Oxidation Communications 35, No 1, 182–189 (2012) Lipid peroxidations, hydrolysis, storage
Effect of Cucumber Mosaic virus on the Contents of Chlorophyll, Proline, the Degree of Lipid Peroxidation and Phenotypic Expression of Pepper Lines with Different Susceptibility to Virus D. Petrovaa*, G. Chanеvaa, E. Stoimenovab, V. Kapchina-Totevaa Department of Plant Physiology, Faculty of Biology, University of Sofia, 1164 Sofia, Bulgaria E-mail:
[email protected] b Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria a
ABSTRACT Cucumber mosaic virus (CMV) infection of pepper plants is characterised by the development of leaf epinasty, rugosity and chlorosis associated with marked metabolic changes. Symptom appearance after CMV inoculation was different for the investigated resistant (L113 and L57) and susceptible (Okal) to CMV pepper lines. It was established appearance of necrotic local lesion and chlorotic pots on the inoculated leaves of L113 and L57 lines, respectively. Next these leaves fell down. On the inoculated leaves of the susceptible line Okal were not observed symptoms. Typical mosaic symptoms for systemic virus spread appeared on the young Okal leaves. The influence of CMV on metabolic changes in the investigated lines was examined. Results showed that the contents of chlorophyll a, b and carotenoids, proline, the degree of lipid peroxidation and membrane permeability were altered insignificantly in the leaves of the resistant pepper lines in comparison with control plants. There are reasons to suppose that the resistant pepper lines have good development defense mechanisms against virus infections. Keywords: CMV, resistance, pepper plants, proline, pigments, lipid peroxidation, membrane leakage. AIMS AND BACKGROUND Virus-infected plants show strong morphological and physiological alterations, with symptoms such as chlorosis and necrosis associated with changes in chlorophyll content. Different analyses have revealed that plants viruses reduce the photosynthetic *
For correspondence.
182
rates of their hosts1–3. Thus, the loss of photosystem II electron transport is a common phenomenon under biotic stress, as is also observed under abiotic stress condition. Plants respond to necrotising pathogens and/or to abiotic stresses by altering their cellular metabolism and invoking various defense mechanisms4,5. For example, proline accumulation is one of the most frequently reported modifications induced by water deficit and salt stress in plants, and it is often considered to be involved in stress-resistance mechanisms6. Plants recognise and resist many invading phytopathogens by inducing a rapiddefense response, term – the hypersensitive response (HR). HR resulted in localised cell and tissue death at the infection site, which further constrained the spread of the infection. Programmed cell death (PCD) that occurred during HR, is accompanied by an increase in production of reactive oxygen species (ROS) and lipid peroxidation7,8. Lipid peroxidation in plants is an important feature of the hypersensitive cell death, a typical defense reaction displayed during incompatible or non-host interactions of plants with pathogens9. Membrane damage by peroxidation of polyunsaturated fatty acids can be initiated by reactive oxygen species (ROS), lipid radicals, or enzymatically by the action of lipoxygenases (LOXs). Hot pepper is one of the most important vegetable crops in Bulgaria. Its importance is also increasing throughout the world. Cucumber mosaic virus (CMV) is one of the most destructive pathogens of pepper crops. In Capsicum annuum, resistance against CMV is characterised by the induction of HR, which is manifested by the appearance of necrotic local lesions at the infection site10. The aim of this work was the study effect of the CMV-infection on the content of some biochemical parameters during pathogenesis on lines of C. annuum with different degree of sensibility to CMV. The interaction between 3 lines of C. annuum and Cucumber mosaic virus (CMV) was used as a model system to study some of the plant defense responses. EXPERIMENTAL Pepper seeds (Capsicum annuum L.) were sown in plastic trays containing a stemsterilise soil mixture. Two resistant pepper lines (L113 and L57) and a susceptible line Okal to Cucumber mosaic virus (CMV) were used in the experiments. The plants were grown in the greenhouse at 25–35°/12–18° (day/night), with a 16 h light/8 h dark photoperiod for 30 days. The inoculations were realised with a CMV-PB preparation at the 50 (L113), 25 (L57) and 25 (Okal) μg/ml virus in 0.01 M phosphate buffer pH 7. The pepper plants at the stage 3rd and 4th leaves were mechanically inoculated on carborundum-dusted 1st and 2nd true leaves with 20 μl inoculums per leaf. The mock-inoculated plants were rubbed by same manner, but the inoculum is virus free. Mock inoculated plants were used as controls. For each experiment 50 plants were used.
183
The readings of symptoms on the inoculated (local) and the systemic (all no rubbed, young) leaves of the plants were performed on the 7th, 17 th and 30th days post-inoculation (dpi). Analyses of the various biochemical parameters were carried out at different post-inoculation times in virus-infected plants and in their corresponding controls, both in symptomatic and asymptomatic leaves. Total chlorophylls and carotenoids were extracted from leaves with 80% acetone and determined according to Arnon11. Malonyl dialdehyde (MDA) contents were measured according to Heath and Packet12. Free proline was extracted, obtained with acid ninhydrin and absorbance read according to Bates et al. methods13. Membrane permeability of leaves was measured by electrolyte leakage14. Inoculated leaves on the 1st, 3rd, 7th and 17th and young systemic leaves on the 7th, 17th and 30th dpi were analysed. Data are averages of triplicate measurements. The significance of differences between control and each treatment was determined using the Student t-test, p ≤ 0.05. RESULTS AND DISCUSSION The response of plants to CMV infection can be estimated by changes in pigment contents, proline concentration, lipid peroxidation and membrane permeability in green plants tissues. Symptomatology and pigment content. In leaves of resistant lines (L113 and L57) developed after inoculation (symptomatic leaves), first symptoms were evident at 17th dpi. Visible necrotic local lesions with light to yellow core, surrounded from black ring appeared on inoculated leaves of L113 line. Chlorotic mottles were observed on inoculated leaves of L57 line. Next these leaves fell down as the petiole picked off in the place where it is attached to the stem after 21st dpi. No symptoms were observed in systemic (asymptomatic) leaves both in L113 and L57 lines to end of the experiment (Fig. 1a,b). On the inoculated leaves of the susceptible line Okal were not observed symptoms and only some of these leaves fell down. Typical mosaic symptoms for systemic virus spread appeared on young Okal leaves up to 30th dpi.
Fig. 1. Health leaf and inoculated leaf with necrotic lesions of L113 line (a) and health leaf and inoculated leaf with chlorotic mottles of L57 line (b)
184
Analysis of chlorophyll (Chl) contents in leaves from healthy (mock-inoculated) and infected L113 and L57 plants indicated that infection with CMV resulted in insignificant changes on Chla and Chlb contents in comparison with the controls (Fig. 2a–d). All virus-infected plants show very slight decrease in the Chla and Chlb contents of asymptomatic (systemic, young) leaves, but the reduction is stronger in the leaves showing symptoms. Reduction in chlorophyll pigments in the resistant lines (L57 and L113) occurred dominantly at the time of symptoms appearance about 17th dpi (Fig. 2a–d). 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.0
1st
c
3rd
dpi
7th
0.6 0.4 0.2
1st
3rd
dpi
7th
17th
young leaves
b
1.4 1.2 1.0 0.8 0.6 0.4 0.2
1.0
inoculated leaves
0.8
0.0
1.6
0.0
17th
chlorophyll b (mg/g FW)
chlorophyll b (mg/g FW)
1.8
inoculated leaves
a
chlorophyll a content (mg/g FW)
chlorophyll a content (mg/g FW)
1.8
7th
17th dpi
30th
young leaves
d
0.8 0.6 0.4 0.2 0.0
7th
17th dpi
30th
Fig. 2. Effect of CMV-treatment on Chla and Chlb contents in the leaves of C. annuum (a, b, c, d) Okal-mock; Okal-CMV; L113-mock; L113-CMV; L57-mock; L57-CMV
The experimental results showed that the CMV infection caused the reduction of Chla and Chlb contents of susceptible pepper leaves (Okal). Chla and Chlb contents were significant decreased (above 50%) in CMV-treated pepper plants in comparison with the mock-control samples in both inoculated and young leaves (Fig. 2a–d). Significant reduction of chlorophyll (a,b) contents start to show up at 17th dpi. Significant increase in carotenoid contents was observed after 17th dpi in inoculated and systemic leaves of Okal in comparison with the controls (Fig. 3a, b). These data coincide with the period of decreased chlorophyll contents in those leaves (Fig. 2a–d). Carotenoid contents in both resistant lines (L113 and L57) was the same in 185
infected and control plants. Insignificant increase in carotenoid contents was observed at the time of symptom expression. More visible changes were in L57 line infected by CMV than in L113 line (Fig. 3a, b). inoculated leaves
a
0.3 carotenoid content [mg/g FW]
carotenoid content (mg/g FW)
0.3
0.2
0.1
0.0
1st
3rd
dpi
7th
17th
young leaves
b
0.2
0.1
0.0
7th
17th dpi
30th
Fig. 3. Effect of CMV-treatment on carotenoid contents in the leaves of C. annuum (a, b) Okal-mock; Okal-CMV; L113-mock; L113-CMV; L57-mock; L57-CMV
The experimental result showed that the CMV-infection caused reduction of the contents of Chla and Chlb and enhancement of the contents of carotenoids in the leaves of susceptible line Okal. A decrease in chlorophyll contents and slight increase of carotenoids in the leaves of resistant lines (L113, L57) occurred simultaneously at the time of symptom appearance. These results are in accordance with those of Edreva et al.15 and Rahoiteli et al.16, who found chlorophyll loss in tobacco leaves after virus-infection, TMV and PMMoV and PaMMoV, respectively. The reduction of the chlorophyll contents has negative effect on plant photosynthetic efficiency. Since it has been reported that photosynthesis is dependent on the light harvesting properties on the chlorophyll17. Recent studies have shown that carotenoids serve a protective function against biotic stress. Changes in proline contents, lipid peroxidation and membrane permeability. In this study, proline contents were significantly elevated in systemic, upper leaves of susceptible line Okal in comparison with controls plants. A more than a 2-fold increase of proline contents was recorded at 30th dpi in young Okal leaves. In contrast, a localised infection with CMV did not induce proline in inoculated leaves. Marked increase of proline contents was detected at 17th dpi in local leaves of Okal. A slow increase of proline contents occurred in inoculated L113 plants in comparison with control plants. Variation in the dynamics of proline contents during pathogenesis was more marked at the time of symptom appearance. The rapid induction of hypersensitive cell death was also accompanied by rapid accumulation of proline in the inoculated leaves of L113 line at 17th dpi (Fig. 4a, b). This is probably a response of the plants to pathogen-inoculation leading to hypersensitive response (HR). Induction of HR potentially limits the spread of disease from the infection points18. In asymptomatic leaves of L113 line were not observed changes in proline level in comparison 186
with control leaves. Insignificant increase of proline contents was found in inoculated leaves of L57 line. The trend of a slight increase of proline was observed as well as in young leaves of L57 line to the end of the experiment (Fig. 4a, b). These observations correlated with differences in degree of resistance of pepper lines to CMV. 5
proline content (µM/g FW)
proline content (µM/g FW)
3.5
inoculated leaves
a 4 3 2 1 0
1st
3rd
dpi
7th
3.0 2.5 2.0 1.5 1.0 0.5 0.0
17th
young leaves
b
7th
17th dpi
30th
Fig. 4. Proline contents in the leaves of C. annuum treated by CMV (a, b) Okal-mock; Okal-CMV; L113-mock; L113-CMV; L57-mock; L57-CMV
Oxidative damage can be detected by lipid peroxidation. Hydroxyl radicals and singlet oxygen can react with lipids and form lipid peroxy radicals and hydroperoxide19,20. An increase of MDA contents was detected in inoculated leaves of susceptible Okal line after infection by CMV in comparison with mock-controls leaves. The observed trend of changes (increased MDA contents) was the same for systemic leaves of Okal line. Substantial changes of MDA contents during pathogenesis were not detected both in inoculated and systemic leaves of L113 and L57 lines, except enhanced MDA levels in inoculated leaves of L113 line at 17th dpi (Fig. 5a, b). A relationship between the MDA contents changes and susceptibility of pepper lines to CMV was observed. Change in MDA was the first evidence that under our experiment CMV-infection induced oxidative stress in the susceptible Okal line (Fig. 5a, b). 0.14
0.16
inoculated leaves
a
MDA concentration (mM/g FW)
MDA concentration (mM/g FW)
0.16
0.12 0.10 0.08 0.06 0.04 0.02 0.00
1st
3rd
dpi
7th
17th
0.14
young leaves
b
0.12 0.10 0.08 0.06 0.04 0.02 0.00
7th
17th dpi
30th
Fig. 5. Effect of CMV-treatment on lipid peroxidation in the leaves of C. annuum (a, b) Okal-mock; Okal-CMV; L113-mock; L113-CMV; L57-mock; L57-CMV
187
200
inoculated leaves
a
150 100 50 0
1st
3rd
dpi
7th
17th
leaf electrolyte leakage (% of mock-control)
leaf electrolyte leakage (% of mock-control)
The peroxidation of membrane lipids leads to the breakdown of their structure and function20,21. The CMV-infection strongly induced electrolyte leakage from the inoculated and systemic leaves of susceptible Okal line, by 50 and 80%, respectively in comparison with the controls (Fig. 6a, b). A mock-inoculation of the primary leaves of L113 and L57 lines with carborund induced a negligible amount of electrolyte leakage from both the local (primary) and systemic (secondary) leaves (date not shown). The visual HR and necrotic symptoms in L113 line were corroborated by measuring the amount of electrolyte leakage, which is indicative of plant cell death. In particular, local infection with CMV on the primary leaves did not induce electrolyte leakage from the systemic, uninoculated leaves in both L113 and L57 lines. Similar situation was also observed in the other biotic stress-stimuli systems18,22. 200
young leaves
b
150 100 50 0
7th
17th dpi
30th
Fig. 6. Effect of CMV-treatment on leakage of electrolytes from leaves of C. annuum (a, b) Okal; L113; L57
CONCLUSIONS A relationship between changes of pigment and proline contents, lipid peroxidation and electrolyte leakage in infected pepper plant and their degree of susceptibility to CMV is evidenced. An increase of the contents of carotenoids, proline, MDA and electrolytes was observed in all inoculation experiments of susceptibility line Okal in comparison with control plants. Variation in the dynamics of studied indices during pathogenesis was more marked at the time of symptoms appearance in the resistance line (L113 and L57). This observation correlated with differences in degree of resistance of pepper genotypes to CMV. ReferenceS 1. C. Tu, R. Ford, C. Kraus: Comparison of Chloroplast and Photosynthetic Rates of Plant Infected and Non Infected by Maize Dwarf Mosaic Virus. Phytopathology, 58, 285 (1967). 2. R. Goodman, Z. Kirali, K. Wood: Photosynthesis. In: The Biochemistry and Physiology of Plant Diseases. University of Missouri Press, Columbia, MO, 1986, p. 46.
188
3. M. Zatlin, R. Hull: Plant Virus–Host Interactions. Ann. Rev. Plant Physiol., 38, 291 (1987). 4. A. Enyedi, N. Yalpani, P. Silverman, I. Raskin: Signal Molecules in Systemic Plant Resistance to Pathogen and Pests. Cell, 70, 879 (1992). 5. L. Mehdy: Active Oxygen Species in Plant Defense against Pathogens. Plant Physiol., 105, 467 (1994). 6. S. Lutts, V. Majerus, J. Kinet: NaCl-induced Senescence in Leaves of Rice (Oryza sativa L.) Cultivars Different in Salinity Resistance. Ann. Bot., 78, 389 (1999). 7. K. Hammond-Kosack, J. Jones: Resistance Gene-depended Plant Defense Responses. Plant Cell, 8, 1773 (1996). 8. Y. Yang, J. Ahah, D. Klessig: Signal Perception and Transduction in Plant Defenses Response. Genes Dev., 11, 1621 (1997). 9. M. Heath: Hypersensitive Response-related Death. Plant Molecular Biology, 44, 321 (2000). 10. D. Petrova, G. Marinova, V. Kapchina-Toteva, E. Stoimenova: Physiological and Biochemical Responses of Susceptible and Resistant Pepper Plants to Cucumber Mosaic Virus Infection. In: Proc. of the International Science Conference, Stara Zagora, Vol. I (Plant studies), 2007, 267–273. 11. D. Arnon: Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta vulgaris. J. Plant Physiol., 24, 1 (1949). 12. R. Heath, L. Packet: Photoperoxidation in Isolated Chloroplasts. Kinetics and Stechiometry of Fatty Acid Peroxidation. Arch. Biochem. Biophys., 125, 189 (1968). 13. L. Bates, R. Waldren, I. Teare: Rapid Determination of Free Proline for Water-stress Studies. Plant and Soil, 39, 205 (1973). 14. R. Dhindsa, P. Plump-Dhindsa, T. Thorpe: Leaf Senescence: Correlated with Increased Levels of Membrane Permeability and Lipid Peroxidation, and Decreases Levels of Superoxide Dismutase and Catalase. J. Exp. Bot., 32, 93 (1981). 15. A. Edreva, I. Georgieva, N. Cholakova: Pathogenic and Non-pathogenic Stress Effects on Peroxidases in Leaves of Tobacco. Envir. Exp. Bot., 29 (3), 365 (1989). 16. J. Rahoiteli, I. Garcia-Luque, M. BARON: Inhibition of Photosynthesis by Viral Infection: Effect on PSII Structure and Function. Physiol. Plantarum, 110, 286 (2000). 17. W. Gao, Y. Zheng, J. Slusser, G. Heisier, F. Grant, J. Xu, D. He: Effects of Supplementary Ultraviolet B Irradiance on Maize Yield and Qualities. A Field Experiment. Photochem. and Photobiol., 80, 127 (2004). 18. S. Lee, B. Hwang: Induction of Some Defense-related Genes and Oxidative Capsicum annuum. Planta, 221, 790 (2005). 19. O. Blokhina, E. Virolinen, K. Fagerstedt: Antioxidant, Oxidative Damage and Oxygen Deprivation Stress. Ann. Rev. Bot., 91, 179 (2003). 20. F. Hollosy: Effect of Ultraviolet Radiation on Plant Cells. Micron, 33, 179 (2002). 21. L. Yuan, Z. Yunqun, C. Jianium, H. Zhide: Intraspecific Responses in Crop Growth and Yield of 20 Wheat Cultivars to Enhanced Ultraviolet–B Radiation under Field Condition. Field Crops Res., 67, 25 (2000). 22. K. Mahdavian, M. Ghorbanli, K. Kalantari: The Effect of Ultraviolet Radiation on the Contents of Chlorophyll, Flavonoid, Anthocyanin and Proline in Capsicum annuum L. Turk. J. Bot., 32, 25 (2008). Received 30 November 2009 Revised 6 December 2009
189
