Alterations in Lignin Peroxidase and Ascorbate Peroxidase Activities in Crocus sativus L. Corms Exposed to Copper
J. Keyhani, E. Keyhani / L. ArziLaboratory for Life Sciences
19979 Tehran
Iran / Institute of Biochemistry and Biophysics
University of Tehran
13145 Tehran
Iran
Keywords: ABTS, ferulic acid, metal toxicity, oxidative stress, rooting
Abstract
Excessive copper uptake induces toxicity that has been linked to the production of reactive oxygen species (ROS), thereby inducing DNA mutations, lipid peroxidation, damage to proteins structure and function. Peroxidases scavenge ROS while also performing other functions, such as lignification in plants. In this research, the activity of ascorbate peroxidase, as well as that of lignin peroxidase, was investigated in extracts prepared from Crocus sativus L. corms cultivated for 6 days in distilled water and in water supplemented with copper sulfate concentrations ranging from 0.0006 mM to 12 mM. Ascorbate peroxidase activity was assayed by monitoring the H2O2-mediated oxidation of ascorbate at 290 nm, while lignin peroxidase activity was assayed by monitoring the H2O2-mediated oxidation of ferulic acid at 310 nm as well as the H2O2-mediated oxidation of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 414 nm. Results showed that ascorbate peroxidase activity decreased as the copper ion concentration increased in the cultivation medium: from a 10 % drop in 0.0006 mM Cu2+ to a 50 % drop in 12 mM Cu2+. Lignin peroxidase activity measured with ferulic acid as the reducing substrate increased in extracts from corms cultivated in the presence of low copper ion concentrations, reaching three times the control value in 0.0012 mM Cu2+ and being still twice the control value in 0.006 mM Cu2+. The activity progressively dropped to one third of the control value in 0.3 mM Cu2+ and then increased again to one and a half times the control value in 12 mM Cu2+. When ABTS was used as the reducing substrate, more moderate increases and more progressive decreases in enzymatic activity were observed with no new increase at higher Cu2+ concentrations. Results revealed a fine tuning in the relative activities of various peroxidases in response to metal stress and in relation with rooting.
INTRODUCTION
It has become common knowledge that, for most living systems, a number of transition metals, referred to as “trace elements”, are essential for good health in minute amounts but become toxic upon excessive uptake. The transition metals beneficial effects often stem from the key role they play in the active site of a number of enzymes (Linder, 1991), while their toxicity most often stems from their ability to promote or exacerbate oxidative cell damage (Baccouch et al., 1998; Hegedüs et al., 2001).Copper is a typical example. It forms the essential redox-active center in a variety of metalloproteins (Linder, 1991), and thereby it is an indispensable part in a wealth of biochemical reactions. On the other hand, excess copper has been identified as a cause of a number of ailments, from cancer to hepatic and neurologic disorders, and even Alzheimer’s and prion diseases (Hartwig, 1995; Lee et al., 2006; Barnham et al., 2006).
In plants, excess copper has been shown to cause alterations in lipid composition and protein content, depression in biomass production, inhibition of root elongation and plant growth, and blocking of the photosynthetic electron transport (Ouariti et al., 1997; Garcia et al., 1999; Cuypers et al., 2005; Fernandez and Henriques, 1991). The most important mechanism of copper toxicity is the production of reactive oxygen species (ROS), causing injuries by oxidative stress. The major ROS-scavenging mechanisms in plants rely on a series of enzymes, including peroxidases. Plants have a large number of peroxidase isoenzymes performing a variety of biological functions while being scavengers of H2O2. Lignin peroxidases for example play an important role in lignin degradation as well as in the strengthening of cell wall (Ward et al., 2001), while ascorbate peroxidases have a high specificity for ascorbate as reducing agent and are mostly involved in the defense against oxidative stress (Shigeoka et al., 2002).
Copper occurs naturally in rock, soil, water, sediment and air and it is thus an ubiquitous potential contamination threat. Our interest has been focused on Crocus sativus L. physiology and, especially, on enzymatic activities detectable in the corm at various stages of the plant development under standard and stressful conditions (Keyhani et al., 2000; Keyhani and Keyhani, 2004; Keyhani et al., 2004). In this research, the activities of specific peroxidases such as ascorbate peroxidase and lignin peroxidase, were investigated in extracts prepared from Crocus sativus L. corms cultivated for 6 days in distilled water and in water supplemented with copper sulfate concentrations ranging from 0.0006 mM to 12 mM. The effect of Cu2+ on rooting was also examined. Results showed that each enzyme reacted in its own specific way and that root elongation was stimulated at low metal concentration, but was aborted when a critical metal concentration was reached.
MATERIALS AND METHODS
Corms and Cultivation Conditions
Crocus sativus corms obtained from the University of Tehran farm located in Karaj, near Tehran, were cultivated as previously described (Keyhani et al, 2004). Briefly, unearthed corms were depleted from their sheathing leaves, cleaned from any dirt particle and cultivated in glass jars 10 cm-deep and 6.5 cm in diameter, containing 50 ml liquid medium. Each corm was placed in an individual jar and cultivated for 6 days. Care was taken to maintain the liquid level constant throughout cultivation. Liquid media consisted of double distilled water (control) and double distilled water supplemented with copper sulfate concentrations ranging from 0.00001 % to 0.2 % (corresponding to 0.0006 mM to 12 mM).
Extract Preparation
Extracts were prepared according to the method described in Attar et al. (2006). Briefly, corms were collected after 6 days cultivation, depleted of any roots or shoots, and homogenized in phosphate buffer 0.01 M, pH 7, containing 0.02 % phenylmethanesulfonyl fluoride as protease inhibitor. After centrifugation at 10,000 g for 10 minutes, then at 35,000 g for 30 minutes, a clear, transparent supernatant termed “crude extract” was obtained and used for our studies. Protein concentrations were determined by the Lowry method.
Enzyme Activity Assays
All assays were carried out at room temperature (~ 22-25 °C). The specific assay procedure for each enzyme is described below. Results are averages of at least three assays.
1. Ascorbate Peroxidase. Ascorbate peroxidase activity was measured by following the H2O2-mediated oxidation of ascorbate at 290 nm (extinction coefficient: 2.8 mM-1cm-1) as described in Keyhani et al. (2000). One unit was defined as the amount of enzyme needed for the oxidation of 1 μmol of substrate per minute.
2. Lignin Peroxidase. Lignin peroxidase activity was measured by following the H2O2-mediated oxidation of ferulic acid at 310 nm (extinction coefficient: 8.68 mM-1cm-1), and also by following the H2O2-mediated oxidation of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) at 414 nm (extinction coefficient: 31.1 mM-1cm-1). One unit was defined as the amount of enzyme needed for the oxidation of 1 μmol of substrate per minute.
RESULTS AND DISCUSSION
Effect of Cu2+ on Rooting
Figure 1 shows Crocus sativus L. corms cultivated for 6 days in distilled water and in selected Cu2+ concentrations. Root elongation was severely hampered in 0.06 mM Cu2+, and further inhibited in higher concentrations. Corms rooted in 0.06 and 0.3 mM Cu2+ exhibited a brownish tissue discoloration in the rooting zone, indicative of stress; corms rooted in 12 mM Cu2+ exhibited a more extensive brownish discoloration and corms rooted in 120 mM Cu2+ exhibited a blue color in addition. The remainder of these studies was conducted for Cu2+ concentrations ranging from as low as 0.0006 mM until no more than 12 mM.
The average root length and the average root number per corm were recorded daily for the 6 days of cultivation. At low copper concentrations, root elongation was moderately stimulated so that, after 6 days cultivation, the average root length was 106 % of the control in 0.0006 mM Cu2+ and 118 % of the control in 0.0012 mM Cu2+. In 0.006 mM Cu2+, root elongation was slowed and the average length at day 6 was 70 % of the control. In 0.06 mM and higher Cu2+ concentrations, root elongation was aborted after the first day of cultivation; the root length obtained at the first day decreased with increasing Cu2+ concentrations, going from 68 % of the control in 0.06 mM Cu2+ to 28 % in 12 mM Cu2+ (Fig. 2).
The average root number per corm remained the same in up to 0.006 mM Cu2+. It decreased to approximately 80 % of the control in 0.06 to 12 mM Cu2+ (Fig. 3).
Thus root elongation took place and was even stimulated in selected Cu2+ concentrations, but once a critical Cu2+ concentration was reached, root elongation was aborted after one day. Similarly, study of the interaction between Ni2+ and horseradish peroxidase led to the conclusion that there was a critical metal concentration causing immediate inhibition of enzymatic activity (Keyhani et al., 2005). Interestingly, the number of roots per corm dropped to 80 % of the control value for the same Cu2+ concentrations at which root elongation was aborted.
Effect of Cu2+ on Lignin and Ascorbate Peroxidases Activities
Lignin peroxidase activity determined by following the H2O2-mediated oxidation of ferulic acid was found to be stimulated in corms cultivated for 6 days in up to 0.006 mM Cu2+, reaching 180 % of the control in 0.0006 mM Cu2+, 250 % in 0.0012 mM Cu2+ and 184 % in 0.006 mM Cu2+. It decreased to 75 % of the control in 0.06 mM Cu2+ and further decreased to 57 % in 0.12 mM Cu2+ and 37 % in 0.3 mM Cu2+. Thereafter, an increase in the enzymatic activity was observed and it reached 162% of the control in corms cultivated in 12 mM Cu2+ (Fig. 4A). Ferulic acid is a widespread cinnamic acid derivative that has been chosen as a model substrate for studying lignin peroxidase-catalyzed polymerization of phenolic compounds in vitro (Ward et al., 2001). The first burst in lignin peroxidase activity was observed at those Cu2+ concentrations where root elongation was either stimulated or maintained, albeit at a reduced pace; it was followed by a drastic drop in enzymatic activity at those Cu2+ concentrations where root elongation was severely inhibited. This would be in accordance with findings by others of a relationship between peroxidase activity, lignin formation and rooting (Syros et al., 2004). The second surge in lignin peroxidase activity occurred at higher Cu2+ concentrations where root elongation was most severely hampered; it was likely related to the anti-oxidative stress role of lignin peroxidase (Belinky et al., 2003).
The peroxidase activity detectable using ABTS as the reducing substrate was stimulated in corms cultivated for 6 days in up to 0.06 mM Cu2+, reaching 138 %, 135 %, 141 % and 115 % of the control in, respectively, 0.0006, 0.0012, 0.006 and 0.06 mM Cu2+. It was at the control level in 0.12 and 0.3 mM Cu2+. It decreased to 78 % of the control in 0.6 and 1.2 mM Cu2+ and to 42 % of the control in 12 mM Cu2+ (Fig. 4B). Thus the activity detectable with ABTS was stimulated mainly when root elongation proceeded for 6 days.
The ascorbate peroxidase activity detectable in corms cultivated for 6 days in increasing Cu2+ concentrations was found to decrease in a stepwise manner. It was 84 %, 84 % and 96 % of the control value in, respectively, 0.0006, 0.0012 and 0.006 mM Cu2+, when root elongation was stimulated or maintained; it was 56 %, 64 % and 74 % of the control value in, respectively, 0.06, 0.12 and 0.3 mM Cu2+, when lignin peroxidase activity was mostly inhibited; it was 44 %, 57 % and 47 % of the control value in, respectively, 0.6, 1.2 and 12 mM Cu2+, when lignin peroxidase activity increased again (Fig. 4C). Results revealed a fine tuning in the relative activities of various peroxidases in response to metal stress.
ACKNOWLEDGEMENTS
This work was supported in part by the University of Tehran (Interuniversities Grant # 31303371), Tehran, Iran, and in part by the J. and E. Research Foundation, Tehran, Iran.
Literature Cited
Attar, F., Keyhani, E. and Keyhani, J. 2006. A comparative study of superoxide dismutase activity assays in Crocus sativus L. corms. Appl. Biochem. Microbiol. 42:101-106.
Baccouch, S., Chaoni, A. and El Ferjani, E. 1998. Nickel-induced oxidative damage and antioxidant responses in Zea mays shoots. Plant Physiol. Biochem. 36:689-694.
Barnham, K.J., Cappai, R., Beyreuther, K., Masters, C.L. and Hill, A.F. 2006. Delineating common molecular mechanisms in Alzheimer’s and prion diseases. Trends Biochem. Sci. 31:465-472.
Belinky, P.A., Flikshtein, N., Lechenko, S., Gepstein, S. and Dosoretz, C.G. 2003. Reactive oxygen species and induction of lignin peroxidase in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 69:6500-6506.
Cuypers, A., Koistinen, K.M., Kokko, H., Karenlampi, S., Auriola, S. and Vangronsveld, J. 2005. Analysis of bean (Phaseolus vulgaris L.) proteins affected by copper stress. J. Plant Physiol. 162:383-392.
Fernandez, J.C. and Henriques, F.S. 1991. Biochemical and structural effect of excess copper in plants. Bot. Rev. 57:246-273.
Garcia, A., Baquedano, F.J., Navarro, P. and Castillo, F.J. 1999. Oxidative stress induced by copper in sunflower plants. Free Radic Res. 31:S45-50.
Hartwig, A. 1995. Currents aspects in metal genotoxicity. Biometals 8:3-11.
Hegedüs, A., Erdeis, S. and Horváth, G. 2001. Comparative studies of H2O2 detoxifying enzymes in green and greening barley seedlings under cadmium stress. Plant Sci. 160:1085-1093.
Keyhani, E., Veissizadeh, M. and Keyhani, J. 2000. Kinetics of isoperoxidases and their differential sensitivity to inhibitors in saffron (Crocus sativus L.) bulb. p. 37-42. In: J.H.S. Hofmeyr, J.M. Rohwer and J.L. Snoep (eds.), BioThermoKinetics 2000: Animating the Cellular Map. StellenboschUniversity Press, Stellenbosch.
Keyhani, E and Keyhani, J. 2004. Hypoxia/anoxia as signaling for increased alcohol dehydrogenase activity in saffron (Crocus sativus L.) corm. Ann.N.Y. Acad. Sci. 1030:449-457.
Keyhani, E., Keyhani, J, Hadizadeh, M., Ghamsari, L. and Attar, F. 2004. Cultivation techniques, morphology and enzymatic properties of Crocus sativus L. Acta Hort. 650:227-246.
Keyhani, J., Keyhani, E., Zarchipour, S., Tayefi-Nasrabadi, H. and Einollahi, N. 2005. Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity. Biochim. Biophys. Acta 1722:312-323.
Lee, V.D., Northup, P.G. and Berg, C.L. 2006. Resolution of decompensated cirrhosis from Wilson’s disease with zinc monotherapy: a potential therapeutic option? Clin. Gastroenterol. Hepatol. 4:1069-1071.
Linder, M.C. 1991. Nutritional biochemistry and metabolism. Elsevier, New York.
Ouariti, O., Boussama, N., Zarrouk, M., Cherif, A. and Ghorbal, M.H. 1997. Cadmium- and copper-induced changes in tomato membrane lipids. Phytochemistry 45:1343-1350.
Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y. and Yoshimura, K. 2002. Regulation and function of ascorbate peroxidase isoenzymes. J. Exptl. Bot. 53:1305-1319.
Syros, T., Yupsanis, T., Zafiriadis, H. and Economou, A. 2004. Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L. J. Plant Physiol. 161:69-77.
Ward, G., Hadar, Y., Bilkis, I., Konstantinovsky, L. and Dosoretz, C. 2001. Initial steps of ferulic acid polymerization by lignin peroxidase. J. Biol. Chem. 276:18734-18741.