Mustafa YILDIZ, Hakan TERZİ, Süleyman CENKCİ, Behiye YILDIZ

Chromium(VI)-ınduced alterations in 2-D protein profiles and antioxidant defence systems of barley cultivars

Krom birikimi, büyüme, d-aminolevülinik asit dehidrataz (ALAD) aktivitesi, fotosentetik pigment içeriği, prolin içeriği, malondialdehit (MDA) içeriği, antioksidant enzim aktivitesi ve 2-D protein profilleri üzerine Cr(VI) stresinin (0, 75, 150 ve 225 μM) etkisi Cr-toleranslı (Zeynelağa) ve Cr-hassas (Orza-96) arpa çeşitleri kullanılarak araştırılmıştır. Orza-96’ya göre Zeynelağa gövde dokuları önemli düzeyde daha yüksek Cr biriktirmiştir. Orza-96’ya göre Zeynelağa gövde kuru ağırlığı, klorofil ve karotenoid içeriği daha düşük azalma göstermiştir. Cr(VI), ALAD aktivitesini önemli düzeyde azaltmış olmasına rağmen, arpa çeşitlerinin ALAD aktiviteleri arasında tutarlı bir farklılık bulunmamıştır. Cr(VI) prolin ve MDA içeriğini arttırmış; fakat bu etki Orza-96’da daha belirgin olarak belirlenmiştir. Cr(VI) stresi süperoksit dismutaz (SOD), guaiakol peroksidaz (POD), askorbat peroksidaz (APX) ve katalaz (CAT) aktivitelerinde artışa neden olmuştur. Genel olarak, artan antioksidant enzim aktiviteleri açısından iki arpa çeşidi arasında belirgin farklılık belirlenmiştir. Bu araştırmanın sonuçları, Orza-96’ya göre Zeynelağa’nın Cr(VI) stresine karşı daha iyi savunma mekanizmalarına sahip olduğunu göstermektedir. Diğer taraftan, iki-yönlü jel elektroforezi ile analiz edilmiş protein profilleri 26 proteinin farklı şekilde eksprese edildiğini göstermiştir.

Arpa çeşitlerinin 2-D protein profilleri ve antioksidant savunma sistemindeki krom(VI) teşvikli değişimler

The effects of Cr(VI) stress (0, 75, 150 and 225 μM) on Cr accumulation, growth, d-aminolevulinic acid dehydratase (ALAD) activity, photosynthetic pigments content, proline content, malondialdehyde (MDA) content, antioxidant enzymes activity and 2-D protein profile were investigated using Cr-tolerant (Zeynelağa) and Cr-sensitive (Orza-96) barley cultivars. Zeynelağa accumulated significantly higher Cr in shoots than Orza-96. Zeynelağa had a lower reduction in shoot dry weight, chlorophyll and carotenoid contents than Orza-96. Although Cr(VI) significantly reduced the ALAD activity, there was no consistent difference between ALAD activities of barley cultivars. Cr(VI) increased proline and MDA contents, but this effect was more pronounced in Orza-96. Cr(VI) stress caused an increase in the activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT). There was, in general, a marked difference between two barley cultivars in the extent of increased antioxidant enzyme activity. The results of the present study indicated that Zeynelağa seems to have better defense mechanisms to Cr(VI) stress compared to Orza-96. On the other hand, protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of 26 proteins.

PDF

___

1. AK. Shanker, C. Cervantes, H. Loza-Tavera, S. Avudainayagam, Chromium toxicity in plants, Environ. Int., 31 (2005) 739.
2. J. Kotas, Z. Stasicka, Commentary: chromium occurrence in the environment and methods of its speciation, Environ. Pollut., 107 (2000) 263.
3. EWD. Huffman, WH. Allaway, Growth of plants in solution culture containing low levels of chromium, Plant Physiol., 52 (1973) 72.
4. FT. Davies, JD. Puryear, RJ. Newton, JN. Egilla, JAS. Grossi, Mycorrhizal fungi increase chromium uptake by sunflower plants: influence on tissue mineral concentration, growth, and gas exchange, J. Plant. Nutr., 25 (2002) 2389.
5. AK. Shanker, G. Pathmanabhan, Speciation dependent antioxidative response in roots and leaves of sorghum (Sorghum bicolor (L) Moench cv CO 27) under Cr(III) and Cr(VI) stress, Plant Soil, 265 (2004) 141.
6. C. Cervantes, J. Campos-Garcia, S. Devars, F. Gutierrez-Corona, H. Loza-Tavera, JC. Torres- Guzman, R. Moreno-Sanchez, Interactions of chromium with microorganisms and plants, FEMS Microbiol. Rev., 25 (2001) 335.
7. S. Samantaray, Biochemical responses of Cr-tolerant and Cr-sensitive mung bean cultivars grown on varying levels of chromium, Chemosphere, 47 (2002) 1065.
8. P. Vajpayee, RD. Tripati, UN. Rai, MB. Ali, SN. Singh, Chromium (VI) accumulation reduces chlorophyll biosynthesis, nitrate reductase activity and protein content in Nymphaea alba L., Chemosphere, 41 (2000) 1075.
9. P. Vernay, C. Gauthier-Moussard, L. Jean, F. Bordas, O. Faure, G. Ledoigt, A. Hitmi, Effect of chromium species on phytochemical and physiological parameters in Datura innoxia, Chemosphere, 72 (2008) 763.
10. A. Metwally, VI. Safronova, AA. Belimov, KJ. Dietz, Genotypic variation of the response to cadmium toxicity in Pisum sativum L., J. Exp. Bot., 56 (2005) 167.
11. V. Pandey, V. Dixit, R. Shyam, Chromium effect on ROS generation and detoxification in pea (Pisum sativum) leaf chloroplasts, Protoplasma, 236 (2009) 85.
12. AJM. Baker, RR. Brooks, Terrestrial higher plants which hyperaccumulate metallic elements: a review of their distribution, ecology and phytochemistry, Biorecovery, 1 (1989) 81.
13. M. Yıldız, B. Uruşak, H. Terzi, A preliminary study on chromium(VI) tolerance at early seedling stage of some barley cultivars grown in Turkey, AKU-J. Sci., 10 (2010) 1.
14. K. Naito, T. Ebato, Y. Endo, S. Shimizu, Effect of benzyladenine on d-aminolevulinic acid synthetic ability and d-aminolevulinic acid dehydratase: differential responses to benzyladenine according to leaf age, Z. Pflanzenphysiol., 96 (1980) 95.
15. HAW. Schneider, Activities and properties of d-aminolevulinic acid dehydratase in greening tissue cultures of Nicotiana tabacum cv. Samsum, Z. Pflanzenphysiol., 62 (1970) 133.
16. D. Mauzerall, S. Granick, The occurrence and accumulation of d-aminolevulinic acid and porphobilinogen in urine, J. Biol. Chem., 219 (1956) 435.
17. MM. Bradford, A rapid and sensitive method for quantitation of microgram quantities of proteins utilizing the principle of protein dye binding, Anal. Biochem., 72 (1976) 248.
18. AR. Wellburn, The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution, J. Plant Physiol., 144 (1994) 307.
19. S. Bates, RP. Waldren, ID. Teare, Rapid determination of the free proline in water stress studies, Plant Soil, 39 (1973) 205.
20. RL. Heath, L. Packer, Photoperoxidation in isolated chloroplasts, Arch. Biochem. Biophys., 125 (1968) 189.
21. C. Beauchamp, I. Fridovich, Superoxide sismutase: improved assay and an assay applicable to acrylamide gels, Anal. Biochem., 44 (1971) 276.
22. HU. Bergmeyer, Methods of enzymatic analysis I. 2nd edn. Academic Press, New York, 1974.
23. Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloro plasts, Plant Cell Physiol., 22 (1987) 867.
24. H. Aebi, Catalase in vitro, Methods Enzymol., 105 (1984) 121.
25. CD. Damerval, D. de Vienne, M. Zivy, H. Thiellement, Technical improvements in two-dimensional electro- phoresis increase the level of genetic variation detect ed in wheat-seedling proteins, Electrophoresis, 7 (1986) 52.
26. LS. Ramagli, LV. Rodriguez, Quantitation of micro gram amounts of protein in two-dimensional poly acrylamide gel electrophoresis sample buffer, Electrophoresis, 6 (1985) 559.
27. SMS. Naqvi, VC. Ozalp, HA. Oktem, M. Yucel, Two dimensional electrophoresis of proteins with a different approach to isoelectric focusing, Analyst, 119 (1994) 1341.
28. DF. Hochstrasser, MG. Harrington, AC. Hochstrasser, MJ. Miller, CR. Merril, Methods for increasing the resolution of two-dimensional protein electrophoresis, Anal. Biochem., 173 (1988) 424.
29. UK. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680.
30. H. Blum, H. Beier, HJ. Gross, Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels, Electrophoresis, 8 (1987) 93.
31. H. Diwan, I. Khan, A. Ahmad, M. Iqbal, Induction of phytochelatins and antioxidant defence system in Brassica juncea and Vigna radiata in response to chromium treatments, Plant Growth Regul., 61 (2010) 97.
32. S. Ali, P. Bai, F. Zeng, S. Cai, IH. Shamsi, B. Qiu, F. Wu, G. Zhang, The ecotoxicological and interactive effects of chromium and aluminum on growth, oxidative damage and antioxidant enzymes on two barley genotypes differing in Al tolerance, Environ. Exp. Bot., 70 (2011) 185.
33. DDK. Prasad, ARK. Prasad, Altered d-aminolevulinic acid metabolism in germinating seedling of Bajra (Pennisetum typhoideum), J. Plant Physiol., 127 (1987) 241.
34. H. Diwan, A. Ahmad, M. Iqbal, Genotypic variation in the phytoremediation potential of Indian mustard for chromium, Environ. Manag., 41 (2008) 734.
35. CH. Foyer, M. Lelandais, KJ. Kunert, Photooxidative stress in plants, Physiol. Plant., 92 (1994) 696.
36. V. Dixit, V. Pandey, R. Shyam, Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria, Plant Cell Environ., 25 (2002) 687.
37. V. Pandey, V. Dixit, R. Shyam, Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium, Chemosphere, 61 (2005) 40.
38. HD. Willekens, M. Inzé, M. Van Montagu, W. Van Camp, Catalase in plants, Mol. Breeding, 1 (1995) 207.
39. K. Asada, Ascorbate peroxidase-a hydrogen peroxide scavenging enzyme in plants, Physiol. Plant., 85 (1992) 235.
40. Y. Ekmekçi, D. Tanyolaç, B. Ayhan, Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars, J. Plant Physiol., 165 (2008) 600.
41. M. Labra, E. Gianazza, R. Waitt, I. Eberini, A. Sozzi, S. Regondi, F. Grassi, E. Agradi, Zea mays L. protein changes in response to potassium dichromate treatments, Chemosphere, 62 (2006) 1234.