Академический Документы
Профессиональный Документы
Культура Документы
P U B L I S H I N G
AUSTRALIAN JOURNAL OF
PLANT PHYSIOLOGY
Volume 27, 2000
CSIRO 2000
w w w. p u b l i s h . c s i r o . a u / j o u r n a l s / a j p p
All enquiries and manuscripts should be directed to
Australian Journal of Plant Physiology
CSIRO PUBLISHING
PO Box 1139 (150 Oxford St)
Collingwood
Telephone: 61 3 9662 7625
Vic. 3066
Facsimile: 61 3 9662 7611
Australia
Email: laurie.martinelli@publish.csiro.au
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
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
975
700
Biomass (g DW tissue1)
600
Root
Leaf
500
400
300
200
100
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.
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
***
976
E. Snchez et al.
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.
References
NH4NO3 (mM)
5.4
10.8
16.2
21.6
27.0
Significance
SOD
H2O2
CAT
630.7 7.67
696.4 10.8
704.3 10.9
710.5 7.70
717.3 8.20
***
378.6 16.4
391.1 15.7
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
***
977
978
E. Snchez et al.
Wolf B (1982) A comprehensive system of leaf analysis and its use for
diagnosing crop nutrients status. Communications in Soil Science
and Plant Analysis 13, 10351059.
Yu Q, Osborne L, Rengel Z (1998) Micronutrient deficiency changes
activities of superoxide dismutase and ascorbate peroxidase in
tobacco plants. Journal of Plant Nutrition 21, 14271437.
Zucker M (1965) Induction of phenylalanine deaminase by light and its
relation to chlorogenic acid synthesis in potato tuber tissue. Plant
Physiology 40, 779784.
http://www.publish.csiro.au/journals/ajpp