Scientia Horticulturae 124 (2010) 274–279

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Hypoxia tolerance and adaptation of anaerobic respiration to hypoxia stress in

two Malus species
Cuiying Li a,b,1, Tuanhui Bai a,b,1, Fengwang Ma a,b,*, Mingyu Han a
a
College of Horticulture, Northwest A & F University, Yangling, Shaanxi 712100, China
b
Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Yangling, Shaanxi 712100, China

A R T I C L E I N F O A B S T R A C T

Article history: Seedlings of two Malus species (M. hupehensis and M. toringoides) were hydroponically grown in
Received 2 August 2009 normoxic and hypoxic nutrient solutions to determine their hypoxia tolerance and adaptation of root
Received in revised form 12 December 2009 anaerobic respiration to hypoxia stress. The growth of both species was inhibited under hypoxia stress.
Accepted 17 December 2009
During hypoxia stress, the generation of superoxide anion radical (O2) and content of hydrogen
peroxide (H2O2) in roots were significantly increased in both species with similar trends, which led to an
Keywords: increase of malondialdehyde (MDA) content and relative membrane permeability (RMP). The degree of
Hypoxia stress
growth inhibition and the levels of O2, H2O2, MDA and RMP were greater in M. toringoides than in M.
Malus
hupehensis. Under hypoxia stress, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and
Anaerobic respiration
Hypoxia tolerance lactate dehydrogenase (LDH) activities in roots were increased during the first 12 days of hypoxia stress
and then gradually decreased in both species. The contents of acetaldehyde, alcohol and lactate in roots
were also increased and showed similar trends as the activities of anaerobic respiration enzymes. The
increases in PDC and ADH activities and lactate content in M. hupehensis under hypoxia conditions were
greater than those of M. toringoides, but alcohol and acetaldehyde contents showed opposite trends.
These data suggest that M. hupehensis is more tolerant of hypoxia and had less damage from oxidative
stress than M. toringoides under hypoxia stress. The capability for anaerobic respiration is up-regulated
in roots of Malus in response to hypoxia stress to minimize damage, and the higher hypoxia tolerance of
M. hupehensis may be partly due to the higher enzyme activity of PDC, ADH and LDH and lower
accumulation of acetaldehyde and alcohol.
ß 2010 Elsevier B.V. All rights reserved.

1. Introduction
A well-oxygenated root-zone environment is essential for a
healthy root system, but frequently they experience root-zone
hypoxia mainly due to soil waterlogging, soil compaction, over-
irrigation or poor drainage (Drew, 1997; Garnczarska and Bed-
narski, 2004; Mainiero and Kazda, 2004; Wang et al., 2009).
Oxygen deprivation results in arrest of aerobic respiration, leading
quickly to an energy deficit in plants (Horchani et al., 2008).
Hypoxia stress is considered as one of the main environmental
factors limiting plant growth and yield worldwide, especially in
higher rainfall region areas. Previous studies have shown that
different plants have different abilities to tolerate hypoxia, even for
* Corresponding author at: College of Horticulture, Northwest A & F University,
No. 3 Taicheng Road, Yangling, Shaanxi 712100, China. Tel.: +86 29 87082648;
fax: +86 29 87082648.
E-mail address: fwm64@sina.com (F. Ma).
1
These authors contributed equally to this work.
cultivars belonging to the same plant species but with different
genotypes (Setter et al., 1994; Methode and Larry, 1999; Kato-
Noguchi and Morokuma, 2007).
Most environmental stresses including hypoxia result in the
production of reactive oxygen species (ROS) in plants, causing
oxidative stress (Mittler et al., 2004; Garnczarska and Bednarski,
2004). ROS such as superoxide radicals (O2), hydroxyl radicals
(OH) and hydrogen peroxide (H2O2) are all very reactive and cause
severe damage to membranes (Bowler et al., 1992; Foyer et al.,
1997). Numerous studies have shown that hypoxia stress triggers
the formation of ROS and induces oxidative stress in plants
(Geigenberger, 2003; Garnczarska and Bednarski, 2004; Bai et al.,
2008). Under hypoxia conditions, the induction of fermentation
metabolism is regarded as an adaptive phenomenon to maintain
the capacity for ATP synthesis (Davies, 1980; Kennedy et al., 1992;
Good and Muench, 1993). The main end product of fermentation
metabolism in plants is ethanol, and the two enzymes alcohol
dehydrogenase (ADH) and pyruvate decarboxylase (PDC), involved
in the ethanol biosynthetic pathway, are induced in such
conditions (Kennedy et al., 1992; Rivoal and Hanson, 1994).

0304-4238/$ – see front matter ß 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2009.12.029
 C. Li et al. / Scientia Horticulturae 124 (2010) 274–279
Anoxia tolerance of plants was also associated with greater
concentrations of ATP and total adenylates and with sustained
levels of ADH and PDC activities (Davies, 1980; Jackson et al., 1982;
Drew, 1997). In maize, rice and Arabidopsis, ADH and PDC loss-of-
function mutants rapidly succumbed to oxygen deprivation,
confirming the importance of alcohol fermentation for stress
tolerance (Kennedy et al., 1992; Rahman et al., 2001). However,
the anaerobic respiration response to hypoxia conditions can be
different due to species and genotype diversity.
Apple (Malus) is one of the most economically important fruits
worldwide. But apple tree often encounters root-zone hypoxia
stress in production. Malus hupehensis and Malus toringoides are
frequently used as rootstocks for apple cultivation in China.
Previous studies showed that M. hupehensis from a higher rainfall
region was more tolerant to hypoxia than M. toringoides from a low
rainfall region (Bai et al., 2008). However, hypoxia tolerance and
adaptation of both Malus species are not well known. In this study,
we compared the growth, hypoxia injury and the anaerobic
respiration metabolism of M. hupehensis and M. toringoides under
normoxic and hypoxic conditions to investigate the difference in
hypoxia tolerance and adaptation of anaerobic respiration.
2. Materials and methods
2.1. Plant material and experimental design
M. hupehensis (Chinese name is Pingyitiancha, P) and M.
toringoides (Chinese name is Bianyehaitang, B) originated from
different climate regions in China. The experiment was conducted
at the Northwest A & F University, Yangling (348200 N, 1088240 E),
China. M. hupehensis and M. toringoides seeds were stratified in
sand for 50 days at 0–4 8C and relative humidity of 60–70%. Three
germinated seeds were planted in each plastic pot (12 cm  12 cm)
filled with sand. The plastic pots were then placed in a greenhouse
under natural light and temperature conditions. From the 2nd
true-leaf stage, the seedlings were watered with 1/2 Hoagland
nutrient solution (pH 6.5  0.1) every other day. Forty days later,
seedlings with similar size (7–8 leaves, about 5 cm in height) were
transferred into plastic tubs (52 cm  37 cm  15 cm, 50 seedlings/
tub) containing 20 L 1/2 Hoagland nutrient solution. The seedlings
were grown in a growth chamber, in which the temperature was
controlled to 20–25 8C in the day and 15–20 8C at night and the light
intensity was 140–160 mmol m2 s1 with a light period of 14 h. The
solution was continuously aerated with an air-pump (20 min aeration
per hour). Seedlings were cultured for 10 days so that these plants
could adapt to ambient culture conditions.
Treatments were initiated after 10 days of solution culture in
the growth chamber. A total of 400 plants were used in one-time
experiment (normoxic and hypoxia treatment) for each species. (1)
Control (normoxic): the nutrient solution was continuously
aerated with an air-pump and maintained dissolved oxygen
(DO) concentration at 8.0–8.5 mg L1 by a DO controller (FC-680,
Corporation of Super, Shanghai, China). (2) Hypoxia: aeration with
compressed air was substituted by N2 to maintain DO concentra-
tions at 1.5–2.0 mg L1 using the other DO controller. Each
treatment was performed in 4 replicates and there are 50 seedlings
used for each treatment in one replicate. At 0, 4, 8, 12, 16 and 20
days of hypoxia treatment, root samples were collected, immedi-
ately frozen in liquid nitrogen, then stored at 70 8C until use.
Each treatment was performed in 4 replicates and there were 50
seedlings used.
2.2. Growth measurements
At 20 days of treatment, plant height and root length were
measured with a ruler and new leaf number was manually counted
275
on 10 plants per treatment, and then each plant was divided into
shoot and root. All the tissues were oven-dried at 70 8C for at least
72 h and then weighed.
2.3. Assay of O2 generation rate
Assay of O2 generation rate followed Elstner and Heupel
(1976) with some modifications. Root tissue (1 g) was ground in
65 mM phosphate buffer (pH 7.8) and centrifuged at 5000  g for
10 min. Supernatant of 1 mL was mixed with 65 mM phosphate
buffer (pH 7.8) and 10 mM hydroxylamine hydrochloride, and
placed at 25 8C for 20 min. Then 17 mM sulfanilamide and 7 mM a-
anaphthylamine were added to the mixture. The absorbance of the
solution at 530 nm was measured after standing for 20 min at
25 8C. A standard curve with nitrogen dioxide radical (NO2) was
used to calculate the O2 generation rate.
2.4. Assay of H2O2 content
The H2O2 content was determined according to a method
described by Patterson et al. (1984). Frozen roots were
homogenized in cold acetone in the ratio of 1 g samples to 2 mL
ice-cold acetone. Titanium reagent (2% TiSO4) was added to a
known volume of extract supernatant to give a Ti concentration of
2%. The Ti–H2O2 complex, together with unreacted Ti, was then
precipitated by adding 0.2 mL 17 M ammonia solution for each mL
of extract. The precipitate was washed five times with ice-cold
acetone by resuspension, drained, and dissolved in 2 M H2SO4
(3 mL). The absorbance of the solution was measured at 410 nm
against blanks, which had been prepared similarly but without
plant tissue.
2.5. Assay of MDA content
Malondialdehyde (MDA) content was measured by the method
of Heath and Packer (1968). Root tissue (0.5 g) was homogenized in
0.3% 2-thiobarbituric acid (TBA) and 10% trichloroacetic acid. After
heating for 30 min at 100 8C, the mixture was centrifuged at
10,000  g for 10 min. Absorbance of the supernatant was
measured at 532 and 600 nm. MDA content was calculated using
extinction coefficient = 155 mM1 cm1.
2.6. Assay of RMP
RMP was measured following the method described by
Dionisio-Sese and Tobita (1998). Roots of equal size and weight
were placed in test tubes containing 10 mL distilled water. The
tubes were incubated in a water bath at room temperature for 2 h
and the initial electrical conductivity of the medium (L1) was
analyzed using an electrical conductivity analyzer (DDS-307,
Shanghai Precision Scientific Instrument Co., Ltd., China). The
samples were autoclaved at 100 8C for 20 min to release all
electrolytes; cooled to 25 8C, and then the final electrical
conductivity (L2) was measured. RMP was calculated using the
formula: RMP = L1/L2  100.
2.7. Extraction and assay of PDC, ADH and LDH
All procedures were carried out at 0–4 8C. The extraction buffer
contained 50 mM Tris–HCl (pH 6.8), 5 mM MgCl2, 5 mM b-
mercaptoethanol, 15% (w/v) glycerol, 1 mM ethylenediaminete-
traacetic acid (EDTA), 1 mM ethyleneglycol-bis(b-aminoethy-
lether)-N,N0 -tetraacetic acid (EGTA), 0.1 mM phenylmethyl
sulfanyl fluoride (PMSF) (Mustroph and Albrecht, 2003). Root
sample (0.5 g fresh weight) was ground in a mortar for 2 min using
the extraction buffer (3 mL) and a small amount of quartz sand,
276 C. Li et al. / Scientia Horticulturae 124 (2010) 274–279

then centrifuged at 12,000  g for 20 min. The supernatant was Table 1

New leaf number (NLN), root length (RL), plant height (PH), fresh weight (FW), dry
used for the following enzyme assays. Change in absorbance of the
weight (DW), and root/shoot (R/S) ratio of two Malus species grown in normoxic
reaction solution at 340 nm was read every 20 s. The unit of and hypoxic nutrient solution for 20 days. Data are mean  S.D., n = 10, Data with
enzyme activity was mmol g1 FW min1. different letters in the same column indicate a significant difference at P < 0.05, LSD’s
Assays for PDC and ADH followed Waters et al. (1991). For PDC, test.
the assay mixture contained 0.01 mL extract, 50 mM 2-[N- Malus hupehensis Malus toringoides
morpholino]-ethane-sulfonic acid (MES) (pH 6.8), 25 mM NaCl,
CK HY HY/CK CK HY HY/CK
10 U ADH, 1 mM MgCl2, 0.5 mM thiamine pyrophosphate (TPP),
2 mM dithiothreitol (DTT), 0.17 mM NADH, 50 mM aminooxo- NLN 3.75 a 3.25 a 86.67% 2.50 a 1.25 b 50.00%
RL (cm) 14.50 a 12.29 a 84.75% 24.63 a 15.53 b 63.05%
acetic acid monosodium salt, and 10 mM pyruvate to start the
PH (cm) 8.75 a 8.06 a 92.11% 7.66 a 5.91 b 77.15%
reaction. For ADH, the assay mixture contained 0.05 mL extract, FW (g) 1.05 a 0.96 a 91.42% 1.69 a 0.92 b 54.43%
50 mM N-tris(hydroxymethyl) methyl-2 amino ethanesulfonic DW (g) 0.37 a 0.31 b 83.78% 0.52 a 0.31 b 59.61%
acid (TES) (pH 7.5), 0.17 mM NADH, and 40% (v/v) acetaldehyde to S/R 0.42 a 0.39 a 92.86% 0.45 a 0.33 b 73.33%
start the reaction. CK: control; HY: hypoxia stress.
Assays for LDH followed Bergmeger (1983a). The assay mixture
contained 0.15 mL extract, 80 mM Tris–NaCl–NADH buffer (pH 7.5,
containing 0.22 mM NADH), and 80 mM Tris–NaCl–pyruvate The reduction in new leaf number was more drastic in M.
solution (containing 0.01 mM pyruvate) to start the reaction. toringoides (50%) than M. hupehensis (13.73%) under hypoxia stress.
Root elongation was inhibited for M. toringoides (37.95%). Root
2.8. Extraction and assay of acetaldehyde, alcohol and lactate elongation for M. hupehensis, however, was not inhibited (Table 1).
At the final harvest, the fresh and dry mass was reduced by 8.58
All procedures were carried out at 0–4 8C. The root tissue was and 16.22% in M. hupehensis, and by 45.57 and 40.39% in M.
ground in 0.6 M HClO4, and let it sit for 30 min before toringoides, respectively, as compared with the controls. The lower
centrifugation at 10,000  g for 10 min (Good and Muench, root/shoot ratio was also observed in M. hupehensis and M.
1993). The ratio of tissue to HClO4 was 1:5. The supernatant toringoides under hypoxia stress, especially in M. hupehensis from
was adjusted to pH 4.5 using 3 M KOH, kept for 15 min, and the humid climate showed a lower degree of growth inhibition and
centrifuged for 10 min at 10,000  g. Then the supernatant was higher dry mass than M. toringoides from the dry climate. These
kept sealed at 70 8C, and assayed for acetaldehyde, alcohol and data showed that hypoxia stress more drastically inhibited the
lactate according to Bergmeger (1983b). growth in roots than in shoots and of M. toringoides than that of M.
For alcohol, the assay mixture contained 0.1 mL extract, hupehensis.
114 mM K4P2O7 (pH 9.0), 1.86 mM NAD+ and 568 U L1 aldehyde
dehydrogenase (AIDH), and the absorbance (A1) was determined at
340 nm after 2 min. After adding ADH to start the reaction, the
absorbance (A2) was determined at 340 nm after 30 min.
For acetaldehyde, the assay mixture contained 0.1 mL extract,
96 mM K4P2O7 (pH 9.0), 1.57 mM NAD+, the absorbance (A1) was
then determined at 340 nm after 3 min. After adding 75 U mL1
AIDH to start the reaction, the absorbance (A2) was determined at
340 nm after 5 min.
For lactate, the assay mixture contained 0.1 mL extract,
116 mM glutamate buffer (pH 8.9), 0.93 mM NAD+ and 1.3 KU L1
1 alanine aminotransferase (ALT), kept at 25 8C for 10 min, and the
absorbance (A1) was determined at 340 nm. After adding
600 KU L1 LDH to start the reaction, the absorbance (A2) was
determined at 340 nm after 30 min.
The content of acetaldehyde, alcohol and lactate was calculated
based on the difference between A2 and A1 and expressed as
mmol g1 FW.

2.9. Statistical analysis

Statistical analyses were carried out by analysis of variance

(ANOVA) using SPSS-11 for Windows statistical software package.
Results were represented as the means  standard deviation (S.D.).
Differences between treatments were separated by the least
significant difference (LSD) test at a 0.05 probability level.

3. Results

3.1. Plant growth

At 20 days of hypoxia stress, new leaf number, root length and

plant height were decreased in both Malus species (Table 1). There
were significant differences in new leaf number and plant height
between the controls and the treatments in M. toringoides,
however, there were no significant differences in M. hupehensis.
Fig. 1. O2 generation rate (A) and H2O2 content (B) in roots of two Malus species
grown in normoxic and hypoxic nutrient solution for 20 days. Data are
means  S.D., LSD’s test. PC: Malus hupehensis control; PH: M. hupehensis hypoxia
stress; BC: Malus toringoides control; BH: M. toringoides hypoxia stress.
 C. Li et al. / Scientia Horticulturae 124 (2010) 274–279
3.2. O2 generation rate and H2O2 content
Compared to controls, O2 generation rate and H2O2 content in
roots of both Malus species were significantly increased under
hypoxia stress, and showed similar trends with increasing duration
of hypoxia treatment (Fig. 1A and B). However, differences were
observed in the two species. O2 generation rate increased greater
in M. toringoides than M. hupehensis. Root H2O2 content was higher
in M. hupehensis than in M. toringoides in the aerated control plants,
but H2O2 content was increased to the same level after 4-day
hypoxia treatment in both species. At the end of the treatment
period, O2 generation rate was increased by 117 and 183% in M.
hupehensis and M. toringoides under hypoxia stress, respectively;
H2O2 content was increased by 53% and 71% in M. hupehensis and
M. toringoides, respectively, as compared with the controls.
3.3. MDA content and RMP
Hypoxia stress remarkably increased MDA content and RMP in
roots of M. hupehensis and M. toringoides (Fig. 2A and B). At 12 days
of hypoxia stress, MDA content was increased by 79.0 and 112% in
M. hupehensis and M. toringoides, relative to their respective
controls, and then dropped at the end of the treatment period. RMP
was increased throughout the entire hypoxia stress period in both
species, and at 20 days of hypoxia stress, RMP was 138 and 177% of
the controls in M. hupehensis and M. toringoides, respectively.
3.4. PDC, ADH and LDH activities
Under hypoxia treatment, the activities of PDC and ADH in roots
of both Malus species were increased relative to the respective
Fig. 2. MDA content (A) and RMP (B) in roots of two Malus species grown in
normoxic and hypoxic nutrient solution for 20 days. Data are means  S.D., LSD’s
test. PC: M. hupehensis control; PH: M. hupehensis hypoxia stress; BC: M. toringoides
control; BH: M. toringoides hypoxia stress.
277
controls. The enzyme activities reached their maximum values
after 8 days of hypoxia stress, and then gradually decreased, but
were still slightly higher than the controls at 20 days (Fig. 3A and
B). Substantial differences were observed between M. hupehensis
and M. toringoides in the activities of PDC and ADH during the same
periods of hypoxia stress. After hypoxia stress for 8 days, PDC
activity in M. hupehensis was increased by 83% relative to
continuously aerated roots, whereas PDC activity in M. toringoides
was increased only by 49%. At 20 days, PDC activity had gradually
declined to 18% of the aerated controls in M. hupehensis, and 11% in
M. toringoides. After hypoxia stress for 8 days, ADH activity in M.
hupehensis was increased to 79% of the aerated controls and 45% in
that of M. toringoides. ADH activity at 20 days was decreased to 45%
in M. hupehensis, whereas, 8% in M. toringoides. In response to
hypoxia stress, LDH activity first increased then decreased in roots
of both Malus species (Fig. 3C). Overall, both species had similar
LDH activities except for the beginning and end of the hypoxia
treatment.
3.5. Acetaldehyde, alcohol and lactate content contents
In response to hypoxia stress, the content of acetaldehyde and
alcohol in roots of both species increased first, reached a
maximum, and then decreased (Fig. 3D and E). During hypoxia
stress, acetaldehyde content in the roots of M. toringoides was
always higher than in M. hupehensis. At 16 days of hypoxia
treatment, M. toringoides had slightly higher alcohol level than M.
hupehensis. In response to hypoxia stress, root lactate content in
both species increased during the first 12 days of stress, and then
decreased. At each sampling point during hypoxia stress except at
12 days, lactate levels in roots of M. hupehensis were higher than in
M. toringoides (Fig. 3F).
4. Discussion
4.1. Hypoxia tolerance
Molecular-oxygen deficiency leads to altered cellular metabo-
lism, poor roots and plant performance and can dramatically
reduce crop productivity (Chérif et al., 1997; Fukao and Bailey-
Serres, 2004). There are numerous studies reporting inhibition in
growth and reduction in dry matter accumulation under hypoxia.
Sojka et al. (1975) found an 83% decrease in leaf area of wheat after
25 days of root hypoxia. Our results showed that all the measured
growth parameters of M. toringoides were significantly reduced
under hypoxia stress when compared with the controls, but there
were no significant differences in new leaf number, root length,
plant height, fresh weight and root/shoot between the controls and
the treatments in M. hupehensis (Table 1). The result of growth
analysis demonstrated that M. hupehensis was relatively more
tolerant to hypoxia, while M. toringoides was more sensitive to
hypoxia.
Excessive generation of ROS results from many stresses,
including hypoxia. ROS are dangerous because they cause lipid
peroxidation, which can seriously disrupt the normal metabolism
of plants through oxidative damage of cellular component (Mittler
et al., 2004). It is well documented that MDA is a product of cell
membrane lipid peroxidation and its content in vivo can indicate
the extent of oxidative stress in plants and cell membrane
homeostasis (Bailly et al., 1996). In the present study, hypoxia
stress led to an increase in O2 generation and H2O2 content in
roots of two species (Fig. 1A and B), which caused the dramatic
increase in MDA content and RMP (Fig. 2A and B). Our data clearly
showed that the increases in O2 generation rate, H2O2 content,
MDA content and RMP under hypoxia stress were greater in M.
toringoides than in M. hupehensis. All these results showed that
278 C. Li et al. / Scientia Horticulturae 124 (2010) 274–279

Fig. 3. PDC (A), ADH (B) and LDH (C) activity, acetaldehyde (D), alcohol (E) and lactate (F) content in roots of two Malus species grown in normoxic and hypoxic nutrient
solution for 20 days. Data are means  S.D., LSD’s test. PC: M. hupehensis control; PH: M. hupehensis hypoxia stress; BC: M. toringoides control; BH: M. toringoides hypoxia stress.

hypoxia stress inhibited the growth of two Malus, and excessive
accumulation of ROS under hypoxia stress led to membrane lipid
peroxidation. Compared with M. toringoides, M. hupehensis had less
growth inhibition and less oxidative damage. Thus, these suggest
that M. hupehensis may be more tolerant of hypoxia than M.
toringoides.
4.2. Adaptation response of anaerobic respiration to hypoxia stress
In the present study, we found that activities of PDC, ADH and
LDH, the key enzymes in alcohol fermentation and lactate
fermentation, were enhanced in roots of both Malus species under
hypoxia conditions (Fig. 3A–C). This indicates that anaerobic
respiration was up-regulated under hypoxia conditions. The
enhanced alcohol fermentation and lactate fermentation would
compensate to some extent for the energy deficit as a result of the
inhibition of aerobic respiration because of hypoxia, and regen-
erated NAD+. These results were similar to some previous reports
on other plants such as rice (Bailey-Serres and Freeling, 1990) and
Arabidopsis (Fukao et al., 2003). The fact that M. hupehensis had
higher PDC, ADH and LDH activity and less growth inhibition than
M. toringoides under hypoxia conditions suggests a positive
relationship between active alcohol fermentation and lactate
fermentation and hypoxia tolerance for Malus.
Up-regulation of anaerobic respiration in response to hypoxia
stress helps plants adapt to hypoxia conditions in the short term,
but it has some limitations. This was shown by the decrease in PDC,
ADH and LDH activity as the hypoxia stress progressed up to 20
days. When hypoxia conditions were first applied, the Malus
seedlings did up-regulate alcohol fermentation and lactate
fermentation to adapt to hypoxia, but when the stress period
exceeded a certain limit, these compensatory functions were
weakened.
Alcohol fermentation is a main anaerobic respiration pathway,
and ADH is an enzyme induced by anoxia or hypoxia. So the
activity and location of ADH is very important for plants to survive
hypoxia stress (Kennedy et al., 1992). Both alcohol fermentation
and lactate fermentation depend on the cytoplasmic pH and for
plants under anoxic conditions, lactate fermentation may contrib-
ute to increasing cytoplasmic acidification (Roberts et al., 1984)
and thus a decrease the cytoplasmic pH, which may progressively
inhibit LDH and activate PDC and ADH, promote alcohol
fermentation (Davies, 1980; Rivoal and Hanson, 1994). Since
lactate fermentation led to a decrease in cytoplasmic pH, the
proton pumps might be also involved in the process (Germain et
al., 1997). In our study, M. hupehensis could maintain higher LDH
activity than M. toringoides during the stress. This indicates that
lactate fermentation could play a relatively important role in root
for Malus to hypoxia stress, especially at the late stress.
Alcohol is the main end product of anaerobic metabolism in
plants (Good and Muench, 1993). It is a relatively nontoxic end
product (Jackson et al., 1982) and a neutral compound which can
 C. Li et al. / Scientia Horticulturae 124 (2010) 274–279
diffuse to the external environment and does not lead to the
acidification of the cytoplasm, a major determinant in intolerance
to O2 deficiency (Roberts et al., 1985). Alcohol is less harmful to
plants, but the long-term accumulation of alcohol can lead to cell
injury because of the breakage of semi-permeable cell protoplasts.
Acetaldehyde, which is generated during alcohol fermentation, is
highly toxic to plant cells (Perata et al., 1992). However lactate,
which is also generated under O2 deficiency, causes cytoplasmic
acidification as it accumulates and thereby can destroy the
tonoplast and mitochondrial structures of plants (Roberts et al.,
1984). In our study, the contents of acetaldehyde, alcohol and
lactate increased considerably in both Malus species under hypoxia
stress, and by 8 days the acetaldehyde and lactate contents were at
the highest, but alcohol accumulated steadily until it attained a
maximum value at 16 days of hypoxia stress (Fig. 3D and E). This
indicates that the anaerobic respiration metabolites could be
further metabolized or diffused out of the cell as the stress
progressed and as a result they accumulated less. However, the
decrease in anaerobic respiration metabolites during the extended
period of hypoxia may also be partly due to the release of
metabolites into the nutrient solution, as a result of cell breakage
when the metabolites accumulated excessively, especially acetal-
dehyde and alcohol. In this study, M. toringoides accumulated more
alcohol and acetaldehyde and less lactate in roots under hypoxia
stress. And it was the stage of hypoxia stress that these plants were
damaged most seriously from ROS. Thus this suggests that
acetaldehyde and alcohol would be more harmful to the seedlings
than lactate generated by the fermentation pathways when they
were under hypoxia conditions.
In conclusion, hypoxia stress inhibited plant growth and
increased accumulation of ROS in both M. hupehensis and M.
toringoides. M. hupehensis exhibited less growth inhibition and
oxidative damage than M. toringoides under hypoxia stress, and
therefore M. hupehensis is more tolerant of hypoxia than M.
toringoides. In response to hypoxia stress, anaerobic respiration
was up-regulated in both species to cope with the stress. M.
hupehensis had more active alcohol fermentation and lactate
fermentation and accumulated less acetaldehyde and alcohol but
more lactate in roots than those of M. toringoides during hypoxia
stress.
Acknowledgments
This work was supported in part by Chinese Ministry of
Agriculture (2006-G28) and China Postdoctoral Science Founda-
tion (20060390310). The authors wish to thank Mr. Kun Wang for
providing seeds.
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