Öz

Bakır (Cu), bitki büyümesi için gerekli olan temel eser metallerden biridir. Ancak, ortamdaki yüksek miktarda Cu, bitkide toksik etki göstererek büyümeyi olumsuz yönde etkilemektedir. Diğer bir yandan, Cu akümülatörü olan Brassica nigra L., sahip olduğu özel mekanizmalar ile yüksek Cu miktarlarını akümüle edebilmekte ve bu metali farklı hücre bölümlerine taşıyarak sitoplazmadan uzaklaştırabilmektedir. Bu çalışmada, B. nigra’nın düşük Cu seviyesindeki gen anlatım profilinin tespiti amaçlanmıştır. Düşük Cu seviyesinde yetiştirilen B. nigra'nın yapraktaki Cu ile indüklenen gen anlatım profili Arabidopsis ATH1 genom çipi kullanılarak tespit edilmiştir. Elde edilen sonuçlara göre, B. nigra’da 95 gen yukarı regüle ve 72 gen aşağı regüle olarak tanımlanmıştır. Glutatyon S-transferaz, glutatyon redüktaz, ağır metal taşıyıcılar, doğal dirençle ilişkili makrofaj proteinleri, sitokrom p450, MYB-gibi transkripsiyon faktörü, bakır/çinko ve Fe süperoksit dismutazlar gibi Cu ile ilişkili bazı genlerin ve bazı protein kinazların yüksek oranda anlatımının olduğu saptanmıştır. Bu çalışma, fakültatif metalofit olarak tespit edilen B. nigra' nın global gen anlatım profilini sunmakta olup, moleküler bir araç olarak ileriki fitoremediasyon çalışmaları için yardımcı olacaktır. 

IDENTIFICATION OF GENES REGULATED IN RESPONSE TO Cu EXPOSURE IN Brassica nigra L.

Abstract

Copper (Cu) is one of the essential trace metals required for plant growth. High amount of Cu in the media inhibits plant growth and is toxic to the plants. Brassica nigra L., a Cu accumulator, can tolerate a high amount of Cu and have specific mechanisms to relocate Cu within the cell compartments and keep the toxic amount of Cu away from the cytoplasm. This study aimed to evaluate the Cu-induced gene expression pattern of B. nigra Diyarbakir ecotype subjected to low Cu treatment. The Arabidopsis ATH1 genome array was used to determine the Cu-induced gene expression in the leaves of B. nigra grown at 25 µM Cu. Ninety-five genes were upregulated, and seventy-two genes were downregulated in the leaves of plants grown under 25 µM Cu. Cu responsive genes, such as glutathione S-transferase, glutathione reductase, heavy metal transporters, natural resistance-associated macrophage proteins, cytochrome p450, MYB-like transcription factor, copper/zinc, and Fe superoxide dismutases, and some protein kinases were highly expressed in the leaves of Cu-treated plants. The present work provides the global gene expression pattern in facultative metallophyte B. nigra, which could serve as a molecular tool for future phytoremediation studies. 

Keywords

• copper
• microarray
• Cu-induced genes
• B. nigra Diyarbakir ecotype

References

1. 1. Afzal, Z., Howton, T.C., Sun, Y. & Mukhtar, M.S. 2016. The roles of aquaporins in plant stress responses. Journal of Developmental Biology, 4(1): 9.
2. 2. Ahanger, M.A., Morad-Talab, N., Abd-Allah, E.F., Ahmad, P. & Hajiboland, R. 2016. Plant growth under drought stress. 649-668. In: Ahmad, P. (ed.). Water Stress and Crop Plants, Wiley-Blackwell, 784 pp.
3. 3. Andrés‐Colás, N., Sancenón, V., Rodríguez‐Navarro, S., Mayo, S., Thiele, D.J., Ecker, J.R., Puig, S. & Peñarrubia, L. 2006. The Arabidopsis heavy metal P‐type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots. The Plant Journal, 45: 225-236.
4. 4. Andresen, E., Peiter, E. & Küpper, H. 2018. Trace metal metabolism in plants. Journal Experimental Botany, 69: 909-954.
5. 5. Asada, K. 1999. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual review of Plant Physiology and Plant Molecular Biology, 50: 601-639.
6. 6. Bosnić, D., Bosnić, P., Nikolić, D., Nikolić, M. & Samardžić, J. 2019. Silicon and Iron Differently Alleviate Copper Toxicity in Cucumber Leaves. Plants, 8: 554.
7. 7. Brahim, L. & Mohamed, M. 2011. Effects of copper stress on antioxidative enzymes, chlorophyll and protein content in Atriplex halimus. African Journal of Biotechnology, 10: 10143-10148.
8. 8. Burkhead, J.L., Gogolin, Reynolds, K.A., Abdel-Ghany, S.E., Cohu, C.M. & Pilon, M. 2009. Copper homeostasis. New Phytologist, 182: 799-816.

9. 9. Cevher-Keskin, B., Yıldızhan, Y., Yüksel, B., Dalyan, E. & Memon, A.R. 2019. Characterization of differentially expressed genes to Cu stress in Brassica nigra by Arabidopsis genome arrays. Environmental Science and Pollution Research, 26: 299-311. 10. Chaumont, F. & Tyerman, S.D. 2014. Aquaporins: Highly Regulated Channels Controlling Plant Water Relations. Plant Physiology, 164: 1600-1618.
10. 11. Chiou, W.Y. & Hsu, F.C. 2019. Copper Toxicity and Prediction Models of Copper Content in Leafy Vegetables. Sustainability, 11: 6215.
11. 12. Colangelo, E.P. & Guerinot, M.L. 2006. Put the metal to the petal: metal uptake and transport throughout plants. Current Opinion in Plant Biology, 9: 322-330.
12. 13. Deng, F., Yamaji, N., Xia, J. & Ma, J.F. 2013. A Member of the Heavy Metal P-Type ATPase OsHMA5 Is Involved in Xylem Loading of Copper in Rice. Plant Physiology, 163: 1353-1362.
13. 14. Dey, S. & Corina Vlot, A. 2015. Ethylene responsive factors in the orchestration of stress responses in monocotyledonous plants. Frontiers in Plant Science, 6: 640.
14. 15. Dresler, S., Hanaka, A., Bednarek, W. & Maksymiec, W. 2014. Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper. Acta Physiologiae Plantarum, 36: 1565-1575.
15. 16. Ducic, T. & Polle, A. 2005. Transport and detoxification of manganese and copper in plants. Brazilian Journal of Plant Physiology, 17: 103-112.
16. 17. Fernandes, J.C. & Henriques, F.S. 1991. Biochemical, physiological, and structural effects of excess copper in plants. The Botanical Review, 57: 246-273
17. 18. Ghori, N.H., Ghori, T., Hayat, M.Q., Imadi, S.R., Gul, A., Altay, V. & Ozturk, M. 2019. Heavy metal stress and responses in plants. International Journal of Environmental Science and Technology, 16: 1807-1828.
18. 19. Guo, R., Lim, W-A. & Ki, J-S. 2016. Genome-wide analysis of transcription and photosynthesis inhibition in the harmful dinoflagellate Prorocentrum minimum in response to the biocide copper sulfate. Harmful Algae, 57: 27-38.
19. 20. Hachez, C., Zelazny, E. & Chaumont, F. 2006. Modulating the expression of aquaporin genes in planta: A key to understand their physiological functions? Biochimica et Biophysica-Acta-(BBA)-Biomembranes, 1758: 1142-1156.
20. 21. Hall, J.L. & Williams, L.E. 2003. Transition metal transporters in plants. Journal of Experimental Botany, 54: 2601-2613.
21. 22. Hoagland, D.R. & Arnon, D.I. 1938. The water culture method for growing plants without soil. California Agricultural Experiment Station Circular, 347: 1-39.
22. 23. Hossain, M.A., Piyatida, P., da Silva, J.A.T. & Fujita, M. 2012. Molecular Mechanism of Heavy Metal Toxicity And Tolerance in Plants: Central Role of Glutathione in Detoxification of Reactive Oxygen Species and Methylglyoxal and in Heavy Metal Chelation. Journal of Botany, 2012: 1-37.
23. 24. Kabata-Pendias, A. & Pendias, H. 2001. Trace elements in soils and plants. CRC, Boca Raton, FL Kapoor D, Singh S, Kumar V, Romero R, Prasad R, Singh J (2019) Antioxidant enzymes regulation in plants in reference to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Plant Gene, 19: 100182.
24. 25. Kopsel, D.E. & Kopsell, D.A. 2007. Copper. pp. 293-328. In: Barker, A.V. & Pilbeam, D.J. (eds). Handbook of plant nutrition. Taylor and Francis Group, Boca Raton, 662 pp.
25. 26. Krämer, U., Talke, I.N. & Hanikenne, M. 2007. Transition metal transport. FEBS Letters, 581: 2263-2272.
26. 27. Küpper, H., Šetlík, I., Šetliková, E., Ferimazova, N., Spiller, M. & Küpper, F.C. 2003. Copper-induced inhibition of photosynthesis: limiting steps of in vivo copper chlorophyll formation in Scenedesmus quadricauda. Functional Plant Biology, 30: 1187-1196.
27. 28. Liu, W., Zhang, X., Liang, L., Chen, C., Wei, S. & Zhou, Q. 2015. Phytochelatin and Oxidative Stress Under Heavy Metal Stress Tolerance in Plants. pp. 191-217. In: Gupta D., Palma J. & Corpas F. (eds). Reactive Oxygen Species and Oxidative Damage in Plants Under Stress. Springer International Publishing, Switzerland, 370 pp.
28. 29. Maksymiec, W. 1997. Effect of copper on cellular procceses in higher plants. Photosynthetica, 34: 321-342.
29. 30. Maksymiec, W. & Krupa, Z. 2006. The effects of short-term exposition to Cd, excess Cu ions and jasmonate on oxidative stress appearing in Arabidopsis thaliana. Environmental and Experimental Botany, 57: 187-194.
30. 31. Marschner, H. 1995. Mineral Nutrition of Higher Plants. pp. 333-347. In: Marschner, H. (ed.). Academic Press, Inc., London, 906 pp.
31. 32. Meharg, A.A. 1994. Integrated tolerance mechanisms: constitutive and adaptive plant responses to elevated metal concentrations in the environment. Plant, Cell and Environment, 17: 989-993.
32. 33. Memon, A.R., Itô, S. & Yatazawa, M. 1979. Absorption and accumulation of iron, manganese and copper in plants in the temperate forest of central Japan. Soil Science and Plant Nutrition, 25: 611-620.
33. 34. Memon, A. R., Yildizhan, Y. & Cevher-Keskin, B. 2008. Phytoremedıatıon of heavy metals from contamınated areas of Turkey. 1-4. Paper presented at the 4th European Bioremediation Conference, Sept 3-6, Chania, Crete, Greece, ISBN 978-960-8475-12-0.
34. 35. Memon, A.R. & Schröder, P. 2009. Implications of metal accumulation mechanisms to phytoremediation. Environmental Science and Pollution Research, 16: 162-175.
35. 36. Memon, A.R. & Zahirovic, E. 2014. Genomics and Transcriptomics Analysis of Cu Accumulator Plant Brassica nigra L. Journal of Applied Biological Sciences, 8: 01-08.
36. 37. Memon, A. R. 2016. Metal hyper-accumulators: Mechanism of hyper-accumulation and metal tolerance. pp. 239-268. In: Ansari, A.A., Gill, S.S., Gill, R., Lanza, G.R. & Newman, L. (eds). Phytoremediation: Management of Environmental Contaminants, Vol 3. Springer-Verlag, 576 pp. ISBN 978-3-319-40146-1
37. 38. Memon, A.R. 2020. Heavy Metal–Induced Gene Expression in Plants. In: Naeem, M., Ansari, A. & Gill, S. (eds). Contaminant in Agriculture. Springer, Switzerland, 443 pp. ISBN 978-3-030-41551-8
38. 39. Merakli, N. & Memon, A. R. 2019. Farkli bakir (Cu) seviyelerinde yetistirilen Brassica nigra ve Brassica juncea’da HMA1, P1B ATPaz’nin anlatimi. pp. 381-388. In: Karagöz, A. (ed.). Congress Book of 1th International Congress of Medical Sciences and Biotechnology, ISBN-978-605-7607-42-3.
39. 40. Mohanty, N., Vass, I. & Demeter, S. 1989. Copper toxicity affects photosystem II electron transport at the secondary quinone acceptor, QB. Plant Physiology, 90:175-179.
40. 41. Mourato, M., Moreira, I., Leitão, I., Pinto, F., Sales, J. & Martins, L. 2015. Effect of Heavy Metals in Plants of the Genus Brassica. International Journal of Molecular Sciences,16: 17975-17998.
41. 42. Mukherjee, I., Campbell, N.H., Ash, J.S. & Connolly, E.L. 2006. Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper. Planta, 223: 1178-1190.
42. 43. Murphy, A. & Taiz, L. 1995. A new vertical mesh transfer technique for metal-tolerance studies in Arabidopsis (ecotypic variation and copper-sensitive mutants). Plant Physiology, 108: 29-38.
43. 44. Nazir, F., Hussain, A. & Fariduddin, Q. 2019. Hydrogen peroxide modulate photosynthesis and antioxidant systems in tomato (Solanum lycopersicum L.) plants under copper stress. Chemosphere, 230: 544-558.
44. 45. Newton, G.L. & Fahey, R.C. 1995. Determination of biothiols by bromobimane labeling and high-performance liquid chromatography. Biothiols Part A Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals, Methods in Enzymology, 251: 166.
45. 46. Noctor, G. & Foyer, C.H. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Reviews of Plant Physiology and Plant Molecular Biology, 49: 249-279.
46. 47. Norvell, W.A. & Lindsay, W.L. 1969. Reactions of EDTA Complexes of Fe, Zn, Mn, and Cu with Soils1. Soil Science Society of America Journal, 33: 86.
47. 48. Pätsikkä, E., Aro, E.M. & Tyystjarvi, E. 2001. Mechanism of copper-enhanced photoinhibition in thylakoid membranes. Physiologia Plantarum, 113: 142-150.
48. 49. Pätsikkä, E., Kairavuo, M., Šeršen, F., Aro, E.-M. & Tyystjärvi, E. 2002. Excess Copper Predisposes Photosystem II to Photoinhibition in Vivo by Outcompeting Iron and Causing Decrease in Leaf Chlorophyll. Plant Physiology, 129: 1359-1367.
49. 50. Pilon, M., Abdel-Ghany, S.E., Cohu, C.M., Gogolin, K.A. & Ye, H. 2006. Copper cofactor delivery in plant cells. Current Opinion in Plant Biology, 9: 256-263.
50. 51. Pilon-Smits, E. & Pilon, M. 2002. Phytoremediation of Metals Using Transgenic Plants. Critical Reviews in Plant Sciences, 21: 439-456.
51. 52. Pollard, A.J., Reeves, R.D. & Baker, A.J.M. 2014. Facultative hyperaccumulation of heavy metals and metalloids. Plant Science, 217-218: 8-17.
52. 53. Prasad, M.N.V. & Strzalka, K. 1999. Impact of heavy metals on photosynthesis. pp. 117-138. In: Prasad M.N.V. & Hagemeyer J. (eds). Heavy Metal Stress in Plants. Springer-Verlag, Berlin Heidelberg, New York Dordrecht London, 245 pp.
53. 54. Printz, B., Lutts, S., Hausman, J.F. & Sergeant, K. 2016. Copper Trafficking in Plants and Its Implication on Cell Wall Dynamics. Frontiers in Plant Science, 7: 601.
54. 55. Puig, S., Andrés-Colás, N., García-Molina, A. & Peñarrubia, L. 2007. Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactionsb and biotechnological applications. Plant, Cell & Environment, 30: 271-290.
55. 56. Puig, S. 2014. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins. Advances in Botany, 2014: 1-9.
56. 57. Sancenón, V., Puig, S., Mira, H., Thiele, D.J. & Peñarrubia, L. 2003. Identification of a copper transporter family in Arabidopsis thaliana. Plant Molecular Biology, 51: 577-587.
57. 58. Schutzendubel, A. & Polle, A. 2002. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany, 53: 1351-1365.
58. 59. Shahid, M., Khalid, S., Abbas, G., Shahid, N., Nadeem, M., Sabir, M., Aslam, M. & Dumat, C. 2015. Heavy metal stress and crop productivity. pp. 1-25. In: Hakeem K. R. (ed.). Crop Production and Global Environmental Issues. Springer International Publishing. Switzerland, 598 pp.
59. 60. Shahbaz, M., Stuiver, C.E.E., Posthumus, F.S., Parmar, S., Hawkesford, M.J. & De Kok, L.J. 2013. Copper toxicity in Chinese cabbage is not influenced by plant sulphur status, but affects sulphur metabolism-related gene expression and the suggested regulatory metabolites. Plant Biology, 16(1): 68-78.
60. 61. Schröder, W.P., Arellano, J.B., Bittner, T., Barón, M., Eckert, H.J. & Renger, G. 1994. Flash-induced absorption spectroscopy studies of copper interaction with photosystem II in higher plants. Journal of Biological Chemistry, 269: 32865-32870.
61. 62. Štolfa, I., Pfeiffer, T.Ž., Špoljarić, D., Teklić, T. & Lončarić, Z. 2015. Heavy Metal-Induced Oxidative Stress in Plants: Response of the Antioxidative System. pp.127-163. In: Gupta, D.K., Palma, J.M. & Corpas, F.J. (eds). Reactive Oxygen Species and Oxidative Damage in Plants Under Stress. Springer International Publishing, Switzerland, 373 pp.
62. 63. Weber, M., Trampczynska, A. & Clemens, S. 2006. Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri. Plant, Cell and Environment, 29: 953-960.
63. 64. Williams, L.E., Pittman, J.K. & Hall, J.L. 2000. Emerging mechanisms for heavy metal transport in plants. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1465: 104-126.
64. 65. Wintz, H. & Vulpe, C. 2002. Plant copper chaperones. Biochemical Society Transactions, 30: 732-735.
65. 66. Wintz, H., Fox, T., Wu, Y.Y., Feng, V., Chen, W., Chang, H.S., Zhu, T. & Vulpe, C. 2003. Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis. Journal of Biological Chemistry, 278: 47644-47653.
66. 67. Xiang, C. & Oliver, D.J. 1998. Glutathione Metabolic Genes Coordinately Respond to Heavy Metals and Jasmonic Acid in Arabidopsis. The Plant Cell, 10: 1539-1550.
67. 68. Yang, X.E., Jin, X.F., Feng, Y. & Islam, E. 2005. Molecular Mechanisms and Genetic Basis of Heavy Metal Tolerance/Hyperaccumulation in plants. Journal of Integrative Plant Biology, 47: 1025-1035.
68. 69. Yruela, I. 2005. Copper in plants. Brazilian Journal of Plant Physiology, 17: 145-156.
69. 70. Yruela, I. 2009. Copper in plants: acquisition, transport and interactions. Functional Plant Biology, 36: 409-430.
70. 71. Zhang, D., Liu, X., Ma, J., Yang, H., Zhang, W. & Li, C. 2019. Genotypic differences and glutathione metabolism response in wheat exposed to copper. Environmental and Experimental Botany, 157: 250-259.