J Chem Ecol (2009) 35:488–494

DOI 10.1007/s10886-009-9615-7

Phthalic Acid Induces Oxidative Stress and Alters

the Activity of Some Antioxidant Enzymes in Roots
of Malus prunifolia
Ru Bai & Fengwang Ma & Dong Liang & Xin Zhao

Received: 5 December 2008 / Revised: 20 February 2009 / Accepted: 10 March 2009 / Published online: 8 April 2009
# Springer Science + Business Media, LLC 2009

Abstract Apple replant is a widespread agricultural prob- Keywords Antioxidant enzymes . Growth inhibition .
lem documented in all of the major fruit-growing regions of Malus prunifolia . Membrane damage . Oxidative stress .
the world. In order to better understand the phytotoxic Phthalic acid
mechanisms induced by allelochemicals involved with this
problem, Malus prunifolia plants were grown hydroponi-
cally to the six-leaf-stage in the presence of phthalic acid (0 Introduction
or 1 mM) for 5, 10, or 15 days. Apple plants were evaluated
for: shoot and root length, fresh and dry weight, malon- Allelopathy is widely reported in agroecosystem and
dialdehyde (MDA) content, hydrogen peroxide (H2O2) silviculture, and is implicated in problems such as exotic
content, superoxide radical (O2·−) generation rate, and plant invasion, replant problems, and soil sickness (Lee et
antioxidant enzyme activities. Shoot and root lengths and al. 2006; Hao et al. 2007; Fernandez et al. 2008).
fresh and dry weights of M. prunifolia decreased in plants Allelopathic plants exert detrimental effects via the release
exposed to phthalic acid. MDA and H2O2 content increased of plant compounds (allelochemicals) through leaching,
in phthalic acid-treated plants as did the generation rate of root exudation, volatilization, and/or decomposition of
O2·− in M. prunifolia roots. The activities of superoxide plant materials (Weir et al. 2004) and can interfere with
dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase the metabolism of other plants. If the effect of such
(EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), gluta- compounds is harmful to plant growth and development,
thione reductase (EC 1.6.4.2), dehydroascorbate reductase it becomes a biotic or allelochemical stress (Romero-
(EC 1.8.5.1), and monodehydroascorbate reductase (EC Romero et al. 2005).
1.6.5.4) increased in phthalic acid-stressed roots compared Under normal growth conditions, a dynamic equilibrium
with control roots. These results suggest that activation of exists between the production and detoxification of free
the antioxidant system by phthalic acid led to the formation radicals in cell organelles. Biotic and abiotic stresses may
of reactive oxygen species that resulted in cellular damage cause the formation of reactive oxygen species (ROS) such
and the decrease of M. prunifolia growth. as superoxide radical (O2·−), hydroxyl radical (OH·−), and/or
hydrogen peroxide (H2O2) that are commonly generated
and accumulated in cells (Cho and Seo 2005). Enhanced
ROS production can affect membrane permeability, dam-
R. Bai : F. Ma : D. Liang : X. Zhao age DNA and proteins, induce lipid peroxidation, and
College of Horticulture, Northwest A&F University,
Yangling, Shaanxi 712100, China
ultimately lead to programmed cellular death (Ding et al.
2007). Plants have evolved mechanisms that protect cell
R. Bai : F. Ma (*) : D. Liang and subcellular systems from the effects of ROS by using
Key laboratory of horticultural plant germplasm resource antioxidant systems such as superoxide dismutase (SOD),
utilization in Northwest China,
Yangling, Shaanxi 712100, China
peroxidase (POD), catalase (CAT), ascorbate peroxidase
e-mail: [email protected] (APX), glutathione reductase (GR), dehydroascorbate
e-mail: [email protected] reductase (DHAR), and monodehydroascorbate reductase
J Chem Ecol (2009) 35:488–494 489

(MDHAR). Tolerance to allelochemicals by various crop growth room with a L/D regime of 12/12 h, 25/20°C, and a
plants has been associated with an increase in the activity photon flux density of 140–160 μmol m−2 s−1. Seedlings
of antioxidant enzymes (Yu and Matsui 1997). were allowed to acclimate to the hydroponic conditions for
The apple replant problem is widespread. Studies have 5 days. Phthalic acid (purchased from Yifang S&T Ltd.
shown that allelochemicals in root exudates or decomposi- Tianjin, China), dissolved in ethanol, was added to the
tion of residues may play a role in this agricultural problem nutrient solution to concentrations of 0 or 1 mM. In the
(Borner 1959; Zhang et al. 2007; Bai et al. 2009). However, preliminary experiment, thinner M. prunifolia leaves and
the mechanisms involved have not been investigated brownish root apices in parallel with a large amount of
extensively. Reports suggest that allelochemicals cause mucilage was secreted from roots 15 days after application
increased membrane leakage, and enhance H2O2 and of 1 mM phthalic acid. This indicates that phthalic acid at
MDA levels in the target plant tissues. For instance, this concentration is lethal to M. prunifolia seedlings. The
cinnamic acid enhanced the generation of free radicals, final concentration of ethanol in control and treatment
increased lipid peroxidation, and oxidative membrane solutions was 0.1% (v/v). The nutrient solutions in the
damage in cucumber and figleaf gourd plants (Ding et al. containers were aerated continuously by air pumps. The
2007). Similarly, 2-benzoxazolinone inhibited lettuce solution in the plastic container was kept at the same level
growth, caused membrane damage, and increased MDA by adding half Hoagland nutrient solution at 24 h intervals.
and H2O2 production (Sanchez-Moreiras and Reigosa Each treatment was replicated three times in a completely
2005). Most studies in this regard focused on vegetables randomized design. Root samples were taken from both
and agronomic crops (Baziramakenga et al. 1995; Yu et al. control and phthalic acid-treated plants on days 5, 10, and
2003; Ye et al. 2006). However, it is not known whether the 15 after treatment, and the tissue was frozen in liquid
inhibitory effects were mediated through production of nitrogen and stored at −70°C until analysis. At the end of
ROS in apple roots under allelochemical stress. Phthalic the experiment (15 days after treatment), root and shoot
acid is a potent allelochemical and inhibits the growth of a length and fresh and dry weight of seedlings were
number of plants such as Lactuca sativa L. (Lee et al. measured. The seedlings were dried in an forced-air oven
2006), Zea mays L. (Chai and Feng 2007), and Malus at 60°C until constant mass was reached.
prunifolia (Bai et al. 2009). Accordingly, its effect on plant
growth, ROS generation rate, and ROS-antioxidant enzyme Measurement of MDA, H2O2, and O2·− Generation Lipid
activity were investigated in M. prunifolia seedlings. peroxidation was followed by measuring MDA accumulation
using the method of Baziramakenga et al. (1995) with some
modifications. Root samples (0.2 g) were homogenized in
Methods and Materials 0.1% of trichloroacetic acid in phosphate buffer (5 ml;
pH 7.8) and centrifuged at 12,000×g for 15 min. Supernatant
Plant Materials and Phthalic Acid Treatment Seeds of (1 ml) was added to 0.5% thiobarbituric acid in 20%
apple rootstock, M. prunifolia, were obtained from Fuping trichloroacetic acid (4 ml). The mixture was placed in a
County (34°75′ N, 109°15′ E), Shaanxi Province. Seed water bath at 100°C for 10 min and then quickly cooled in
sterilization was done according to Zhang et al. (2007) and an ice-bath for 15 min. Samples were centrifuged at
involved surface-sterilization in 0.3% (v/v) H2O2 for 12,000×g for 5 min, and then the absorbance of the
20 min, followed by several rinses with sterile H2O. The supernatant was measured at 450, 532, and 600 nm.
sterilized seeds were stratified at 4°C for 85 days. Sprouted H2O2 in the supernatant was measured according to
seeds were sown in plastic pots (9 cm in diameter, 12 cm Patterson et al. (1984). Root samples (0.5 g) were
high; three seeds per pot) filled with sterilized sand. All homogenized in 5-ml pre-cooled acetone and centrifuged
pots were placed in a greenhouse at the College of for 10 min at 1,500×g. Titanium chloride (0.1%, w/v) and
Horticulture, Northwest A&F University, Yangling (34°20′ concentrated ammonia (0.2 ml) were added into the
N, 108°24′ E). Plants were grown without supplementary supernatant (1 ml), the mixture was allowed to react
illumination with night and day temperatures at 20 to 25°C (10 min at 25°C) and the reaction mixture was centrifuged
and relative humidity at 65–80%. Seedlings were watered at 1,500×g for 10 min. Absorbance at 410 nm was
once a week with Hoagland nutrient solution (Hoagland measured, and the H2O2 concentration was calculated
1920), pH 6.0±0.2. When the seedlings reached the six- according to a standard curve.
leaf-stage, batches of 45 uniform seedlings were transferred The rate of O2·− generation was measured as described
into a hydroponic system (plastic container; 45×37× by Elstner and Heupel (1976) with some modifications.
22.5 cm) filled with 5 l half Hoagland nutrient solution at Root tissue (1 g) was homogenized in 65 mM potassium
pH 6.0±0.2 and electrical conductivity at 1.2 ms/cm, phosphate buffer (3 ml; pH 7.8). The homogenate was
respectively. The containers were placed in a controlled centrifuged at (10,000×g for 15 min). The supernatant
490 J Chem Ecol (2009) 35:488–494

(0.5 ml) was added to 65 mM potassium phosphate buffer Glutathione reductase activity was monitored at A340 nm in a
(0.5 ml; pH 7.8) containing 10 mM hydroxylammonium- 1 ml reaction mixture containing Tris–HCl (100 mM; pH 8.0),
chloride (0.1 ml) and incubated (25°C for 20 min). ethylenediaminetetraacetic acid (1 mM), oxidized glutathione
Sulphanilic acid (58 mM; 1 ml) and α-naphthyl amine (GSSG;1 mM), and NADPH (0.2 mM). The reaction was
(7 mM; 1 ml) were added to the mixture, and it was initiated by adding NADPH (Grace and Logan 1996).
allowed to incubate (25°C for 20 min). The final solution Monodehydroascorbate reductase activity was assayed at
was mixed with an equal volume of chloroform and the 340 nm in a 1 ml reaction mixture containing Hepes–KOH
absorbance of the pink phase was measured at 530 nm. (50 mM; pH 7.6), NADH (0.1 mM), AsA (0.25 mM), and
AsA oxidase (EC 1.10.3.3; 0.25 U). The reaction was
Extraction and Assay of Enzyme Activities Antioxidant initiated by adding AsA oxidase (Miyake and Asada 1992).
enzymes (SOD, POD, and CAT) were extracted according Dehydroascorbate rductase activity was measured at
to the method of Yu et al. (2003) with some modifications. 265 nm in a 1 ml assay solution containing Hepes–KOH
Root samples (0.5 g) were homogenized in phosphate (100 mM; pH 7.0), ethylenediaminetetraacetic acid (1 mM),
buffer (8 ml; 0.1 M; pH 7.5) containing 2% (w/v) GSH (2.5 mM), and DHA (0.2 mM). The reaction was
polyvinylpyrrolidone. The homogenate was centrifuged initiated by adding DHA (Dalton et al. 1986).
(12,000×g for 20 min) and the supernatant was used for
enzyme analysis. All assays were carried out at 2–4°C. Statistical Analysis All data were subjected to analysis of
Superoxide dismutase activity was measured according variance, followed by Tukey’s Studentized Range Test
to Beauchamp and Fridovich (1971) with minor modifica- (SAS Statistical package, version 8.2). Results are pre-
tion. The assay medium (3 ml) contained phosphate buffer sented as the means±standard deviation (SD).
(50 mM; pH 7.8), EDTA-Na (0.1 mM), L-methionine
(12 mM), riboflavin (2 μM), and nitrotetrazolium blue
chloride (75 μM). Riboflavin was added last. The tubes (A) 18
Control Phthalic acid
were shaken and placed at a photosynthetic photon flux of 16
50 μmol m−2 s−1 for 15 min. The reaction was initiated and 14
a
a
terminated by turning the light on and off, respectively. The
Length (cm plant-1)

12 b
A560 was measured on a spectrophotometer and tubes
10 b
containing the assay mixture, but without the root extract
(control), were illuminated to determine maximum A560. 8
Peroxidase activity was measured according to Sofo et 6
al. (2004) with some modification. The reaction solution 4
(3 ml) contained phosphate buffer (2.9 ml, 50 mM; pH 7.0),
2
guaiacol (50 μl; 10 mM), H2O2 (10 μl; 40 mM), and crude
enzyme extract (40 μl). The Increase in A470nm due to the 0
Shoot Root
oxidation of guaiacol was measured at 20°C.
Catalase activity was assayed by monitoring the decrease in (B) 4.50
Control Phthalic acid
A240nm (Aebi 1984). The reaction mixture contained phos- 4.00
phate buffer (50 mM; pH 7.0) and H2O2 (30% w/v) and was 3.50 a
started by adding the reaction solution to crude extract (10 μl).
Weight (g plant-1)

3.00 b
AsA-related enzymes were extracted according to Nakano
and Asada (1981). Generally, each 0.5 g of roots material 2.50

was homogenized in KH2PO4-KOH (6 ml, 50 mM; pH 7.5) 2.00 a

containing ethylenediaminetetraacetic acid (0.1 mM), Triton 1.50
X-100 (0.3% v/v), and insoluble polyvinylpolypyrrolidone 1.00
b
(4% w/v). The homogenate was centrifuged (16,000×g for
0.50
15 min at 2°C) and the supernatant was used for APX, GR,
MDHAR, and DHAR analyses. 0.00
Fresh Dry
Ascorbate peroxidase was measured by monitoring the
decrease in A290 nm (Nakano and Asada 1981). The assay Fig. 1 Shoot and root length (a) and fresh and dry weight (b) of Malus
mixture (1 ml) contained Hepes–KOH (50 mM; pH 7.6), prunifolia plants grown at 0 (control) or 1 mM phthalic acid. Samples
were taken 15 days after treatment. Values are means of three replicates±
ethylenediaminetetraacetic acid (0.1 mM), H2O2 (0.2 mM, standard error (SE). Significant difference (P<0.05 level) was tested
AsA 0.5 mM), and enzyme extract. The reaction was between the control and 1 mM phthalic acid treatment for each dependent
initiated by adding H2O2. variable and indicated by different letters above the bars
J Chem Ecol (2009) 35:488–494 491

Results (A) 18
Control Phthalic acid b
15
Effect on Plant Growth The toxic effect of 1 mM phthalic b

SOD activity
12

(U g-1 FW)
acid appeared after 15 days following treatment. The length
of shoot and root of M. prunifolia plants had reduced shoot 9 b
and root length compared to lower than controls (Fig. 1a).
6 a
Fresh and dry weights were 3.09 and 1.61 g plant−1 in a a
control plants compared to 2.63 and 0.79 g plant−1 for 3
plants treated with 1 mM phthalic acid (Fig. 1b). 0
5 10 15
(A) 10 Time of treatment (d)
Control Phthalic acid
8 b (B) 4.0
b
(µmol g-1 FW)

Control Phthalic acid

MDA content

3.5
6 b

(µmol h-1 g-1 FW)

3.0

POD activity
b 2.5
4 a a b
a
2.0
b
2 1.5 a a
a
0 1.0
5 10 15 0.5
Time of treatment (d)
0.0
5 10 15
(B) 0.5
Control Phthalic acid Time of treatment (d)
b
0.4 b (C) 0.55
Control Phthalic acid
(µmol g-1 FW)
H2O2 content

a a a 0.45
a
(µmol h-1 g-1 FW)

0.3 b
CAT activity

0.35
0.2 b
b a
0.25 a a
0.1
0.15
0.0
5 10 15 0.05
5 10 15
Time of treatment (d)
Time of treatment (d)
(C) 1.4
Control Phthalic acid Fig. 3 SOD (a), POD (b), and CAT (c) activities of Malus prunifolia
1.2 plants grown at 0 (control) or 1 mM phthalic acid. The values are
O2•– genaration rate

b means of three replicates±standard error (SE). Significant difference

(µmol h-1g-1 FW)

1.0
b (P<0.05 level) was tested between the control and 1 mM phthalic acid
0.8 a a a a treatment at each sampling date separately and indicated by different
letters above the bars
0.6

0.4 Effect on MDA, H2O2, and O2·− Generation MDA concen-

tration in M. prunifolia roots increased significantly after 5,
0.2
10, and 15 days of treatment (Fig. 2a). The maximum
0.0 increase of 125.4% was observed after 15 days. Exposure
5 10 15
to phthalic acid also resulted in an increase in the H2O2
Time of treatment (d)
content and O2·− generation rate, respectively (Fig. 2b, c).
Fig. 2 MDA (a), H2O2 (b) content, and O2·− generation rate (c) in The maximum increases of 37.2% and 29.9%, respectively,
Malus prunifolia plants grown at 0 (control) or 1 mM phthalic acid. were observed after 15 days.
Values are means of three replicates±standard error (SE). Significant
difference (P<0.05 level) was tested between the control and 1 mM
phthalic acid treatment at each sampling date separately and indicated Effect on Enzyme Activity In response to phthalic acid,
by different letters above the bars activities of SOD, POD, and CAT, as well as enzymes in
492 J Chem Ecol (2009) 35:488–494

ascorbate-glutathione cycle, e.g., APX, GR, MDHAR, and within cells (Masia 2003). MDA accumulation due to lipid
DHAR, all showed similar trends in the activity of root peroxidation has been reported in response to a variety of
enzymes in response to phthalic acid. In general, enzyme abiotic and biotic stresses (Dhindsa et al. 1981; Apel and
activities increased progressively during the treatment Hirt 2004). In the present study, enhanced MDA (Fig. 2a)
period (days 5–15) compared to enzymes in the roots of suggests that phthalic acid probably induces oxidative
plants not exposed to phthalic acid (Figs. 3 and 4). stress, and, as a result, disrupts cellular membrane structure,
and causes a loss of cellular integrity. Similar results with
other allelochemicals have been found (Wu et al. 2002;
Batish et al. 2006; Ye et al. 2006). Many studies show that
Discussion increased MDA content is associated with increased O2·−
and H2O2 production following biotic and abiotic stresses
Phthalic acid treatment significantly decreases the growth (Forman et al. 2002; Lara-Nunez et al. 2006). Here,
of M. prunifolia plants (Fig. 1). This finding is in phthalic acid induced O2·− and H2O2 production in M.
agreement with studies on L. sativa L. (Lee et al. 2006) prunifolia roots (Fig. 2b, c), which suggests that phthalic
and Z. mays L. (Chai and Feng 2007), and consistent with acid could trigger ROS generation and induce oxidative
the inhibitory effects of other allelochemicals on plant stress in M. prunifolia roots. This observation is consistent
growth reported previously (Lin and Kao 2000; Asao et al. with that obtained with mung bean treated with 2-
2004; Batish et al. 2008). benzoxazolinoen (Batish et al. 2006). Increases in SOD
The formation of free radials in cells results in damage to activity (Fig. 3a) also indicate that excessive generation of
cell membranes due to lipid peroxidation. Thus, the level of O2·− has been triggered by pthalic acid treatment, and,
MDA, produced during lipid peroxidation, is a good consequently, that SOD activity was up-regulated to
indicator of oxidative damage that could be occurring mitigate the oxidative damage. Similar responses have been

(A) 10 (B) 8
Control Phthalic acid Control Phthalic acid
7
8 b b
(µmol min-1g-1 FW)

(µmol min-1g-1 FW)

6
APX activity

GR activity

6 b 5 b
a 4
a a a a a a
4 a
3

2
2
1
0 0
5 10 15 5 10 15
Time of treatment (d) Time of treatment (d)

(C) 9 (D)
8 Control Phthalic acid
b 10
(µmol min-1g-1 FW)

7 Control Phthalic acid

MDHAR activity

b
(µmol min-1g-1 FW)

6 8
DHAR activity

a b
5 b
a a 6
4 a a
a a
3 4 a
2
2
1
0 0
5 10 15 5 10 15
Time of treatment (d) Time of treatment (d)

Fig. 4 APX (a), GR (b), MDHAR (c), and DHAR (d) activities of Significant difference (P<0.05 level) was tested between the control
Malus prunifolia plants grown with 0 (control) or 1 mM phthalic acid. and 1 mM phthalic acid treatment at each sampling date separately
The values are means of three replicates±standard error (SE). and indicated by different letters above the bars
J Chem Ecol (2009) 35:488–494 493

observed in plants treated with other allelochemicals (Yu et ASAO, T., KITAZAWA, H., TOMITA, K., SUYAMA, K., YAMAMOTO, H.,
HOSOKI, T., and PRAMANIK, M. H. R. 2004. Mitigation of
al. 2003; Batish et al. 2006; Wu et al. 2002; Lin et al.
cucumber autotoxicity in hydroponic culture using microbial
2007). strain. Sci. Hortic 99:207–214.
Accumulation of H2O2 in M. prunifolia roots in response BAI, R., ZHAO, X., MA, F. W., and LI, C. Y. 2009. Identification and
to phthalic acid treatment enhances lipid peroxidation and bioassay of allelopathic substances from the root exudates of
Malus prunifolia. Allelopathy J. in press.
causes a severe oxidative stress resulting in disruption of
BATISH, D. R., SINGH, H. P., SETIA, N., KAUR, S., and KOHLI, R. K. 2006.
metabolic activities in the cell. Enhanced H2O2 levels are 2-Benzoxazolinone (BOA) induced oxidative stress, lipid peroxida-
removed by the antioxidant enzymes such as CAT, APX, tion and changes in some antioxidant enzyme activities in mung
and POD (Blokhina et al. 2003), and glutathione-ascorbate bean (Phaseolus aureus). Plant Physiol. Biochem. 44:819–827.
BATISH, D. R., SINGH, H. P., KAUR, S., KOHLI, R. K., and YADAV, S.
cycle (Nakano and Asada 1981). In the present study, S. 2008. Caffeic acid affects early growth, and morphogenetic
increases in the activities of these antioxidant enzymes response of hypocotyl cuttings of mung bean (Phaseolus aureus).
paralleled the accumulation of MDA and H2O2 in M. J. Plant Physiol 165:297–305.
prunifolia root after exposure to phthalic acid (Figs. 2–4). BAZIRAMAKENGA, R., LEROUX, G. D., and SIMARD, R. R. 1995.
Effects of benzoic and cinnamic acids on membrane permeability
Our observations are consistent with those of Cruz-Ortega
of soybean roots. J. Chem. Ecol. 21:1271–1285.
et al. (2002), who reported that allelochemical stress causes BEAUCHAMP, C., and FRIDOVICH, I. 1971. Superoxide dismutase:
increases in the level of free radicals and the activity of Improved assays and an assay applicable to acrylamide gels.
antioxidant enzymes and suggests that increased induction Anal. Biochem 44:276–287.
BLOKHINA, O., VIROLAINEN, E., and FAGERSTEDT, K. V. 2003.
of these enzymes is necessary to prevent lipid peroxidation
Antioxidants, oxidative damage and oxygen deprivation stress:
(i.e., to counter the higher MDA and H2O2 in roots). A review. Ann. Bot 91:179–194.
Although there were increased levels of antioxidants in BORNER, H. 1959. The apple replant problem. I. The excretion of
roots after exposure to phthalic acid, oxidative stress still phlorizin from apple root residues. Contrib. Boyre Thompson Inst
20:39–54.
occurred. Our data are consistent with those of Batish et al. CHAI, Q., and FENG, F. X. 2007. Identification of root exudation of
(2006), who showed that exposure of mung bean to 2- Zea mays L. and allelopathy of 1,2-benzenedicarboxylic acid. J.
benzoxazolinone increased APX, GR, and CAT activity. An Gansu Agric. Univ 5:43–48(in Chinese).
increased POD activity in response to allelochemicals also CHO, U. H., and SEO, N. H. 2005. Oxidative stress in Arabidopsis
thaliana exposed to cadmium is due to hydrogen peroxide
has been demonstrated in cucumber root (Yu et al. 2003).
accumulation. Plant Sci 168:113–120.
Oracz et al. (2007) observed an increase in the cell CRUZ-ORTEGA, R., AYALA-CORDERO, G., and ANAYA, A. L. 2002.
membrane permeability, MDA level, H2O2 concentration, Allelochemical stress produced by the aqueous leachate of
and SOD and CAT activity in mustard treated by sunflower Callicarpa acuminata: Effects on roots of bean, maize, and
tomato. Physiol. Plant 116:20–27.
extract. Furthermore, the activity and expression of most
DALTON, D. A., RUSSELL, S. A., HANUS, F. J., PASCOE, G. A., and
antioxidant enzymes is stimulated by ROS accumulation EVANS, H. J. 1986. Enzymatic reactions of ascorbate and
(Apel and Hirt 2004). glutathione that prevent peroxide damage in soybean root
In summary, phthalic acid induces oxidative stress in M. nodules. PNAS 83:3811–3815.
DHINDSA, R. S., PLUMB-DHINDSA, P., and THORPE, T. A. 1981. Leaf
prunifolia roots through the generation of ROS and
senescence: Correlated with increased levels of membrane
decreases plant growth, despite the concomitant increase in permeability and lipid peroxidation, and decreased levels of
antioxidant enzymes. This might be one of the mechanisms superoxide dismutase and catalase. J. Exp. Bot 32:93–101.
responsible for the apple replant problem. The increase in DING, J., SUN, Y., XIAO, C. L., YAN, K. S., ZHOU, H., and YU, J. Q.
2007. Physiological basis of different allelopathic reactions of
antioxidant enzymes could reflect a defensive response to the
cucumber and figleaf gourd plants to cinnamic acid. J. Exp. Bot
cellular damage provoked by phthalic acid treatment. 58:3765–3773.
However, this increase was not strong enough to eliminate ELSTNER, E. F., and HEUPEL, A. 1976. Inhibition of nitrite formation
all the deleterious effects provoked by phthalic acid, only from hydroxylammoniumchloride: A simple assay for superoxide
dismutase. Anal. Biochem 70:616–620.
alleviated the impact of stress.
FERNANDEZ, C., VOIRIOT, S., MEVY, J. P., VILA, B., ORMENO, E.,
DUPOUYET, S., and MELOU, A. B. 2008. Regeneration failure of
Acknowledgements This work was supported by the Natural Pinus halepensis Mill.: The role of autotoxicity and some abiotic
Science Fund of Shaanxi province and Talent Support Program of environmental parameters. For. Ecol. Manage 255:2928–2936.
Northwest A&F University. FORMAN, H. J., TORRES, M., and FUKUTO, J. 2002. Redox signaling.
Mol. Cell. Biochem 234:49–62.
GRACE, S. C., and LOGAN, B. A. 1996. Acclimation of foliar
antioxidant systems to growth irradiance in three broad-leaved
References
evergreen species. Plant Physiol 112:1631–1640.
HAO, Z. P., WANG, Q., CHRISTIE, P., and LI, X. L. 2007. Allelopathic
AEBI, H. 1984. Catalase in vitro. Meth. Enzymol. 105:121–126. potential of watermelon tissues and root exudates. Sci. Hortic.
APEL, K., and HIRT, H. 2004. Reactive oxygen species: Metabolism, 112:315–320.
oxidative stress and signal transduction. Annu. Rev. Plant Biol. HOAGLAND, D. R. 1920. Optimum nutrient solutions for plants.
55:373–399. Science 52:562–564.
494 J Chem Ecol (2009) 35:488–494

LARA-NUNEZ, A., ROMERO-ROMERO, T., VENTURA, J. L., BLANCAS, ROMERO-ROMERO, T., SANCHEZ-NIETO, S., SANJUAN-BADILLO, A.,
V., ANAYA, A. L., and CRUZ-ORTEGA, R. 2006. Allelochemical AMAUA, A. L., and CRUZ-ORTEGA, R. 2005. Comparative effects
stress causes inhibition of growth and oxidative damage in of allelochemical and water stress in roots of Lycopersicon
Lycopersicon esculentum Mill. Plant Cell Environ 29:2009–2016. esculentum Mill. (Solanaceae). Plant Sci 168:1059–1066.
LEE, J. G., LEE, B. Y., and LEE, H. J. 2006. Accumulation of SANCHEZ-MOREIRAS, A. M., and REIGOSA, M. J. 2005. Whole plant
phytotoxic organic acids in reused nutrient solution during response of lettuce after root exposure to BOA (2(3H)-Benzox-
hydroponic cultivation of lettuce (Lactuca sativa L.). Sci. Hortic azolinone). J. Chem. Ecol 31:2689–2703.
110:119–128. SOFO, A., DICHIO, B., XILOYNNIS, C., and MASIA, A. 2004. Effects of
LIN, C. C., and KAO, C. H. 2000. Effect of NaCl stress on H2O2 different irradiance levels on some antioxidant enzymes and on
metabolism in rice leaves. Plant Growth Regul 30:151–155. malondialdehyde content during rewatering in olive tree. Plant
LIN, R. Z., WANG, X. R., LUO, Y., DU, W. C., GUO, H. Y., and YIN, D. Sci 166:293–302.
Q. 2007. Effects of soil cadmium on growth, oxidative stress and WEIR, T. L., PARK, S. W., and VIVANCO, J. M. 2004. Biochemical and
antioxidant system in wheat seedlings (Triticum aestivum L.). physiological mechanisms mediated by allelochemicals. Curr.
Chemosphere 69:89–98. Opin. Plant Biol 7:472–479.
MASIA, A. 2003. Physiological effects of oxidative stress in relation to WU, F. Z., HUANG, C. H., and ZHAO, F. Y. 2002. Effects of phenolic
ethylene in post-harvest produce, pp. 165–197, in D. M. Hodges acids on growth and activities of membrane protective enzymes
(ed.). Postharvest Oxidative Stress in Horticultural CropsFood of cucumber seedlings. Agric. Sci. China 35:821–825.
Products Press, New York. YE, S. F., ZHOU, Y. H., SUN, Y., ZOU, L. Y., and YU, J. Q. 2006.
MIYAKE, C., and ASADA, K. 1992. Thylakoid-bound ascorbate Cinnamic acid causes oxidative stress in cucumber roots, and
peroxidase in spinach chloroplasts and photoreduction of its promotes incidence of Fusarium wilt. Environ. Exp. Bot 56:255–
primary oxidation product monodehydroascorbate radicals in 262.
thylakoids. Plant Cell Physiol 33:541–553. YU, J. Q., and MATSUI, Y. 1997. Effects of root exudates of cucumber
NAKANO, Y., and ASADA, K. 1981. Hydrogen peroxide is scavenged (Cucumis sativus) and allelochemicals on ion uptake by
by ascorbate specific peroxidase in spinach chloroplasts. Plant cucumber seedlings. J. Chem. Ecol 23:817–827.
Cell Physiol 22:867–880. YU, J. Q., YE, S. F., ZHANG, M. F., and HU, W. H. 2003. Effects of
ORACZ, K., BAILLY, C., GNIAZDOWSKA, A., COME, D., CORBINEAU, root exudates and aqueous root extracts of cucumber
F., and BOGATEK, R. 2007. Induction of oxidative stress by (Cucumis sativus) and allelochemicals, on photosynthesis and
sunflower phytotoxins in germinating mustard seeds. J Chem antioxidant enzymes in cucumber. Biochem. Syst. Ecol 31:129–
Ecol 33:251–264. 139.
PATTERSON, B. D., MACRAE, E. A., and FERGUSON, I. B. 1984. ZHANG, J. H., MAO, Z. Q., WANG, L. Q., and SHU, H. R. 2007.
Estimation of hydrogen peroxide in plant extracts using titanium Bioassay and identification of root exudates of three fruit tree
(IV). Anal. Biochem 139:487–492. species. J. Integr. Plant. Biol 49:257–261.