Peroxidase activity and total phenol content in citrus cuttings treated with different copper sources

The proper citrus cutting choice is considered of basic importance for a healthy and productive citriculture. Substrates composed of organic materials are widely used for the production of citrus seedlings but this can lead to plant copper de ﬁ ciency, mainly caused by substrate high chelating properties. Copper is an essential heavy metal and is involved in many different plant physiological processes. This experiment was carried out in order to evaluate the behavior of ‘ Rangpur lime ’ lemon rootstock ( Citrus limonia Osbeck) grafted with cv. ‘ Valencia ’ ( Citrus sinensis L. Osbeck), when treated with different copper formulations, as cupric oxychloride (50% Cu — Novartis Biociências ™ ), cuprous oxide (32.8% Cu — Yara Vita ™ ), chelated copper (5% Cu — Stoller ™ ) and copper sulphate (25% Cu — Microsal ™ ) analyzing the activity of peroxidase (POD) and the total content of phenols. The different copper formulations did not show signi ﬁ cant differences about plant height, diameter, leaf and root dry weight. The comparison among copper treatments shows that cuprous oxide promoted the antioxidant system (POD activity and phenolic content) while chelated copper was not effective.


Introduction
Brazil is considered the major producer of orange in the world. According to Fundecitrus, in 2009 in Sao Paulo State 517 citrus nurseries were screened with a production of about 8.6 million rootstocks and 17.6 million of citrus seedlings (Amaro and Baptistalla, 2010).
The citrus seedlings quality is one of the production factor influencing the success of commercial crops, since plants only reveal their maximum productivity potential six or eight years after planting (Zanetti et al., 2004).
Copper deficiency is common in plants that grow in rich organic matter soils, where this element form insoluble complexes, unavailable to the plants (Turvey, 1984). In citrus, the deficiency symptoms show dark-green leaves in vigorous branches, exaggerated size, exanthema, and the veins may protrude. It can cause dieback (Malavolta, 2006;Rodriguez and Gallo, 1960).
At supra-optimal concentrations, copper disturbs photosynthetic and mitochondrial electron transport, nitrogen assimilation, cell wall metabolism and many other plant functions (Maksymiec, 1997). Moreover, it affects different parameters of plant metabolism as dry mass accumulation (Ali et al., 2002;Zheng et al., 2004), chlorophyll (Liu et al., 2004;Lou et al., 2004), water content (Burzynski and Klobus, 2004) and the balance in macro and micronutrient levels (Ali et al., 2002;Bernal et al., 2007). Its toxicity depends not only on the metal concentration and exposure duration, but on the developmental stage and physiological state of plants, as well (Ahsan et al., 2007).
Several substances act as protection system in plants against these reactive oxygen species, including peroxidases, catalase, superoxide dismutase and phenolic compounds. The antioxidant activity of phenolic compounds is due to their ability to scavenge free radicals, donating hydrogen atoms or electrons, or chelating metal cations (Amarowicz et al., 2004). On the other hand, peroxidases are considered to protect cells from heavy metal stress (Karataglis et al., 1991) and are used as stress markers in metal poisoning situations (Chaoui et al., 2004).
The aim of the present study was the assessment of the effect of different copper source additions on the growth of citrus seedlings. Further aim of the present study was to investigate the induction of POD activity and phenolic compounds in citrus seedlings when treated with different copper sources.

Plant material
The experiment was carried out in a commercial citrus nursery located in Botucatu, Sao Paulo State, Brazil, 22°54′13.8″ S and 48°27′32.8″ W, 786 m altitude. The average annual temperature was 21°C, and accordingly to Köppen the region climate is humid subtropical (cwa), temperate/mesothermal. The experiment started in July 2006, with the sowing of 'Rangpur lime' seeds (Citrus limonia Osbeck) in coconut fiber substrate, donated by the Citriculture Center "Sylvio Moreira", Cordeiropolis -SP. This variety has been chosen because it represents the major choice of Brazilian citrus growers (65%) (Amaro and Baptistalla, 2010), due to the superior horticultural characteristics such as earliness to bearing and high fruit yield (Pompeu Junior, 1991). In March, plants were grafted with cv. 'Valencia' (Citrus sinensis L. Osbeck).

Copper treatments
Copper treatments were applied in January, February, March and April, 2007. Control plants did not receive any kind of treatment. The second treatment was treated with cupric oxychloride (50% Cu -Novartis Biociencias™) provided two times per week by foliar spray. The third treatment was treated with cuprous oxide (32.8% Cu -Yara Vita™), two times per week by foliar spray, as well. The fourth treatment with chelated copper (5% Cu -Stoller™) was used, applied two times per week, via fertirrigation, the fifth treatment was carried out with copper sulphate (25% Cu -Microsal™), once a week, with irrigation water, to avoid phytotoxicity, since according to previous experiments, the damage occurred when copper sulphate was applied two times per week. Every week all the seedlings received insect control pulverization, as recommended for citrus.

Copper determination
Plant material (leaves) were thoroughly washed with distilled water and desiccated, in a paper bag, at 60-65°C for 48 h. Samples were then finely ground for chemical analyses. Copper content was determined by atomic absorption according to Malavolta et al. (1997).

Plant growth measurement
Growth parameters were evaluated as plant height (cm), rootstock diameter (mm) and dry weight of leaves and roots (g). Plants were measured monthly, starting in February (1st), and ending in May (4th), before treatment application. Plants were randomly collected and were taken out of the plot, measured, frozen in liquid nitrogen and kept in deep freezer until biochemical analysis.

POD activity determination
For POD assay, leaves were ground in liquid nitrogen and the finely ground material (0.3 g) was dispersed in cold 0.2 M potassium phosphate buffer, pH 6.7, and centrifuged at 12,000 ×g for 10 min at 4°C. Peroxidase activity was determined according to by Lima et al. (1999) on 250 μL of the supernatant, by addition of 10 μmol of hydrogen peroxide, 35 μL of phenol and 2 μmol of aminoantipyrine. The reaction was carried out during 5 min in water bath at 27°C and the reaction was stopped with 2 mL of ethanol. The reaction occurrence was measured at 505 nm and the results are expressed in μmol of H 2 O 2 reduced minute −1 g −1 fresh material.

Total phenol content
The amount of total phenolics in leaf extracts was determined according to the Folin-Ciocalteu method (Singleton and Rossi, 1965). Dry material (50 g) was introduced into test tubes and 2.5 mL of Folin-Ciocalteu's reagent and 2.0 mL of sodium carbonate (20% w/w) were added. The tubes were mixed and allowed to stand for 45 min. The total phenolics content were measured at 765 nm and were expressed as mg gallic acid equivalent g −1 dry material (GAE).

Statistical analysis
The experiment was conducted with five treatments and three replicates. The analysis of variance (ANOVA) was carried out on experimental data and in the case of significance; means were compared by t test (LSD) at 5% probability by SISVAR Software.

Copper levels
Copper content in sample leaves was determined as described in methods and reported in Fig. 1. From the data reported in Fig. 1, it can be noted that the treatment with cuprous oxide lead to an excessive copper accumulation, accordingly with Teofilo Sobrinho et al. (1994), in all the determinations, while the treatment with cupric oxychloride produced high copper levels in 1st, 3rd and 4th evaluations.

Plant growth measurement
Average plant height (cm) ( Table 1), showed significant differences among treatments only in the 2nd evaluation, where the cupric oxychloride lead to the highest height, 64.08 cm, while, the control had 58.50 cm. In the 3rd evaluation, plant height resulted lower than the previous, because samples came from the measurements of the aerial part of the grafted variety.
The average rootstock diameter (mm) ( Table 1) was generally unaffected by treatments and increased during plant development, as expected, until the 2nd evaluation. In the 3rd evaluation differences in treatments were observed, treatment with copper sulphate, cupric oxychloride and copper sulphate induced increase in the rootstock diameter. Normally, the ideal rootstock diameter for grafting is around 0.50-0.60 mm, and taking into account samples in the grafting season, all treatments, included the control reached the ideal rootstock diameter for grafting.
The leaves were from Rangpur lime in the 1st and 2nd evaluations and in the 3rd and 4th evaluations were from cv 'Valencia', after grafting (Table 2). It can be observed that in the fourth evaluation a significant difference among treatments on dry leaf weight, meanwhile significant differences on dry root weight among treatments were shown in the third evaluation.
The dry leaf weight showed, when compared with controls (4th evaluation), a decrease of 6.68% in cupric oxychloride treatment, 5.07% in cuprous oxide treatment, 7.82% in chelated copper treatment and 12.90% in copper sulphate treatment. As regards the dry roots weight, the chelated copper treatment increased by 6.17%, but the other treatments decreased by 2.28% in cupric oxychloride treatment, 4.89% in cuprous oxide treatment and 16.07% in copper sulphate treatment.

Total phenol content
The total phenol content is presented in Table 4 for the different copper treatments in citrus seedlings leaves. During the evaluations period (February-May/2007) significant differences were observed among treatments in the 1st, 2nd and 3rd evaluation. As observed for POD activity, the total phenol content presented the highest values in the treatment with cuprous oxide (34.84, 36.58 and 37.45 mg GAE g DM −1 ). Cupric oxychloride treatment showed 34.79 mg GAE g DM −1 as phenol content, higher than other treatments in the 4th evaluation. Meanwhile, control showed the lowest results, when compared with other treatments in the 1st, 2nd and 3rd (22.89, 26.95 and 27.74 mg GAE g DM −1 ).

Discussion
A common problem in Brazilian plant nurseries is represented by copper deficiency in citrus seedlings. However, foliar treatments lead to progressive copper accumulation in the leaf cuticle and was considered excessive according to Teofilo Sobrinho et al. (1994). In this experiment, the average Cu content found in the leaves were about 6 times higher using cupric oxychloride, 22 times higher for cupric oxide, 4 times higher for chelated copper (Fig. 1), with respect to controls. High Cu levels were also observed by Ferreira (2007) in leaves of olives trees treated with copper oxychloride, copper hydroxide and Bordeaux mixture. Therefore, the secondary aim of the experiment was to observe the effect of excessive levels of Cu in citrus seedlings and how the plants manage to control the stress.
Copper is an essential micronutrient for plant growth, metabolism and enzyme activity; but if in excess, it is also a proven inhibitor of various physiological functions. Cu excess can interfere with enzyme function and can decrease protein synthesis and carbohydrate, nucleic acid and lipid metabolism (Marschner, 1986).
Among the treatments used in the present work, significant differences were observed only in the 3rd evaluation for dry root weight, and in the 4th evaluation for dry leaf weight (Table 2). It appears that treated plants presented a lower average values when compared to controls, indicating a possible toxic effect of the copper sources.
As Cu can affect membrane integrity, this could lead to a reduction of root water content, affecting pressure potential and   growth (Jouili and Ferjani, 2003). The same copper effect was observed in Matricaria chamomilla, which showed a significant and visible growth depression in leaf rosettes and roots (Kovácik et al., 2008). Mourato et al. (2009) noticed a root growth reduction at the maximum copper doses applied in Lupinus luteus and Chaoui et al. (2004) in Pisum sativum L. According to Jiang et al. (2001) and Wojcik and Tukiendorf (2003) this is a commonly described effect of toxic concentrations of copper. Meanwhile, despite the toxic copper concentrations found in citrus leaves, there was no evidence of toxicity; this fact can be attributed to the protection offered by the plant antioxidant system. Copper, at high concentrations, causes oxidative stress due to overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can be cytotoxic and can damage important cell compounds (Kovácik and Backor, 2007;Maksymiec and Krupa, 2006;Posmyk et al., 2009). The generation of ROS is considered to be the primary event under a variety of stress conditions. Consequences of ROS formation depend on the intensity of the stress and on the physicochemical conditions in the cell (i.e. antioxidant status, redox state and pH) (Posmyk et al., 2009).
This can be seen in plants treated with cuprous oxide, since they had the highest copper content (Fig. 1). The high level of copper caused an oxidative stress and resulted in a fast and high production of antioxidant components, such as POD and phenols. The increase of POD activity can also be observed with copper sulphate treatment. The same effect was observed on L. luteus by Mourato et al. (2009) and on Len culinaris Medic. by Janas et al. (2010) where a high Cu amount was accumulated in the roots, inducing oxidative stress and excessive ROS production, resulting in the rapid deterioration of membrane lipids. Reports on copper sulphate effects in increasing POD activity were described by Fang and Kao (2000) in rice, and this effect was attributed to the generation of lipid hydroperoxides, that are toxic for cells and should be scavenged by POD as soon as possible. Thus, free radical generation is likely to be involved in induction of POD activity by Cu 2 + . It can be also noted that the increase in POD activity depended on the different copper treatment (Table 3).
POD activity showed a peak in the 2nd evaluation (except in the control) probably due to the initiation of plant lignification. Baccouch et al. (1998), Weckx and Clijsters (1996) and Chen et al. (2002) observed an increase in POD isoenzymes is in part responsible for lignin synthesis in radish (Raphanussativus) roots and may remove the excess of hydrogen peroxide caused by Cu, thus serving as a detoxifying system during Cu treatment. According to Lin et al. (2005), Cu strongly enhanced POD activity in soybean roots, which was correlated with root growth inhibition and increased lignin content.
Lignin is a highly branched polymer of phenylpropanoid compounds, generally formed from three distinct phenylpropanoid alcohols, including coniferyl, coumaryl, and sinapyl alcohols. The polymerization of these lignin precursors is catalyzed by POD in the presence of H 2 O 2 (Mäder and Füssl, 1982) and by laccases in the presence of O 2 (Sterjiades et al., 1993). Or it can be due to the increase in POD content, which also oxidizes IAA in response to the presence of copper ions. This could decrease the endogenous presence of auxins in roots of metal-treated plants and thereby reduce their growth (Chaoui et al., 2004). This can be observed as the reduction of dry mass of shoots and roots in the last evaluation for all treatments, when compared to control (Table 2).
Thus, high copper concentrations promoted an elevated ROS production, initializing the antioxidants systems to fight against these reactive species and to balance the redox plant status. We noted that copper, in the sulphate or oxide formulation induces oxidative damage in plants, promoting an increase in peroxidase content. In this case, this enzyme could be an indicator of the copper use in citrus seedlings production.
It had been reported that the antioxidant activity of plant materials is well correlated with the content of phenolic compounds (Skerget et al., 2005). The biological and antioxidant activities of phenolic compounds (PhC) can be, at least in part, attributed to their high tendency to chelate heavy metals, in particular, the hydroxyl and carbonyl groups of PhC can strongly bind Cu and Fe. This chelating behavior makes PhC strong candidates in preventing metal-catalyzed free radical formation (Lopes et al., 1999).
In the second evaluation, for all copper treatments, total phenol content increased with respect to controls, being increased also in the third evaluation with cuprous oxide and chelated copper treated plants (Table 4). However, in the 4th evaluation, with the exception of control plants, cited treatments did not promote the same behavior. An explanation of the increase of peroxidase activity in the 2nd evaluation can be due to the concomitant increase of phenol content, as enzyme substrate, indicating that plants are subjected to a lignification process. This is in accord with stem diameter data.
These observations agree with reports of Rice-Evans et al. (1996), proposing that phenolic antioxidants appear more powerful than peroxidase, and even suggested that peroxidases can use them as substrates, along with reduced ascorbate, as an efficient H 2 O 2 scavenging system in plant vacuoles (Zancani and Nagy, 2000). In this case, phenols acted as reducing agents, scavenging ROS and chelating copper, thus reducing the metal toxicity in cells (Gordon and Roedig-Penmam, 1998), even in the presence of low copper doses, where plants had no toxicity symptoms and adequate growth.
The response of phenolic compounds to Cu 2+ can vary among plant species and in different tissues, as well as with metal concentration (Ali et al., 2006;Caldwell, 2002;Gordon and Roedig-Penmam, 1998). Various plants species react differently to excess Cu 2+ , but differences in plant responses seem to depend not only on copper concentration but also on the capability of plants to increase their antioxidant protection system against heavy metal stress.
The experiment carried out by testing different sources of copper is in accordance with reported literature about the increase of peroxidase activity and phenol content relate to growth and lignification process as well as plant performance against metal induced free radical damage. The resulting plant behavior leads to an asymptomatic high copper content and normal growth.
It can be concluded that the different copper treatments do not interfere significantly with the growth and development of Rangpur 'lime' seedlings, despite being at toxic levels. The comparison among copper treatments shows that cuprous oxide promoted the antioxidant system (POD activity and phenolic content) while chelated copper was not effective.