Salicylic acid alleviates growth inhibition and oxidative stress caused by zucchini yellow mosaic virus infection in Cucurbita pepo leaves

ARTICLE IN PRESS

Physiological and Molecular Plant Pathology 69 (2006) 172–181 www.elsevier.com/locate/pmpp

Salicylic acid alleviates growth inhibition and oxidative stress caused by zucchini yellow mosaic virus infection in Cucurbita pepo leaves Deya Eldeen Mohammed Radwana,b, Khalaf Ali Fayezb, Sabry Younis Mahmoudc, Ahmed Hamadd, Guoquan Lua, a

College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China b Botany Department, Faculty of Science, Sohag University, Sohag 82524, Egypt c Virology Department, Faculty of Agriculture, Sohag University, Sohag 82786, Egypt d Botany Department, Faculty of Science, Assiut University, Assiut, Egypt Accepted 18 April 2007

Abstract The effects of zucchini yellow mosaic virus (ZYMV) infection and pretreatments with salicylic acid (SA) on biomass accumulation of pumpkin (Cucurbita pepo cv. Eskandarani) were investigated. The response of photosynthesis, transpiration and the activities of antioxidant enzymes in leaves was also considered. Significant reductions in growth parameters (i.e. leaf area, biomass and shoot height), photosynthesis and chlorophyll a and b content were detected in ZYMV-infected leaves in comparison to healthy controls. Antioxidant enzyme activities were increased up to 3-fold for peroxidase (POD), 2-fold for ascorbate peroxidase (APX) and catalase (CAT) activities and 1.3-fold for SOD activity by virus infection. ZYMV infection also caused increases in H2O2 and malondialdehyde (MDA) contents. These results suggest that ZYMV infection causes oxidative stress in pumpkin leaves leading to the development of epidemiological symptoms. Interestingly, spraying pumpkin leaves with SA led to recovery from the undesirable effects of ZYMV infection. Leaves treated with 100 mM SA three days before inoculation had the appearance of healthy leaves. No distinct disease symptoms were observed on the leaves treated with 100 mM SA followed by inoculation with ZYMV. In non-infected plants, SA application increased activities of POD and superoxide dismutase (SOD) and inhibited APX and CAT activities. In contrast, SA treatment followed by ZYMV inoculation stimulated SOD activity and inhibited activities of POD, APX and CAT. In addition, MDA displayed an inverse relation, indicating inhibition of lipid peroxidation in cells under SA treatment. It is suggested that the role of SA in inducing plant defense mechanisms against ZYMV infection might have occurred through the SA-antioxidant system. Such interference might occur through inhibition or activation of some antioxidant enzymes, reduction of lipid peroxidation and induction of H2O2 accumulation following SA application. r 2007 Elsevier Ltd. All rights reserved. Keywords: Leaf growth; Photosynthesis rate; Pigments; Pumpkin; Salicylic acid; Zucchini yellow mosaic virus; Antioxidant enzyme activity; Gas exchange

1. Introduction Zucchini yellow mosaic virus (ZYMV) is one of the most important viruses affecting cucurbit production globally. It Abbreviations: Chl, chlorophyll; POX, peroxidase; SA, salicylic acid; SA+V, treated with salicylic acid then inoculated with virus; ZYMV, Zucchini yellow mosaic virus; E, transpiration rate; Ci, intercellular CO2 concentration; Gs, stomatal conductance; WUE, water use efficiency Corresponding author. Tel./fax: +86 571 86971117. E-mail addresses: [email protected] (D.E.M. Radwan), [email protected] (G. Lu). 0885-5765/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2007.04.004

causes destructive diseases on a large variety of economically important cucurbits including zucchini squash (Cucurbita pepo) [1]. It is efficiently transmitted by several aphid species in a non-persistent mode. The disease can completely destroy the crop under favorable conditions leading to yield loss. Moreover, ZYMV is widely spread in many countries including Egypt and is considered a serious problem in the major cucurbit producing regions worldwide. Resistance to ZYMV has not yet been established in C. pepo germplasm collections. Therefore, ZYMV resistance has been introduced into zucchini squash

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commercial cultivars through crosses with Cucurbita moschata [2]. Several chemicals have been reported as resistance inducers in plants [3]. Among these chemicals, salicylic acid (SA) is considered one of the key components of defense signal transduction, inducing the full set of systemic acquired resistance genes [4]. Moreover, exogenous application of SA may influence some physiological processes such as seed germination and fruit yield [5], transpiration rate [6], stomatal closure [7], membrane permeability [8], growth and photosynthesis [9–13] and antioxidant capacity [14]. Thus, SA may affect plant defense mechanisms against harmful diseases. SA generally, can control both biotic and abiotic defense programs [15]. Nowadays, the importance of SA in pathogen-induced disease resistance is well documented [4,16]. To our knowledge, the use of SA against viral infections in zucchini squash (C. pepo) is still limited. Certain biochemical and physiological changes associated with virus infection include the oxidative burst [17], growth inhibition, changes in pigment content, chlorophyll synthesis and antioxidant enzyme systems [18]. Altered active oxygen species (AOS) formation occurs under biotic and abiotic stress [19]. This alteration causes direct or indirect damage to plants leading to the appearance of epidemiological symptoms. Plants have a set of enzymes and redox metabolites that carry out AOS detoxification and known as antioxidant systems. The main antioxidant enzymes are superoxide dismutase (SOD) catalyzing the dismutation of O 2 to H2O2, catalase (CAT) that dismutates H2O2 to oxygen and water, and ascorbate peroxidase (APX) that reduces H2O2 to water by utilizing ascorbate as specific electron donor. Moreover, other enzymes involved in the ascorbate– glutathione ASC–GSH cycle [glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR)] as well as glutathione peroxidase (GPX) and glutathione-S-transferases, are important in protecting cells against oxidative stress. These enzymes are present in different isoforms in several cell compartments and their expression is genetically controlled and regulated both by developmental and environmental stimuli, according to the necessity to remove AOS produced in cells [20–22]. The balance between SOD and APX or CAT activities in cells is crucial for determining the steady-state level of O 2 and H 2O 2. Reported data indicate that under abiotic and biotic stress conditions, which induce an over-production of AOS in cells, plant resistance is due to the capability to increase the activity of AOS-detoxifying enzymes or the biosynthesis or regeneration of antioxidant metabolites [19,21,23,24]. It is well established that plant viruses affect many physiological processes such as photosynthesis [20] by decreasing the photosynthesis rate (depending on the infection stage), decreasing pigment contents [18,25],

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increasing or decreasing the respiration rates and altered gas exchange relations [26,27]. Although there are many reports concerning the change in physiological properties after virus infection, there is limited information on physiological responses in plants infected by ZYMV. To our knowledge, no information is available on the effects of ZYMV infection and exogenous SA on transpiration, gaseous exchange, photosynthesis rate and antioxidant enzyme activities. Therefore, the main objectives of the present study are to investigate the effects of ZYMV infection and SA treatments on plant growth, oxidative stress, the response of certain antioxidant enzymes, photosynthetic properties and biomass accumulation in pumpkin (C. pepo cv. Eskandarani) leaves. 2. Materials and methods 2.1. Plant materials Seeds of pumpkin (C. pepo cv. Eskandarani) were sown in a mixture of sand and clay (1:2, v/v) in plastic pots (10 cm in diameter) in separate growth chambers under a photoperiod of 12 h. Day and night temperatures were 24 and 18 1C, respectively; the relative humidity was about 70%. The plants were kept at 100% water holding capacity. 2.2. SA treatments and virus inoculation After 21 days of growth, plants were divided into seven groups. Each group comprised four replications. The first group was considered as a control without any treatment. Second, third and fourth groups were treated with 10, 50 and 100 mM of SA respectively by spraying the leaves until run-off. The control was sprayed with water. To improve spread on leaves, two drops of Tween 80 were added prior to spraying. Inoculation of virus was performed 3 days after spraying. All leaves were mechanically inoculated. The fifth group was inoculated with ZYMV at the same time of inoculation of the other groups. The sixth group (SA+V ino) was inoculated with a mixture of virus inoculum and 100 mM SA solution (1:1). The last group was sprayed with 100 mM SA without inoculation. The inoculum was prepared from infected top leaves, ground in a mortar containing 0.1 M phosphate buffer pH 7.0 (1:2, w/v); the homogenate was filtrated through two layers of muslin, and the leaves of healthy plants were dusted with carborundum, and rubbed gently with a cotton swab previously dipped into the suspension of virus inoculum. Two weeks after inoculation, the youngest fully developed leaves from both control and treated plants were collected for analysis of biochemical changes. 2.3. Chlorophyll content and photosynthetic gas exchange analyses Fifteen days after inoculation with virus, the leaves with similar age were harvested and ground in 85% acetone to

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extract the pigments. Contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) were determined spectrophotometrically according to Metzner et al. [28]. Chlorophyll contents of infected leaves, SA treated and inoculated after treatment with SA were then compared to the control. Photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (E) were determined by using an Infra Red Analyzer (LI6400 System, Li-COR Company Lincoln, NE). The values presented represent the means of eight replicates. 2.4. Antioxidant enzyme activity analyses For analyses of enzyme activity, leaves were homogenized with a mortar and pestle under chilled conditions in a buffer solution specific for each enzyme. The homogenate was filtered through four layers of muslin cloth and centrifuged at 12 000 rpm for 10 min at 4 1C. The supernatants were used for enzyme assays. CAT (EC 1.11.1.6) and peroxidase (POD, EC 1.11.1.7) activities were measured according to Chandlee and Scandalios [29] and Zhang [30], respectively, with some modifications. For POD, the reaction mixture consisted of 50 mM potassium phosphate buffer (pH 6.1), 1% guaiacol, 0.4% H2O2 and enzyme extract. Increase in the absorbance due to oxidation of guaiacol ðE ¼ 25:5 mM1 cm1 Þ was measured at 470 nm. Enzyme activity was calculated in terms of mmol of guaiacol oxidized min1 g1 fresh weight at 2572 1C. CAT activity was measured by monitoring the disappearance of H2O2 by measuring the decrease in absorbance at 240 nm (E ¼ 0.036 mM1 cm1) of a reaction mixture consisting of 25 mM potassium phosphate buffer (pH 7.0), 10 mM H2O2 and enzyme extract. APX (EC 1.11.1.11) was determined according to Nakano and Asada [31] and with modified procedure of Hema`ndez et al. [32]. The decrease in ascorbate concentration was followed by decline in absorbance at 290 nm and activity was calculated using the extinction coefficient (E ¼ 2.8 mM1 cm1 at 290 nm) for ascorbate. SOD (EC 1.15.1.1) activity was assayed according to Beauchamp and Fridovich [33] with some modifications. The samples (0.5 g) were homogenized in 5 ml extraction buffer consisting of 50 mM phosphate, pH 7.8, 0.1% (w/v), BSA, 0.1% (w/v) ascorbate, 0.05% (w/v) b-mercapto ethanol. The assay mixture in 3 ml contained 50 mM phosphate buffer (pH 7.8), 9.9 mM L-methionine, 57 lM NBT, 0.025% (w/v) Triton X-100 and 0.0044% (w/v) riboflavin. The photo-reduction of NBT (formation of purple formazan) was measured at 560 nm. One unit of SOD activity was defined as extract volume that caused 50% inhibition of the photo-reduction of NBT. 2.5. H2O2, MDA contents and antioxidant activity H2O2 content was determined colorimetrically as described by Jana and Choudhuri [34]. H2O2 was extracted by homogenizing 0.5 g leaf tissue with 3 ml phosphate

buffer (50 mM, pH 6.5). The homogenate was centrifuged at 6000 rpm for 25 min. To determine H2O2 level, 3 ml of extracted solution was mixed with 1 ml of 0.1% titanium sulfate in 20% H2SO4. The mixture was then centrifuged at 6000 g for 15 min. The intensity of the yellow color of the supernatant at 410 nm was measured. H2O2 level was calculated using the extinction coefficient (E ¼ 0.28 mmol cm1). Lipid peroxidation was expressed as malondialdehyde (MDA) content and was determined as 2thiobarbituric acid (TBA) reactive metabolites according to Zhang [30]. Lipid peroxidation was expressed as nmol (g fresh weight)1 by using an extinction coefficient (E ¼ 155 mM cm1). Antioxidant activity (AOA) was calculated according to Arabshahi et al. [35] and expressed as percentage inhibition of lipid peroxidation relative to the control using absorbance at 532 nm obtained from MDA analysis according to the following equation: AOA activity ð%Þ absorbance of control  absorbance of sample  100. ¼ absorbance of control

2.6. Statistical analysis All data were subjected to ANOVA test and means were compared by two conventional methods of analysis. The LSD values for significant mean differences at levels Po0.05 and Po0.01 were separated. The data were also statistically analyzed by Duncan’s range test to estimate the differences among the variants [36,37]. All statistics tests were carried out using SPSS program. 3. Results 3.1. Morphological and growth parameters Tables 1 and 2 show the effects of ZYMV infection and SA treatments with or without virus inoculation on the leaf area, plant height, biomass and water contents of pumpkin leaves (C. pepo cv. Eskandarani). Growth inhibition was detected in response to ZYMV infection which was indicated by reduction of leaf area, biomass and plant height by 81%, 28.9% and 37%, respectively of corresponding control values. In contrast, leaf water content seemed to be unaffected by ZYMV infection. SA application aided recovery from the pathological effects caused by virus infection especially with 100 mM (SA+V) level (Fig. 1). Plants treated with 100 mM SA had leaf area and plant height more or less similar to controls. Moreover, biomass accumulation seemed to be induced by SA application regardless of viral infection. From the results in Table 2, we can observe the maximum increase in biomass was detected in the plants treated with 100 SA without virus inoculation. At the same time, plants treated with 100 SA+V showed an increase in biomass

ARTICLE IN PRESS D.E.M. Radwan et al. / Physiological and Molecular Plant Pathology 69 (2006) 172–181 Table 1 Effect of ZYMV infection and SA treatments on leaf area (cm2 plant1) and plant height (cm) of C. pepo (cv. Eskandarani) leaves Treatments

Control Infected 10 mM SA+V 50 mM SA+V 100 mM SA +V 100 mM SA +V ino 100 mM SA

Leaf area (cm2 plant1)

Plant height (cm)

M7SD

%

M7SD

%

161.03a732.67 30.36**c72.53 63.10**c79.37 104.04*b716.19 106.36*b725.50 127.15a,b721.95 155.10a73.56

100 18.86 39.19 64.61 66.05 78.96 96.32

19.11a,b,c72.04 12.01**d70.57 16.02c70.69 17.18b,c71.25 20.64a,b72.27 14.01c70.62 21.19a73.23

100 62.84 86.42 89.92 108.05 87.50 110.93

Statistical significance of differences compared to control: *Po0.05; **Po0.01. Duncan’s composition between variants at a ¼ 0.05. Variants with the same letters show the same reaction. The values are means (M) of four replicates7standard deviation (SD).

Table 2 Effect of ZYMV infection and SA treatments on biomass (g plant1) and water content (%) of C. pepo (cv. Eskandarani) leaves Treatments

Control Infected 10 mM SA+V 50 mM SA+V 100 mM SA +V 100 mM SA +V ino 100 mM SA

Leaf dry weight (g plant1)

Water content (%)

M7SD

%

M7SD

0.38b,c70.02 0.27**d70.004 0.33c,d70.03 0.38b,c70.05 0.41b,c70.05 0.43a,b70.04 0.48**a70.06

100 69.83 87.04 97.83 106.26 111.22 125.57

93.98a70.93 93.86a70.83 91.42b70.63 92.18a,b70.07 90.85*b71.10 90.81*b70.85 91.76b70.97

Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0.05. Variants with the same letters show the same reaction. The values are means (M) of four replicates7standard deviation (SD).

accumulation. These results indicate that ZYMV could cause serious disease leading to growth reduction and morphological leaf modification which could be alleviated by exogenous application of SA. 3.2. Chlorophyll content and photosynthesis rate Pumpkin leaves infected with ZYMV showed a significant reduction in photosynthetic rates (Pn) and chlorophyll content (see Table 3). The reduction in chlorophyll content clearly appeared as the development of leaf mosaics and disease symptoms which was confirmed by the chemical analysis of the chlorophyll (a+b) concentration and Pn. Chlorophyll (a+b) concentration and Pn were reduced by 45% and 52.5%, respectively, in comparison to corresponding controls. An alleviation of ZYMV severe infection effects was evidenced by alteration in Pn and chlorophyll contents at 50 and 100 mM (SA+V) levels. Compared with control, the values of Pn showed no significant difference for 50 and

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100 mM (SA+V) treated leaves. Similarly, chlorophyll (a+b) concentration decreased slightly but not significantly at 50 mM (SA+V) level. In all SA treatments, chlorophyll a/b ratio was higher than healthy controls. The increase in a/b ratio is due to the decrease in Chl b content. So, Chl b is more sensitive to ZYMV infection and SA treatments than Chl a. Moreover, leaf Pn and chlorophyll (a+b) concentration seemed to be unaffected by 100 mM SA without further virus inoculation (Table 3). 3.3. Antioxidant enzyme activities The analyses of four antioxidant enzymes activities, SOD, CAT, POD and APX, revealed many changes due to ZYMV infection and SA treatments. ZYMV infection caused significant increases in enzyme activities up to 3-fold in the case of POD, 2-fold for APX and CAT activities and 1.3-fold greater than controls for SOD activity (Tables 4 and 5). On the other hand, the effect of SA levels on antioxidant enzyme activities differed markedly with or without virus inoculation. For example, with increasing SA dose (10–100 mM SA+V), the activities of POD, APX and CAT decreased markedly while SOD activity increased. Interestingly, although there was a decrease in POD and APX activities, the values of POD were still higher than controls, although APX was lower than the control. In contrast, SOD activities increased gradually with increasing SA dose and the values were significantly higher than respective controls. It seems that spraying SA and virus inoculation counteracted the enhancement of SOD and inhibition of CAT activities. Without virus inoculation, spraying 100 mM SA to the leaves increased POD and SOD activities by 141.37% and 111.78%, respectively. In contrast, the APX and CAT were inhibited in response to SA spraying and their activities were reduced by 36.19% and 46.25% of the control, respectively. 3.4. MDA and H2O2 contents Due to ZYMV infection, MDA and H2O2 concentrations increased significantly in pumpkin (C. pepo cv. Eskandarani) leaves. This increase led to increases of 54.2% and 30.08% in H2O2 and MDA contents, respectively, compared with the control (Table 6). Regardless of inoculation, SA dramatically increased H2O2 while resulting in decreased MDA contents in pumpkin. After virus inoculation, SA-treated plants showed decreases in MDA for all SA concentrations, which were lower than respective controls. The highly significant decrease of MDA was noticed for 100 mM (SA+V) and 100 mM (SA+V inoculated together) treatments. All (SA+V) treatments caused increases in H2O2 content. For instance, 100 mM (SA+V) increased of

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Fig. 1. Effect of ZYMV infection SA and (SA+V) treatments on leaf morphology of pumpkin (C. pepo cv. Eskandarani). ZYMV-infected old leaf (a) showing severe mosaic and young leaf (b) showing chlorosis, green blisters, size reduction and malformation. (c–e) leaves treated with 10, 50 and 100 mM (SA+V) respectively, (f) sprayed with 100 mM (SA+V ino). All SA treatments caused decrease in disease severity and the symptoms totally disappeared following treatment with 100 mM (SA+V). Control leaf (g) without any treatment and (h) treated with 100 mMSA.

H2O2 content by 36.13% in comparison with the control. In other words, H2O2 and MDA contents were SA-level dependent. Without virus inoculation, SA treatment increased H2O2 by 27.73% compared to controls, while MDA content

decreased by 11.81% compared to controls (Table 6). The AOA of leaf extracts were calculated on the basis of relative amounts of MDA, which indicated lipid peroxidation, with respect to control MDA concentration. It was found that AOA varied greatly among the treatment levels.

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Table 3 Effect of ZYMV infection and SA treatments on photosynthetic rate (mmol m2 s1) and chlorophyll content (mg g1 FW) of C. pepo (cv. Eskandarani) leaves Treatments

Control Infected 10 mM SA+V 50 mM SA+V 100 mM SA +V 100 mM SA +V ino 100 mM SA

Net photosynthesis rate (Pn) (mmol m2 s1)

Chlorophyll content (mg g1 FW)

M7SD

%

M7SD

%

a/b ratio

17.48a70.94 8.30**d70.73 12.53**c72.81 15.18a,b,c72.59 16.85a,b71.26 14.35*b,c72.60 16.55a,b70.35

100 47.50 71.67 86.84 96.42 82.12 94.71

0.60a70.08 0.27**c70.01 0.35**c70.03 0.48*b70.02 0.54a,b70.08 0.60a70.01 0.62a70.01

100.00 45.00 58.30 80.00 90.00 100.00 103.33

2.30 2.75 2.52 3.88 2.09 3.00 2.79

Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0.05. Variants with the same letters show the same reaction. The values are means (M) of eight replicates7standard deviation (SD). Table 4 Effect of ZYMV infection and SA treatments on POD and APX activities (unit g1 FW) of C. pepo (cv. Eskandarani) leaves Treatments

Control Infected 10 mM SA+V 50 mM SA+V 100 mM SA +V 100 mM SA +V ino 100 mM SA

APX (unit g1 FW)

POD activity (unit g1 FW)

Treatments

M7SD

%

M7SD

%

41.67g78.124 128.62**d711.10 202.19**a77.82 161.51**b71.24 143.77**c712.02 74.50**e71.90 58.91*f79.96

100 308.69 485.26 387.63 345.04 178.79 141.39

22.38b70.18 47.07**a71.10 11.19b,c71.46 7.33*b,c70.27 4.63*c70.27 13.89b,c71.17 8.10*b,c70.50

100 210.34 50.00 32.76 20.69 62.07 36.21

Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0.05. Variants with the same letters show the same reaction. The values are means (M) of four replicates7standard deviation (SD).

Table 5 Effect of ZYMV infection and SA treatments on CAT and SOD activities (unit g1 FW) of C. pepo (cv. Eskandarani) leaves Treatments

Control Infected 10 mM SA+V 50 mM SA+V 100 mM SA +V 100 mM SA +V ino 100 mM SA

Table 6 Effect of ZYMV infection and SA treatments on H2O2 concentration (mmol g1 FW) and MDA content (nmol g1 FW) of C. pepo (cv. Eskandarani) leaves

CAT activity (unit g1 FW)

SOD activity (unit g1 FW)

M7SD

%

M7SD

%

40.00b,c70.49 79.00**a72.12 45.25b70.04 35.00b,c70.80 30.25b,c71.73 25.00b,c71.72 18.50*c70.44

100 197.5 113.1 87.5 75.63 62.5 46.25

35.56b,c70.04 47.06a,b70.40 31.66c77.84 47.99a,b78.13 50.91*a74.52 49.01*a,b71.77 39.75 a,b,c78.02

100 132.33 89.027 134.97 143.16 137.82 111.78

Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0.05. Variants with the same letters show the same reaction. The values are means (M) of four replicates7standard deviation (SD).

For example, ZYMV infection showed very low negative AOA. Moreover, with the increase of SA treatments the AOA increased and this increase was SA concentration-

Control Infected 10 mM SA+V 50 mM SA+V 100 mM SA +V 100 mM SA +V ino 100 mM SA

H2O2 (nmol g1 FW)

MDA (mmol g1 FW)

AOA

M7SD

%

M7SD

(%)

14.28d70.52 22.02**a71.17 16.00*c70.62 17.20*c70.35 19.44**b70.51 20.13**a70.27 18.24**b70.64

100 147.99 114.37 119.25 132.32 140.69 128.86

12.70b70.20 16.52**a70.09 12.79b70.61 11.42*c71.01 9.56**d70.40 10.06**d70.47 11.20*c70.08

30.08 0.71 10.08 24.72 20.79 11.81

Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0.05. Variants with the same letters show the same reaction. The values are means (M) of four replicates7standard deviation (SD).

dependent. Interestingly, the highest increase in AOA was obtained from leaves treated with 100 mM SA followed by ZYMV inoculation. 3.5. Gas exchange ZYMV infection and SA treatments strongly affected gas exchange parameters, i.e. transpiration rate (E), intercellular CO2 concentration (Ci), stomatal conductance (Gs) and water use efficiency (WUE) (Figs. 2–5). Plants infected with ZYMV showed a significant reduction in both transpiration rate (E) and stomatal conductance (Gs) in comparison with healthy plants. Furthermore, intercellular CO2 concentration (Ci) and water use efficiency (WUE ¼ Pn/E) seemed to be unaffected by ZYMV infection. Exogenous SA application recovered the undesirable effects of ZYMV infection. Thus, transpiration rates (E) and stomatal conductance (Gs) were almost similar to control in all (SA+V) treatments. The pattern of increase or decrease in E and Gs was inconsistent with increasing

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178 12

a

Transpiration rate a

10

a

a

a

0.8 a 0.7

a

a

a

Gs (mmol m-2 S-1)

E(mmol m-1S-1)

0.6 8 b **

6 4

a Stomatal Conductance a a,b

0.5 0.4 b **

0.3 0.2

2

0.1 0 Control

Infected

10 SA+V

50 SA+V 100 SA+V 100SA+V ino

0

SA only

Control

Treatments

Fig. 2. Effect of ZYMV infection and SA treatments on transpiration rate (mmol m2 s1) of C. pepo (cv. Eskandarani) leaves. The values are means of eight replicates7standard deviation. Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0:05, variants with different letters are considered significantly different. 250

a *

240

1.8

210 200 190 180 50 SA+V

100 SA +V

100SA+V ino

SA only

Treatments

Fig. 3. Effect of ZYMV infection and SA treatments on intercellular CO2 concentration (mol mol) of C. pepo (cv. Eskandarani) leaves. The values are means of eight replicates7standard deviation. Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0:05, variants with different letters are considered significantly different.

SA levels. WUE and Ci were quite similar to control in 50 and 100 (SA+V) treatments. Only 10 mM (SA+V) treatment altered the Ci, which was 8% higher, and WUE which was 17.6% lower, than controls. No significant changes were noted for simultaneous application of 100 mM SA and virus inoculation. In absence of virus inoculation, 100 mM SA treatment caused no significant alterations in any of gas exchange parameters except for Ci which decreased by 8% compared to controls. 4. Discussion Virus infection leads to the appearance of epidemiological symptoms characteristic to specific host–virus interactions. The appearance of these symptoms occurs due to changes in metabolism, alteration of some enzyme activities, photosynthetic capacity and accumulation or reduc-

WUE (umol CO2/ mmol H2O)

Ci (mol mol)

a

c *

10 SA+V

Water use effeciency

2

b,c

Infected

SA only

Fig. 4. Effect of ZYMV infection and SA treatments on stomatal conductance (mmol m2 s1) of C. pepo (cv. Eskandarani) leaves. The values are means of eight replicates7standard deviation. Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0:05, variants with different letters are considered significantly different.

b

220

Control

50 SA+V 100 SA+V 100SA+V ino Treatments

a,b b

10 SA+V

Intercellular CO2 concentration

230 b

Infected

a,b

b *

a,b

a

a,b a,b

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Control

Infected

10 SA+V

50 SA+V 100 SA+V 100SA+V ino

SA only

Treatments

Fig. 5. Effect of ZYMV infection and SA treatments on water use efficiency (mmol CO2/mmol H2O) of C. pepo (cv. Eskandarani) leaves. The values are means of eight replicates7standard deviation. Statistical significance of differences compared to control: *, significant at Po0.05; **, significant at Po0.01. Duncan’s composition between variants at a ¼ 0:05, variants with different letters are considered significantly different.

tion in certain metabolites such as caroteniods, ascorbate, glutathione and tocopherols. To our knowledge, little is known about the physiology underlying plant virus interactions characterized by systemic infection, such as ZYMV infection. In the present work, infection of pumpkin (C. pepo cv. Eskandarani) leaves with ZYMV caused the appearance of definite disease symptoms including severe mosaic, green blisters, size reduction and deformation of the new leaves resulting in a tendril-like appearance. In addition to the reduction of plant height and stunting, a highly significant reduction of biomass was also noticed 2 weeks after ZYMV inoculation (Fig. 1). Shoot stunting and reduction of leaf area and biomass strongly affected plant health and finally lead to yield loss,

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although the leaf water content remained unaffected by ZYMV infection. The observed morphological changes were similar to those described previously with Styrian pumpkin [1] and other cucurbit plants infected with ZYMV [38,39]. It is known that photosynthesis plays an important role in plant growth and productivity. The net photosynthetic rate is positively associated with the crop yield in asparagus plants [9]. Any kind of stress, such as ZYMV disease, affecting photosynthesis rate can directly affect plant growth. In this experiment, the net photosynthesis rate and chlorophyll content under ZYMV stress were significantly reduced to reach half values of the control. From chlorophyll a/b ratios, it can be concluded that ZYMV infection might affect chlorophyll molecules by degradation or synthesis inhibition especially chlorophyll b which was more sensitive to disease stress. Wood [40] demonstrated that a decrease in net photosynthetic rate was accompanied by a decrease in chlorophyll contents in virus-infected plants. Photosynthetic properties and growth of infected plants have been shown negatively influenced by virus infection [41]. In contrast, SA-treated plants exhibited photosynthesis rates and chlorophyll concentrations similar to those of controls, especially with the concentration of 100 mM SA with or without virus. This indicates that SA might play a certain role in alleviating photosynthesis inhibition caused by virus infection. This role might be through conservation of chlorophyll molecules and prevention from further degradation during symptom development. In the present work, carotenoid content was unaffected by SA application (data not shown). Thus SA might have protected the carotenoid molecules from degradation. The production of AOS in plant cells in response to virus infection is well documented [32,42,43]. AOS metabolism induces many changes in antioxidant enzyme systems which are still under discussion. Many reports describe contradictory results with both induction and inhibition of antioxidant enzyme activities involved in free radical scavenging [44–46]. We determined the activities of some antioxidant enzymes that decreased or increased in response to virus infection. Moreover, changes in these antioxidant enzymes are known to be directly involved in the activation of plant defense responses [32,42,46]. The present results show that the activities of POD, APX, CAT and SOD enzymes increased significantly (up to 2-fold or greater) as a result of ZYMV infection. Our results concerning POD and APX activities are in agreement with those reported by Riedle-Bauer [43] who found a highly significant increase in POD, APX, CAT and SOD in both Cucumis sativus and C. pepo infected by ZYMV, while Herna`ndez et al. [32] reported no changes in antioxidant enzymes in peach plants infected with long-term plum pox virus. Furthermore, in this work, exogenous application of SA decreased the POD, APX and CAT activities but increased SOD activity with increasing SA level. Suppression of POD, APX and CAT activities and induction of

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SOD reflects the role of SA in defense responses against virus infection. Formation of H2O2 in the cells during disease development is controlled by the activities of antioxidant enzymes which are directly or indirectly controlled by hormones such as SA [45]. Because of this phenomenon, it is convenient to analyze H2O2 level in both ZYMV-infected and SA-treated leaves. Interestingly, an induction of H2O2 was recorded in our experiments under SA treatment and ZYMV infection. It is known that SA acts as electron donor for peroxidase isoforms, causes inhibition of the enzyme and leads to the production of SAfree radicals. Inhibition of peroxidase leads to enhanced H2O2 accumulation causing an alteration in the cellular redox state. So, application of SA induces at least one of the cellular protective mechanisms through potentiation of an oxidative burst. The mode of SA was proposed to increase H2O2 level by inhibition of CAT activity during plant–pathogen interactions. Moreover, pretreatment with SA alone specifically inhibited CAT activity and increased H2O2 level in rice roots. H2O2 accumulation affected by SA has also been observed in other plant species [47,48]. During pathogen attack in plants, AOS, including H2O2 accumulation, can act with at least one of three mechanisms against pathogens. First, H2O2 can act directly by killing the pathogen. Second, H2O2 can react with transition metals leading to enhanced generation of reactive hydroxyl radicals, the devastating effect on biomolecules of which is well known. Third, H2O2 also hinders penetration of plants by micro-organisms. It contributes to cell wall stiffening by facilitating peroxidase reactions catalyzing intra- and inter-molecular cross-links between structural components of cell walls and lignin polymerization. As a consequence, an increase in mechanical barriers slows down pathogen penetration allowing plant cells to arrange defenses which require more time to be activated. In addition, H2O2 can diffuse easily through biological membranes, so it acts as intercellular signal which is able to activate defense response against pathogen attack [42]. Oxidative stress can be demonstrated by MDA concentration which is considered a general indicator of lipid peroxidation [49]. Generally, high MDA contents were detected in ZYMV-infected leaves while all SA treatments caused a noticeable reduction in MDA contents. Increased lipid peroxidation can be observed both in plants with mosaic symptoms and in plants showing leaf-yellowing and malformations. In other words, lipid peroxy-radicals are known to oxidize several pigment molecules leading to bleaching of these pigments. This co-oxidation of pigment molecules might be responsible for yellowing of virusinvaded tissues. Kluge [50] reported that yellowing of virusinfected leaves of Beta vulgaris has been attributed to this co-oxidation. Moreover, the present results suggest that application of exogenous SA could increase the AOA of leaf extracts under ZYMV infection. Many reports indicate that plant growth mainly depends on photosynthesis capacity [9]. Stomatal closure and gas exchange parameters are defined as more or less controlling

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photosynthesis capacity. A noticeable reduction in E (transpiration rate), stomatal number (data not shown) and Gs was observed in the present study in response to ZYMV infection, agreeing with a previous report by Rysˇ lava` et al. [41] in tobacco leaves. Although a reduction of Gs had been noticed in ZYMV-infected leaves, the Ci values remained unchanged. This may be due to decreasing photosynthetic CO2 fixation or enhancing photorespiration which can keep CO2 concentration almost similar to control while stomatal conductance is variable. To our knowledge, there are few reports concerning gas exchange relations in virus-infected plants. Guo et al. [26] reported reductions in transpiration rate and stomatal conductance in Brassica juncea under turnip mosaic virus infection. Furthermore, we found that SA treatment could help to recover the reduction in transpiration rate occurring under ZYMV infection, since SA reversed stomatal closure [7]. The changes in photosynthesis after exogenous SA application under environmental stresses were due to either stomatal or non-stomatal limitations [51,52]. In conclusion, the following points can be drawn from the present investigation:









ZYMV infection negatively affected various physiological and metabolic processes of pumpkin leaves. These negative effects could be reversed by exogenous SA application. Plant health and normal growth, as well as the predicted yield of the plant, were affected by ZYMV infection, which in turn influenced the whole physiological network of pumpkin leaf cells. Because no apparent symptoms were noticed on the leaves, the 100 mM SA concentration seemed to be the most suitable concentration to alleviate growth and photosynthesis inhibition caused by ZYMV infection. The role of SA in inducing plant defense mechanisms against virus infection might be described through SA interfering with the antioxidant systems. Such interference occurred by either inhibition or activation of some antioxidant enzymes, reduction of lipid peroxidation or induction of H2O2 accumulation.

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