Access the full text.
Sign up today, get DeepDyve free for 14 days.
O. Tanaka, Yutaka Nasu, A. Takimoto, M. Kugimoto (1982)
Absorption of Copper by Lemna as Influenced by Some Factors Which Nullify the Copper Effect on Flowering and GrowthPlant and Cell Physiology, 23
S. Mishra, S. Srivastava, R. Tripathi, R. Govindarajan, S. Kuriakose, M. Prasad (2006)
Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L.Plant physiology and biochemistry : PPB, 44 1
A. Teramura, J. Sullivan (1994)
Effects of UV-B radiation on photosynthesis and growth of terrestrial plantsPhotosynthesis Research, 39
P. Hasselt (1974)
PHOTO‐OXIDATION OF UNSATURATED LIPIDS IN CUCUMIS LEAF DISCS DURING CHILLING, 23
D. Wetherell (1961)
Culture of Fresh Water Algae in Enriched Natural Sea WaterPhysiologia Plantarum, 14
U.K. Laemmli (1970)
Cleavage of structural proteins during assembly of the head of bacteriophage T4Nature, 227
M. Rao, G. Paliyath, D. Ormrod (1996)
Ultraviolet-B- and Ozone-Induced Biochemical Changes in Antioxidant Enzymes of Arabidopsis thaliana, 110
Yuying He, D. Häder (2002)
UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine.Journal of photochemistry and photobiology. B, Biology, 66 2
U. Laemmli (1970)
Cleavage of structural proteins duringNature
S. Mano, M. Nishimura (2005)
Plant peroxisomes.Vitamins and hormones, 72
G. Venkataraman (1969)
The cultivation of algae.
F. Moore, Frank Kramer, R. Knowles (1951)
Statistics for Medical StudentsThe Yale Journal of Biology and Medicine, 24
C. Foyer (1997)
Oxygen Metabolism and Electron Transport in PhotosynthesisCold Spring Harbor Monograph Archive, 34
J. Scandalios (1992)
Molecular biology of free radical scavenging systems
Kiyoshi Tanaka, H. Mitsuhashi, N. Kondo, K. Sugahara (1982)
Further Evidence for Inactivation of Fructose-1,6-bisphosphatase at the Beginning of SO2 Fumigation. Increase in Fructose-1,6-bisphosphate and Decrease in Fructose-6-phosphate in SO2-Fumigated Spinach LeavesPlant and Cell Physiology, 23
C. Bestwick, A. Ádám, N. Puri, J. Mansfield (2001)
Characterisation of and changes to pro and anti-oxidant enzyme activities during the hypersensitive reaction in lettuce (Lactuca sativa L.)Plant Science, 161
R. Heath, L. Packer (1968)
Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation.Archives of biochemistry and biophysics, 125 1
(1995)
Response of plants to UV-B radiation: Some biochemical and physiological effects
N. Nedunchezhian, K. Annamalainathan, G. Kulandaivelu (1992)
Induction of heat shock‐like proteins in Vigna sinensis seedlings growing under ultraviolet‐B (280‐320 nm) enhanced radiationPhysiologia Plantarum, 85
M. Ehling-Schulz, S. Scherer (1999)
UV PROTECTION IN CYANOBACTERIAEuropean Journal of Phycology, 34
S. Madronich, R. McKenzie, M. Caldwell, L. Björn (1994)
Changes in ultraviolet-radiation reaching the earths surfaceAMBIO: A Journal of the Human Environment, 24
J.F. Bornman, C. Sundby-Emanuelsson (1995)
Environmental and Plant Metabolism: Flexibility and Acclimation
N. Mallick, F. Mohn (2000)
Reactive oxygen species: response of algal cellsJournal of Plant Physiology, 157
G.W. Prescott (1962)
Algae of western great lakes area
A. Abo-Shady, M. El-sheekh, A. El-Naggar, A. Abomohra (2008)
Effect of UV-B radiation on growth, photosynthetic activity and metabolic activities ofChlorococcum sp.Annals of Microbiology, 58
K. Mopper, X. Zhou (1990)
Hydroxyl radical photoproduction in the sea and its potential impact on marine processes.Science, 250 4981
(1962)
Zur Physiologic der Speicherung Organischer Phosphate in Chlorella
M. Kapoor, G. Sreenivasan, N. Goel, J. Lewis (1990)
Development of thermotolerance in Neurospora crassa by heat shock and other stresses eliciting peroxidase inductionJournal of Bacteriology, 172
Misako Kato, S. Shimizu (1987)
Chlorophyll metabolism in higher plants. VII. Chlorophyll degradation in senescing tobacco leaves; phenolic-dependent peroxidative degradationBotany, 65
G.W. Prescott (1975)
Algae of the western great lakes area
L. Sandalio, H. Dalurzo, M. Gómez, M. Romero‐Puertas, L. Río (2001)
Cadmium-induced changes in the growth and oxidative metabolism of pea plants.Journal of experimental botany, 52 364
R. Sinha, M. Klisch, A. Gröniger, D. Häder (2000)
Mycosporine-like amino acids in the marine red alga Gracilaria cornea — effects of UV and heatEnvironmental and Experimental Botany, 43
P. Bhargava, N. Atri, A. Srivastava, L. Rai (2007)
Cadmium mitigates ultraviolet-B stress in Anabaena doliolum: Enzymatic and non-enzymatic antioxidantsBiologia Plantarum, 51
M. Prather, P. Midgley, F. Rowland, R. Stolarski (1996)
The ozone layer: the road not takenNature, 381
G. Gerloff, G. Fitzgerald, F. Skoog (1950)
THE ISOLATION, PURIFICATION, AND CULTURE OF BLUE–GREEN ALGAEAmerican Journal of Botany, 37
A. Schützendübel, P. Nikolova, C. Rudolf, A. Polle (2002)
Cadmium and H2O2-induced oxidative stress in Populus × canescens rootsPlant Physiology and Biochemistry, 40
H. Shibata, K. Baba, H. Ochiai (1991)
Near-UV Irradiation Induces Shock Proteins in Anacystis nidulans R-2; Possible Role of Active OxygenPlant and Cell Physiology, 32
R. Larson (1988)
The antioxidants of higher plantsPhytochemistry, 27
T. Yuasa, K. Ichimura, T. Mizoguchi, K. Shinozaki (2001)
Oxidative stress activates ATMPK6, an Arabidopsis homologue of MAP kinase.Plant & cell physiology, 42 9
B. Halliwell (1991)
Reactive oxygen species in living systems: source, biochemistry, and role in human disease.The American journal of medicine, 91 3C
I. Fridovich (1986)
Biological effects of the superoxide radical.Archives of biochemistry and biophysics, 247 1
B. Halliwell, J. Gutteridge (1985)
Free radicals in biology and medicine
C. Beauchamp, I. Fridovich (1971)
Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.Analytical biochemistry, 44 1
Annals of Microbiology, 58 (2) 195-201 (2008) Impact of UV-B radiation on antioxidant enzymes and protein electrophoretic pattern of the green alga Chlorococcum sp. Atef Mohmed ABO-SHADY*, Amal Hamed EL-NAGGAR, Mostafa Mohamed EL-SHEEKH, Abd El-Fatah ABOMOHRA. Phycology Research Unit, Botany Department, Faculty of Science, Tanta University, Tanta, Egypt. Received 17 December 2007 / Accepted 3 April 2008 -2 Abstract - The impact of two intensities (2.5 and 5 W m ) of ultraviolet-B (UV-B) radiation on lipid peroxidation, protein pattern and some antioxidant enzymes including superoxide dismutase, catalase and peroxidase has been studied in Chlorococcum sp. isolated from El-Kased freshwater canal, Tanta, Egypt. Exposure of Chlorococcum sp. for 2 h to 2.5 and -2 5Wm increased malondialdehyde (MDA) content by 238 and 274%, respectively. The activity of superoxide dismutase and peroxidase were increased at all exposure times at both intensities. In contrast, the activity of catalase was inhibit- ed by increasing UV-B intensity as well as exposure time compared to the control. The reduction in catalase activity -2 reached 71.8 and 95.9% after 2 h of exposure to 2.5 and 5 W m , respectively. With regard to protein pattern, expo- sure of Chlorococcum sp. to UV-B stress induced marked changes in protein synthesis patterns. Three types of alterations were noticed, i) significant reduction in the biosynthesis of certain proteins, ii) induction of specific proteins biosynthesis and iii) changes in band intensities of some proteins. Key words: Antioxidant enzymes, Chlorococcum sp., lipid peroxidation, oxidative stress, protein pattern, ultraviolet-B radiation. INTRODUCTION ROS were generated in peroxisomes during pho- torespiration and in glyoxysomes during ß-oxidation Depletion of the ozone layer in the stratosphere of lipids (Huang et al., 1983). In addition, ROS pro- owing to increasing emission of chlorinated fluoro- duction was found to be stimulated by various envi- carbons (CFCs) and other man-made chlorocarbons ronmental stresses such as exposure to UV radia- is today accepted as a reality (Madronich et al., tion (Bornman and Sundby-Emanuelsson, 1995; 1995; Prather et al., 1996). The ozone absorbs UV- Rao et al., 1996). It was suggested that interaction B radiation (280-320 nm), and the thinning of the between UV-B radiation, oxygen, and certain organ- ozone layer has created a concern for the potential ic compounds, e.g. dissolved organic matter (DOM) impact of increased UV-B radiation on the earth’s or humic substances, can induce a production of ecosystems. reactive oxidants, for example superoxide (O ), Increasing doses of UV-B radiation reaching the hydrogen peroxide (H O ), and singlet oxygen ( O ) 2 2 2 earth’s surface as a consequence of stratospheric (Mopper and Zhou, 1990). ozone depletion may also be deleterious for plants Plants are well adapted for minimising the dam- (Teramura and Sullivan, 1994; Bornman and age that could be induced by the ROS under natu- Sundby-Emanuelsson, 1995). In the plant system, ral growth conditions. However, O2 toxicity emerges reactive oxygen species (ROS) are formed by when the production of these ROS exceeds the inevitable leakage of electrons onto molecular oxy- quenching capacity of the protective systems due to gen from the electron transport activities of chloro- environmental adverse conditions such as high solar plast and mitochondria (Foyer, 1997). Furthermore, radiation, drought, heat and other stresses (Yuasa et al., 2001). The first step in formation of the ROS occurs when molecular oxygen (O ) accepts one * Corresponding author. Phone: 20403344352 (ext. 367); electron to form the superoxide radical (O ) Fax: 20403350804; E-mail: atefaboshady@yahoo.com A.M. ABO-SHADY et al. (Mallick and Mohn, 2000). Superoxide radicals are 1975). Axenic cultures of the organism were toxic by-products of oxidative metabolism obtained by repeated subculturing, antibiotic treat- (Fridovich, 1986). When the antioxidant defence ment according to Venkataraman (1969) and ultra- system becomes unable to remove these toxic free violet irradiation according to Gerloff et al. (1950). radicals, a series of the cytotoxic ROS will be The alga was cultured in a medium described by formed, including H O and OH radicals. The intra- Kuhl (1962) at room temperature (25 ± 2 °C). 2 2 cellular H O is potentially more harmful than O Cultures were illuminated by means of fluorescent 2 2 2 because H O can diffuse rapidly across biological white tubes (40 W.F. 7 day light), which gave light 2 2 membranes; consequently it can cause oxidative intensity of about 12 kilolux. The cultures were aer- stress far from the site of its formation (Mallick and ated with a mixture of 97% air and 3% CO .The Mohn, 2000). Also, the lipid peroxides derived from algal growth was monitored by measuring the opti- H O were found by Kapoor et al. (1990) to be cal density of the cell suspension spectrophotomet- 2 2 membrane damaging agents, causing severe per- rically at 560 nm (Wetherell, 1961). UV-B irradiation turbations of the membrane permeability proper- was applied to the cultures at logarithmic phase ties. If H O produced in the chloroplast as a result having optical density from 0.15-0.20 at 560 nm. 2 2 of dismutation of O is not removed, it causes inhi- bition of the Calvin cycle enzymes (Tanaka et al., Exposure to UV-B radiation. The tested organism 1982). H O is a strong oxidant that can initiate grown in liquid culture was transferred into a steril- 2 2 localized oxidative damage leading to disruption of ized Petri dish (17 x 2.5 cm) and exposed to artifi- -2 metabolic functions and losses of cellular integrity cial UV-B radiation (2.5 and 5 W m , separately) for at sites where it accumulates (Mallick and Mohn, 30, 60, 90 and 120 min in addition to control (unir- 2000). radiated culture). The UV-B radiation system com- On the other hand, the OH radicals are the most prised an array of three ultraviolet long lamps, UV- reactive species known to chemistry (Halliwell and A, UV-B and UV-C. The UV-B lamp (T-8 M) manufac- Gutteridge, 1989). This OH radical is able to cause tured by Vilbar-Lourmat, France. The spectral emis- lipid peroxidation, denaturation of proteins and sion of UV-B source range from 280-320 nm with a mutation of DNA. In addition, singlet oxygen, which peak at 312 nm. The suspension was gently agitat- can be formed when excitation energy is transferred ed by a magnetic stirrer during irradiation to facili- to oxygen, produces also deleterious effects (Yuasa tate uniform exposure. et al., 2001). Generally, plant cells have evolved defences, Determination of lipid peroxidation. Lipid per- antioxidant mechanisms, to contrast the possible oxidation was estimated by the concentration of negative effects due to the presence of ROS. These malondialdehyde (MDA), as a product of unsaturat- include several enzymatic and non-enzymatic ed fatty acids peroxidation. MDA concentration was mechanisms. The non-enzymatic antioxidant estimated by the method described by Heath and defence system consists of low molecular weight Packer (1968). A definite fresh weight of algae was antioxidants such as ascorbate, glutathione, α toco- extracted in 10 ml 5% (w/v) trichloroacetic acid pherol, and carotenoids (Alscher and Hess, 1993). using a homogenizer. The homogenate was then While the enzymatic antioxidant defence system centrifuged at 4000 rpm for 10 min. The super- includes antioxidant enzymes such as superoxide natant (2 ml) was mixed with 2 ml of 0.67% (w/v) dismutase (SOD), catalase (CAT), peroxidase thiobarbaturic acid (TBA), and incubated at 100 °C (POD), glutathione reductase (GR) and ascorbate in a water-bath for 20 min then cooled immediate- peroxidase (APX) (Mishra et al., 2006). ly. MDA formation was determined spectrophoto- The present work is intended to throw some metrically by measuring the absorbance of the reac- light on the impact of UV-B radiation on green algae tion product of MDA with TBA in the supernatant at represented by Chlorococcum sp. as model organ- 532 nm, corrected for non-specific turbidity by sub- ism. So the effect of UV-B on protein patterns, lipid tracting the absorbance at 600 nm. MDA concentra- peroxidation and activity of some antioxidant tion (µM/g fresh weight) was calculated using the -1 -1 enzymes including SOD, CAT and POD of extinction coefficient (155 mM cm ). Chlorococcum sp. was studied. Extraction of soluble proteins and enzymatic assays. A sample of 0.2 g fresh weight (harvested MATERIALS AND METHODS immediately after UV irradiation) was frozen, then homogenised in 4 ml of 50 mM cold phosphate Experimental organism and growth condi- buffer at pH 7 (modified from Beauchamp and tions. The unicellular green alga Chlorococcum sp. Fridovich, 1971). The homogenates were cen- was isolated from El-Kased freshwater canal, Tanta, trifuged at 4000 rpm for 20 min. The supernatant Egypt and identified according to Prescott (1962, was used as a raw extract for enzymatic assay. Ann. Microbiol., 58 (2) 195-201 (2008) 197 Catalase (EC 1.11.1.6) activity was measured (1970). Gel was stained with Coomassie brilliant by recording the decomposition of H O as blue R 250. The gel was run at 10 mA/cm in the 2 2 expressed by the decrease in the absorbance at stacking gel and when the bromophenol blue track- 240 nm according to the method described by Kato ing dye enters the separating gel, the current was and Shimizu (1987). A sample of 3 ml of reaction raised to 15 mA/cm. The current was shut off when mixture contained 0.1 M sodium phosphate buffer the bromophenol blue tracking reaches the bottom at pH 7, 2 mM H O and 0.1 ml enzyme extract. The of the separating gel. 2 2 decrease in H O was followed as a decline in the 2 2 absorbance at 240 nm and the activity was calcu- Statistical analysis. Results are presented as -1 -1 lated using the extinction coefficient (40 mM cm mean ± standard deviation (SD) from three differ- at 240 nm) for H O . The activity was expressed in ent readings. The statistical analyses were carried 2 2 units of µM of the substrate converted per minute out using SPSS 10.0 and Minitab 14. Data obtained per mg chlorophyll. were analysed statistically to determine the degree Peroxidase (EC 1.11.1.7) activity was measured of significance between treatments using one way according to Kato and Shimizu (1987). The assay and two way analysis of variance (ANOVA). medium contained 0.1 M sodium phosphate buffer Additionally, the LSD test was used to determine at pH 5.8, 7.2 mM guaiacol, 11.8 mM H O and 0.1 treatment differences comparing with control at P ≤ 2 2 ml enzyme extract and the final assay volume was 0.001 level of significance. 3 ml. The reaction was initiated by the addition of H O , and the change in the absorbance was meas- 2 2 ured at 470 nm. Activity was calculated using the RESULTS -1 -1 extinction coefficient (26.6 mM cm at 470 nm) for tetraguaiacol. Enzyme activity was expressed in Lipid peroxidation units of µM of the substrate converted per min per Exposure to UV-B increased the malondialdehyde mg chlorophyll. (MDA) content at all doses compared to the control. Superoxide dismutase (SOD) (EC 1.15.1.1) The most pronounced increase was detected at 5 W -2 activity was assayed on the basis of its ability to m (Fig. 1). The increase in MDA content attained inhibit the photochemical reduction of nitro blue 238 and 274% with respect to control after 2 h of -2 tetrazolium by superoxide (Beauchamp and exposure to 2.5 and 5 W m , respectively. One way Fridovich, 1971). Three millilitres of the reaction analysis of variance showed that, exposure to 2.5 -2 mixture contained 50 mM sodium phosphate and 5 W m UV-B revealed high significant effect on buffer at pH 7.8, 13 mM methionine, 75 µM nitro MDA content at all the exposure times (Table 1). blue tetrazolium, 2 µM riboflavin, 100 µM EDTA and 0-200 µl enzyme extract. Riboflavin was added last, and then tubes were shaken and Antioxidant enzymes As a general trend, the activity of SOD and peroxi- placed 30 cm below light bank consisting of two dase increased with increasing UV-B exposure time 8-w fluorescent tubes. The reaction was started -2 (Fig. 2A, 2B). The intensity of 2.5 W m increased by switching on light and allowed to run for 10 the activity of SOD and peroxidase at all exposure min before being stopped by switching off light. Absorbance of the reaction mixture was read at 560 nm. A non-irradiated reaction mixture has an absorbance of zero at 560 nm, the reaction medi- Control 2.5 W 5W um-lacking enzyme develops an intense colour 0.35 and this decreases with increasing volumes of enzyme extract added (in light). Log A was 0.3 plotted as a function of the volume of enzyme 0.25 extract (0-200 µl) in the reaction mixture. The volume of enzyme extract producing 50% inhibi- 0.2 tion of the reaction was read from the resultant 0.15 curve. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of 0.1 the initial rate of the reaction in the absence of 0.05 enzyme. 030 60 90 120 Qualitative characteristic of protein using gel Exposure time (min) electrophoresis. SDS-PAGE was carried out in a IG. 1 - Effect of UV-B irradiation on MDA content ( M/gm FW) estimated in vertical system with gels of 155 x 130 mm, 0.5 mm -2 FIG. 1 - Effect of pH on chitinase activity from Bacillus licheniformis A1. Chlorococcum sp. exposed to two UV-B intensities (2.5 and 5 W m )at thick, using the method described by Laemmli Assays were performed in 50 mM of different buffers between different exposure times. pH 5.0 and 9.0 at 65 °C. MDA content (μM/g FW ) A.M. ABO-SHADY et al. A A Control 2.5 W 5W TABLE 1 - Effect of UV-B exposure on MDA content in cul- times and the maximum activities were 368 and tures of Chlorococcum sp. exposed to two UV-B -2 intensities (2.5 and 5 W m ) for 30, 60, 90 and 12 120 min 030 60 90 120 Exposure time (min) Each value is the mean of three readings ± standard devi- B Control 2.5 W 5W ation. * Highly significant at P ≤ 0.001 using one way analysis of 7.5 variance (ANOVA). 6.5 5.5 4.5 671% compared to the control after 2 h of expo- sure, respectively. However, SOD and peroxidase 3.5 activities showed their maximum (434 and 123%, 2.5 -2 respectively) after 90 min of exposure to 5 W m . 2 1.5 In contrast to such response of SOD and peroxidase 0.5 to UV-B exposure, the activity of catalase was reduced with increasing UV-B intensity as well as 0 306090 120 Exposure time (min) exposure time compared to the control (Fig. 2C). The reduction in catalase activity reached 71.8 and C Control 2.5 W 5W -2 95.9% after 2 h of exposure to 2.5 and 5 W m , 1.4 1.3 respectively. Two way analysis of variance (data not 1.2 shown) showed that, UV-B intensities, exposure 1.1 times and interaction between them revealed high 0.9 significant effect (P ≤ 0.001) of UV-B radiation on 0.8 the three studied enzymes. One way analysis of 0.7 0.6 variance showed that, exposure to 2.5 and 2 0.5 5Wm UV-B revealed high significant effect on the 0.4 0.3 activity of the three enzymes at all the exposure 0.2 times (Table 2). 0.1 030 60 90 120 Exposure time (min) FIG. 2 - Effect of UV-B radiation on the activity of antioxidant enzymes of Chlorococcum sp. TABLE 2 - Effect of UV-B radiation on the activity of super- exposed to 2.5 and 5 Wm-2 at different oxide dismutase (SOD), peroxidase and catalase -2 exposure times. A: Effect on superoxide dis- in Chlorococcum sp. exposed to 2.5 and 5 W m mutase (SOD). B: Effect on peroxidase. C: at different exposure times Effect on catalase. Each value is the mean of three readings ± standard deviation. Units of different enzymes are defined in Materails and Methods. * Highly significant at P ≤ 0.001 using one way analysis of variance (ANOVA). -1 -1 Catalase activity (µM min mg chl a) Catalase activity (?M/min.mg Chl a) -1 -1 Peroxidase activity (µM min mg chl a) -1 SOD activity (µ µl enzyme extract) Peroxidase activity (?M/min.mg Chl a) SOD activity (Unit/?l enzyme extract) Ann. Microbiol., 58 (2) 195-201 (2008) 199 Protein electrophoretic pattern ides in a membrane disrupts its function and can Results in Fig. 3 show the changes in protein elec- cause it to collapse (Halliwell, 1991). trophoretic pattern of Chlorococcum sp. after UV-B Malondialdehyde (MDA) formation is caused by -2 exposure to 2.5 and 5 W m for 30, 60, 90 min and oxidative degradation of polyunsaturated fatty 2 h in addition to unirradiated control. acids, in particular linolenic acid (van Hasselt, Chlorococcum sp. responded to UV-B stress by 1974). The present study revealed that, exposure of inducing the synthesis of two new proteins having Chlorococcum sp. to UV-B determined a significant molecular weights about 119 and 91 KDa which increase in MDA content with increasing irradiation were not originally seen in the control lane (Fig. 3). time and intensity. These results are in agreement In addition, there was a gradual loss in the intensi- with those obtained by He and Häder (2002) on ty of one protein band with molecular weight about cyanobacteria. Thus, it could be concluded that the 73 KDa with the increase of exposure time at 2.5 W increase in membrane leakage, which was observed -2 m until the complete disappearance after 2 h of in Chlorococcum sp. exposed to UV-B radiation -2 exposure. At 5 W m , bands having molecular (Abo-Shady et al., 2008), may be mainly ascribed weights about 145, 136, 106, 73, 56, and 52 KDa to the peroxidation of the membrane lipids due to were gradually disappearing until complete loss increased ROS production during UV-B irradiation. after 2 h of exposure. On the other hand, there was Plant cells have evolved defence antioxidant mech- a gradual increase in the intensity of protein bands anisms to combat the damage posed by the pres- having molecular weights about 145, 136, 119, 79, ence of the ROS. These include several enzymatic and 50 KDa by increasing the exposure time at 2.5 and non-enzymatic mechanisms. The main -2 Wm ; the maximum intensity of these bands enzymes involved in ROS scavenging, during nor- appeared after 2 h of exposure. Protein bands with mal and stress conditions, are superoxide dismu- molecular weights about 79 and 50 KDa showed the tases (SODs), peroxidases and catalases. -2 same trend at 5 W m . Our results showed that the activity of SOD increased significantly in Chlorococcum sp. with increasing UV-B intensity and exposure time to cer- KDa tain limits, beyond which the activity decreased, which may be due to the denaturation of the enzyme at high doses of UV-B or to an inactivation of the enzyme by H O , which is produced in differ- 2 2 116 ent cellular compartments and also from a number of non-enzymatic and enzymatic processes in cells (Schützendübel et al., 2002; Mishra et al., 2006). Peroxidase is one of the most widely distributed antioxidant enzymes in the plant cells. Present results showed that peroxidase responded to UV-B irradiation in the same manner as SOD. Rao et al. (1996) observed in Arabidopsis thaliana that, gels stained for peroxidase activity revealed only one isoform in the control plants and UV-B exposure enhanced the intensity of the existing isoform and caused the synthesis of a new isoform of peroxi- FIG. 3 - Vertical SDS-PAGE profile of the isolated proteins dase. Peroxidases and catalase are involved in the of Chlorococcum sp. following UV-B exposure for -2 conversion of H O into water and molecular oxy- different time periods at 2.5 and 5 W m .Lane 1: 2 2 marker proteins, lane 2: unirradiated control, lane gen. Peroxidase functions in chloroplasts whereas -2 3: 30 min at 2.5 W m , lane 4: 60 min at 2.5 W catalase is present in peroxisomes and mitochondria -2 -2 m , lane 5: 90 min at 2.5 W m , lane 6: 2 h at (Mishra et al., 2006). -2 -2 2.5 W m , lane 7: 30 min at 5 W m , lane 8: 60 In the present study, catalase activity decreased at -2 -2 min at 5 W m , lane 9: 90 min at 5 W m and lane 2 -2 all exposure times under the two UV-B intensities 10: h at 5 W m . and reduction of catalase depends on UV-B dose. These results are in agreement with those obtained DISCUSSION by Rao et al. (1996), who showed that UV-B radia- tion decreased catalase activities of Arabidopsis Lipid peroxidation occurs when the OH radicals are thaliana. Also, Bhargava et al. (2007) reported that generated close to the cell membranes and attack UV-B radiation decreased catalase activities of the fatty acid side chains of membrane lipids, espe- Anabaena doliolum.Mishra et al. (2006) reported cially the unsaturated ones, resulting in the forma- that catalase is sensitive to O radicals and thus tion of lipid hydroperoxides (Bestwick et al., 2001; stress may result in inactivation of the enzyme. The Yuasa et al., 2001). Accumulation of the lipid perox- decrease in catalase activity may also be associated A.M. ABO-SHADY et al. Ehling-Schulz M., Scherer S. (1999). UV protection in with degradation caused by induced peroxisomal cyanobacteria. Eur. J. Phycol., 34: 329-338. proteases or may be due to photoinactivation of the Foyer C.H. (1997). Oxygen metabolism and electron trans- enzyme (Sandalio et al., 2001). port in photosynthesis. In: Scandalios J., Ed., Molecular An induction of UV-shock proteins in response to Biology of Free Radical Scavenging Systems. Cold high intensities of UV-B and in response to UV-C Spring Harbor Laboratory Press, New York, pp. 587- irradiation has been reported in algae by Sinha et al. (2000). Shibata et al. (1991) indicated that Fridovich I. (1986). Biological effects of the superoxide some of the UV-shock proteins were also inducible radical. Arch. Biochem. Biophysi., 247: 1-11. by heat shock. Furthermore, Nedunchezhian et al. Gerloff G.G., Fitzgerald G.P., Skooge F. (1950). The isola- (1992) concluded that heat-shock-like proteins have tion, purification and culture of blue-green algae. been found to be UV-B inducible in plant seedlings. Amer. J. Bot., 37: 216-218. The present results of protein pattern obtained from Halliwell B. (1991). Reactive oxygen species in living sys- gel electrophoresis indicated that Chlorococcum sp. tems. Source: biochemistry and role in human disease. Am. J. Medicine, Sept. 30, v91, n3C, p 14S (9). responded to UV-B shock by the induction of sever- al proteins, with disappearance of some existing Halliwell B., Gutteridge J.M.C. (1989). Free Radicals on Biology and Medicine. Oxford, Clarendon Press. proteins, especially at higher doses of UV-B. At 2.5 -2 Wm , Chlorococcum sp. responded to UV-shock by He Y.Y., Häder D.-P. (2002). UV-B-induced formation of reactive oxygen species and oxidative damage of the induction of two new proteins (119 and 91 KDa). cyanobacterium Anabaena sp.: Protective effects of Whereas one protein band (73 KDa) completely dis- ascorbic acid and N-acetyl-L-cysteine. J. Photochem. appeared after a 2-h exposure. Chlorococcum sp. Photobiol. B: Biol., 66: 115-124. -2 responded to exposure to 5 W m by the induction Heath R.L., Packer L. (1968). Photoperoxidation in isolat- of two proteins (119 and 91 KDa) and six protein ed chloroplasts. I. Kinetics and stoichiometry of fatty bands (145, 136, 106, 73, 56, and 52 KDa) gradu- acid peroxidation. Arch. Biochem. Biophys., 125: 189- ally disappeared with increasing exposure time. Synthesis of other existing proteins (e.g. 79 and 50 Huang A.H.C., Trelease R.N., Moore T.S. (1983). Plant KDa) was increased by increasing of UV-B irradia- Peroxisomes. Academic Press, New York. tion dose. These results agree with those of Ehling- Kapoor M., Sreenivasan M.G., Goel N., Lewis J. (1990). Schulz and Scherer (1999) who classified proteins Development of thermotolerance in Neurospora crassa by heat shock and other stresses eliciting peroxidase into three categories by their response to UV-B irra- induction. J. Bacteriol., 172: 2798-2801. diation: 1) proteins whose synthesis is stimulated Kato M., Shimizu S. (1987). Chlorophyll metabolism in by UV-B; 2) proteins which are repressed and/or higher plants. VII. Chlorophyll degradation in senesc- degraded in response to UV-B; and 3) proteins ing Tobacco leaves; phenolic-dependent peroxidative which are not affected by UV-B. degradation. Can. J. Bot., 65: 729-735. Kuhl A. (1962). Zur Physiologic der Speicherung Organischer Phosphate in Chlorella. Biet. Physiol. REFERENCES Morphol. Algen. Gustav. Fresh Verlag. Stuttgart, Germany. Abo-Shady A.M., El-Naggar A.H., El-Sheekh M.M., Laemmli U.K. (1970). Cleavage of structural proteins dur- Abomohra A. (2008). Effect of UV-B radiation on ing assembly of the head of bacteriophage T4. Nature, growth, photosynthetic activity and metabolic activities 227: 680-685. of Chlorococcum sp. Ann. Microbiol., 58 (1): 21-27. Madronich S., Mckenzie R.L., Caldwell M.M., Björn L.O. Alscher R.G., Hess J.L., Eds (1993). Antioxidants in Higher (1995). Changes in ultraviolet radiation reaching the Plants. CRC Press, Boca Raton, FL. earth’s surface. Ambio, 24: 143-152. Beauchamp C., Fridovich I. (1971). Superoxide dismu- Mallick N., Mohn F.H. (2000). Reactive oxygen species: tases: improved assays and an assay applicable to response of algal cells. J. Plant Physiol., 157: 183-193. acrylamide gels. Anal. Biochem., 44: 276-287. Mishra S., Srivastava S., Tripathi R.D., Govindarajan R., Bestwick C.S., Adam A.L., Puri N., Mansfield J.W. (2001). Kuriakose S.V., Prasad M.N.V. (2006). Phytochelatin Characterization of and changes to pro- and anti-oxi- synthesis and response of antioxidants during cadmi- dant enzyme activities during the hypersensitive reac- um stress in Bacopa monnieri L. Plant Physiol. tion in Lettuce (Lactuca sativa L.). Plant Sci., 161: 497- Biochem., 44: 25-37. Mopper K., Zhou X.L. (1990). Hydroxyl radical photopro- Bhargava P., Atri N., Srivastava A.K., Rai L.C. (2007). tection in the sea and its potential impact on marine Cadmium mitigates ultraviolet-B stress in Anabaena processes. Science, 250: 810-812. doliolum: Enzymatic and non-enzymatic antioxidants. Nedunchezhian N., Annamalainathan K., Kulandaivelu G. Biol. Plantarum, 51 (3): 546-550. (1992). Induction of heat shock-like proteins in Vigna Bornman J.F., Sundby-Emanuelsson C. (1995). Response sinensis seedlings growing under ultraviolet-B (280- of plants to UV-B radiation: Some biochemical and 320 nm) enhanced radiation. Physiol. Plant., 85: 503- physiological effects. In: Smirnoff N., Ed., Environmental and Plant Metabolism: Flexibility and Acclimation, Bios Scientific, Oxford, pp. 245-262. Ann. Microbiol., 58 (2) 195-201 (2008) 201 Prather M., Midgley P., Rowland F.S., Stolarski R.S. (1996). Sinha R.P., Klisch M., Gröniger A., Häder D.-P. (2000). The ozone layer: the road not taken. Nature, 381: 551- Mycosporine-like amino acids in the marine red alga 554. Gracilaria cornea – effects of UV and heat. Environ. Exper. Botany, 43: 33-43. Prescott G.W. (1962). Algae of western great lakes area. W.M.C. Brown Co. Inc., Dubuque, Iowa, USA. Tanaka K., Mtsuhashi H., Kondo N., Sugahara K. (1982). Further evidence for inactivation of fructose-1,6- Prescott G.W. (1975). Algae of the western great lakes biphosphate at the beginning of SO2 fumigation: area. Department of Botany and Pathology, Michigan increase in fructose-1,6-biphosphate and decrease in State Univ. East Lansing, Michigan. fructose-6-phosphate in SO -fumigated spinach Rao M.V., Paliyath G., Ormrod D.P. (1996). Ultraviolet-B- leaves. Plant Cell Physiol., 23: 1467-1470. and ozone-induced biochemical changes in antioxidant Teramura A.H., Sullivan J.H. (1994). Effects of UV-B radi- enzymes of Arabidopsis thaliana. Plant Physiol., 110: ation on photosynthesis and growth of terrestrial 125-136. plants. Photosynth. Res., 39: 463-473. Sandalio L.M., Dalurzo H.C., Gómez M., Romero-Puertas van Hasselt P.R. (1974). Photo-oxidation of unsaturated M.C., del Rio L.A. (2001). Cadmium induces changes in lipids in cucumber leaf discs during chilling. Acta Bot. the growth and oxidative metabolism of pea plants. J. Neerl., 23: 159-169. Exp. Bot., 52: 2115-2126. Venkataraman G.S. (1969). The Cultivation of Algae. Schützendübel A., Nikolova P., Rudolf C., Polle A. (2002). Indian Council of Agricultural Research, New Delhi. Cadmium and H O -induced oxidative stress in 2 2 Populus canescens roots. Plant Physiol. Biochem., 40: Wetherell D.F. (1961). Culture of freshwater algae in 577-584. enriched natural seawater. Plant Physiol. (Copenh.), 14: 1-6. Shibata H., Baba K., Ochiai H. (1991). Near-UV irradiation induces shock proteins in Anacystis nidulans R-2: Yuasa T., Ichimjura K., Mizoguchi T., Shinozaki K. (2001). Possible role of active oxygen. Plant Cell Physiol., 32: Oxidative stress activates ATMPK6, an Arabidopsis 771-776. homologue of AMP kinase. Plant Cell Physiol., 42 (9): 1012-1016.
Annals of Microbiology – Springer Journals
Published: Nov 21, 2009
You can share this free article with as many people as you like with the url below! We hope you enjoy this feature!
Read and print from thousands of top scholarly journals.
Already have an account? Log in
Bookmark this article. You can see your Bookmarks on your DeepDyve Library.
To save an article, log in first, or sign up for a DeepDyve account if you don’t already have one.
Copy and paste the desired citation format or use the link below to download a file formatted for EndNote
Access the full text.
Sign up today, get DeepDyve free for 14 days.
All DeepDyve websites use cookies to improve your online experience. They were placed on your computer when you launched this website. You can change your cookie settings through your browser.