sulphuric acid
reagent (1310±7 m3 concentrated H2SO4 plus 5310±6 m3 acetic acid
containing 5310±8 m3 anisaldehyde).
To determine the contents of starch in the stem segments, ethanolinsoluble fractions were extracted with 1310±6 m3 of hot water at
100 °C for 10 min and then treated with 1310±6 m3 of 9.23103 mol
m±3 perchloric acid at 20 °C for 15 min. After the addition of 33
10±6 m3 deionized water, the supernatant was collected by
centrifugation. The precipitate was twice extracted with 2310±6
m3 of 4.83103 mol m±3 perchloric acid. The ®nal precipitate was
kept for the analysis of polysaccharides except for starch. The
combined extract was hydrolysed at 100 °C for 2 h and then
neutralized with 2.53103 mol m±3 NaOH to adjust the pH to around
5.4. The amount of reducing sugar in the hydrolysate was measured
by the method of Somogyi±Nelson. The amount of starch is
expressed as a value obtained by multiplying the measured amount
of reducing sugars by 0.9.
To determine the amount of polysaccharides except for starch, the
residue obtained by extraction with perchloric acid was hydrolysed
with 2310±6 m±3 of 0.53103 mol m±3 HCl at 100 °C for 2.5 h. The
hydrolysate was neutralized with 0.5310±3 mol m3 NaOH and
centrifuged to collect the supernatant. Reducing sugars in the
hydrolysate were measured by the method of Somogyi±Nelson. The
amount of polysaccharides, except for starch, is expressed as a value
obtained by multiplying the amount of reducing sugars by 0.9.
Analysis of amino acids: The amount of amino acids in the fraction
eluted from a Dowex 50W-X8 column was determined with
ninhydrin after the complete removal of the ammonia used to elute
the amino acids. Then each amino acid was separated with a
Cosmosil 5C18-MS column (nacalai tesque, Tokyo, Japan) after
dabsylation with 4-dimethylaminoazobenzene-4¢-sulphonyl chloride
according to the method of Stocchi et al. (1989). Dabsylated amino
acids were detected at the absorbance of 436 nm. The amount of
each amino acid was determined by using a standard mixture of
amino acids (Wako Pure Chemical Industries, Osaka, Japan).
Growth and glycolysis in anoxic pondweeds 1849
Total nitrogen contents: To estimate total content of nitrogenous
compounds in stem segments, dried segments were degraded by the
Kjeldahl method (Hambrñus et al., 1976). The amount of ammonia
produced by Kjeldahl degradation was determined using Nessler's
reagent (Thompson and Morrison, 1951).
Lactate contents: After incubation, the segments (about 120 mg FW)
were frozen in liquid nitrogen, powdered with a mortar and pestle,
and extracted with 6310±7 m3 of ice-cold 0.63103 mol m±3
perchloric acid for 10 min and then centrifuged to collect the
supernatant. The extraction with perchloric acid was repeated again.
The combined extract (1.2310±6 m3) was neutralized with 43
103 mol m±3 KOH after the addition of 0.05% methyl orange of
2.5310±9 m3 as a pH indicator. After cooling on ice for 15 min, the
extract was centrifuged at 15 000 g for 10 min to collect the
supernatant.
The amount of lactate in the extract was determined by an
enzymatic method with lactate dehydrogenase and alanine aminotransferase (Noll, 1985).
14
C-glucose and 14C-pyruvate feeding experiments
After incubation of stem segments in the gas-¯ow system, the stop
valve attached to the lid of the growth chamber was closed to shut off
the supply of gas. After separation of the growth chamber, D-(U14
C)-glucose of 0.37 MBq (ARC, speci®c activity, 300 mCi mmol±1)
or (2-14C)-pyruvate of 0.11 MBq (ARC, speci®c activity, 5 mCi
mmol±1) was injected into the small vial with stem segments in the
growth chamber by a syringe through a rubber septum (5 mm
double-skirted rubber stopper, Sigma±Aldrich Japan, Tokyo, Japan)
attached to the lid of the growth chamber. The hole of the rubber
septum was sealed with adhesive vinyl tape. The segments treated
with 14C-glucose and those treated with 14C-pyruvate were incubated
in the dark at 25 °C for a further 8 h and 1.5 h, respectively. After
incubation, the segments were washed with deionized water three
times, weighed, and plunged into liquid nitrogen. Ethanol (53
10±6 m3) was added to three frozen segments and heated at 80 °C for
2 h. An aliquot of the ethanol-soluble fraction was used to distill
ethanol with a small distillatory apparatus. The ethanol-insoluble
materials were further extracted with 3310±6 m3 of 50% ethanol and
2310±6 m3 of deionized water at 80 °C for 2 h and 1 h, respectively.
The combined ethanol- and water-soluble fraction was dried under
reduced pressure and redissolved in deionized water as an ethanolsoluble fraction. An aliquot of the ethanol-soluble fraction was
applied to a Dowex1-X8 column and a Dowex 50W-X8 column as
described in the Extraction section and separated into acidic, basic
and neutral substances. The ethanol-soluble fraction and the acidic,
basic and neutral substances were analysed by two-dimensional thinlayer chromatography (2-D TLC) with a cellulose thin-layer plate
(Avicel SF 20320 cm, Asahi Kasei Co., Tokyo, Japan) and
developed according to the method described in a previous paper
(Ishizawa and Esashi, 1988). The ®rst solvent was isobutylic
acid:water:n-propanol:n-butanol:isopropanol:25% NH4OH:0.053
103 mol m±3 EDTA (50:18:7:1.5:1.5:2:1, by vol.), and the second
solvent was sec-butanol:98% formic acid:water (6:1:2, by vol.).
Before the second development, three different amounts of 14Cglucose were applied to the plate as a standard for calculating
radioactivity incorporated into each separated metabolite. The
chromatogram was exposed to X-ray ®lm (Konica Medical ®lm,
New A) for 8 d at ±80 °C. To estimate radioactivity of each
metabolite separated by 2-D TLC, the density of each spot on the
autoradiogram was determined by NIH Image using an Apple
Macintosh computer. For identi®cation of metabolites separated by
2-D TLC, the metabolites were eluted from the plate and again
analysed together with authentic compounds by cellulose and silica
gel G TLC. Radioactivities in distilled ethanol, in the ethanol-
soluble fraction and ethanol-insoluble fraction, and in 43103 mol
m±3 KOH as an absorbent of carbon dioxide were determined using a
liquid scintillation spectrometer (LSC-300, Aloka, Tokyo, Japan).
Measurement of alanine aminotransferase activity
Fresh weights of stem segments were measured after incubation. The
segments were frozen in liquid nitrogen and powdered with a mortar
and pestle with cooling by liquid nitrogen. Ten volumes of extract
buffer (100 mol m±3 TRIS±HCl, pH 8.0, 15% glycerol and 10 mol
m±3 dithiothreitol) were added to the powder. The supernatant was
collected by centrifugation at 15 000 g for 15 min and fractionated
by the addition of solid (NH4)2SO4. The fraction precipitated with
80% saturated (NH4)2SO4 was dissolved in extraction buffer and
desalted with Centricorn (Amicon, USA). All extract procedures
were conducted at 4 °C.
The activity of alanine aminotransferase was measured by the
method of Hùrder and Rej (1983). The reaction mixture was
composed of 2310±5 m3 of alanine/TRIS buffer (615 mol m±3
alanine plus 110 mol m±3 TRIS, pH 7.3), 1310±7 m3 of lactate
dehydrogenase (speci®c activity:33109 U m±3), 5310±7 m3 of
500 mol m±3 pyridoxal 5-phosphate, and 3.3 mg NADH. A crude
enzyme solution of 3310±8 m3 was added to 2310±6 m3 of reaction
mixture was pre-incubated at 30 °C for 10 min. After addition of
2310±7 m3 of 2-oxoglutarate/TRIS buffer (180 mol m±3 2-oxoglutarate, 110 mol m±3 TRIS, pH 7.3), the decrease in absorbance at
340 nm was measured at 30 °C. The content of proteins in the sample
was measured by the method of Bradford (1976).
Preparation of specimens for light microscopic observation
Stem segments of pondweed turions were incubated in aerobic and
anaerobic conditions and ®xed with 2.5% glutaraldehyde in 10 mol
m±3 MES buffer (pH 6.5) overnight, post-®xed with 1% OsO4 in the
same buffer for 2 h at 4 °C, dehydrated in a graded ethanol series,
and in®ltrated with Spurr's resin (Spurr, 1969). Thin sections were
stained with 0.1% toluidine blue O in 100 mol m±3 phosphate buffer
(pH 7.4) for observation under an Olympus Vanox light microscope.
Results
Effects of anaerobic conditions on metabolism in
elongating stem segments
Effects of anaerobic treatments on growth were examined
in stem segments excised from pondweed turions. In the
absence of oxygen, length (Fig. 1A) and fresh weight
(Fig. 1B) of segments increased, but the dry weight
(Fig. 1C) decreased. These results show that some
materials in stem tissues are consumed for the stimulated
elongation in anaerobic conditions. To examine which
components of the stem are used during anaerobic growth,
each component was extracted from the growing segments.
Over a period of 6 d, neither contents of nitrogenous
compounds (Fig. 2A) nor contents of polysaccharides
except for starch (Fig. 2B) in segments changed in
anaerobic conditions. However, contents of starch
(Fig. 2C) decreased in anaerobic conditions. Ethanolsoluble sugars (Fig. 2D) decreased after 1 d of anaerobic
incubation and then slightly increased. The amounts of
starch that had been consumed after 6 d of anaerobic
incubation corresponded to about 60% of the decreases
in dry weights of segments shown in Fig. 1C. Figure 3
1850 Sato et al.
Fig. 1. Time-courses of aerobic and anaerobic growth of stem
segments excised from pondweed turions. Length (A), fresh weight
(B) and dry weight (C) of the segments were measured after
incubation in air and nitrogen gas. The fresh and dry weights are
means of three segments and the data are means 6SE of nine
replicates and six replicates, respectively. The length is the mean 6SE
of 27 segments in nine replicates.
shows cross-sections of stem segments after 3 d in aerobic
(Fig. 3A) and anaerobic (Fig. 3B) conditions. Large
aerenchymas developed in stem tissues. Both parenchymatous and epidermal cells contained many amyloplasts.
After 3 d of incubation, a number of amyloplasts in the
cells had drastically decreased in anaerobic conditions.
These results show that storage starch in pondweed stems
is the main substrate for anaerobic respiration. On the other
hand, ethanol-soluble sugars of pondweed turions were
analysed by cellulose TLC and it was found that the main
sugar was sucrose (data not shown).
Figure 2E shows that total contents of amino acids
signi®cantly increase throughout the incubation period in
anaerobic conditions. The content of each free amino acid
in stem tissues was determined by HPLC analysis
Fig. 2. Changes in contents of nitrogenous compounds (A),
polysaccharides except for starch (B), starch (C), ethanol-soluble
sugars (D), and amino acids (E) in pondweed turions in aerobic and
anaerobic conditions. Analytical methods for each substance are
described in the Materials and methods. Data are means 6SE of three
replicates.
Growth and glycolysis in anoxic pondweeds 1851
Fig. 3. Decrease in the number of amyloplasts after anaerobic elongation of the stem in pondweed turions. Cross-sections of stems were prepared
from segments that had been incubated in air (A) and nitrogen gas (B) for 3 d and stained with 0.1% toluidine blue O. Bars show 25 mm.
Table 1. Changes in contents of amino acids in stem segments incubated in aerobic and anaerobic conditions
Amino acids
Asp
Glu
Ser
Thr + Arga
Gly
Ala
Pro+GABAa
Val
Ile
Leu
Phe
Cys
Lys
His
Tyr
a
Treatment
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Air
N2
Contents of amino acids (nmol mg±1 DW)
Initial
1d
3d
6d
1.9361.44
5.9860.25
0.0360.03
1.7160.51
1.3160.31
0.4760.07
0.2560.06
1.4660.11
1.3160.31
0.2360.05
0.1660.06
1.4260.14
6.7562.37
0.7560.29
0.7860.19
0.4160.05
0.5660.03
0.1460.02
0.3260.03
0.1660.03
0.3260.03
0.8360.67
1.2661.07
nd
nd
0.3260.24
0.3760.17
0.0760.07
0.0460.04
0.1860.24
0.2360.10
2.4261.82
0.8860.88
1.4360.60
0.6960.35
0.4560.12
0.3660.06
2.1160.26
1.4260.33
0.2460.11
0.2560.16
1.2360.24
6.4962.76
0.7760.12
1.8660.34
0.8160.08
1.1360.05
0.3060.03
0.6460.04
0.0960.05
0.7460.01
0.2660.11
1.0460.78
nd
0.3760.22
0.4660.11
0.5160.19
0.1060.10
0.0460.04
0.1860.13
0.4260.13
8.8160.17
2.5862.32
0.5360.12
1.3760.08
0.3260.03
0.4860.02
1.1460.46
2.2360.21
0.1760.04
0.4560.14
0.7860.12
18.363.38
0.5160.21
3.5060.70
0.7560.30
1.2860.05
0.3260.07
1.0060.19
0.1260.04
1.2160.28
0.6960.49
7.6667.08
nd
0.2160.12
0.2860.06
0.5860.10
0.1260.12
0.0960.09
0.1660.06
0.5560.18
0.7460.44
0.3060.15
1.7260.06
0.1460.06
1.4160.70
0.2360.10
0.3860.12
0.1760.09
0.0360.03
0.5060.34
nd
0.1460.14
nd
0.0660.04
Threonine and arginine, and proline and g-aminobutylic acid were not separated.
(Table 1). Contents of glutamine, asparagine, methionine,
and tryptophan in the tissues were not determined by this
method. Moreover, threonine and arginine, and proline and
g-aminobutyric acid (GABA) were not completely separated. The content of aspartate in anoxic stems was less than
that in normoxic stems. Contents of alanine, valine,
isoleucine, and leucine were increased by anaerobic
conditions. Contents of GABA plus proline also tended
to increase in anaerobic conditions. The increase in alanine
content was the most prominent, and its content after 6 d of
anaerobic conditions was 10-fold greater than that of initial
tissues. In aerobic conditions, contents of amino acids
except for that of aspartic acid remained almost constant
throughout the incubation period.
1852 Sato et al.
Determination of metabolic fate of glucose
incorporated into stem tissues
To determine the characteristics of anaerobic metabolism
in pondweed stems, 14C-labelled metabolites were analysed after 14C-glucose feeding in an aerobic and an
anaerobic closed system for 8 h to pondweed stems that
had been incubated for various periods in the gas-¯ow
apparatus. The total incorporation of 14C into stem tissues
(Fig. 4A) is expressed as the sum of 14C radioactivities in
the ethanol-soluble fraction (Fig. 4B), ethanol-insoluble
fraction (Fig. 4C), CO2 absorbed by KOH (Fig. 4D), and
ethanol extracted from segments (Fig. 4E). Total incorporation of 14C into stem segments gradually decreased in
both aerobic and anaerobic conditions. The amount of 14Cglucose incorporated in aerobic conditions was 2.0±3.1fold greater than that in anaerobic conditions. In both
aerobic and anaerobic conditions, 14C incorporated into
soluble fractions (Fig. 4B) was more than 75% of the total
incorporation. Incorporation of 14C into ethanol-insoluble
fractions (Fig. 4C) was inhibited by anaerobic conditions,
suggesting that anabolism is depressed in anoxia. The
amounts of 14C released as CO2 in air were 2.3±3.7-fold
higher than those in nitrogen gas (Fig. 4D). On the other
hand, over 15% of 14C-glucose incorporated into stem
segments in anaerobic conditions was converted into
ethanol, but there was almost no 14C incorporation into
ethanol in air. The results show that activation of ethanol
fermentation continues throughout the anaerobic incubation period.
14
C-labelled metabolites of stem segments were
separated by 2-D TLC. Figure 5 shows autoradiograms
of 2-D TLC analysis of ethanol-soluble materials extracted
from stem segments in air or nitrogen gas for 3 d and 6 d.
Sucrose was heavily radiolabelled regardless of the
presence or absence of oxygen, indicating that sucrose
synthesis is always active in stem tissues. Radioactivity of
many metabolites was decreased in anaerobic conditions,
but 14C incorporated into alanine was signi®cantly
increased. Figure 6 shows changes in the percentages
of 14C incorporated into alanine, ethanol and sucrose to
total 14C incorporation, which were calculated from the
data in Figs 4 and 5. In air, 14C incorporation into ethanol
was negligible, and the ratio of 14C incorporation into
alanine gradually decreased. In anaerobic conditions,
ratios of 14C incorporation into ethanol were high, and
they continued to increase with incubation time. The
amount of 14C-labelled ethanol released into the gas space
of the incubation chamber could not be measured.
Therefore, the actual ratio of 14C incorporation into
ethanol might be greater than the value shown in Fig. 6.
On the other hand, both ratios of 14C incorporated into
sucrose and alanine remained at about 10% in anaerobic
conditions, and they were always greater than those in
aerobic conditions.
Fig. 4. Changes in 14C incorporation into the ethanol-soluble fraction
(B), ethanol-insoluble fraction (C), CO2 (D), and ethanol (E) in
pondweed turions incubated with (U-14C)-glucose in aerobic and
anaerobic conditions. Total incorporation of 14C into tissues (A) is the
sum of radioactivities in all fractions. Data are means 6SE of three
replicates.
14
C incorporation into lactate in the 14C-glucose feeding
experiments could not be determined because lactate was
not detected by 2-D TLC. Changes in contents of lactate in
Growth and glycolysis in anoxic pondweeds 1853
Fig. 5. Autoradiogram of two-dimensional TLC. Ethanol-soluble compounds were extracted from pondweed turions that had been incubated in
aerobic and anaerobic conditions for 3 d. Authentic Glc shows spots of 14C-glucose (17, 50 and 170 Bq) that were applied as a standard for
quantitative analysis to the TLC plate before the second development. Ala, alanine; Suc, sucrose; Glc, glucose; Fru, fructose. Arrows show the
®rst and second dimensions.
stem tissues were therefore measured by enzymatic
analysis (Fig. 7). The lactate content increased after 1 d
of incubation in anaerobic conditions, and the increase had
almost stopped after 3 d of incubation.
transferase, which is involved in the conversion of
pyruvate to alanine (Fig. 8). Anaerobic conditions activated alanine aminotransferase throughout the incubation
period until the sixth day of incubation.
Enhancement of alanine aminotransferase activity by
anaerobic conditions
Table 1 and Fig. 6 clearly show that alanine synthesis is
promoted by anaerobic treatments. An experiment was
carried out to determine whether the increase in alanine
synthesis is accompanied by activation of alanine amino-
Metabolism of (2-14C)-pyruvate fed to stem segments
14
C-labelled metabolites in stem segments fed (214
C)pyruvate for 90 min in anaerobic conditions were
analysed after anaerobic incubation for 1 d. Ethanolsoluble materials extracted from the segments were
applied to a Dowex 50W-X8 column. The metabolites
1854 Sato et al.
Fig. 7. Changes in contents of lactate in pondweed turions incubated
in aerobic and anaerobic conditions. Data are means 6SE of three
replicates.
Fig. 6. Distribution of 14C incorporated into pondweed turions among
ethanol, sucrose and alanine. Total 14C incorporation and 14C
incorporated into ethanol were the same as data shown in Fig. 4. 14C
incorporation into sucrose and alanine was estimated from the density
and size of each spot on the autoradiogram shown in Fig. 5. Data are
means 6SE of three replicates.
bound to the resins were eluted with ammonia and
separated by 2-D TLC analysis. Only two radioactive
metabolites were detected on the autoradiogram. The two
compounds eluted from the plate were identi®ed as
glutamic acid and GABA by cellulose TLC with a solvent,
phenol:water (3:1, w/w), and by silica gel G TLC with a
solvent, ethanol:28% ammonia water (77:23, v/v), respectively (data not shown).
Discussion
Anaerobic elongation of pondweed turions is supported by
a metabolic system to keep the energy status at the same
level as that in air (Ishizawa et al., 1999). Findings in this
study show that storage starch is the main substrate for
anaerobic respiration. Consumption of starch was promoted in anaerobic conditions, but inhibited in aerobic
conditions (Figs 2C, 3). In a previous paper (Ishizawa et al.,
1999), it was reported that the rate of ethanol production is
greatly enhanced by anaerobic conditions. 14C-glucose
incorporated into turion tissues was ef®ciently converted
into ethanol (Figs 4, 6). These results show that ethanol
fermentation is the main metabolic pathway for production
of ATP which is necessary for anaerobic growth of
pondweed turions.
Summers et al. (2000) reported that anaerobic elongation of Potamogeton pectinatus stems is associated with an
unusually large Pasteur effect. They suggested that starch
is a source of energy in anaerobic conditions. Both
Potamogeton distinctus and P. pectinatus seem to have a
similar mechanism for regulation of energy production
through glycolysis and ethanol fermentation in anaerobic
growth. In the case of Potamogeton distinctus, the
consumption of starch was clearly prevented under aerobic
Fig. 8. Changes in the activities of alanine aminotransferase in
pondweed turions incubated in aerobic and anaerobic conditions. Data
are means 6SE of three replicates.
conditions compared with anaerobic conditions (Fig. 2C),
suggesting a manifestation of the Pasteur effect. The
occurrence of the Pasteur effect should show an enhancement of CO2 released under anaerobic conditions.
However, the ratios of 14C incorporation into CO2 in
aerobic conditions to that in anaerobic conditions remained
around 3.0 (Fig. 4D), suggesting that a Pasteur effect does
not occur. The incompatible result seemed to be attributed
to potential problems of the method to estimate a rate of
CO2 production. Since the uptake of 14C-glucose by
turions was signi®cantly inhibited in anoxia (Fig. 4A), it
was unsuitable to compare the amounts of 14C incorporation into CO2 in anaerobic conditions directly with those
in aerobic conditions. On the other hand, SchluÈter and
Crawford (2001) reported that long-term anoxia tolerance
in leaves of Acorus calamus and Iris pseudacorus is
associated with down-regulation of glycolysis. Whether a
Pasteur effect is brought about in the growth of anoxic
pondweed turions warrants further investigation.
Figures 5 and 6 show that the active biosynthesis of
sucrose continues in anoxic pondweed turions. Sucrose is
the main sugar in ethanol-soluble fractions of pondweed
stems. Figure 2D shows that ethanol-soluble sugars
decrease at an early stage of anaerobic incubation,
Growth and glycolysis in anoxic pondweeds 1855
suggesting that sucrose is actively used. These results
suggested that sucrose biosynthesis is enhanced by
anaerobic conditions. Also, in the case of Potamogeton
pectinatus stems (Summers et al., 2000), sucrose accumulates in anaerobic conditions. In both cases, the conversion
from starch to sucrose may be necessary in order to use it
as a substrate for glycolysis. The meaning of sucrose
conversion remains unclear. Sugars produced by starch
degradation may be transported to a site of glycolysis after
the conversion of sucrose. Alternatively, the conversion to
sucrose may be involved in the regulation of carbohydrate
metabolism just as the futile cycle of sucrose metabolism
operates in sink organs (Nguyen-Quoc and Foyer, 2001).
Further study on the degradation process of storage starch
in amyloplasts in anaerobic conditions (Fig. 3) is needed to
determine the meaning of sucrose biosynthesis.
Many plant tissues in anaerobic conditions produce
lactate in addition to ethanol (Davies, 1980). In the case of
pondweed turions, lactate accumulation almost stopped at
1 d after anaerobic incubation (Fig. 7), whereas the
conversion from glucose to ethanol increased after 3 d of
anaerobic incubation (Fig. 6). These results suggest that
lactate fermentation precedes alcoholic fermentation in
pondweed turions exposed to anaerobic conditions. Davies
et al. (1974) and Roberts et al. (1984) proposed that a shift
from lactate fermentation to alcoholic fermentation occurs
by the operation of a biochemical pH-stat. This metabolic
change in pondweed turions may be regulated by the
biochemical pH-stat mechanism. However, it should be
noted that the pH-stat hypothesis is debatable (Ratcliffe,
1997; Vartapetian and Jackson, 1997).
In addition to ethanol and lactate, there was a signi®cant
accumulation of alanine as an end-product of anaerobic
metabolism in pondweed turions (Figs 5, 6; Table 1).
Several plants subjected to anaerobic stress are known to
produce alanine (Streeter and Thompson, 1972; Smith and
ap Rees, 1979; Menegus et al., 1988). These alanine
accumulators are not always anaerobic-tolerant plants.
Therefore, the degree of anaerobic tolerance is unlikely to
be determined by the accumulation of alanine itself. In
maize root tips (Roberts et al., 1992), alanine as well as
lactate is produced at the early stage of hypoxia, but
alanine itself does not seem to contribute to cell acidosis.
In pondweed turions, however, a high rate of alanine
conversion from glucose was maintained throughout the
period of anaerobic elongation (Fig. 6). Unlike ethanol
fermentation, the rate of alanine production did not change
during the 6-d anaerobic period (Fig. 6). Crawford (1978)
pointed out that diversity of end-products in anaerobic
metabolism is important to prevent the accumulation of a
toxic metabolite, such as lactate or ethanol. Alanine
production may contribute to the ef®ciency of glycolysis
by dispersing the end product. The metabolic fate of
alanine, which accumulated in anaerobic conditions, was
not examined after anoxic turions had been transferred to
aerobic conditions. Lactate and ethanol, but not alanine,
are wasteful end-products of anaerobic metabolism. The
physiological signi®cance of the accumulation of alanine
may be a temporary store of carbon and nitrogen in a state
of anoxia.
The activity of alanine aminotransferase was enhanced
in pondweed turions in anaerobic conditions (Fig. 8). The
activities of aminotransferase in barley roots (Muench and
Good, 1994) and in corn roots (Muench et al., 1998) were
reported to be greatly enhanced by anaerobic treatments. In
the case of barley (Muench and Good, 1994), a speci®c
isozyme of alanine aminotransferases is increased in
anaerobic conditions. On the other hand, valine and
leucine, which are usually produced from pyruvate,
signi®cantly increased in anoxic turions, whereas aspartate
signi®cantly decreased (Table 1). Such a signi®cant
decrease in asparatate content by anaerobic treatment has
been found in maize root tips (Roberts et al., 1992), pea
roots (Smith and ap Rees, 1979) and radish leaves (Streeter
and Thompson, 1972). In these cases, aspartate is thought
to be the main source of the amino group of alanine. All
these facts suggest that alanine is produced from pyruvate
by an action of alanine aminotransferase.
In this study, however, 14C incorporation into alanine
could not be detected when 14C-pyruvate was fed to
pondweed turions in anaerobic conditions, but 14C
incorporation into GABA and glutamate was detected.
Table 1 suggests the increase in GABA contents by
anaerobic treatments. Stimulation of GABA production by
anaerobic conditions has been reported in many plants
(Shelp et al., 1999). GABA synthesis is thought to
contribute to the prevention of cytoplasmic acidosis in
anaerobic conditions because it occurs through the H+consuming a-decarboxylation of glutamate by an action
of glutamine decarboxylase (EC 4.1.1.15). Pyruvatedependent 4-aminobutylate transaminase has been reported to be involved in GABA synthesis (Van Cauwenberghe
and Shelp, 1999). Therefore, 14C-pyruvate may be exclusively used for GABA synthesis rather than alanine
synthesis. Moreover, the results suggest that alanine
synthesis in pondweed turions occurs in a special compartment where access by exogenous pyruvate is dif®cult.
However, the reason why 14C-pyruvate is not converted to
alanine remains unknown.
Acknowledgement
We thank Mr Kenichi Satoh for help with the cultivation of
Potamogeton plants.
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