Exposure of Azolla plants to UV-B radiation for 6 h resulted in a decrease in biomass and relative growth rate (RGR), which coincided with an increase in doubling time (DT) as compared with the control. Also, the protein content decreased. On the other hand, hydrogen peroxyde (H2O2) and malondialdehyde (MDA) accumulated significantly in UV-treated Azolla plants. Conversely, the addition of selenium (Se) at 1 ppm resulted in a significant increase in biomass and protein content of untreated and UV-treated Azolla plants, and a significant reduction in both H2O2 and MDA. Moreover, the addition of Se to UV-treated and untreated Azolla plants resulted in a significant increase in total ascorbate and total glutathione (GSH) contents compared with the control and UV-stressed Azolla plants. Also, glutathione redox potential (GSH/TG) increased significantly in UV-treated Azolla plants in the presence of Se. There also was a significant increase (38%) in ascorbate peroxidase (APX) activity in UV-treated plants compared with the control. APX activity in the presence of Se did not change significantly compared with the control. Glutathione reductase (GR) activity increased significantly in UV-treated Azolla, while glutathione peroxidase (GSH-PX) activity did not. On the other hand, both GSH-PX and GR activity in untreated and UV-treated Azolla plants were significantly enhanced by the application of Se to the nutrient media at a concentration of 1 ppm. Therefore, we can conclude that Se protects Azolla plants from UV-B stress.
- Azolla caroliniana,
- glutathione peroxidase,
- UV-B radiation
L’exposition de plants d’Azolla au rayonnement UV-B pendant 6 h a provoqué une diminution de la biomasse et du taux de croissance relative (TCR), coïncidant avec une augmentation du temps de doublement (TD) comparativement au témoin. Aussi, le contenu en protéines a diminué. Par contre, le peroxyde d’oxygène (H2O2) et le malondialdéhyde (MDA) se sont accumulés de façon significative dans les plants d’Azolla traités par rayonnement UV. À l’inverse, l’ajout de 1 ppm de sélénium (Se) a provoqué une augmentation importante de la biomasse et du contenu en protéines des plants d’Azolla exposés ou non au rayonnement UV, ainsi qu’une réduction importante du H2O2 et du MDA. De plus, l’ajout de Se a provoqué une augmentation importante du contenu total d’ascorbate et de glutathion chez les plants d’Azolla exposés ou non au rayonnement UV comparativement au témoin et aux plants d’Azolla exposés au rayonnement UV en présence de Se. Aussi, le potentiel d’oxydoréduction de glutathion (NrH) a augmenté significativement chez les plants d’Azolla exposés au rayonnement UV en présence de Se. Il y a également eu une augmentation importante (38 %) de l’activité d’ascorbate peroxydase (APX) chez les plants exposés au rayonnement UV comparativement au témoin. L’activité APX en présence de Se n’a pas changé de façon significative comparativement au témoin. L’activité de glutathion réductase (GR) a augmenté significativement chez l’Azolla exposé au rayonnement UV, mais ce n’était pas le cas pour l’activité de glutathion peroxydase (GSH-PX). Toutefois, l’activité de GSH-PX et l’activité de GR dans les plants d’Azolla traités ou non par rayonnement UV ont été rehaussées de façon significative par l’application de Se aux milieux nutritifs à une concentration de 1 ppm. Ainsi, nous pouvons conclure que le Se protège les plants d’Azolla du stress relié au rayonnement UV-B.
- Azolla caroliniana,
- glutathion peroxydase,
- rayonnement UV-B,
Selenium (Se) is an important element for human and animal nutrition, due to its role in a series of biochemical reactions enhancing antioxidant activity (Rayman 2002). In contrast, Se is not considered essential for plants (Terry et al. 2000; Kápolna et al. 2009). Nevertheless, several studies have shown that at low concentrations, Se exerts a beneficial effect on the growth and stress tolerance of plants by enhancing their antioxidative capacity (Xue and Hartikainen 2000; Xue et al. 2001; Djanaguiraman et al. 2005; Kong et al. 2005; Lyons et al. 2009).
Many studies have shown deleterious UV effects, such as reduced photosynthesis and protein contents, impaired chloroplast function, and DNA damage (Agrawal 1992; Lesser et al. 1994). Ultraviolet radiation also produces oxidative stress that arises from the deleterious effects of active oxygen species (Costa et al. 2002; Ibrahim and Mostafa 2007) that react with lipids, pigments, proteins and nucleic acid (Dai et al. 1997; Nasibi and M-Kalantari 2005).
The addition of Se at low concentrations alleviated the oxidative stress caused by UV irradiation in lettuce (Lactuca sativa L.) and ryegrass (Lolium perenne L.) (Hartikainen and Xue 1999; Hartikainen et al. 2000), and in strawberry (Fragaria x ananassa Duch.) (Valkama et al. 2003). Pennanen et al. (2002) reported that Se, in addition to increasing the growth of plants, delayed the death of plants subjected to severe UV stress. Furthermore, at an optimal level, Se was able to increase the antioxidative capacity of senescing plants and delay senescence in lettuce, ryegrass and soybean (Glycine max L.) (Xue et al. 2001; Djanaguiraman et al. 2005). Selenium also improved the recovery of potato plants from light and chilling stress (Seppänen et al. 2003), and enhanced salt resistance in sorrel (Rumex patientia L.) seedlings (Kong et al. 2005).
Several studies have shown that the protective role of Se against oxidative stress in higher plants coincided with enhanced glutathione peroxidase (GSH-PX) activity and decreased lipid peroxidation (Hartikainen et al. 2000; Xue et al. 2001; Djanaguiraman et al. 2005). Cartes et al. (2005) demonstrated that selenite was more efficient than selenate at promoting GSH-PX activity in ryegrass plants. In addition, Se affected the activity of catalase, superoxide dismutase, and glutathione S-transferase (Xue and Hartikainen 2000).
The addition of Se can influence activity and antioxidant levels in stressed plants and thus regulate reactive oxygen species (ROS) levels. In plants, GSH-PX is a powerful scavenger of H2O2 and lipid peroxide with the help of glutathione. GSH-PX is believed to be a key enzyme that can be widely and robustly activated by Se in various plants exposed to diverse environmental stresses (Feng et al. 2013).
GSH-PX and glutathione reductase (GR) appear to be a complementary pair of enzymes. While GSH-PX reduces toxic hydroperoxides that are formed as a result of oxidative stress, GR converts oxidized glutathione to its reduced form.
The role of Se in mitigating environmental stresses has been extensively investigated in animals and humans and, to a lesser extent, in plants. The aim of this study is to investigate the role of Se in mitigating UV-B radiation effect on biomass and on the antioxidant system in Azolla caroliniana Willd. plant.
Materials and methods
Plant material and growth condition
Azolla caroliniana (known as water velvet) was provided in 1982 by Prof. Weam El-Aggan from the Catholic University of Louvain (Louvain, Belgium); it was identified by Prof. G.A. Peters of Kettering Laboratory (Yellow Springs, OH, USA). The plants were acclimated in the greenhouse of the Faculty of Science of Alexandria University (Alexandria, Egypt), in 2500 mL polyethylene vessels that were filled with a nitrogen-free, modified Hoagland solution (2/5 con-centration, pH 5.1) in which KNO3 and Ca(NO3)2 were replaced by KCl and CaCl2, respectively. About 5 g (fresh mass) of Azolla from the stock material were inoculated in each vessel to make a new subculture, and so on. The plants were freed from epiphytic microorganisms by thorough washing with distilled water. The cultures were grown in a growth chamber under a 16-h photoperiod at irradiance of 1200 µmol m-2 s-1 (cool white fluorescent tubes) and light/dark temperatures of 28-30/20-25°C (stock culture). Before being used, the plants were surface-sterilized with 0.2% Clorox (El-Aggan 1982), then thoroughly washed with distilled water.
Selenium treatment and growth estimation
5 g of Azolla plants were transferred to 250 mL vessels containing 2/5 modified Hoagland solution and treated with 1 ppm Se in the form of sodium selenite (Hassan and Mostafa 2015) for 7 d, and were then exposed to UV-B radiation supplied by three UV-emitting tubes (TL12/100W/01, Philips, Holland) positioned 50 cm above leaf level. Rack height, lamp spacing and lamp power were adjusted as described by Ibrahim and Mostafa (2007) to maintain a total daily flux of biologically effective UV-B radiation doses of 5.75 KJ m-2d-1. Plants were rotated under the lamp banks to minimize potential effects resulting from microenvironment variations for 6 h. Samples were taken for chemical analyses. The number of generations and doubling time (DT) were determined from the fresh mass and duration of experiment using the equation given in Peters et al. (1979): n (final mass) = n° × 2G, where G = number of generations, n° = initial mass of Azolla plants (mass of inoculum); DT = duration of experiment per generation. Relative growth rate (RGR) was calculated by using the formula of Subudhi and Watanabe (1981): RGR [kg kg-1d-1] = 0.693 DT-1.
Estimation of hydrogen peroxide (H2O2)
Hydrogen peroxide content was determined according to Velikova et al. (2000). Plant tissues (50 mg) were homogenized in an ice bath with 5.0 mL of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 rpm for 15 min. The supernatant was used in the assay for H2O2. The reaction mixture contained 0.5 mL of plant extract, 0.5 mL of 10 mM potassium phosphate buffer (pH), and 0.1 mL 1 M KI. The content of H2O2 was calculated by comparing it with a standard calibration curve using different concentrations of H2O2, and the results were expressed as μmol H2O2 g-1 f.m.
Estimation of lipid peroxidat
Estimation of lipid peroxidat was measured as described by Zhang et al. (2007). About 1.5 mL of plant extract was homogenized in 2.5 mL of 5% thiobarbituric acid (TBA) dissolved in 5% TCA. The mixture was heated at 95°C for 15 min, then quickly cooled on ice and centrifuged at 5,000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm. Correction of non-specific turbidity was made by subtracting the absorbance value measured at 600 nm. The concentration of malondialdehyde (MDA) was calculated in μmol g-1 f.m.
Estimation of ascorbate and glutathione levels
Reduced glutathione was determined using the method described in Moron et al. (1979). A homogenate was prepared with 0.5 g of plant extract with 5 mL of 5.0% TCA, and it was then centrifuged at 1,000 rpm for 10 min. The supernatant was used for the estimation of GSH with 2 mL of freshly prepared 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) solution to 0.1 mL of plant extract and 0.9 mL sodium phosphate buffer (pH 8). The intensity of the resulting yellow colour was measured using a spectrophotometer at 412 nm after 10 min. The values are expressed as nmol GSH g-1. GSH estimation was done using the method of Griffith (1980) by adding 0.1 mL of neutralized supernatant to a standard solution mixture consisting of 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.5) containing ethylenediaminetetraacetic acid (EDTA), 0.2 mL of 6 mM DTNB, 0.1 mL of 2 mM nicotinamide adenine dinucleotide phosphate (NADPH), and 0.1 mL of 1 U yeast GR. Changes in absorbance at 412 nm were monitored at 25 ± 2°C until the absor-bance reached 5 U.
Extraction and estimation of ascorbate were done using the method described by Oser (1979). The reaction mixture consisted of 2% sodium molybdate, 0.2 mL 0.15 N H2SO4, 0.1 mL 1.5 mM Na2HPO4, and 1 mL of tissue extract. The mixture was incubated at 60°C in a water bath for 40 min. After centrifugation at 3,000 g for 10 min, absorbance was read at 660 nm.
Preparation of enzyme extract and assay of enzyme activity
Leaf tissues (0.5 g) were ground to a fine powder in liquid N2 and then homogenized in 0.2 mL of 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM D-isoascorbic acid, 2% (w/v) polyvinylpyrrolidone (PVP) and 0.05% (w/v) Triton X-100 using a chilled pestle and mortar, following the method of Gossett et al. (1994). The homogenate was centrifuged at 10,000 x g for 10 min at 4°C, and the supernatant was collected and used for assays of ascorbate peroxidase (APX), GSH-PX and GR. Protein concentrations in the enzyme extract and plant extract were determined according to the method of Bradford (1976) using defatted bovine serum albumin (BSA) (Sigma, fraction V) as a standard.
Ascorbate peroxidase (EC 220.127.116.11)
APX was assayed as described by Nakano and Asada (1981). The reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.2 mM EDTA, 0.5 mM ASA and 0.25 mM H2O2. The reaction was started at 25°C by adding H2O2 after having added the enzyme extract. The decrease in absorbance at 290 nm was recorded for 1 min and the amount of ascorbate oxidized was calculated from the extinction coefficient 2.8 mM-1 cm-1. Enzyme activity was expressed in terms of μmol ascorbate oxidized mg-1 protein min-1.
Glutathione peroxidase (EC 18.104.22.168)
GSH-PX activity was modified from Flohé and Günzler (1984). For the enzyme reaction, 0.2 mL of the super-natant was mixed with 0.4 mL GSH (Sigma product), and the mixture was put in an ice bath for 30 min. The mixture was then centrifuged for 10 min at 3,000 rpm; 0.48 mL of the supernatant was placed in a cuvette, and 2.2 mL of 0.32 M NaHPO4 and 0.32 mL of 1.0 mM 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, Sigma) were added for colour development. The absorbance at 412 nm was measured with a spectrophotometer after 5 min. Enzyme activity was calculated as a decrease in GSH with reaction time compared with that of the non-enzyme reaction.
Glutathione reductase (EC 22.214.171.124)
GR activity was determined at 25°C by measuring the rate of NADPH oxidation as the decrease in absorbance at 340 nm (extinction coefficient = 6.2 mM-1cm-1) according to the method of Halliwell and Foyer (1978). The reaction mixture (0.1 mL) consisted of 100 mM Tris-HCl (pH 7.8), 21 mM EDTA, 0.005 mM NADPH, 0.5 mM oxidized glutathione (GSSG), and the enzyme. NADPH was added to start the reaction. For APX, GPX and GR, enzyme activity was expressed in μmol mg-1 protein min-1.
A statistical analysis was done using the Statistical Package for Social Sciences (SPSS Statistics 20.0, IBM Corporation, Armonk, NY, USA) software. Arithmetic mean, standard deviation, and ANOVA for more than two groups were used, followed by Duncan’s multiple range test (MRT) to detect significant differences between the two groups. The level of significance was set at 0.05.
Results and discussion
The exposure of Azolla plants to UV-B radiation for 6 h resulted in a decrease in biomass and RGR, which coincided with an increase in DT as compared with the control. Also, the protein content decreased. Compared with the control, the reduction in biomass and protein content was 13% and 22%, respectively. Similarly, several studies have reported that exposure of plants to UV radiation resulted in a marked decrease in fresh mass and protein content (Agrawal 1992; Nasibi and M-Kalantari 2005). It has been reported that a reduction in fresh mass under UV radiation might be related to the enhancement of ROS generation (Mahdavian et al. 2008) and hence to the destruction of plasma membranes (Dai et al. 1997; Selvakumar 2008). Thus, the reduction in biomass in UV-treated Azolla plants might be related to a decrease in water absorption resulting from the destruction of plasma membranes and proteins due to generation of ROS. Nasibi and M-Kalantari (2005) reported that ROS destroy macromolecules such as proteins, polysaccharides, lipids and DNA.
Conversely, the addition of Se at a concentration 1 ppm resulted in a significant increase in biomass and protein content of untreated and UV-treated Azolla plants (Table 1). With Se, UV-treated Azolla plants’ biomass and protein content were 1.8 to 1.6-fold those of UV-treated plants without Se, respectively (Table 1). Similarly, some studies have demonstrated the benefits of adding a small amount of Se, including increased tuber yield and starch concentration in young potato leaves (Turakaine et al. 2004), and increased growth in ryegrass and lettuce exposed to UV-B radiation (Hartikainen and Xue 1999; Pennanen et al. 2002).
The positive synergistic effect of Se and UV radiation was found to be partly associated with the anti-oxidative role of Se in increasing GSH-PX activity (Xue and Hartikainen 2000). In this investigation, the addition of Se at the optimum concentration (1 ppm) to UV-treated Azolla growth media coincided with the promotion of plant growth and protein content, thus indicating that Se may have particular biological functions in the alteration of the antioxidant defense system.
Data in Table 2 show a significant accumulation of H2O2 and MDA in UV-treated Azolla plants. H2O2 and MDA contents were 2.6 and 1.9-fold those of the control, respectively. It has been shown that the induction of ROS generation was an early effect of UV radiation in plants (Kakani et al. 2003). In this study, the exposure of Azolla plants to UV radiation enhanced the accumulation of H2O2 and MDA contents. In accordance with these observations, Wang et al. (2010) reported that the production of O2- and H2O2 in Zea plants increased with UV-B radiation and that their continuous accumulation resulted in lipid peroxidation. Also, Nasibi and M-Kalantari (2005) suggested that peroxy radicals could abstract hydrogen from other unsaturated fatty acids, thus leading to a chain reaction of peroxidation and breakdown of the structure and function of membrane lipids of rapeseed (Brassica napus L.) exposed to UV-B radiation.
On the other hand, in this study, the addition of Se at a concentration of 1 ppm to the nutrient medium of UV-treated or untreated Azolla plants significantly reduced H2O2 and MDA contents, thus revealing that the addition of Se results in a marked reduction in ROS generation, which is possibly due to the reactivation of antioxidants, especially the H2O2 quencher (e.g. GSH-PX) in Azolla plants. Other studies have shown that an optimal Se concentration inhibits lipid peroxidation in many stressed plants (Cartes et al. 2005; Yao et al. 2010; Kumar et al. 2012).
In the present study, the addition of Se to UV-treated and untreated Azolla plants resulted in a significant increase in total ascorbate and GSH contents compared with the control and UV-stressed Azolla plants (Table 3). Also, glutathione redox potential (GSH/TG) increased significantly with the addition of Se to UV-treated Azolla plants. These observations could reflect the enhancement of GR activity to allow the reducing agent (GSH) to eliminate H2O2 through APX or GSH-PX. Reduced glutathione plays an important role in the antioxidant defense system of Azolla since it participates in the regeneration of ascorbate through dehydroascobate reductase, and it can also react with singlet oxygen and OH radical to protect protein thiol groups (Asada 1994). The enhancement of ascorbate and glutathione levels by Se compared with the control and UV-stressed Azolla plants reflects the capacity of GSH to synthesize organic Se. This conclusion is supported by Hasanuzzaman and Fujita (2011) who found that ASA and GSH contents were enhanced by Se in both salt- and drought-stressed rapeseed seedlings.
There was a significant (38%) increase in APX activity in UV-treated plants compared with the control. APX activity did not change significantly in the presence of Se compared with the control. GR activity increased significantly, while GSH-PX activity did not change significantly in UV-treated Azolla plants compared with the untreated control. On the other hand, both GSH-PX and GR activities were significantly enhanced by the application of Se at a concentration of 1 ppm to the nutrient media of untreated and UV-treated Azolla plants. There was a 2.7-fold increase in GSH-PX activity with Se application in UV-treated plants compared with the control (Table 4).
GSH-PX, with the help of GSH, is a powerful scavenger of H2O2 and lipid hydroperoxides. GSH-PX is believed to be a key enzyme that can be widely and robustly activated by Se in various plants exposed to diverse environmental stresses (Feng et al. 2011). In this study, under UV-stress and in the absence of Se, H2O2 in Azolla plants may be quenched by APX, while in the presence of Se, H2O2 is primarily scavenged by GSH-PX instead of APX. Similar results were obtained with Chlamydomonas cells (Takeda et al. 1997). The increased activity of the GSH-PX enzyme in response to Se suggests a unique role for this enzyme in counteracting oxidative stress in plants. Se-pretreated rapeseed seedling exhibited increased ASA levels and significantly enhanced APX, dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), GSH-PX and catalase activity compared with drought-stressed plants without Se (Hasanuzzaman and Fujita 2011). The positive synergistic effect of Se and UV radiation was found to be associated with the antioxidative role of Se through increased GSH-PX and catalase activity, whereas APX responded negatively (Xue and Hartikainen 2000).
The insignificant changes in ascorbate content of UV-treated Azolla plants in the absence of Se may be due to its participation in reducing H2O2, which is catalyzed by a high level of APX activity. Ascorbate is the principal electron donor and it is oxidized to dehydroascorbate in the reaction of APX with H2O2, then reduced back to ascorbate in the presence of GR (Zhang and Kirkham 1996). Hence, the levels of ascorbate and GSH play an important role in oxidative defense. These insignificant changes in ascorbate level might be correlated with the increased activity of APX and GR or could be due to its participation in the ascorbate-glutathione oxidative pathway. It was shown that there was a decrease in ascorbate level in Bruguiera parviflora (Roxb.) Wight & Arn., which is likely due to its role in reducing H2O2 to H2O (Parida et al. 2004).
Several studies reported that the addition of Se enhanced the biosynthesis of glutathione fractions and activity of peroxidases (Hasanuzzaman and Fujita 2011; Feng and Wei 2012; Malik et al. 2012) as well as GR activity (Mittler 2002). As shown in this study, the addition of Se caused a significant increase in GSH-PX, GSH/TG as well as GR activity. These observations were accompanied with a significant decrease in H2O2 and MDA contents. These results might indicate that the application of Se can influence the activity of GSH-PX for scavenging ROS through the ascorbate-glutathione cycle. Therefore, Se might protect Azolla plants from UV stress.
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