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AUSTRALIAN JOURNAL OF
PLANT PHYSIOLOGY
Volume 27, 2000
CSIRO 2000

An international journal of plant function

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Aust. J. Plant Physiol., 2000, 27, 973978

Phenolic compounds and oxidative metabolism in green bean plants


under nitrogen toxicity
Esteban SnchezA, Juan M. SotoB, Pablo C. GarcaA, Luis R. Lpez-LefebreA, Rosa M. RiveroA,
Juan M. RuizA and Luis RomeroAC
A

Department of Plant Biology, Faculty of Sciences, University of Granada, 18071-Granada, Spain.


B
Department of Agrotechnology Science, University of Chihuahua, 31170-Chihuahua, Mxico.
C
Corresponding author; email: lromero@goliat.ugr.es

Abstract. The objective of the present work was to determine the effect of nitrogen toxicity on the metabolism of
phenolic compounds and of oxidative stress in Phaseolus vulgaris L. cv. Strike. The nitrogen was applied to the nutrient solution as NH4NO3 at 5.4, 10.8, 16.2, 21.6 and 27 mM. The results indicate that the application of 27 mM N can
be defined as toxic, as it drastically depressed growth of the green bean plants in our experiment. In addition, the
abiotic stress from the application of this N dosage inhibited the enzymes polyphenol oxidase, peroxidase and catalase, and stimulated phenylalanine ammonia-lyase and superoxide dismutase activities. The result was foliar
accumulation of phenolic compounds and hydrogen peroxide (H2O2). The accumulation of H2O2 also apparently
caused a reduction in biomass production.
Keywords: green bean, Leguminosae, nitrogen toxicity, oxidative metabolism, Phaseolus vulgaris, phenolic
bioactivity.
Introduction
Phenolic compounds are among the most widely distributed
secondary products in the plant kingdom. Many of these are
physiologically and ecologically important, being involved
in such diverse processes as rhizogenesis (Curir et al. 1990),
vitrification (Kevers et al. 1984), resistance to different types
of stress (Delalonde et al. 1996), and redox reactions
(Takahama and Oniki 1992). Nevertheless, the processes that
have been most thoroughly studied and those that most
directly involve phenolic compounds are related to pest and
disease resistance (Dbeler et al. 1997).
The metabolism of phenolic compounds in plants implies
both the synthesis and oxidation of those compounds.
Phenylalanine ammonia-lyase (PAL) is the key enzyme of
the biosynthetic pathway of most phenolic compounds. PAL
catalyses the elimination of ammonium from L-phenylalanine, giving rise to trans-cinnamate (Hao et al. 1996), this
being the first step in the biosynthesis of derivatives of
specific plant phenylpropanoids such as lignin, suberin,
flavonoids, coumarins and amides (Solecka and Kacperska
1995; Rsler et al. 1997). PAL activity is affected by many
factors, including light, temperature, growth regulators,
RNA inhibitors, protein synthesis, tissue damage, pathogen

attack, fungicide and herbicide application, and the nutritional status of certain nutrients (Jones 1984; Ruiz et al.
1998, 1999).
Meanwhile, the enzymes involved in the oxidation processes include polyphenol oxidase (PPO) and peroxidase
(POD), which catalyse the oxidation of phenols to quinones
(Thipyapong et al. 1995). Numerous works have demonstrated the activity of both enzymes to be affected by different types of biotic and abiotic stress (Sderhll 1995;
Kwak et al. 1996), notably among the latter being the nutritional status of certain elements such as boron and calcium
(Cakmak and Rmheld 1997; Tomasbarberan et al. 1997;
Ruiz et al. 1998, 1999).
On the other hand, the oxidation of phenolic compounds
generally leads to the production of quinones (Thipyapong
et al. 1995), which are highly toxic compounds responsible
for the generation of reactive oxygen species (ROS) (O2,
OH and hydrogen peroxide (H2O2); Pillinger et al. 1994).
One of the enzymes involved in the synthesis of these compounds is superoxide dismutase (SOD). Both the enzymatic
activities of SOD and H2O2-metabolizing enzymes have
received much attention, given their essential role as defence
mechanisms against different types of stress (Bowler et al.
1992).

Abbreviations used: BSA, bovine serum albumin; CAT, catalase; DTT, 1,4-dithio-DL-threitol; EDTA, ethylenediaminetetraacetic acid; Fe-EDDHA,
ethylenediamine-di(o-hydroxyphenylacetic acid); H2O2, hydrogen peroxide; NBT, nitroblue tetrazolium; PAL, phenylalanine ammonia-lyase; PMSF,
phenylmethanesulfonyl fluoride; POD, peroxidase; PPFD, photosynthetic photon flux density; PPO, polyphenol oxidase; PVP, polyvinylpyrrolidone;
ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase.
CSIRO 2000

10.1071/PP00008

0310-7841/00/0100973

974

Under normal conditions, the cellular levels of ROS are


maintained at a low level by a protective mechanism based
on the compartmentalization of SOD, catalase and peroxidase. However, in some cases, and especially under stress
conditions, this protective mechanism is overwhelmed by
oxidation, during which high levels of ROS are produced,
reaching cellular concentrations of more than 1 M (Bolwell
and Wojtaszek 1997; Wojtaszek 1997a, b).
In present-day agriculture, the main types of stress commonly generated as a consequence of the heavy use of inorganic fertilizers are related to the nutritional status of certain
nutrients, primarily nitrogen (N), given its extensive use
(Sisson et al. 1991). An adequate N supplement is a key
factor for growth and productivity in most crops. Generally,
plant growth slows under N supply exceeding 10 mM, a value
considered on the threshold of toxicity for some species
(Benton Jones 1997; Cao and Tibbitts 1998). It appears that
the relationship between the availability of N and the
accumulation of phenolic compounds is usually positive
(Keller and Hrazdina 1998; Stout et al. 1998). Nevertheless,
the relationship between N supply and the response of oxidative metabolism has been scarcely treated. Thus, the objective of the present work was to assess the influence of N
toxicity on two of the key metabolic processes in the adaptation of plants to any type of stress, such as the bioactivity
of phenolics and oxidative metabolism. Our ultimate aim
was to define these processes as bioindicators of this type of
abiotic stress.
Materials and methods
Crop design and plant sampling
Seeds of Phaseolus vulgaris cv. Strike, sown in April 1999 in southeastern Spain (Granada), were transferred at 8 d to a growth chamber
under controlled environmental conditions, with relative humidity of
6080%, temperature 30/20C (day/night), and a photoperiod of 16/8 h
in a photosynthetic photon flux density (PPFD) of 350 mol m2 s1
(measured at the top of the plants with a 190 SB quantum sensor, LICOR Inc., Lincoln, NE, USA). Four plants were grown in 8-L pots
(25 cm upper diameter, 17 cm lower diameter, 25 cm height), filled with
vermiculite. For 30 d (including 8 d for germination) before the experimental treatments, the plants received a nutrient solution of 5.4 mM
NH4NO3, 1.6 mM K2HPO4, 0.3 mM K2SO4, 4 mM CaCl2.2H2O, 1.4 mM
MgSO4.H2O, 5 M Fe-EDDHA, 2 M MgSO4.H2O, 1 M ZnSO4.7H20,
0.25 M CuSO4.5H2O, 0.3 M Na2MoO4.2H2O and 0.5 M H3BO3. The
nutrient solution (pH 6.06.1) was renewed every 3 d.
At 30 d after sowing, the different N treatments (5.4, 10.8, 16.2, 21.6
and 27 mM N, in the form of NH4NO3) were applied for 30 d (until
harvest). The experimental design was a complete randomized block
with six replicates (individual pots) with 24 plants per treatment.
The plants were sampled at 60 d after sowing, at full pod development. All the leaf samples were taken in the mature state. Leaves were
picked at about one-third of the plant height from the plant apex. The
material was rinsed three times in distilled water after disinfecting with
non-ionic detergent at 1% (Wolf 1982), then blotted on filter paper. The
leaves were used fresh for the analysis of PAL, PPO, POD, total
phenols, SOD, H2O2 and catalase (CAT), with triplicate assays for each
extraction.

E. Snchez et al.

Plant analysis
Extraction and assay of PAL
Extraction of PAL (EC 4.3.1.5) was carried out following the
method of Lister et al. (1996). Fresh plant material was ground at 4C
in buffer (50 mM Na2HPO4/KH2PO4, pH 7.0, 5% polyvinylpyrrolidone
(PVP) (Mr 44 000), 50 mM Na ascorbate, 18 mM mercaptoethanol, 0.1%
(v/v) Triton X-100). The homogenate was filtered through four layers of
cheesecloth and centrifuged at 20 000 g for 10 min. (NH4)2SO4 was
added to the supernatant (to 35% saturation), which was then centrifuged for 20 min at 20 000 g to remove the PVP. More (NH4)2SO4 was
added to this supernatant to reach 80% saturation. This fraction was
centrifuged at 20 000 g for 20 min and the pellet resuspended in extraction buffer (without PVP and Triton). This solution was used for PAL
assays. Protein was estimated by the method of Bradford (1976) using
bovine serum albumin (BSA) as a standard. All these procedures were
carried out at 04C.
PAL activity was assayed by the methods of Zucker (1965) and
McCallum and Walker (1990). PAL activity was determined from the
yield of cinnamic acid, estimated from absorbance at A290 in the presence and absence of phenylalanine. To determine whether the reaction
was enzymatic, a control extract was boiled and assayed.
Extraction and assay of PPO
The PPO (EC 1.14.18.1) extraction method used was that proposed
by Thipyapong et al. (1995), with some modifications. Leaves were
ground to a fine powder in a mortar and pestle and extracted at a ratio
of 150 mg fresh weight to 1 mL extraction buffer (100 mM TrisHCl,
pH 7.0, 100 mM KCl, 1 mM phenylmethanesulfonyl fluoride (PMSF)
and 3% (w/v) PVP), containing sodium dodecyl sulfate (SDS) at 0, 0.5,
1, 2 or 4% (w/v). The homogenates were centrifuged at 12 000 g for
15 min, and the supernatant was used for PPO assay. Protein concentration was measured by the method of Bradford (1976) using BSA as
standard. All these procedures were carried out at 04C.
PPO activity was assayed as described by Nicoli et al. (1991), with
some modifications. Optimum activity was reached using SDS at 2%
(data not shown). PPO activity was measured by the change in A370 of
the assay mixture (30C) based on the measurement of the disappearance of caffeic acid by enzymatic oxidation. To determine whether the
reaction was enzymatic, a control extract was boiled and assayed.
Extraction and assay of peroxidase and catalase
The method used for extraction and assay of peroxidase
(EC 1.11.1.7) and catalase (EC 1.11.1.6) was a modified version of
those proposed by Kalir et al. (1984) and Badiani et al. (1990). Fresh
plant material was ground with 50 mM Trisacetate buffer, pH 7.5, 5 mM
2-mercaptoethanol, 2 mM 1,4-dithio-DL-threitol (DTT), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM PMSF, and 1% (w/v) PVP. The
homogenate was filtered through two layers of Miracloth and centrifuged for 30 min at 37 000 g. The pellet was discarded and the supernatant used for POD and CAT assays, and to measure the protein
concentration by the method of Bradford (1976), using BSA as standard. All these procedures were carried out at 04C.
POD activity was determined by following the change of A485 due to
guaiacol oxidation (Kalir et al. 1984; Ruiz et al. 1998). CAT activity
was determined by following the consumption of H2O2 at A240 for 5 min
in 3 mL of reaction mixture, due to H2O2 oxidation (Kalir et al. 1984;
Rao et al. 1997). To determine whether the reactions were enzymatic,
the control extracts were boiled and assayed.
Extraction and assay of SOD
SOD (EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT),
according to the methods of Giannopolitis and Ries (1977) and Beyer
and Fridovich (1987), with some modifications (Yu et al. 1998). Frozen

Phenolic compounds and oxidative metabolism under N toxicity

leaf samples were weighed and homogenized on ice in a mortar and


pestle for 2 min with 1.5 g of quartz sand and 10 mL of homogenizing
solution containing 50 mM HEPES buffer and 0.1 mM Na2EDTA
(pH 7.6). The homogenate was centrifuged at 4C for 15 min at
15 000 g, then filtered through qualitative filter paper (MN 617,
Macherey-Nagel, Germany) to produce the crude extract, which was
used for SOD assay and to measure the protein concentration by the
method of Bradford (1976), using BSA as standard. All these
procedures were carried out at 04C.
For total SOD assay, a 5-mL reaction mixture was used, containing
50 mM HEPES (pH 7.6), 0.1 mM EDTA, 50 mM Na2CO3 (pH 10.0),
13 mM methionine, 0.025% (w/v) Triton X-100, 63 M NBT, 1.3 M
riboflavin and an appropriate aliquot of enzyme extract. The reaction
mixtures were illuminated for 15 min; PPFD was 380 mol m2 s1.
Identical reaction mixtures that were not illuminated were used to
correct for background absorbance. One unit of SOD activity was
defined as the amount of enzyme required to cause 50% inhibition of
the reduction of NBT as monitored at 560 nm.

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The enzymes PPO and POD catalyse the oxidation of


phenols to quinones (Thipyapong et al. 1995). In this study,
the N dosage also significantly influenced the activities of
PPO (P < 0.001; Table 1) and POD (P < 0.001; Table 1), both
enzymes presenting the highest activities values with the
5.4 mM N treatment and the lowest activities with the highest
dosages of N, with a difference of 37 and 36%, respectively.
Thus, the lowest activities for these two enzymes, together
with the highest PAL activity, with the application of
27 mM N appears to have caused the foliar accumulation of
phenolics at this treatment level. We propose that the application of high N dosages in general favours the synthesis and
accumulation of phenolics, and also inhibits the oxidative
processes of these compounds.

Extraction and quantification of total phenols

Extraction and assay of total H2O2


The methods used were those proposed by MacNevin and Uron
(1953) and Brennan and Frenkel (1977). Hydroperoxides form a specific complex with titanium (Ti4+), which can be measured by colorimetry at 415 nm. The concentration of the peroxide in the extracts was
determined by comparing the absorbance against a standard curve
representing a titaniumH2O2 complex from 0.1 to 1 mM. The hydroperoxides represent the total peroxides.
Statistical analysis
Analysis of variance was used to assess the significance of treatment
means. The data shown are mean values s.e. A correlation analysis
was also made between the different variables. Levels of significance
were represented by * at P < 0.05, ** at P < 0.01, *** at P < 0.001 and
NS, not significant.

Results and discussion


The effectiveness of the N treatments in our experiment is
reflected in the production of root (P < 0.001) and foliar
biomass (P < 0.001), which diminished sharply as the N
dosage increased (Fig. 1). That is, the application of 27 mM N
resulted in markedly less root and foliar biomass (60 and
70% of the control, respectively) than did the application of
5.4 mM N, indicating that the greater dosage applied (27 mM)
was toxic, whereas the lesser dosage (5.4 mM) stimulated
growth.
PAL is the key enzyme of the biosynthetic pathway of
most phenolic compounds (Hao et al. 1996). In our experiment, the application of the highest N dosage boosted PAL
activity (P < 0.001; Table 1) to its highest value, this being
41% higher than the lowest value registered with the lowest
dosage of N. The foliar levels of total phenolics (Table 1)
showed a similar trend; the plants treated with 27 mM N
showing the highest foliar accumulation of total phenolics,
exceeding the values of the lowest N dosage by 27%
(P < 0.001; Table 1).

700

Biomass (g DW tissue1)

The phenolic compounds of the plant material were extracted with


methanol. Total phenolic content was assayed quantitatively by A765
with Folin-Ciocalteau reagent (Singleton and Rossi 1965; Singleton
et al. 1985).

600

Root

Leaf

500
400
300
200
100
0
5.4 10.8 16.2 21.6 27.0

5.4 10.8 16.2 21.6 27.0

N treatments
Fig. 1. Root and foliar biomass in green bean plants in response to N
treatment (5.4, 10.8, 16.2, 21.6 and 27 mM of NH4NO3). Data are means
s.e. (n = 6).

Table 1.

Effect of N treatment on bioactivity of phenolic


compounds
Phenylalanine ammonia-lyase (PAL) expressed as cinnamic acid produced [mol (mg protein)1 min1]; total phenols, expressed as caffeic
acid mol (g FW)1]; polyphenol oxidase (PPO) expressed as caffeic
acid oxidized [mol (mg protein)1 min1]; peroxidase (POD) expressed
as guaiacol oxidized [mol (mg protein)1 min1]. * P < 0.05,
** P < 0.01, *** P < 0.001 by ANOVA at P = 0.05. Data are means
s.e. (n = 6)
NH4NO3 (mM)
5.4
10.8
16.2
21.6
27.0
Significance

PAL

Total phenols

PPO

POD

2.96 0.22
3.22 0.25
3.46 0.25
3.93 0.34
4.18 0.33
***

29.5 0.56
30.9 0.61
30.1 0.64
33.5 1.15
37.4 1.08
***

4.51 0.02
5.59 0.04
5.61 0.02
4.56 0.02
2.86 0.05
***

4.63 0.03
5.41 0.02
3.48 0.04
3.32 0.01
2.94 0.01
***

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E. Snchez et al.

On the other hand, it has been observed that oxidative


stress can be activated in response to a variety of stimuli,
such as high and low temperatures, exposure to UV rays,
nutrient deficiency, drought, herbicides and pathogen attack
(Inz and Van Montagu 1995; Paranhos et al. 1999). As we
indicated in the Introduction, one of the ways in which H2O2
is generated is through SOD (Bowler et al. 1992; Foyer et al.
1994). In our experiment, SOD activity significantly
increased with the N dosage (P < 0.001), presenting its
highest value at 27 mM N, this being 12% higher than at
5.4 mM N (Table 2).
As might be expected, H2O2 also showed its highest
concentrations at the highest N dosage, with an increase of
19% with respect to the lowest N dosage (P < 0.001; Table 2).
Therefore, the application of high N dosages stimulates
oxidative metabolism, causing a higher SOD activity and
therefore a greater concentration of foliar H2O2.
The accumulation of H2O2 in tissues is due, on the one
hand, to the action of SOD and, on the other hand, to the inhibition of CAT and POD (Gaspar et al. 1991; Rao et al. 1996).
As indicated above, POD had its lowest activity with the
application of the highest amount of N, as did CAT
(P < 0.001), which showed a drastic reduction (82%)
(Table 2). Therefore, our results indicate that H2O2 accumulates in the leaves of plants treated with 27 mM N, and point
to the positive action of SOD and principally the negative
effect of N on POD and CAT enzymes.
In the Introduction, we noted that the oxidation of
phenolic compounds generally leads to the production of
quinones (Thipyapong et al. 1995), which are highly toxic
compounds that generate ROS. In our experiment, the oxidation of the phenolics was substantial at 5.4 mM N applied,
since this treatment showed the highest PPO activity
(Table 1) and the lowest foliar phenolic concentrations
(Table 1). Therefore, this treatment should give high H2O2
contents. However, as shown in Table 2, the lowest N dosage
gave the lowest foliar concentrations for this compound,
perhaps because the highest activities of POD and CAT were

at this level (Tables 1 and 2), thereby impeding the accumulation of H2O2 and thus its toxic effects. On the contrary, at
27 mM N, H2O2 was generated possibly by SOD, given that
this treatment significantly reduced the oxidation of the
phenolics. Therefore, as indicated above, the accumulation
of H2O2 in the highest N treatment was due to a direct effect
of N increasing the SOD activity and mainly inhibiting POD
and CAT activities.
The mechanisms governing foliar accumulation of phenolics and H2O2 upon the application of 27 mM N appear to
be the following:
Firstly, the toxic and harmful accumulation of NH4+,
resulting both from the heavy supply of NH4NO3 (27 mM N)
and from the strong PAL activity found in this treatment,
could explain the accumulation of phenolics and H2O2, given
that these compounds normally accumulate as protective
responses against different types of both biotic and abiotic
stress (Dixon and Paiva 1995; Willekens et al. 1997).
In addition, N toxicity increases the susceptibility of
plants to pathogens (Benton Jones 1997; Barker 1999). This
fact could also explain the accumulation of phenolics and
H2O2, since both compounds form part of preventive as well
as initial responses of plants attempting to avoid or counteract pathogen infection (Smith-Becker et al. 1998).
Finally, the foliar accumulation of H2O2 at the highest N
dosage could also explain the trend of the production of root
and foliar biomass in our experiment (Fig. 1), since the production of biomass diminished with N dosage. This reduction could be due also to the toxic effects from H2O2
accumulation in the plants treated with 27 mM N (Okuda
et al. 1991).
In conclusion, our results indicate that the application of
27 mM N can be defined as toxic, as it drastically depressed
growth of green bean plants in our experiment. In addition,
the abiotic stress from the application of this N dosage inhibited the enzymes PPO, POD and CAT, and stimulated PAL
and SOD activities. The result was foliar accumulation of
phenolic compounds and H2O2, the accumulation of which
also apparently caused a reduction in biomass production.

Table 2. Effect of N treatment on oxidative metabolism


Superoxide dismutase (SOD) expressed as SOD activity [units (mg
protein)1 min1]; hydrogen peroxide (H2O2) expressed as peroxide
[mol (g FW)1]; catalase (CAT) expressed as H2O2 oxidized [mol (mg
protein)1 min1]. * P < 0.05, ** P < 0.01, *** P < 0.001 by ANOVA at
P = 0.05. Data are means s.e. (n = 6)

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NH4NO3 (mM)
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16.2
21.6
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704.3 10.9
710.5 7.70
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***

378.6 16.4
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345.1 15.6
399.0 13.6
450.4 14.7
***

0.21 0.04
0.18 0.02
0.10 0.03
0.11 0.02
0.04 0.00
***

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Manuscript received 5 January 2000, accepted 25 May 2000

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