ACTIVITY OF PEROXIDASES AND PHENYLALANINE AMMONIA-LYASE IN LUPINE AND SOYBEAN
59
BIOLOGICAL LETT. 2008, 45: 59–67
Available online at http://www.biollett.amu.edu.pl
Activity of peroxidases and phenylalanine ammonia-lyase
in lupine and soybean seedlings treated with copper
and an ethylene inhibitor
JAGNA CHMIELOWSKA1,2, JOANNA DECKERT1, JOSÉ DÍAZ2
1
Department of Plant Ecophysiology; Faculty of Biology,
Adam Mickiewicz University, ul. Umultowska 89; 61-614 Poznañ, Poland
2
Departamento de Bioloxia Animal, Bioloxia Vexetal e Ecoloxia, Universidade da Coruña,
Campus da Zapateira s/n E-15071 Coruña, Spain
e-mail: [email protected] (J. Deckert)
(Received on 28 May 2008, Accepted on 20 March 2009)
Abstract: Plant growth and activity of peroxidases (POX) and phenylalanine ammonia-lyase (PAL) were
investigated in seedlings of yellow lupine (Lupinus luteus L.) and soybean (Glycine max L.) treated with
copper (25 mg/l) and an inhibitor of ethylene biosynthesis (CoCl2). Both lupine and soybean plants stressed
with an excess of copper exhibited a decrease in root length and fresh weight. In lupine plants, an
enhancement of POX and PAL activity was observed. Surprisingly, the ethylene inhibitor had no significant
influence on plant growth and the activity of both enzymes. These results suggest that POX and PAL
play a role in Cu-induced responses in plants.
Keywords: copper, peroxidase, phenylalanine ammonia-lyase, ethylene inhibitor, soybean, lupine
INTRODUCTION
Copper is an essential micronutrient, which is a constituent of many enzymes
taking part in various processes, such as photosynthesis, respiration, nitrate metabolism, and lignification. However, an excess of copper is strongly phytotoxic. It causes
stunted growth, lipid peroxidation, disturbances in intracellular ion balance, decline
in respiratory rates, and net photosynthetic oxygen release (KUPPER et al. 1996, PADUA
et al. 1999, CHEN et al. 2000, DÍAZ et al. 2001, ALOUI-SOSSÉ et al. 2004). One of
the most important mechanisms of copper toxicity is connected with the production
of reactive oxygen species (ROS) (CHEN et al. 2000, RAEYMAEKERS et al. 2003,
DR¥¯KIEWICZ et al. 2004). A complex antioxidant system, which includes peroxidase, superoxide dismutase and catalase, is used by plants to avoid damages caused
by ROS. Peroxidases (POX, EC 1.11.1.7) form a large family of enzymes, which also
60
Jagna Chmielowska, Joanna Deckert and José Díaz
take part in other defense mechanisms – such as resistance to pathogens and salt tolerance – that involve lignification, suberization and cross-linking of cell wall structural proteins (HIRAGA et al. 2001, ROS-BARCELÓ & AZNAR-ASENSIO 2002, LI et al.
2003, PASSARDI et al. 2004, LIN & KAO 2005). Phenylalanine ammonia-lyase (PAL,
EC 4.3.1.5) is a key enzyme in lignin synthesis, because it catalyses the first step of
phenylpropanoid pathway, i.e. desamination of phenylalanine, which results in formation of trans-cinnamic acid (BOUDET 2000, GOUJON et al. 2003). Enhancement
of POX and PAL activity and increase in lignin content is a common plant response
to stress factors (JBIR et al. 2001, MANDRE 2002, EGERT & TEVINI 2003, GULEN &
ATILLA 2003, JOUILI & FERJANI 2003, LIN et al. 2005).
Various processes, which take place in plant organisms, are controlled by plant
hormones. Elevated levels of ethylene in response to stress factors suggest that
this hormone acts as a regulator of plant defense mechanisms (VISSER et al. 1996,
BELTRANO et al. 1999, CONCELLÓN et al. 2005, HAYS et al. 2007). Indeed, ethylene
release is induced by copper in Arabidopsis thaliana (ARTECA & ARTECA 2007),
so a role in regulating its response to the heavy metal is possible.
An earlier study performed in our laboratory showed that heavy metals, such
as cadmium and lead, cause growth inhibition in soybean and lupine, and that this
effect is correlated with various stress responses. These include activation of the
antioxidant system (RUCIÑSKA et al. 1999, SOBKOWIAK et al. 2004), perturbation of
the cell cycle and DNA damages (DECKERT & GWӏD 1999, SOBKOWIAK & DECKERT 2003, 2004, RUCIÑSKA et al. 2004), and induction of various proteins (PRZYMUSIÑSKI & GWӏD 1999, SOBKOWIAK & DECKERT 2006). The function of some
of those proteins in plant response to metals is not known. One of the proteins induced by Cd2+ in soybean was identified as chalcone synthase (CHS; EC 2.3.1.74)
(SOBKOWIAK & DECKERT 2006). This result suggested a possible involvement of
phenylpropanoid pathway in the response of legume plants to metals. The influence
of both the heavy metal and ethylene inhibitor on soybean and lupine plants has not
been examined yet. Moreover, the above-mentioned studies concerned the influence
of lead and cadmium. Therefore, we found it interesting to examine the impact of
another heavy metal – copper – on these two plant species.
The aim of this study was to determine whether POX and PAL take part in
plant response to an excess of copper, and if their activity is modified by ethylene.
MATERIAL AND METHODS
Material, growth conditions and treatment procedures
Seeds of yellow lupine (Lupinus luteus L. cv. Vantus) and soybean (Glycine max
L. cv. Naviko), were surface-sterilized with 75% ethanol for 5 min, followed by 1%
sodium hypochloride for 10 min, washed in water, and germinated in Petri dishes
containing water-moistened filter paper for 48 h. Seedlings were then transferred to
dishes containing 5 ml of either distilled water (control) or aqueous solution of copper
containing 25 mg/l of Cu2+ (391 µM CuSO4) or the aqueous solution of 25 mg/l of Cu2+
supplemented with 10 µM CoCl2. Seedlings were incubated in the dark at 22oC for
48 h. The statistical analysis was based on 3 independent experiments. Each experimental variant was represented by 90 seedlings.
ACTIVITY OF PEROXIDASES AND PHENYLALANINE AMMONIA-LYASE IN LUPINE AND SOYBEAN
61
Peroxidase (POX) activity measurements
Root tips (300 mg of tissue) were ground on ice in a mortar with 400 µl of
Tris-HCl buffer at pH 7.5 and 15 mg of polyvinylpolypyrrolidone (PVPP, PolyclarAT). They were centrifuged at 4°C for 15 min at 12 000 g. Supernatants were used
in measurements of protein concentration (BRADFORD 1976), and POX activity was
measured according to a modified method of CHRISTENSEN et al. (1998). POX activity was estimated by recording absorbance of mixtures containing 40 µl of
samples, 900 µL of 20 mM citrate sodium at pH 5.5, 10 µL of 100 mM solution of
3,3-diaminobenzidine (DAB), and 50 µL of 6 mM H2O2 at λ = 452 nm and 25°C.
The activity of the enzyme is expressed as A452/mg of protein.
Phenylalanine ammonia-lyase (PAL) measurements
PAL extraction and activity measurements were carried out according to a
modified method of CAHILL & MCCOMB (1992). Root tips (300 mg of tissue) were
ground on ice in a mortar with 30 mg of PVPP and 4 ml of 0.1 M Tris-HCl buffer
at pH 8.9, containing 10 mM mercaptoethanol. Extracts were centrifuged at 4°C for
30 min at 12 000 g. Supernatant was used for further measurements. The mixture
containing 500 µl of samples and 1000 µl of 80 mM borate buffer at pH 8.9 with 30 mM
phenylalanine were incubated for 1 h in a water bath at 30°C. Then 1.5 ml of 2M HCl
was added, and the concentration of trans-cinnamic acid was measured spectrophotometrically at λ = 290nm. The activity of the enzyme is expressed as A 290/mg of protein.
Statistical analysis
Statistical analyses were performed using Statgraphics Plus for Windows, 5.1
Professional Version (Statistical Graphics Corp.). Where necessary, transformations
were carried out to normalize the data prior to analysis. Two-way ANOVAs were
performed (P < 0.05).
RESULTS
The excess of copper caused a significant reduction of root length and fresh
weight of both soybean and lupine seedlings (P < 0.05). The reduction was greater
in soybean roots, which were more than 60% shorter and weighed approximately 40%
less than the control. Ethylene inhibitor (CoCl2) did not affect root growth. The growth
parameters are presented in Table 1.
Generally POX activity was higher in the soybean than in lupine roots (Fig. 1).
Copper treatment caused enhancement of POX activity by 110% (ANOVA: F1,8 = 31.26,
P = 0.0005) in lupine plants, whereas it had no significant effect on soybean plants.
Ethylene inhibitor had no significant influence on POX.
A similar tendency can be seen in PAL activity (Fig. 2), which was generally
higher in soybean than in lupine roots. An excess of copper caused an increase in
PAL activity by 110% (ANOVA: F1,8 = 13,65, P = 0.006) in lupine plants but had no
significant effect on soybean seedlings. Ethylene inhibitor did not have any significant effect on PAL activity.
62
Jagna Chmielowska, Joanna Deckert and José Díaz
Table 1. Effect of copper and ethylene inhibitor (CoCl2) on the length and fresh weight of lupine and
soybean roots
Treatment
Root length (mm)
Root fresh weight (mg)
lupine
soybean
lupine
soybean
Control
31.0 ± 7.8
55.2 ± 16.4
91.4 ± 11.6
67.6 ± 7.2
Control +10ìM CoCl2
32.8 ± 6.3
58.3 ± 21.7
99.8 ± 12.7
69.4 ± 4.4
Copper (25 mg/L)
27.4 ± 6.3
20.2 ± 7.0
81.6 ± 9.4
39.1 ± 9.6
Copper (25 mg/L) + 10 ìM
26.8 ± 7.5
20.0 ± 7.6
78.0 ± 9.7
37.4 ± 5.2
CoCl2
Data are means ± SD from 3 (soybean) or 4 (lupine) independent experiments
16
A452/mg of protein
14
12
10
8
6
4
2
0
0 0+I
lupine
Cu Cu+I
lupine
0 0+I
soybean
Cu Cu+I
soybean
Fig. 1. Effect of copper and ethylene inhibitor on the activity of peroxidases (POX) extracted from lupine and soybean roots: 0 = control, Cu = copper (25 mg/l), I = ethylene inhibitor (10 M CoCl2). Data
are means ± SD from 3 independent experiments.
ACTIVITY OF PEROXIDASES AND PHENYLALANINE AMMONIA-LYASE IN LUPINE AND SOYBEAN
63
9
A290/mg of protein
8
7
6
5
4
3
2
1
0
0 0+I
lupine
Cu Cu+I
lupine
0 0+I
soybean
Cu Cu+I
soybean
Fig. 2. Effect of copper and ethylene inhibitor on the activity of phenylalanine ammonia-lyase (PAL)
extracted from lupine and soybean roots: 0 = control, Cu = copper (25 mg/l), I = ethylene inhibitor (10 M
CoCl2). Data are means ± SD from 3 independent experiments.
DISCUSSION
Growth inhibition resulting from cupric stress was noted in several plant species, such as Arabidopsis thaliana, Glycine max, Lycopersicon esculentum, Helianthus annuus, Brassica pekinensis, Raphanus sativus, Zea mays, and Vitis vinifera
(OUZOUNIDOU et al. 1995, MAZHOUDI et al. 1997, CHEN et al. 2000, LLORENS et al.
2000, JOUILI & FERJANI 2003, SGHERRI et al. 2003, WÓJCIK & TUKIENDORF 2003,
LIN et al. 2005, XIONG & WANG 2005). This effect is confirmed by the present study,
in which lupine and soybean plants treated with copper exhibited a reduction of root
length and fresh weight (Table 1).
Copper, as a transition metal, contributes to ROS production through Fenton
reaction. An increase in ROS levels and lipid peroxidation has been reported in plants
treated with this metal (MAZHOUDI et al. 1997, BANDYOPADHYAY et al. 1999, CHEN
et al. 2000, DR¥¯KIEWICZ et al. 2004, LIN et al. 2005). POX are involved in hydrogen peroxide (H2O2) scavenging, so their induction could alleviate oxidative stress
caused by copper. Indeed, we observed an induction of POX activity in lupine roots
(Fig. 1). Such an enhancement of POX enzymes is a response to cupric stress that
has been reported previously in other plant species (CHEN et al. 2000, DÍAZ et al.
2001, LIN et al. 2005, XIONG & WANG 2005). However, it is interesting to point out
that in soybean we did not observe peroxidase induction by copper, but we detected
a strong reduction in root biomass, revealing a degree of stress even higher than in
lupine. A possible explanation is that the copper levels in the soybean root cells were
too high, and the plant reached the stage of exhaustion, where resistance to the stress
64
Jagna Chmielowska, Joanna Deckert and José Díaz
decreases (LICHTENTHALER 1998). According to this explanation, POX would be
initially induced in soybean roots in the first hours of treatment (stage of resistance)
and then would decrease to reach the control levels after 48 h of metal treatment (stage
of exhaustion), when we analyzed POX activity. LIN et al. (2005) observed an increase in POX activity in copper-treated roots, but the concentrations of the metal
applied in their experiment were much lower than that used by us, so it is possible
that in their study the roots were still at the stage of resistance.
The mechanism described above (reaching the stage of exhaustion) could also
explain the phenylalanine ammonia-lyase (PAL) activity results in soybean roots,
which showed no induction of the enzyme by copper. However, in lupine there was
an enhancement of the enzyme caused by the metal stress. PAL catalyses the first
reaction of the phenylpropanoid pathway and is affected by various stress factors
(SARMA & SHARMA 1999, KHAN et al. 2003, LAFUENTE et al. 2003). Several products of the phenylpropanoid pathway, such as salicylic acid, coumarins, anthocyanins
and lignin, are involved in plant defense mechanisms. In our experiment, an excess
of copper caused an enhancement of PAL activity by 110% in lupine plants. An
increase in the activity of the enzymes during cupric stress has also been noted by
other authors (JOUILI & FERJANI 2003).
Both POX and PAL are involved in lignin biosynthesis (B OUDET 2000, GOUJON et al. 2003). As cupric stress causes an enhancement of the activity of both
enzymes, it would be interesting to check whether an excess of copper affects lignin
concentration. In fact, an increase in lignin content was reported in soybean roots
treated with copper by LIN et al. (2005), but the concentration of the applied metal
was lower than that used in our study.
Ethylene is known to inhibit elongation growth (K NOESTER et al. 1997, PIERIK
et al. 1999, NICOLAS et al. 2001, RAJALA et al. 2002). Surprisingly in our study an
inhibitor of ethylene synthesis (CoCl2) had no effect on plant growth. This contrasts
with other experiments, in which CoCl2 caused an increase in barley shoot length
and a slight increase in root length. However, the barley seedlings were treated with
ethylene inhibitors for a longer time (4 days) than the seedlings used in our study
(LOCKE et al. 2000). Lupine seedlings treated with an inhibitor of ethylene perception, silver thiosulfate (STS), initially exhibited an increase in hypocotyl growth but
the final length was similar to the control (NICOLÁS et al. 2001). Another inhibitor
of ethylene action, cyclopropenylmethyl butyl, caused a slight increase in root growth
of canola seedlings but had no effect on shoot length (SALEH-LAKHA et al. 2005).
Those examples show that the effect of ethylene inhibitors depends on inhibitor type,
plant species, and seedling age.
Pine cells characterized by a high ethylene production exhibit a higher POX
activity (IEVINSH & OZOLA 1998). Moreover, ethylene induces type III peroxidase
gene (TcPer-1) in cocoa (BAILEY et al. 2005). These reports suggest that ethylene
may act as a positive regulator of POX. The effect is not confirmed in the present
study, in which ethylene inhibitor had no influence on POX activity.
In conclusion, the present study confirms that copper stress inhibits lupine and
soybean growth and enhances lupine POX and PAL activity. However, the inhibitor
of ethylene synthesis (CoCl2) seems to have no effect on plant growth and the activity of the enzymes.
ACTIVITY OF PEROXIDASES AND PHENYLALANINE AMMONIA-LYASE IN LUPINE AND SOYBEAN
65
REFERENCES
ALAOUI-SOSSÉ B., GENET P., VINIT-DUNAND F., TOUSSAINT M.-L., EPRON D., BADOT P.-M. 2004.
Effect of copper on growth in cucumber plants (Cucmis sativus) and its relationships with
carbohydrate accumulation and changes in ion contents. Plant Science 166: 1213–1218.
ARTECA R. N., ARTECA J. M. 2007. Heavy-metal-induced ethylene production in Arabidopsis thaliana. J. Plant Physiol. 164: 1480–1488.
BAILEY B. A., STREM M. D., BAE H., ANTUNEZ G., MAYOLO G. A., GUILTINAN M. J. 2005. Gene
expression in leaves of Theobroma cacao in response to mechanical wounding, ethylene, and/
or methyl jasmonate. Plant Sci. 168: 1247–1258.
BANDYOPADHYAY U., DAS D., BENERJEE R. K. 1999. Reactive oxygen species: Oxidative damage
and pathogenesis. Current Sci. 77: 658–666.
BELTRANO J., RONCO M. G., MONTALDI E. R. 1999. Drought stress syndrome in wheat is provoked
by ethylene evolution imbalance and reversed by watering, aminoethoxyvinyloglycine, or
sodium benzoate. J. Growth Regul. 18: 59–64.
BOUDET A-M. 2000. Lignins and lignification: Selected issues. Plant Physiol. Biochem. 38: 81–96.
BRADFORD M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
CAHILL D.M., MCCOMB J. A. 1992. A comparison of changes in phenylalanine ammonialyase activity, lignin and phenolic synthesis in roots of Eucalyptus calophylla (field resistant) and
E. marginata (susceptible) when infected with Phytophtora cinnamoni. Physiol. Mol. Plant.
Pathol. 40: 315–332.
CHEN L-M., LIN CH. CH., KAO CH. H. 2000. Copper toxicity in rice seedlings: Changes in antioxidative enzyme activities, H2O2 level, and cell wall peroxidase activity in roots. Bot. Bull. Acad.
Sin. 41: 99–103.
CHRISTENSEN J. H., BAUW G., WELINDER K. G., MONTAGU M., BOERJAN W. 1998. Purification
and characterization of peroxidases correlated with lignification in poplar xylem. Plant Physiol.
118: 125–135.
CONCELLÓN A., AÑÓN M. C., CHAVES A. R. 2005. Effect of chilling on ethylene production in
eggplant fruit. Food Chem. 92: 63–69.
DECKERT J., GWӏD E. A. 1999. The effect of lead on cyclin expression in lupine roots. Acta
Physiol. Plant. 21: 249–256.
DÍAZ J., BERNAL A., POMAR F., MERINO F. 2001. Induction of shikimate dehydrogenase and peroxidase in pepper (Capsicum annuum L.) seedling in response to copper stress and its relation to lignification. Plant Sci. 161: 179–188.
DR¥¯KIEWICZ M., SKÓRZYÑSKA-POLIT E., KRUPA Z. 2004. Copper-induced oxidative stress and
antioxidant defence in Arabidopsis thialana. BioMetals 17: 379–387.
EGERT M., TEVINI M. 2003. Influence of ultraviolet radiation-B on peroxidase activity of Allium
schoenoprasum leaves. Biol. Plant. 47: 265–267.
GOUJON T., SIBOUT R., EUDES A., MACKAY J., JOUANIN L. 2003. Genes involved in biosynthesis
of lignin precursors in Arabidopsis thaliana. Plant Physiol. Biochem. 41: 677–687.
GULEN H., ATILLA E. 2003. Effect of heat on peroxidase activity and total protein content in strawberry plants. Plant Sci. 166: 739–744.
HAYS D. B., DO J. H., MASON R. E., MORGAN G., FINLAYSON S. A. 2007. Heat stress induced
ethylene production in developing wheat grains, induced kernel abortion and increased maturation in susceptible cultivar. Planta Sci. 172: 113–1123.
HIRAGA S., SASAKI K., ITO H., OHASHI Y., MATSUI H. 2001. A large family of class III plant peroxidases. Plant Cell Physiol. 42: 462–468.
IEVINSH G., OZOLA D. 1998. Spatial distribution of ethylene production by individual needles along
a shoot of Pinus sylvestris L.: relationship with peroxidase activity. Ann. Bot. 82: 489–495.
JBIR N., CHAIBI W., AMMAR S., JEMMALI A., AYADI A. 2001. Root growth and lignification of two
wheat species differing in their sensitivity to NaCl, in response to salt stress. Life Sci. 324:
863–868.
66
Jagna Chmielowska, Joanna Deckert and José Díaz
JOUILI H., FERJANI E. E. 2003. Changes in antioxidative and lignifying enzyme activities in sunflower roots (Helianthus annus L.) stressed with copper excess. Compt. Rend. Biol. 326: 639–644.
KHAN W., PRITHIVIRAJ B., SMITH D. L. 2003. Chitosan and chitin oligomers increase phenylalanine
ammonia-lyase and tyrosine ammonia-lyase activities in soybean leaves. J. Plant Physiol. 160:
859–863.
KNOESTER M., LINTHORST H. J. M., BOL J. F., VAN LOON L. C. 1997. Modulation of stress-inducible ethylene biosynthesis by sense and antisense gene expression in tobacco. Plant Sci. 126:
163–173.
KUPPER H., KUPPER F., SPILLER M. 1996. Environmental relevance of heavy metal-substituted
chlorophylls using the example of water plants. J. Exp. Bot. 47: 259–266.
LAFUENTE M. T., ZACARIAS L., MARTINEZ-TELLEZ M. A., SANCHEZ-BALLESTA M. T., GRANELL A.
2003. Phenylalanine ammonia-lyase and ethylene in relation to chilling injury is affected by
fruit age in citrus. Postharv. Biol. Technol. 29: 308–317.
LI Y., KAJITA SH., KAWAI SH., KATAYAMA Y., MOROHOSHI N. 2003. Down-regulation of an anionic
peroxidase in transgenic aspen and its effect on lignin characteristics. J. Plant Res. 116:
175–182.
LICHTENTHALER H. K. 1998. The stress concept in plants: an introduction. In P. Csermely (ed.)
“Stress of life: From molecules to Man” Ann. New York Acad. Sci. 851: 187–198.
LIN CH.-CH., CHEN L.-M., LIU Z.-H. 2005. Rapid effect of copper on lignin biosynthesis in soybean roots. Plant Sci. 168: 855–861.
LIN Y.-C., KAO C.-H. 2005. Nickel toxicity of rice seedlings: cell wall peroxidase, lignin and NiSO4
– inhibited root growth. Crop. Environm. Bioinfo. 2: 131–136.
LLORENS N., AROLA L., BLADE C., MAS A. 2000. Effects of copper exposure upon nitrogen metabolism in tissue cultured Vitis vinifera. Plant Sci. 160: 159–163.
LOCKE J. M., BRYCE J. H., MORRIS P. C. 2000. Contrasting effects of ethylene perception and
biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.).
J. Exp. Bot. 51: 1843–1849.
MANDRE M. 2002. Relationships between lignin and nutrients in Picea abies L. under alkaline air
pollution. Water Air Soil Poll. 133: 361–377.
MAZHOUDI S., CHAOUI A., GHORBAL M. H., FERJAZNI E. 1997. Response of antioxidant enzymes
to excess copper in tomato (Lycopersicon esculentum, Mill.). Plant Sci. 127: 129–137.
NICOLÁS J. L., ECHEVERRIA M. A., SÁNCHEZ-BRAVO J. 2001. Influence of ethylene and Ag+ on
hypocotyl growth in etiolated lupine seedlings. Effects on cell growth and division. Plant
Growth Regul. 33: 95–105.
OUZOUNIDOU G., CIAMPROVA M., MOUSTAKAS M., KARATAGLIS S. 1995. Response of maiz (Zea
mays) plants to copper stress – growth, mineral content and ultrastructure of roots. Environm.
Exp. Bot. 32: 167–176.
PADUA M., AUBERT S., CASIMIRO A., BLIGNY R., MILLAR A. H., DAY D. A. 1999. Induction of
alternative oxidase by excess copper in sycamore cell suspensions. Plant Physiol. Biochem.
37: 131–137.
PASSARDI F., PENEL C., DUNAND C., 2004. Performing the paradoxical: how plant peroxidases
modify the cell wall. Review article. Trends Plant Sci. 9: 534–540.
PIERIK R., VERKERKE W., VOESENEK L. A. C. J., BLOM K. C. W. P. M., VISSER E. J. W. 1999.
Thick root syndrome in cucumber (Cucumis sativus L.): a description of phenomenon and an
investigation of role of ethylene. Ann. Bot. 84: 755–762.
PRZYMUSIÑSKI R., GWӏD E. A. 1999. Heavy metal-induced polypeptides in lupin roots are similar to pathogenesis-related proteins. J. Plant Physiol. 154: 703–708.
RAJALA A., PELTONEN-SAINIO P., ONNELA M., JAKCSON M. 2002. Effects of applying stem-shortening plant growth regulators to leaves on root elongation by seedlings of wheat, oat and barley:
mediation by ethylene. Plant Growth Regul. 38: 51–58.
RAEYMAEKERS T., POTTERS G., ASARD H., GUISEZ Y., HOREMANS N. 2003. Copper-mediated
oxidative burst in Nicotiana tabacum L. vc. Bright Yellow 2 cell suspension cultures. Protoplasma 221: 93–100.
ACTIVITY OF PEROXIDASES AND PHENYLALANINE AMMONIA-LYASE IN LUPINE AND SOYBEAN
67
ROS-BARCELÓ A., AZNAR-ASENSIO G. J. 2002. Basic peroxidases in cell walls of plants belonging
to the Asteraceae family. J. Plant Physiol. 159: 339–345.
RUCIÑSKA R., WAPLAK S., GWӏD E. A., 1999. Free radical formation and activity of antioxidant
enzymes in lupine roots exposed to lead. Plant Physiol. Biochem. 37: 187–194.
RUCIÑSKA R., SOBKOWIAK R., GWӏD E. A. 2004. Genotoxicity of lead in lupin root cells as
evaluated by the comet assay. Cell. Mol. Biol. Lett. 9: 519–528.
SALEH-LAKHA S., GRICHKOV. P., SISLER E. C., GLICK B. R. 2005. The Effect of the Ethylene
Action Inhibitor 1-Cyclopropenylmethyl Butyl Ether on Early Plant Growth. J. Plant Growth
Regul. 23: 307–312.
SARMA A. D., SHARMA R. 1999. Purification and characterization of UV-B induced phenylalanine
ammonia-lyase from rice seedlings. Phytochemistry 50: 165–173.
SGHERRI C., COSI E., NAVERI-IZZO F. 2003. Phenols and antioxidative status of Raphanus sativus
grown in copper excess. Physiol. Plant. 118: 21–28.
SOBKOWIAK R., DECKERT J. 2003. Cadmium-induced changes in growth and cell cycle gene expression
in suspension-culture cells of soybean. Plant Physiol. Biochem. 41: 767–772.
SOBKOWIAK R., RYMER K., RUCIÑSKA R., DECKERT J. 2004. Cadmium-induced changes in antioxidant in suspension culture of soybean cells. Acta Biochim. Pol. 51: 219–222.
SOBKOWIAK R., DECKERT J. 2004. The effect of cadmium on cell cycle control in suspension culture cells of soybean. Acta Physiol. Plant. 26: 335–344.
SOBKOWIAK R, DECKERT J. 2006. Proteins induced by cadmium in soybean cells. J. Plant Physiol.
163:1203–1206.
VISSER E. J. W., BÖGMANN G. M., BLOM C. W. P. M., VOESENEK L. A. C. J. 1996. Ethylene accumulation in waterlogged Rumex plants promotes formation of adventitious roots. J. Exp.
Bot. 47: 403–410.
WÓJCIK M., TUKIENDORF A. 2003. Response of wild type of Arabidopsis thaliana to copper stress.
Biol. Plant. 46: 79–84.
XIONG Z.-T., WANG H. 2005. Copper toxicity and bioaccumulation in Chinese cabbage (Brassica
pekinensis Rupr.). Environm. Toxicol. 20: 188–194.