Journal of Life Sciences 6 (2012) 1307-1316

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
Irada Huseynova and Jalal Aliyev
Department of Fundamental Problems of Biological Productivity, Institute of Botany, Azerbaijan National Academy of Sciences, Baku AZ1073, Azerbaijan Received: July 28, 2012 / Accepted: October 10, 2012 / Published: December 30, 2012. Abstract: Viral diseases are an important limiting factor in many crop production systems in Azerbaijan. Symptomatic plants in main crop-producing areas were tested by ELISA (enzyme-linked immunosorbent assay) using specific monoclonal and polyclonal antibodies. Then RCA (rolling circle amplification) of circular DNA and PCR using different specific primer pairs have indicated that the tested symptomatic plant samples were completely infected by the following viruses: Luteovirus [BLRV (Bean leaf roll virus)], Potyviruses [BCMV (Bean common mosaic virus), BYMV (Bean yellow mosaic virus)], Bromovirus [(AMV) Alfa-alfa mosaic virus], Geminiviruses [CpCDV (Cickpea chlorotic dwarf virus) and TYLCV (Tomato yellow leaf curl virus)] and Nanoviruses [two different FBNYV (Faba bean necrotic yellow virus) and FBNSV (Faba bean necrotic stunt virus)]. At the same time generation sites of superoxide and hydrogen peroxide radicals and activity of antioxidant enzymes were studied in the naturally infected plants. Key words: Food crops, virus-like symptoms, viral diseases, molecular methods, reactive oxygen species, antioxidant enzymes.

1. Introduction
The ability to accurately detect and identify a potential plant pathogenic organism is fundamental to plant pathogen diagnostics and plant disease management for food quality. Increasing international travel and trade of plant materials enhances the risk of introducing new viruses and their vectors into production systems. In addition, changing climate conditions can contribute to a successful spread of newly introduced viruses or their vectors and establishment of these organisms in areas that were previously unfavorable [1, 2]. Viral diseases are also an important limiting factor in many crop production systems in our country. It causes extensive leaf yellowing, stem and leaf deformation, reduced fruit quality, substantial crop loss and shortening the life-span of vegetable crops [3].

Corresponding author: academician, research field: aliyev-j@botany-az.org.

Jalal plant

Aliyev, professor, physiology. E-mail:

The last four years infection incidences were determined for nine viruses on major food crops such as chickpea, lentil, faba bean, tomato, pea and alfa-alfa. The probable cause of decay of virus infected plants is not only the virus activity itself, but also the reduced tolerance to repeated unfavorable environmental situations [4]. Therefore, any little but long lasting defect in the biochemical process might have determinant role in limiting the lifetime of vegetables. Plants have evolved complex antioxidant systems in order to protect cellular membranes and organelles from the damaging effects of ROS (reactive oxygen species) [5]. Antioxidant enzymes and metabolites are located in different plant cell compartments to fulfill their protective function. The key enzymes, SODs (superoxide dismutases; EC 1.15.1.1), are a family of metalloenzymes catalyzing the dismutation of O2 to H2O2. SODs can be found in chloroplasts, mitochondria, peroxisomes, and in cytoplasm. CATs (Catalases; EC 1.11.1.6), heme proteins that catalyze the removal of H2O2, are located

1308

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

in peroxisomes. Enzymes and metabolites of the ascorbate-glutathione cycle (APO (ascorbate peroxidase; EC 1.11.1.11); GR (glutathione reductase, EC 1.8.1.7)) which is important in H2O2 scavenging are located in organelles and cytoplasm [5, 6]. Antioxidant enzymes were often studied at sites of attempted pathogen attack and in connection with immediate responses of invaded cells [7]. The main aim of this study is focused on detection of viral infections of vegetable crops by different molecular techniques, to find out generation sites of superoxide and hydrogen peroxide radicals in the naturally infected plants and to investigate the possible role of antioxidant systems against stress, in order to deepen our knowledge of the plant-virus interaction.

infection, the main crop-producing areas of Azerbaijan, fields of faba bean (Vicia faba L.), pea (Pisum sativum L.), chickpea (Cicer arietinum L.), tomato (Solanum lycoprsicum L.) and lentil (Lens culinaris L.) were surveyed in different regions (including Goychay, Nakhchivan, Masalli and Lerik) during the period of 2009-2011. Samples were collected from plants showing virus-like symptoms, such as leaf rolling, yellowing, mosaic, stunting, wilting, and shortening of the internodes, phloem discoloration, necrosis and stunted growth (Fig. 1). The number of samples collected in each field depended on the number of symptomatic plants observed. Virus-free plants for negative control were collected under same field conditions. Each field was evaluated using a standard format, recording location, conditions, development stage, virus disease symptoms, and presence or absence of the insect populations. Virus disease incidence in each field was determined on the basis of visual symptoms and by

2. Materials and Methods

2.1 Field Visits and Sample Collections To determine the presence or absence of virus

(a)

(b)

(c)

(d)

Fig. 1 Symptomatic Solanum lycoprsicum plants collected from Masalli fields associated with virus infestation and showing virus-like symptoms such as (a) leaf deformation and discoloration; (b) shortening the life-span, extensive leaf yellowing and stunting; (c) leaf curling; (d) destroyed and reduced fruit quality.

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

1309

counting the percentage of infected plants at different, randomly selected locations in the field. Collected plant samples with symptoms of potential virus infection were immediately frozen in liquid N2 and stored at -20 C. 2.2 Detection of Viruses Using ELISA Virus detection was performed with double and triple antibody sandwich ELISA (DAS-ELISA and TAS-ELISA) [8] using polyclonal and monoclonal antibodies (diluted 1:500 and 1:1000) for following viruses: BLRV (Bean leaf roll virus), BCMV (Bean common mosaic virus), BYMV (Bean yellow mosaic virus), AMV (Alfa-alfa mosaic virus), CpCDV (Cickpea chlorotic dwarf virus) and TYLCV (Tomato yellow leaf curl virus). All antibodies were kindly provided by Dr. S. Winter (DSMZ, Braunschweig, Germany). ELISA result was measured by recording its absorbance value using an ELISA plate reader (Stat Fax) at A405. Samples with absorbance values higher than the mean value for non-infected control plants plus two or three standard deviations were considered positive. 2.3 DNA Extraction and Molecular Analyses Plant samples that reacted serologically with the virus antibody were selected for further testing by RCA (rolling circle amplification) and PCR (polymerase chain reaction). Frozen plant samples were ground in liquid nitrogen, and DNA was prepared using the method of Edwards [9], modified as described by Grigoras [10]. The circular viral DNA was amplified by RCA using the TempliPhi Amplification Kit (GE HealthCare, UK). 29 DNA polymerase amplifies single- or double-stranded circular DNA templates by rolling circle amplification. Viral DNA for detection nanovirus infection was amplified by PCR using primer pairs F103/R101 and C5F/C5R, which yielded PCR products of the expected size (770 bp and 660 bp, respectively) [11]. Viral DNA extracted from infected plants was also

amplified by PCR using different specific primer pairs, which yielded PCR products of the expected size. The amplifications were carried out in an Applied Biosystems 2720 Thermal Cycler Biorad (SingAPOre) using the special program. Amplification products were resolved by electrophoresis in a 1.2% agarose gel with TAE buffer and stained with ethidium bromide (0.5 g/mL). The gels were photographed under UV light by Gel Documentation System UVITEK (UK). 2.4 Histochemical Staining of Superoxide Anion Radical Histochemical staining for ROS accumulation was conducted as described previously [12-14] with some modifications. For superoxide determination Petri dishes were used, the leaf samples were immersed in 6 mM NBT solution containing 50 mM sodium phosphate (pH 7.5) and 10 mM sodium azide for 12 h in the dark. ROS reaction was stopped by soaking the leaves with lacto-glycerol-ethanol (1:1:4 by vol.) and boiling in water for 5 min, and the cleared leaves were preserved in 50% ethanol and photographed. 2.5 Histochemical Staining of H2O2 To detect hydrogen peroxide, traverse sections of the leaf stem were cut by hand, and the detached leaves of the virus infected plants were deposited in Petri dishes containing a solution of 5 mM DAB and 10 mM MES at pH 3.8 for 12 h in darkness. To detect hydrogen peroxide, reaction was stopped by soaking the leaves with lacto-glycerol-ethanol (1:1:4 by vol) and boiling in water for 5 min, and the cleared leaves were preserved in 50% ethanol and photographed [12-14]. 2.6 Enzyme Extractions and Determination of the Activity Plants were excised and rapidly weighed (1 g fr wt). For all enzyme extracts leaf material was ground with

1310

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

a pestle and ice-cold mortar using different specific enzyme buffers. The homogenates were filtered through four layers of cheesecloth and then centrifuged at 4 C for 20 min at 15,000 g. The supernatant was collected and used for analyses of enzymatic activities. 2.6.1 CAT The activity of catalase was determined as a decrease in absorbance at 240 nm for 1 min following the decomposition of H2O2 as described by Kumar and Knowles [15]. The reaction mixture contained 50 mM phosphate buffer (pH 7.0) and 15 mM H2O2 and reaction was initiated by adding enzyme extract. 2.6.2 APO The activity of ascorbate peroxidase was assayed according to Nakano and Asada [16]. The assay mixture consisted of 0.05 mM ASA, 0.1 mM H2O2, 0.1 mM EDTA, 50 mM sodium phosphate buffer (pH 7.6), and 0.3 mL enzyme extract. The activity was measured as a decrease in absorbance at 290 nm for 30 s. 2.6.3 GR Glutathione reductase activity was determined at 340 nm for 10 min in 1 mL reaction mixture containing 100 mM potassium phosphate buffer (pH 7.8), 1 mM EDTA, 0.2 mM NADPH and 0.5 mM GSSG [17]. 2.6.4 SOD Superoxide dismutase activity was estimated by using SOD Assay Kit-WST (Sigma-Aldrich, USA). The absorbance was recorded at 450 nm and one enzyme unit of SOD activity was defined as the amount of enzime recuired to cause 50% inhibition of the rate of NBT reduction.

using 29 DNA polymerase and restricted by endonucleases AatII, xBaI, Sau3A, BamHI or HindIII [18-19]. All amplified products were resolved by agarose gel electrophoresis. Detection of virus infection in symptomatic samples was also performed by PCR method using different specific primer pairs [11]. The use of different molecular methods indicated that the tested symptomatic plant samples were completely infected by the following viruses: Luteovirus [BLRV (Bean leaf roll virus)], Potyviruses [BCMV (Bean common mosaic virus), BYMV (Bean yellow mosaic virus)], Bromovirus [AMV (Alfa-alfa mosaic virus)], Geminiviruses [CpCDV (Cickpea chlorotic dwarf virus) and TYLCV (Tomato yellow leaf curl virus)] and Nanoviruses [two different FBNYV (Faba bean necrotic yellow virus) and FBNSV (Faba bean necrotic stunt virus)]. It is important to note that additional pathogens can be expected on these crops. 3.2 Determination of Superoxide Anion As known, the level of plant resistance to viral diseases provides many physiological and biochemical parameters responsible for maintaining the viability and alterations in plant metabolism under stress conditions. On this basis, histochemical study of the possible presence of superoxide anion, H2O2 and activity of antioxidant enzymes in virus infected plant leaves were performed in the present work. ROS generation is a common feature in both incompatible and compatible plant-pathogen interactions. The oxidative burst observed in the initial stages of incompatible interactions [20] is responsible for the induction of defense reactions leading to hypersensitive responses and the development of SAR (systemic acquired resistance) [21]. A major defense mechanism in plants is HR (hypersensitive response) whereby cells infected with pathogens are instructed to self-destruction by the host plant. This is thought to deny nutrients to the invading pathogen. It is proposed that in plants the

3. Results and Discussion

3.1 Virus Infection All collected plant samples firstly were tested by enzyme-linked immunosorbent assay. Then DNA was extracted from the fresh leaves, amplified by RCA

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

1311

process is initiated by a reaction between NO (nitric oxide) and H2O2, which is formed by O2 (superoxide) dismutation by SOD during the HR. Plant-virus interaction may result in a host hypersensitive response or in systemic symptoms [22, 23]. One of the earliest responses of plant cells to pathogens is the production of ROS. The typical ROS detected are O2. (superoxide radicals) and H2O2 [24, 25]. ROS play a crucial role during pathogenesis. They are involved in the hypersensitive response typical for plant-pathogen incompatible interactions. They can limit the spread of pathogen by strengthening plant cell walls and/or by killing pathogens directly [26, 27]. However, ROS act as cytotoxic compounds, too. In this work, the authors detected the presence of superoxide anion O2 in places of infection with the use of NBT. Accumulation of insoluble blue-colored formazan complex (reduced NBT) is an indicator of generation of ROS, in particular of superoxide anion. This accumulation was observed in infected leaves after infiltration. Then staining declined rapidly, preceding the apparition of necrosis (Fig. 2). Histochemical staining for superoxide production in leaves tissues was based on the ability of O2 to reduce NBT and used to detect in situ the production of superoxide radicals [28]. Detached leaets from plants subjected to the viral diseases above described and their respective controls were immersed in sodium phosphate buffer (pH 7.8) containing 0.1% NBT and 10 mM sodium azide. Leaets of healthy plants were also inltrated with 50 mM sodium phosphate buffer (pH 7.8) containing only 10 mM sodium azide and used as control. Superoxide was visualized as a purple discoloration of NBT. Discoloration of leaf was quantified using a digital imaging system (Fig. 3). In typical incompatible interactions, one of the early events of HR is an oxidative burst with the generation of superoxide (O2) and the subsequent accumulation of H2O2 [21].
Fig. 3 Detection of H2O2 using DAB (3,3diaminobenzidine tetrahydrochloride) staining method. The reaction mixture contained 5 mM DAB in 10 mM MES buffer (pH 3.8). (a) Cucumis sativus L.; (b) Zea mays L.; (c) Solanum lycoprsicum L. Healthy leaves shown on the right panels, infected leaves shown on the left panels. The experiment was repeated two times. (c) (a)

(b)

(c)

Fig. 2 Detection of superoxide anion radical with NBT staining. Leaves were infiltrated after being submerging in a reaction mixture containing 6 mM NBT in 50 mM sodium phosphate buffer (pH 7.6) plus 10 mM sodium azide. (a) Cucumis sativus L.; (b) Zea mays L.; (c) Solanum lycoprsicum L. Healthy leaves shown on the right panels, infected leaves shown on the left panels. The experiment was repeated two times.

(a)

(b)

1312

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

3.3 Determination of H2O2 Accumulation of H2O2 was observed using DAB staining method. The reaction mixture contained 5 mM DAB in 10 mM MES (pH 3.8). Leaves were incubated overnight at 25 C, and then cleared twice in 50% (vv) ethanol for 10 min. DAB polymerizes to produce a brown precipitate on contact with H2O2 in the presence of peroxidase, and, thus, provides a useful marker of peroxide accumulation [29]. Superoxide anions are thought to be produced outside the plant cell by a plasma membrane-associated NAD(P)H oxidase, and are usually rapidly converted to H2O2 by superoxide dismutase. To examine whether H2O2 is also accumulated at the site of elicited HR, infected leaves were dipped in a solution of DAB. Fig. 3 clearly shows that H2O2 accumulated during the HR caused by virus infection. In higher plants, production of H2O2 is thought to be driven by increases in the concentrations of superoxide anions. However, a slightly lower level of DAB staining was observed in healthy leaves compared with infected leaves (Fig. 4). Thus, the results indicate that an alteration in the chloroplastic metabolism is produced during the early response to virus infection favoring the accumulation of ROS in the plants. 3.4 Analyses of the Antioxidant Enzymes Activity Plants have evolved complex antioxidant systems in order to protect cellular membranes and organelles from the damaging effects of ROS [30, 31]. Increase in peroxidase activity is also a response to viral infection, and has been reported in tobacco [32], peaches, apricots [33] and beans [34]. In plant cells, enzymes and redox metabolites act in synergy to carry out ROS detoxification. SOD catalyses the dismutation of O2 to H2O2, CAT dismutates H2O2 to oxygen and water, and APO reduces H2O2 to water by utilising ASC (ascorbate) as specific electron donor. These are considered the main enzymatic systems for protecting cells against oxidative damage. The balance between SOD and

APO or CAT activities in cells is crucial for determining the steady-state level of O2 and H2O2. Redox metabolites, such as ASC and the tripeptide GSH (glutathione), also protect plant cells against ROS-induced damage, either by directly removing reactive chemical species or blocking the oxidative chain reactions triggered by ROS. Results reported in the literature indicate that alteration in the expression/activity of ROS-scavenging enzymes could also be a key step in the activation of phytopathogen defence. On this base, the authors also studied the activities of the antioxidant enzymes, CAT, APO, SOD and GR, in viral infected plant leaves. These enzymes are known to be involved in an immediate plant defense response. Samples for activity measurements of the antioxidant enzymes were collected during the early stage of the infection, when the first visible symptoms of the virus infection appeared on the leaves (in early June). As shown in Fig. 4, the activity of antioxidant enzymes in infected leaves generally was higher than that of comparable healthy leaves. Analysis of CAT activity in infected leaves showed that this enzyme in all the samples studied had a significant difference compared with the control. CAT activity was 1.4-fold higher (up to 41%) in infected leaves of Solanum lycopersicum and 1.27-fold higher (up to 32%) in infected leaves of Vicia faba compared to the healthy plants. The most significant differences between the values of CAT activity were observed in infected Cicer arietinum samples, where the activity was 2.6-fold higher (up to 163%) compared to the control plants. In infected Lens culinaris and Pisum sativum samples, CAT activity only was slightly compared with the control. Analysis of CAT activity in infected Lens culinaris leaves showed that this enzyme activity did not differ significantly (only up to 17%) compared to the control and accounting 0.42 mmol/mg min, respectively. As shown in Fig. 4, CAT activity in infected Pisum sativum leaves did not seem to be affected significantly by viral stress.

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

1313

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

CAT activity (mMol/g min) APO activity (mMol/g min) GR activity (mMol/g min) SOD activity (mMol/g min)

1 0.8 0.6 0.4 0.2 0

0.6 0.5 0.4 0.3 0.2 0.1 0

0.4 0.3 0.2 0.1 0

Fig. 4 Activities of catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase in viral infected plant leaves.

APO activity was 1.2-fold higher in infected leaves of Solanum lycopersicum compared to the healthy plants and accounting 0.44 mmoL/mg min. As shown in Fig. 4, APO activity in infected leaves of Vicia faba and Cicer arietinum slightly increased (up to 24% and 16%, respectively), although the activity of CAT

significantly differed compared to the control. The most significant differences between the activities of APO and CAT were observed in infected Lens culinaris and Pisum sativum samples, where the APO activity was 2.2 and 1.5-fold higher compared to the control plants. This may indicate the existing

1314

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

functional relationship and competition between the studied enzymes under these viral diseases. Other works also suggest that, along with the activation of SOD and APO in the leaves, a sharp decrease in activity of CAT is observed, which may be due to inhibition of the enzyme substrateH2O2 Interestingly, it has been reported that in a compatible response between barley and powdery mildew the cytosolic isoenzyme of APO is up-regulated in both epidermal and mesophyll cells. In these cells, that are not able to trigger a response to stop pathogens, the APO increase limits the propagation of oxidative processes allowing cells to maintain their viability, a condition required for the penetration of biotrophic powdery mildew in plant tissues [35]. This up-regulation of APO confirms previous results reporting an increase in APO activity during successful infection of barley leaves by biotrophic compatible pathogens [36-38] and has also been reported to occur in leaves of susceptible apricot infected by plum pox virus [39]. CAT activity has also been reported to decrease in cells undergoing HR. However, the suppression mechanisms of these two H2O2 scavenging enzymes are different. CAT is down-regulated at the transcription level [40], whereas, APO regulation in HR involves both transcription and translation (or posttranslation) processes. In tobacco leaves, inoculated with TMV (tobacco mosaic virus), a rise in APO mRNA occurs [41], probably as an antioxidant response triggered by the increasing presence of H2O2 within cells and similar to that activated under abiotic stress [42]. In spite of the increase in its expression, the activity of the enzyme is strongly suppressed in the TMV-infected cells by a mechanism, still not well characterized, that acts at the transcriptional or post-transcriptional level [43]. In this case, the high activity of antioxidant enzymes can probably be one of the markers of resistance to the pathogen. The effects of a viral infection caused an increase in GR activity about 1.86-fold higher (up to 72%) and

accounting 0.28 mmoL/mg min in infected leaves of Solanum lycopersicum compared to the healthy plants. GR activity in infected samples of Vicia faba increased up to 17% compared to the control. In contrast, the GR activity in infected samples of Cicer arietinum and Lens culinaris did not significantly differ from the control plants. As shown in Fig. 4, the most significant difference between the GR activities were observed in infected Pisum sativum samples, where activity increased approximately 2-fold higher, i.e. up to 96% compared to the control plants. As shown in Fig. 4, the change of SOD activity in infected plant leaves was different, the actives of Cu/Zn-SOD decreased (up to 46% and 22%) in infected samples of Cicer arietinum and Lens culinaris compared to the healthy plants. The most interesting value of SOD activity observed in infected Pisum sativum samples, where the activity did not differ from the control and accounted 0.22 unit/mg proteins. Obviously, the activation of antioxidant defence systems in plants by abiotic and biotic stresses is a general phenomenon and probably contributes to increased resistance against a subsequent stress.

4. Conclusion
The following viruses on major food crops collected from different regions of Azerbaijan: Luteovirus [BLRV (Bean leaf roll virus)], Potyviruses [BCMV (Bean common mosaic virus), BYMV (Bean yellow mosaic virus)], Bromovirus [AMV (Alfa-alfa mosaic virus)], Geminiviruses [CpCDV (Cickpea chlorotic dwarf virus) and TYLCV (Tomato yellow leaf curl virus)] and Nanoviruses [two different FBNYV (Faba bean necrotic yellow virus) and FBNSV (Faba bean necrotic stunt virus)] were detected in this study. ROS generation is a common feature in both incompatible and compatible plant-pathogen interactions. On this basis, histochemical evaluation of the possible presence of superoxide anion, H2O2 and activity of antioxidant enzymes in virus infected plant leaves were performed

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan

1315

in the present work. Results reported in this study also indicate that the alteration in the expression/activity of ROS-scavenging antioxidant enzymes could also be a key step in the activation of phytopathogen defence. The ability to maintain high physiological function in the presence of viruses may be a more important resistance or tolerance mechanism than actual avoidance of infection. The information presented in the current study is important because the better understanding of the mechanisms behind the viral impact on host plant physiology can lead to the development of improved cultivars that either resist viral infection or can better tolerate infection by experiencing less severe symptoms.

[9]

[10]

[11]

[12]

References
[1] V. Ortiz, E. Navarro, S. Castro, G. Carazo, J. Romero, Incidence and transmission of faba bean necrotic yellows virus (FBNYV) in Spain, Spanish Journal of Agricultural Research 4 (2006) 255-260. H.J. Vetten, P.W.G. Chu, J.L. Dale, R. Harding, J. Hu, L. Katul, et al., Nanoviridae, in: C.M. Fauquet, M.A. Mayo, J. Manoloff, U. Desselberger, L.A. Ball (Eds.), Virus Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses, Elsevier, London, 2005, pp. 343-452. A.F. Bent, Plant disease resistance genes: Function meets structure, Plant Cell 8 (1996) 1757-1771. J. Stanley, D.M. Bisaro, R.W. Briddon, J.K. Brown, C.M. Fauquet, B.D. Harrison, et al., Family Geminiviridae, in: C.M. Fauquet, M.A. Mayo, J. Manoloff, U. Desselberger, L.A. Ball (Eds.), Virus Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses, Elsevier, London, 2005, pp. 301-326. S.H. Lee, N. Ahsan, K.W. Lee, D.H. Kim, D.G. Lee, S.S. Kwak, et al., Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses, Journal of Plant Physiology 164 (2007) 1626-1638. J.L. Dangl, J.D.G. Jones, Plant pathogens and integrated defence responses to infection, Nature 411 (2001) 826-833. R. Goldbach, E. Bucher, M. Prins, Resistance mechanisms to plant viruses: An overview, Virus Research 92 (2003) 207-212. M.F. Clark, A.N. Adams, Characteristics of the microplate method of enzyme-linked immunosorbent [13]

[2]

[14]

[15]

[3] [4]

[16]

[17]

[5]

[18]

[19]

[6]

[7]

[20]

[8]

assay for the detection of plant viruses, Journal of General Virology 34 (1977) 475-483. K. Edwards, C. Johnstone, C. Thompson, A simple and rapid method for the preparation of plant genomic DNA for PCR analysis, Nucleic Acids Research 19 (1991) 13-49. Grigoras, T. Timchenko, L. Katul, A. Grande-Prez, H.J. Vetten, B. Gronenborn, Reconstitution of authentic nanovirus from multiple cloned DNAs, Journal of Virology 83 (2009) 10778-10787. I.M. Huseynova, N.F. Sultanova, A.Ch. Mammadov, Detection of single-stranded DNA plant viruses in vegetable plants using PCR method, Proceedings of ANAS 66 (2) (2011) 5-12. M.J. Fryer, L. Ball, K. Oxborough, S. Karpinski, P.M. Mullineaux, N.R. Baker, Control of ascorbate peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves, Plant Journal 33 (2003) 691-705. T. Kariola, G. Brader, E. Helenius, J. Li, P. Heino, E.T. Palva, Early responsive to dehydration 15A negative regulator of ABA-responses in Arabidopsis, Plant Physiology 142 (2006) 1559-1573. R. Mahalingam, N. Shah, A. Scrymgeour, N. Fedoroff, Temporal evolution of the Arabidopsis oxidative stress response, Plant Molecular Biology 57 (2005) 709-730. C.N. Kumar, N. Knowles, Changes in lipid peroxidation and lipolytic and free-radical scavenging enzyme during aging and sprouting of potato (Solanum tuberosum L.) seed-tubers, Plant Physiology 102 (1993) 115-124. Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts, Plant Cell Physiology 22 (1981) 867-880. G.G. Yannarelli, A.J. Fernandez-Alvarez, Glutatione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress, Phytochemistry 68 (2007) 505-512. N.F. Sultanova, I.M. Huseynova, A.Ch. Mammadov, Detection and identification of main virus infection in vegetable plants by sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Reports of ANAS LXVII (5) (2011) 113-122. I.M. Huseynova, A.Ch. Mammadov, N.F. Sultanova, J.A. Aliyev, First report of circular single-stranded DNA viruses of vegetables in Azerbaijan: Fast definition by RCA (rolling circle amplification) method and molecular characterization, Reports of ANAS LXV (6) (2009) 106-114. T. Jabs, A.J. Slusarenko, The hypersensitive response, in: A.J. Slusarenko, R.S.S. Frazer, L.S. van Loon (Eds.), mechanisms of resistance to plant diseases, Dordrecht, Kluwer, 2000, pp. 279-324.

1316

Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan Physiology 84 (1987) 438-442. [33] P. Daz-Vivancos, M. Rubio, V. Mesonero, P.M. Periago, A. Ros Barcelo, P. Martnez-Gomez, et al., The APOplastic antioxidant system in Prunus: Response to long-term plum pox virus infection, Journal of Experimental Botany 57 (2006) 3813-3824. [34] D.E.M. Radwan, K.A. Fayez, S.Y. Mahmoud, G. Lu, Modifications of antioxidant activity and protein composition of bean leaf due to bean yellow mosaic virus infection and salicylic acid treatments, Acta Physiologia Plantarum 32 (2010) 891-904. [35] K. Burhenne, L. Gregersen, Up-regulation of the ascorbate-dependent antioxidative system in barley leaves during powdery mildew infection, Molecular Plant Pathology 1 (2001) 303-314. [36] H.M. El-Zahaby, G. Gullner, Z. Kiraly, Effects of powdery mildew infection of barley on the ascorbate-glutathione cycle and other antioxidant in different host-pathogen interactions, Phytopathology 85 (1995) 1225-1230. [37] E. Kuzniak, M. Sklodowska, The effect of Botrytis cinerea infection on ascorbate glutathione cycle in tomato leaves, Plant Science 148 (1999) 69-76. [38] H.Vanacker, T.L.W. Carver, C.H. Foyer, Pathogen-induced changes in the antioxidant status of the apoplast in barley leaves, Plant Physiology 117 (1998) 1103-1114. [39] J.A. Hernndez, J.M. Talavera, P. Martinez-Gomez, F. Dicenta, F. Sevilla, Response of antioxidative enzymes to plum pox virus in two apricot cultivars, Physiologia Plantarum 111 (2001) 313-321. [40] S. Dorey, F. Bailleul, P. Saindrenan, B. Fritig, S. Kauffmann, Tobacco class I and II catalases are differentially expressed during elicitorinduced hypersensitive cell death and localized acquired resistance, Molecular Plant-Microbe Interactions 11 (1998) 1102-1109. [41] R. Mittler, E. Lam, V. Shulaev, M. Cohen, Signal controlling the expression of cytosolic ascorbate peroxidase during pathogeninduced programmed cell death in tobacco, Plant Molecular Biology 39 (1999) 1025-1035. [42] Kubo, H. Saji, K. Tanaka, N. Kondo, Expression of Arabidopsis cytosolic ascorbate peroxidase gene in response to ozone of sulfur dioxide, Plant Molecular Biology 29 (1995) 479-489. [43] R. Mittler, X. Feng, M. Cohen, Post-transcriptional suppression of cytosolic ascorbate peroxidase expression during pathogen-induced programmed cell death in tobacco, Plant Cell 10 (1998) 461-473.

[21] P. Wojtaszek, Oxidative burst: An early plant response to pathogen infection, Biochemistry Journal 322 (1997) 681-692. [22] K.E. Hammond-Kosack, J.D.G. Jones, Responses to plant pathogens, in: B.B. Buchanan, W. Gruissem, R.L. Jones (Eds.), Biochemistry & Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Maryland, 2000, pp. 1102-1156. [23] J.A. Hernndez, J.M. Talavera, P. Martnez-Gmez, F. Dicenta, F. Sevilla, Response of antioxidative enzymes to plum pox virus in two apricot cultivars, Physiologia Plantarum 111 (2001) 313-321. [24] I.E. Johansen, O.S. Lund, C.K. Hjulsager, J. Laursen, Recessive resistance in Pisum sativum and potyvirus pathotype resolved in gene-for-cistron correspondence between host and virus, Journal of Virology 75 (2001) 6609-6614. [25] E. Kombrink, E. Schmelzer, The hypersensitive response and its role in local and systemic disease resistance, European Journal of Plant Pathology 107 (2001) 69-78. [26] M.R. Sahoo, M. DasGupta, P.C. Kole, J.S. Bhat, A. Mukherjee, Antioxidative enzymes and isozymes analysis of taro genotypes and their implications in Phytophthora blight disease resistance, Mycopathologia 163 (2007) 241-248. [27] S.M. Salazar, A.P. Castagnaro, M.E. Arias, N. Chalfoun, U. Tonello, J.C. Daz Ricci, Induction of a defense response in strawberry mediated by an avirulent strain of Colletotrichum, European Journal of Plant Pathology 117 (2006) 109-122. [28] K.T. Leath, L.J. Rowell, Histological study of resistance of Zea mays to Puccinia graminis, Phytopathology 56 (1966) 305-1309. [29] C. Rusterucci, D.H. Aviv, B.F. Holt, J.L. Dangl, J.E. Parker, The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis, Plant Cell 13 (2001) 2211-2224. [30] V. Paranidharan, A. Palaniswami, P. Vidhyasekaran, R. Velazhahan, Induction of enzymatic scavengers of active oxygen species in rice in response to infection by Rhizoctonia solani, Acta Physiologia Plantarum 25 (2003) 91-96. [31] Z. ubr, S. Novkov, H. Drahovsk, Detection of transgene copy number by analysis of the T1 generation of tobacco plants with introduced P3 gene of Potato virus A, Acta Virology 50 (2006) 135-138. [32] M.L. Lagrimini, S. Rothstein, Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection, Plant