Environmental and Experimental Botany 50 (2003) 29 /40
www.elsevier.com/locate/envexpbot
Anaerobic metabolism in the roots of seedlings of the invasive
exotic Lepidium latifolium
Hongjun Chen *, Robert G. Qualls
Environmental Science and Health Graduate Program, Department of Environmental and Resource Sciences, University of Nevada, Reno,
NV 89557, USA
Received 14 June 2002; received in revised form 25 November 2002; accepted 26 November 2002
Abstract
Lepidium latifolium is an invasive exotic crucifer that is widely distributed in riparian zones and wetlands. In this
study, anoxic carbohydrate metabolism and post-anoxic injury in the roots of L. latifolium seedlings were examined. A
significant increase in the activity of the fermentative enzymes alcohol dehydrogenase (ADH) and lactate
dehydrogenase (LDH) in roots occurred under anoxia and increased with the duration of anaerobic treatment during
7 days. However, pyruvate decarboxylase (PDC) and cytochrome c oxidase (CCO) activity was maintained at relatively
stable levels under anaerobic and aerobic conditions. Soluble protein concentration in anoxic roots was two to three
times that in aerobic roots throughout 7 days of anoxia. The concentration of the fermentation product ethanol in roots
was two times greater under anoxia than under aerobic conditions. The concentration of lactate was much smaller than
that of ethanol, but the trend was similar to that of ethanol. There was no significant difference in the concentration of
malate between aerobic and anaerobic conditions for 9 days. Superoxide dismutase (SOD) activity in roots was two to
three times higher under anoxia than under aerobic conditions throughout 7 days, but this increase in SOD activity
decreased slightly with the duration of anoxia. Compared with aerobic conditions, the concentration of malondialdehyde (MDA), an indicator of free radical damage, increased by two to three times under anoxia. Two days after L.
latifolium seedlings were returned to aerobic conditions, the concentrations of ethanol and MDA in roots were still
significantly higher under the previously anoxic treatment than under continuously aerobic conditions, while no
significant difference in enzyme activities or in concentrations of lactate and malate was found between treatments. The
metabolism of L. latifolium roots under anoxia is characterized by the concurrent activity of both fermentative
pathways and aerobic metabolism. Roots of L. latifolium have metabolically adaptive strategies to anoxia, but there is
evidence of oxidative stress under anoxia and of post-anoxic injury from free radicals upon re-exposure to air. Results
suggest that L. latifolium exhibit a mixture of characteristics typical of hydrophytic, facultative, and anoxia intolerant
species.
# 2003 Elsevier Science B.V. All rights reserved.
Keywords: Anoxic tolerance; Flooding; Metabolic adaptation; Oxidative damage; Weed; Wetlands
* Corresponding author. Present address: Wetland Biogeochemistry Institute, Louisiana State University, Baton Rouge, LA 70803,
USA. Tel.: /1-225-578-6429; fax: /1-225-578-6423.
E-mail address: [email protected] (H. Chen).
S0098-8472/02/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0098-8472(02)00112-0
30
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
1. Introduction
Lepidium latifolium L., perennial pepperweed or
tall whitetop, is an invasive exotic crucifer (Young
et al., 1995) that has been classed as a noxious
weed in the western United States. It is extremely
competitive in many habitats, especially in wetlands and riparian areas, and forms a monospecific stand that can crowd out desirable native
species (Young et al., 1995). It tolerates a wide
range of soil water potential and exhibits morphological adaptability to survive long periods of
flooding by developing extensive aerenchyma in
roots and adventitious roots on the base of
flooded stems (Chen et al., 2002).
Most plants can tolerate anoxia for only short
periods of time before irreparable damage occurs
(Crawford, 1993). Under anaerobic conditions, the
absence of oxygen results in a switch from an
aerobic metabolism to fermentative pathways,
allowing the re-oxidation of NADH and the
production of ATP (Perata and Alpi, 1993). The
fermentation pathways leading to the production
of lactate and ethanol are believed to play a major
role in anaerobic recycling of NADH (Perata and
Alpi, 1993; Armstrong et al., 1994), although other
pathways exist (Kennedy et al., 1992). However,
the anaerobic pathway of metabolism alone is not
able to sustain the cellular extension and division
phases of root growth (Crawford, 1993). Acidification of the cytoplasm is viewed as a cause of
injury and death of most plant roots under anoxia
(Drew, 1997). Davies et al. (1974) has suggested
that lactate dehydrogenase (LDH, EC 1.1.1.17)
and pyruvate decarboxylase (PDC, EC 4.1.1.17)
act as a metabolic pH-regulator in the fermentative metabolism. However, Kennedy et al. (1992)
pointed out that the hypothesis cannot serve as a
unifying theory for flood tolerance because it does
not appear to operate in the same manner in plants
that are truly flood-tolerant, such as rice (Oryza
sativa L.) and Echinochloa phyllopogen (Stapf)
Kos. Anaerobic metabolism in plants, depending
on the species, may not be limited to the induction
of alcoholic or lactic acid fermentation as the only
biochemical responses to anoxia or hypoxia (Kennedy et al., 1992).
Compared with flood-sensitive species, floodtolerant species are better able to regulate their
processes of glycolysis and fermentation to ethanol
(Drew, 1997). Many studies (e.g. John and Greenway, 1976; Rumpho and Kennedy, 1981; Ishizawa
et al., 1999) have indicated that there is a
significant increase in activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) in flood-tolerant species
under anaerobic conditions. This increase in ADH
activity may help flood-tolerant plants avoid the
detrimental effects of the accumulation of highly
toxic acetaldehyde or lactate, which leads to
cytoplasmic acidification (Perata and Alpi, 1993).
Pearson and Havill (1988) have shown the importance of this regulation by comparing the
metabolic changes in roots of barley (Hordeum
vulgare L.) and wetland rice under hypoxia. In
barley under hypoxia, ADH activity increased
about 10-fold whereas cytochrome c oxidase
(CCO, EC 1.9.3.1) activity decreased markedly.
Despite the high carbon flow to ethanol, barley
was not able to maintain energy charge. In
contrast, rice retained high ADH activity and
relatively constant CCO activities. CCO’s high
affinity for O2 may help scavenge any available
oxygen in the roots to maintain a high ATP level.
Re-exposure to oxygen after anoxia causes
severe injury to plant tissues and organs (Crawford, 1993). The main reason is the generation of
superoxide radicals, iron induced-hydroxyl radicals, or other reactive oxygen species (Hendry and
Brocklebank, 1985). The oxygen free radicals react
with cell components and produce a cascade of
oxidative reactions (Scandalios, 1993). Lipid peroxidation, an important oxidative reaction in
biological membranes, causes impairment of membrane functioning, inactivation of membranebound receptors and enzymes, and increased
non-specific permeability to ions (Gutteridge and
Halliwell, 1990). Consequently, lipid peroxidation
is often taken as a stress indicator of oxidative
damage in plants under environmental stresses
(Hendry and Grime, 1993). In anoxia tolerant
species, malondialdehyde (MDA), an important
product of lipid peroxidation, occurs to a much
smaller extent than in intolerant species, as has
been shown for wheat (Albrecht and Wiedenroth,
1994), Brassica napus L. (Leul and Zhou, 1999),
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
and Iris germanica L. (Hunter et al., 1983). Anoxia
tolerant species might be better protected against
oxygen free radicals either by the accumulation of
antioxidants, or by the induction of superoxide
dismutase (SOD, EC 1.15.1.1) (Larson, 1988). In
flood-tolerant species, an increase in SOD activity
is an important protection mechanism in preventing oxidative damage from free radicals during
recovery from anoxia stress (Crawford, 1993).
L. latifolium survives long periods of soil flooding but grows very little (Blank et al., 2002). It also
grows in habitats with large fluctuations in soil
moisture. We hypothesized that L. latifolium
seedlings (1) possess metabolic adaptations to
root anoxia, thus leading to a strong alcoholic
fermentation; (2) suffer from decreased electron
transport activity as indicated by CCO activity;
and (3) increase SOD activity, thus maintaining
the low concentration of MDA in roots, and
avoiding post-anoxic injury from oxygen free
radicals upon re-exposure to air. In order to test
these hypotheses, we examined important fermentative enzymes (ADH, LDH and PDC), the
metabolic end products of pyruvate, CCO, SOD
and MDA in the roots of L. latifolium seedlings.
2. Materials and methods
2.1. Plant material
Seeds of L. latifolium were collected from
University of Nevada Farm at Reno, Nevada,
and germinated in washed sand in a greenhouse.
The seedlings were then transferred to plastic pots
with washed sand and watered using half-strength
Hoagland’s nutrient solution (Lindsay, 1991).
Later, 135 uniform small plants, with three to
four mature leaves and uniform length roots, were
selected and randomly transplanted into 45 4.2-l
plastic containers, three plants per container.
Plants were placed through holes in lids on top
of containers that contained half-strength Hoagland’s solution. Non-toxic putty was used to seal
the holes. Before initiation of the treatment, plants
were allowed to grow in nutrient solutions for 5
days that were flushed by air in order to keep them
under well-aerated conditions.
31
After the initial 5 days period, 25 of the 45
containers were randomly assigned to the anaerobic treatment. The solution in these containers was
flushed by nitrogen gas as the anaerobic treatment.
In order to remove oxygen from the nutrient
solution and provide a redox buffered solution,
titanium(III) citrate was injected into the nutrient
solution in which the roots grew (DeLaune et al.,
1990). The other 20 containers were kept under
aerobic conditions as the control and the solution
in these containers was continuously flushed by air
for 9 days. The anaerobic treatment included the
four anaerobic durations of 1, 3, 5 and 7 days.
After 7 days, all plants in anaerobic treatment
were returned to aerobic conditions for 2 days by
aerating in order to examine post-anoxic injury in
the roots of L. latifolium seedlings. On days 1, 3, 5,
7 and 9 after the initial treatment, the approximately 2 cm apical zone of plant roots was
sampled and then immediately frozen in liquid
N2 and stored at /80 8C until required for
biochemical determinations. The apical zone was
sampled because of its importance in root metabolism and the sensitivity of apical root tissue to
anoxia (John and Greenway, 1976).
Each container contained one platinum electrode and a calomel reference electrode. Redox
potential (Eh) was monitored twice per day using a
millivolt (mV) meter and was calculated by
addling the potential of the calomel reference
electrode (/244 mV) to the mV reading. Redox
potential in solution flushed by N2 was 130 /200
mV and in that flushed by air was 470/560 mV. In
this study, only the roots of seedlings grown in
nutrient solution were exposed to anaerobic conditions whereas the shoots obtained oxygen from
the atmosphere. The experimental treatment mimicked anoxia caused by soil flooding in the
natural environment.
2.2. ADH, LDH, PDC and CCO
Frozen root samples (ca. 0.5 g fresh weight)
were ground in 5 ml of extraction buffer with a
pre-cooled mortar and pestle on ice. The homogenates were filtered through Miracloth, and
centrifuged for 20 min at 30 000/g at 4 8C. The
supernatants were immediately analyzed for en-
32
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
zyme activities. All operations were carried out at
0 /4 8C. The extraction buffer for ADH and LDH
contained 50 mM Tris (pH 6.8), 5 mM MgCl, 5
mM mercaptoethanol, 1 mM EDTA, 1 mM
EGTA, 0.5 mM thiamine pyrophosphate (TPP),
15% (v/v) glycerine and 0.1 mM proteinase inhibitor (4-(2)-aminoethyl-benzensulfonyl fluoride
hydrochloride) (Biemelt et al., 1999). The extraction buffer for PDC was 125 mM 2-(N -morpholino) ethanesulphonic acid (pH 6.8), 5 mM MgCl2,
2 mM EDTA, 0.5 mM TPP, and 2 mM dithiothreitol (Thomson and Greenway, 1991). Protein
concentration in ADH and LDH extracts was
determined according to Bradford (1976).
The activities of ADH, LDH and PDC were
determined spectrophotometrically at 340 nm by
the oxidation or reduction of pyridine nucleotides
at 25 8C. The reaction mixtures (3 ml) were as
follows. ADH, 50 ml of extract, 40 mM Tris /HCl
(pH 9.0), 5 mM MgCl2, 0.10 mM NAD and 50
mM ethanol; LDH, 65 ml of extract, 45 mM Tris /
HCl buffer (pH 7.2), 0.18 mM NADH, and 3.0
mM pyruvate. For PDC assay, extracts were first
incubated at 25 8C for 1 h at pH 6.0 with 0.5 mM
TPP and 1 mM MgCl2 to ensure maximum PDC
activity (Morrell et al., 1990). The reaction mixture
was 600 ml of extract, 80 mM MES buffer (pH 6.0),
30 I.U. yeast ADH, 1 mM MgCl2, 0.5 mM TPP,
0.17 mM NADH and 10 mM sodium pyruvate.
The extraction buffer for CCO was 50 mM
KH2PO4, pH 7.4 with 1 mM EDTA and 0.1% (v/v)
Triton-100 (Pearson and Havill, 1988). The reaction mixture (3 ml) was 50 ml of extract, 50 mM
KH2PO4 (pH 7.4) with 1 mM EDTA, 7.25 mM
ascorbate and 0.05 mmol l 1 cytochrome c. Rates
of cytochrome c oxidation were measured at 550
nm and an extinction coefficient of 20 mM cm 1
was used to calculate CCO activity (Bergmeyer,
1983).
by centrifugation. Neutralized extracts were analyzed immediately. Ethanol, lactate and malate in
roots were enzymatically determined according to
Bergmeyer (1983).
2.4. SOD activity and lipid peroxidation
The extraction buffer for SOD was 50 mM
potassium phosphate (pH 7.8) containing 0.1 mM
EDTA, 1% (w/v) PVP-10 and 0.1% (v/v) Triton X100 (Iturbe-Ormaetxe et al., 1998). The reaction
mixture was 2.4 ml buffer, 0.12 ml NADH
solution, 75 ml EDTA/MnCl solution, 0.3 ml
sample (or buffer) and 0.3 ml mercaptoethanol
solution (Paoletti et al., 1986). The spectrophotometric assay was based on an inhibition of NADH
oxidation by superoxide radicals. One unit of SOD
was expressed as 50% inhibition of NADH oxidation rate.
Lipid peroxides were extracted by grinding
approximately 0.5 g fresh weight of frozen root
samples with 5 ml 50 mM phosphate buffer (pH
7.2) on ice. The homogenates were filtered through
Miracloth and centrifuged for 20 min at 30 000 /
g. MDA, a principal product of lipid peroxidation
in roots was quantified by a photometric determination of the MDA /TBA complex produced in
the reaction between MDA and 2-thiobarbituric
acid (TBA) reagent (Hendry and Grime, 1993).
The absorbance was read at 532 nm, and the
values were corrected by subtracting the nonspecific absorbance at 600 nm. The concentration
of MDA was calculated against 1,1,3,3-tetraethoxypropane standards. Calibration curves were made
using 1,1,3,3-tetraethoxypropane in the range of
0/1.5 nmol. Tetraethoxypropane was stoichiometrically converted into MDA during the acidheating step of the assay.
2.5. Data analysis
2.3. Ethanol, lactate and malate
Frozen root samples (ca. 0.5 g fresh weight)
were ground in 5 ml 30% HClO4 with a pre-cooled
mortar and pestle on ice. The homogenates were
centrifuged for 30 min at 30 000 /g at 4 8C. The
supernatant fraction was neutralized with 69%
K2CO3 and the precipitated protein was removed
The experiment consisted of two factors, Eh
treatment (anaerobic or aerobic) and the time of
treatment (1, 3, 5, 7 and 9 days). Data on all
enzyme activities, soluble protein, ethanol, lactate,
malate and MDA in 1 /7 days of treatment were
analyzed with the two-way analysis of variance
(ANOVA). Variation was partitioned into anae-
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
robic treatment and the duration of treatment as
main effects. Differences between the anaerobic
treatment and aerobic control on each individual
date were also tested using the Student’s t -test.
3. Results
3.1. ADH, LDH, PDC and CCO activities
The ADH activity of the roots of L. latifolium
seedlings under anoxia increased greatly from 2.5
mmol g1 DW min 1 at day 1 to 9.6 mmol g1
DW min1 at day 7 (Fig. 1A). There was no
significant change in ADH activity in roots under
aerobic conditions for 9 days. Compared with the
aerobic control, ADH activity of roots under
anoxia was 6.4 times that of the control at day 7.
Two days after returning to aerobic conditions,
ADH activity of roots decreased from 9.6 to 3.1
mmol g1 DW min 1 and there was no significant
difference in ADH activity under previously anoxic and continuously aerobic conditions (Fig.
1A). ANOVA showed that the effects of the
anaerobic treatment (P B/0.0001) and the time of
treatment (P B/0.001) on ADH activity were
significant.
The LDH activity of roots increased with the
time of anoxic treatment for 7 days while that of
roots in the aerobic condition did not significantly
change for 7 days (Fig. 1B). LDH activity of roots
increased from 1.2 mmol g1 DW min 1 at day 1
to 2.6 mmol g1 DW min 1 at day 7 in response to
anoxia for 7 days and was1.6 times that of the
control at day 7. Response of LDH activity in
roots was similar to that of ADH between anoxic
and aerobic conditions but maximum ADH activity was nearly four times that of maximum LDH
activity. Two days after return to aerobic conditions, LDH activity of roots decreased and there
was no significant difference in LDH activity
between anoxic and aerobic conditions (Fig. 1B).
ANOVA showed a significant effect of the anoxic
treatment (P B/0.0001) and the time of treatment
(P B/0.01) on LDH activity.
ANOVA showed that there was no significant
change in PDC activity of roots of L. latifolium
seedlings between anaerobic and aerobic condi-
33
tions for 9 days (P/0.118) (Fig. 1C). PDC
activity was maintained at a relatively stable level,
1.4 mmol g 1 DW min1. ANOVA showed no
significant effect of the time of treatment (P /
0.60) on PDC activity. While the mean PDC
activity was consistently slightly higher under
anaerobic conditions, there was no significant
difference in PDC activity of roots under aerobic
and anaerobic conditions (Fig. 1C).
The activity of CCO was significantly elevated
by anoxia only at day 7, although average CCO
activity in roots was increased by about 30% under
anoxia, compared with the control plants (Fig. 2).
Two days after seedlings returned to aerobic
conditions, CCO activities of roots between anaerobic treatment and aerobic control were equivalent (Fig. 2). ANOVA showed no significant effect
of the time of treatment (P /0.683).
Also, we observed that there was higher soluble
protein concentration in roots of L. latifolium
under anoxia than that under aerobic conditions
(Fig. 3). Soluble protein concentration of anoxic
roots was two to three times that of aerated plants
throughout 7 days. ANOVA showed a significant
effect of anaerobic treatment (P B/0.001) or the
duration of treatment (P/0.013) on protein
concentration. Two days after returning to aerobic
conditions, there was no significant difference in
soluble protein concentration between anoxic and
aerobic conditions (Fig. 3).
3.2. Ethanol, lactate and malate
The end products of pyruvate fermentation were
measured in roots grown in the two treatments.
Ethanol concentration in the roots was two times
greater under anoxia than under aerobic conditions (Fig. 4A). The concentration of lactate (Fig.
4B) was much smaller than that of ethanol, but the
trend was similar to that of ethanol between
anoxic and aerobic conditions. ANOVA showed
that there was a significant effect of anaerobic
treatment on the concentrations of ethanol (P B/
0.001) and lactate (P B/0.001). No significant
difference in the concentration of malate (Fig.
4C) in the roots was found between aerobic and
anaerobic conditions and the concentration of
malate retained at stable level around 0.3 /0.4
34
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
Fig. 1. Activities (mean9/S.E. of three plants) of (a) ADH, (B) LDH and (C) PDC in roots of L. latifolium seedlings under aerobic and
anoxic conditions. The arrow indicates a return to aerobic conditions. ANOVA showed the following effects of the treatment (AHD,
P B/0.001; LDH, P B/0.001; PDC, P/0.118) and the time of treatment (ADH, P B/0.001; LDH, P B/0.01; PDC, P /0.599).
Asterisks, *, ** and ***, indicate the difference between treatment and control at P B/0.05, 0.01 and 0.001 for a t -test, respectively. The
treatment was 7 days anoxia followed by a 2 days aerobic conditions. The control was continuous 9 days aerobic conditions.
mmol g1 DW min 1, although ANOVA showed
a significant effect of anaerobic treatment on level
of malate (P B/0.05). Two days after returning to
aerobic conditions, the concentration of ethanol
decreased but remained significantly higher in
anoxic roots of plants than that in the control
(Fig. 4A), while there was no significant difference
in the concentration of lactate or malate between
the treatment and control (Fig. 4B and C).
ANOVA showed a significant effect of the time
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
35
3.3. SOD activity and lipid peroxidation
Fig. 2. CCO activity (mean9/S.E. of three plants) in roots of L.
latifolium seedlings under aerobic and anoxic conditions. The
arrow indicates a return to aerobic conditions. ANOVA
showed a significant effect of the treatment (P B/0.003) but
the effect of the time of treatment was not significant (P/
0.683). An asterisk * indicates a significant difference between
treatment and control at P B/0.05 for a t -test. The treatment
was 7 days anoxia followed by a 2 days aerobic conditions. The
control was continuous 9 days aerobic conditions.
of treatment on the production of ethanol (P B/
0.05) and lactate (P B/0.001) but no significant
effect on malate levels was observed (P /0.446).
SOD activity was greater under anoxia throughout the 7 days (Fig. 5A). Compared with aerobic
controls, SOD activity increased by 3.6, 2.0, 2.5
and 2.6 times during 1, 3, 5, and 7 days anoxia,
respectively. The SOD activity of roots decreased
after 2 days post-anoxic recovery phase and there
was no significant difference in SOD activity
between previously anoxic and continuously aerobic conditions (Fig. 5A). ANOVA showed a
significant effect of anaerobic treatment on SOD
activity (P B/0.001), but the effect of the time of
treatment was not significant (P /0.222).
The concentration of MDA in the roots under
anoxia increased from 35 nmol g1 DW at day 1
to 56 nmol g1 DW at day 7, compared with 19.2
nmol g1 in aerated roots (Fig. 5B). There was no
significant change in the concentration of MDA in
the roots grown in aerobic conditions for 9 days. A
rise in the concentration of MDA under anoxia
increased with the time of anoxic treatment. Two
days after plants were returned to aerobic conditions, the concentration of MDA decreased from
56 to 33 nmol g1 DW, but the concentration of
MDA in the roots under anoxia was still significantly higher than in the continuous aerobic
Fig. 3. Soluble protein concentration (mean9/S.E. of three plants) in roots of L. latifolium seedlings under aerobic and anoxic
conditions. The arrow indicates a return to aerobic conditions. ANOVA showed a significant effect of the treatment (P B/0.001) and
the time of treatment (P/0.013). The asterisks * and ** indicate a significant difference between treatment and control at P B/0.05
and 0.01 by a t -test, respectively. The treatment was 7 days anoxia followed by a 2 days aerobic conditions. The control was
continuous 9 days aerobic conditions.
36
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
Fig. 4. Concentrations (means9/S.E. of three plants) of (A)
ethanol, (B) lactate and (C) malate in roots of L. latifolium
seedlings under anoxic and aerobic conditions. The arrow
indicates a return to aerobic conditions. ANOVA showed the
effects of the treatment (ethanol, P B/0.001; lactate, P B/0.001;
malate, P /0.022) and the time of treatment (ethanol, P/
0.024; lactate, P B/0.001; malate, P/0.446). Asterisks * and
** indicate the difference between treatment and control at P B/
0.05 and 0.01 for a t -test. The treatment was 7-day anoxia
followed by a 2 days aerobic conditions. The control was
continuous 9 days aerobic conditions.
conditions (Fig. 5B). ANOVA showed a significant effect of the anaerobic treatment on MDA
concentration (P B/0.0001) but no significant effect of the time of treatment was found (P /
0.151).
4. Discussion
Activities of ADH and LDH in the roots of L.
latifolium seedlings increased by several fold in
Fig. 5. SOD activity (mean9/S.E. of three plants) and concentration (mean9/S.E. of four plants) of MDA, a lipid
peroxidation product in roots of L. latifolium seedlings under
anoxic and aerobic conditions. The arrow indicates a return to
aerobic conditions. ANOVA showed a significant effect of the
treatment (SOD, P B/0.001; MDA, P B/0.001) but effect of the
time of treatment was not significant (SOD, P/0.222; MDA,
P/0.151). The asterisks * and ** indicate a significant
difference between treatment and control at P B/0.05 and
0.01 for a t -test, respectively. The treatment was 7 days anoxia
followed by a 2 days aerobic conditions. The control was
continuous 9 days aerobic conditions.
response to anoxia for 7 days and reached their
maxima on day 7 (Fig. 1A and B), as has been seen
for barnyard grass (Echinochloa crus-galli var.
oryzicola) (Rumpho and Kennedy, 1981), and
marsh plants (Smith and ap Rees, 1979). The
absolute level of ADH activity of roots was 4-fold
higher than that of LDH activity. This increase in
ADH activities is essential in maintaining ATP
production in the absence of O2 because it
enhances the flux through the glycolytic pathway
and compensates for a reduced energy yield in the
fermentative pathway (Dennis et al., 2000). Similarly, we found that the concentration of ethanol
in roots were nearly four times higher than that of
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
lactate or malate under anoxia (Fig. 4A /C). These
results showed that ethanolic fermentation is a
main metabolic pathway in the roots of L.
latifolium seedlings under anoxia, and that lactate
fermentation is an alternative pathway to alcoholic
fermentation under anoxia. This conclusion corresponds to previous studies (Perata and Alpi, 1993;
Armstrong et al., 1994; Vartapetian and Jackson,
1997) showing that alcoholic fermentation is
believed to be the main metabolic pathway for
generating ATP in the roots of plants under
anoxia.
CCO is the final enzyme regulating the flow of
electron transport chain to the terminal electron
acceptor (O2), and safely reducing O2 into H2O in
aerobic respiration (Stryer, 1995). In the absence
of CCO activity, partial reduction generates hazardous compounds such as superoxide anion.
Therefore, CCO activity is a measure of respiratory potential of a plant or tissue (Hendry and
Grime, 1993). Pearson and Havill (1988) have
shown that CCO activity of the roots of Agropyron
pungens (Pers.) Roem. and Schult. and H. vulgare
L., both non-wetland species, subjected to 3 weeks
hypoxia decreased by 80 /92%, compared with
aerated controls, while CCO activity of the roots
of O. sativa L., a wetland crop plant, was little
affected. Our study indicated that CCO activity of
the roots remained relatively stable and was
slightly higher in anaerobic conditions than in
aerobic conditions (Fig. 2), contrary to hypothesis
#2. This result indicates that mitochondrial respiration persisted in the roots of L. latifolium
seedlings grown in O2-free solution. Based on these
results above, we conclude that metabolism in the
roots of L. latifolium seedlings grown in O2-free
nutrient solution was characterized by the concurrent activity of both fermentative pathway and
aerobic metabolism. This metabolic concurrence
may be due to the heterogeneous distribution of
aerenchyma in root tissues that occurs in the
cortex, leading to the heterogeneity of O2 availability. It has been reported that maize (Zea mays
L.) roots in O2-free solution have an aerobic cortex
and an anoxic stele since the aerenchyma provides
adequate O2 for respiration in the cortex but not in
the stele (Thomson and Greenway, 1991). Thus,
this reflects an important mode of metabolic
37
adaptation to survive flooding stress, but the
inability to completely maintain aerobic metabolism via aerenchyma probably restricts its ability
to grow.
Protein biosynthesis in plant roots is immediately inhibited by anoxia (Bailey-Serres and Freeling, 1990). In maize roots, the inhibition at the
early stage of anoxia is partially restored by
inducing the synthesis of a special set of proteins
called anaerobic polypeptides (ANP) such as ADH
proteins (Sachs et al., 1996). In our study, we
observed a much higher soluble protein concentration in anoxic roots than in aerated roots
throughout 7 days (Fig. 3). Studies have shown
that ADH proteins are selectively synthesized
during anoxia in maize (Sachs and Freeling,
1978; Sachs et al., 1996). These soluble proteins
might mainly be ANP. The ANP have been viewed
to be essential for plant survival for a prolonged
period of anoxia (Sachs et al., 1996; Ishizawa et al.,
1999).
In the present study, SOD activity in the roots
of L. latifolium seedlings was two to three times
higher in anoxia than in aerobic conditions
throughout 7 days, but this increase in SOD
activity grew less with the duration of anoxia
(Fig. 5A) as has been seen for Iris pseudacorus L.,
a species tolerant to anoxia (Monk et al., 1987).
The response of L. latifolium resembled something
intermediate between flood-tolerant and intolerant
species (Hunter et al., 1983). The increase in SOD
activity under environmental stress has been
considered to play an important role in controlling
superoxide levels in cellular compartments because
SOD is widely distributed among living organisms
and considered as the major enzymatic defense
against oxidative injury present in cells (Fridovich,
1986). Little is known about the biochemical
mechanism of the increase in SOD activity of
roots in the absence of O2. In view of the
biochemical role of SOD, the enzyme is inducible
by oxygen in plants, animals and bacteria (Halliwell, 1982). However, in terms of evolutionary
adaptations, an increase in SOD activity during
anoxia is of advantage to plants when aerobic
conditions are restored.
Peroxidative damage in plants has been implicated in such processes as leaf senescence, wound-
38
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
Table 1
The responses of metabolic activities in the root to anoxia among different plant species, compared with aerobic conditions
Ratio of concentration, activity or other parameter under
anoxic vs. aerobic conditions
L. latifolium
Obligate
ADH
6.4
4 /10
PDC
Ethanol
1.2
1.9
10
3 /6
CCO
SOD
1.6
2.6
1
3 /13
MDA
2.9
Yielda
0.3
Porosity (%)a
2.0
Porosity under anoxic 43.6
conditions (%)a,b
0.6
1.1 /1.7
1.5 /1.9
13.8 /29.6
Facultative
3.5
No data
6
0.6
0.6 /2.0
0.8 /1.3
0.5
2.2
8.3
Anoxia-intolerant
10 /40
6 /27
7 / /100
0.2
0.5
138
0.3
2.0
4
Biemelt et al., 1999; Ishizawa et al., 1999; Pearson
and Havill, 1988; John and Greenway, 1976
Biemelt et al., 1999; John and Greenway, 1976
Good and Muench, 1993; Pearson and Havill,
1988; Smith and ap Rees, 1979; John and Greenway, 1976
Pearson and Havill, 1988
Leul and Zhou, 1999; Bennicellia et al., 1998;
Monk et al., 1987
Leul and Zhou, 1999; Bennicellia et al., 1998;
Hunter et al., 1983
Chen et al., 2002; Justin and Armstrong, 1987
Chen et al., 2002; Justin and Armstrong, 1987
Chen et al., 2002; Justin and Armstrong, 1987
Classification of the individual species used in the referenced studies was from Fitter (1978) or from US Fish and Wildlife Service
(1996).
a
From literature summary in Chen et al. (2002).
b
Actual porosity under anaerobic conditions instead of ratio.
ing (Thompson et al., 1987), susceptibility to water
stress (Moran et al., 1994) and oxygen stress
(Hunter et al., 1983). In this study, we observed
that the concentration of MDA, a lipid peroxidation product, increased 3-fold under anoxia for 7
days (Fig. 5B), while SOD activity of roots was
two to three times higher under anoxia than under
aerobic conditions (Fig. 5A). Two days after reintroduction to aerobic conditions, the concentration of MDA in the roots remained significantly
higher under anoxia than under aerobic conditions. These results indicate that oxidative damage
existed in the roots under anoxia and during a
recovery phase. Contrary to our hypothesis (#3),
the roots of L. latifolium did not avoid oxidative
damage through SOD defense mechanism alone,
although this species tolerates or survived long
periods of soil flooding (Chen et al., 2002). A
significant rise in MDA level has been reported in
intolerant species such as waterlogged B. napus L.
seedlings (Leul and Zhou, 1999) and flooded I.
germanica L. (Hunter et al., 1983). In contrast, the
MDA level remained the same in I. pseudacorus
L., an anoxia tolerant wetland species (Hunter et
al., 1983). Anoxia-tolerant species may be better
protected against oxygen free radicals either by the
accumulation of antioxidants, or by the induction
of SOD or other active oxygen-species-removing
enzymes (Larson, 1988; Monk et al., 1987).
Table 1 summarizes the ratio of the concentration, activity, or other parameter under anoxic
conditions versus aerobic conditions (anoxic response/aerobic response) from this study and
others from the literature. The ratios for activity
of PDC and the concentration of ethanol were
unusually low, but closer to those typical of
obligate hydrophytes. The ratios of activities for
ADH, CCO, and for the percentage porosity
under anoxic conditions (not the ratio) were
typical of obligate hydrophytes. The ratios of the
activity of SOD and the concentration of MDA,
however, were either intermediate between facul-
H. Chen, R.G. Qualls / Environmental and Experimental Botany 50 (2003) 29 /40
tative and anoxia intolerant species, or similar to
anoxia intolerant species. Additionally, the yield
ratio (from Chen et al., 2002) was typical of anoxia
intolerant species.
L. latifolium exhibited the concurrent activity of
both fermentative pathway and aerobic metabolism like flood-tolerant species such as rice (Fox
and Kennedy, 1991). It also exhibited a higher
activity of ADH, such as occurs in rice, and a
higher activity of SOD, such as is occurs in I.
pseudacorus L. (Hunter et al., 1983) under anoxia.
Despite the increased activity of SOD, and contrary to our expectation, L. latifolium suffered
oxidative stress under anoxia and during the
recovery phase as indicated by high concentration
of MDA in the roots. In saturated soil, we
observed mortality of older roots that might, at
least in part be related to either insufficient
aeration or oxidation damage. The very rapid
response to anaerobic conditions may also indicate
the ability to rapidly adapt, consistent with our
first hypothesis. For example, SOD responses were
seen after only 1 day, and ADH increased rapidly
in the first 2 days. Based on its ability to survive
with inhibited growth in saturated soil, L. latifolium is typical of a facultative hydrophyte (Chen et
al., 2002). According to the classification of
wetness /dryness of habits in which it naturally
occurs, this species is classified as an intermediate
species in Europe (Fitter, 1978). In this study, an
unusual mixture of responses typical of obligate
hydrophytes, facultative species, and anoxia intolerant species characterized the metabolic adaptation of L. latifolium to anoxia.
Acknowledgements
We thank Akiko Takiyama for assistance in
collecting samples and Nikki Vasconellos for
reading the draft of this manuscript. Funding for
this project was provided by a grant from US
Department of Agriculture, National Research
Initiative Competitive Grants Weed Science Program. Funding also provided in part by the
Nevada Agricultural Experiment Station, publication number 5202379.
39
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