Bakır Stresi altında Nohut Bitkisinde (Cicer arietinum L.) Stresle İlişkili Gen Ekspresyon Seviyelerinde Meydana Gelen Değişimlerin Belirlenmesi ve Hücresel H2O2 ile İlişkisi

Year 2020, Volume: 34 Issue: 2, 303 - 315, 01.12.2020

Musa Kar , Nuriye Öztürk

Abstract

Bu çalışmanın amacı, nohut (Cicer arietinum) bitkisinde farklı zaman ve konsantrasyonlarda bakıra maruz kalmış bitki yapraklarındaki stres genlerinin ekspresyon seviyelerindeki değişimlerin belirlenmesidir. Ayrıca; gen ekspresyonlarındaki değişim ile hücresel H2O2 arasındaki ilişki tespit edilmeye çalışılmıştır. Bu bağlamda hücrede oksidatif stresin göstergesi olan melondialdehit (MDA) miktarı ve reaktif oksijen türlerinden (ROS) H2O2 miktarları tespit edilmiştir. Ayrıca antioksidan savunma elemanlarından Metallothionein (MT), Catalase (CAT) ve superoksit dismutaz (Cu/Zn-SOD) enzimlerinin gen ekspresyonlarındaki değişim house-keeping gen olarak seçilmiş aktinin ekspresyon düzeyi baz alınarak tespit edilmiştir. Çalışma sonucunda MDA içeriğinin süre ve konsantrasyona bağlı olarak belirgin bir şekilde arttığı, stres alakalı gen ekpresyonlarının bütün konsantrasyonlarda kontrolden yüksek düzeyde eksprese edildiği ancak en yüksek ekspresyon gerçekleştikten sonra süre ve konsantrasyondan arttıkca ekpsreyon düzeylerinde bir azalma olduğu belirlenmiştir. Bu durum stres anındaki kararlı bir artış gösteren hücresel H2O2 ile ilişkilendirilmiştir. Çalışma sonucunda Cu maruziyetinin oskidatif strese neden olarak stres alakalı genlerin ekpresyonlarını indüklediği tespit edilmiştir. Ayırca hücresel H2O2’nin belirli konsantrasyona kadar gen ekpresyonunu up-regule ederken belirli konsantrasyondan sonra down regule etmiş olduğu düşünülmektedir. bu calısmanın sonucları sayesinde; tarım alanlarındaki bitkiler belirli konsantrasyonda H2O2 ye maruz bırakılarak biyotik ve ya abiyotik streslere karsı oksidadatif stres cevabını daha erken evrede vermesi sağlanabilir. Bu sayede, daha dayanıklı ürünler elde edilmesine bağlı olarak kimyasal zirai ilac kullanımı asgari düzeye indirilebilir.

Keywords

cicer arietinum, Cu, H2O2, Oksidatif Sinyalizasyon, stres alakalı genler

Supporting Institution

Nevsehir Hacı Bektaş Veli Üniversitesi Bilimsel Arastırma ve Projeler Birimi

Project Number

NEUBAP 16/2F21

The Evulation of Stress Related Gene Expression Level and Relationship to Cellular H2O2 in Chickpea (Cicer arietinum L.) Under Copper Stress

Abstract

The aim of this study is to determine the changes in expression levels of stress genes in chickpea (Cicer arietinum) plant leaves exposed to copper (Cu) at different times and concentrations. Also; the relationship between the changes in gene expression and cellular H2O2 was investigated. In this context, the amount of malondialdehyde (MDA) and reactive oxygen species (ROS) hydrogen peroxide (H2O2) levels were determined. Furthermore, the changes in gene expressions of Metallothionein (MT), Catalase (CAT) and superoxide dismutase (Cu / Zn-SOD) enzymes were determined based on the actin expression level that selected as a housekeeping gene. It was determined that MDA content increased significantly due to time and concentration, In all duration and concentrations, the expression of stress-related genes significantly differed from the control group. Hleowever, a decrease has been determined by all gene expressions after the highest expression. This phenomenon is associated with cellular H2O2, which shows a steady increase in stress. At the end of the study, it was concluded that the elevating duration and concentration of Cu induced oxidative stress and caused the expression of stress-related genes. Furthermore, cellular H2O2 might be acting as a signal molecule that, up-regulate gene expressions until a certain concentration and down-regulate until a certain concentration. Thanks to the results of this study; Plants in agricultural areas can be exposed to a certain concentration of H2O2 to provide an earlier response to oxidative stress against biotic or abiotic stresses. In this way, the use of chemical pesticides can be minimized due to obtaining more durable products.

Keywords

Cicer arietinum, Cu, H2O2, Oxidative signaling, Stress-related genes

Project Number

NEUBAP 16/2F21

References

• Ahmad, P. 2014. Oxidative Damage to Plants: Antioxidant Networks and Signaling (Elsevier GmbH.). Apel, K. and Hirt, H. 2004. REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction. Annual Review of Plant Biology 55(1): 373–399.
• Aydin, S. Büyük, I. and Aras, E.S. 2014. Expression of SOD gene and evaluating its role in stress tolerance in NaCl and PEG stressed Lycopersicum esculentum. Turkish Journal of Botany 38(1): 89–98.
• Baloǧlu, M.C. Kavas, M. Aydin, G. Öktem, H.A. and Yücel, A.M. 2012. Antioxidative and physiological responses of two sunflower (Helianthus annuus) cultivrs under PEG-mediated drought stress. Turkish Journal of Botany 36707–714.
• Choudhury, F.K. Rivero, R.M. Blumwald, E. and Mittler, R. 2017. Reactive oxygen species, abiotic stress and stress combination. Plant Journal 90(5): 856–867.
• Dai, Q. lin Chen, C. Feng, B. Liu, T. ting Tian, X. Gong, Y. ya Sun, Y. kun Wang, J. and Du, S. zhang 2009. Effects of different NaCL concentration on the antioxidant enzymes in oiLseed rape (Brassica napus L.) seedlings. Plant Growth Regulation 59273–278.
• Duman, F. Urey, E. Temizgul, R. and Bozok, F. 2010. Biological responses of a non-target aquatic plant (Nasturtium officinale) to the herbicide, tribenuron-methyl. Weed Biology and Management 10(2): 81–90.
• FAO 2004. FAO. 2004. Data sets, indicators, and methods to assess land degradation in drylands. World Soil Resources Report No. 100. Rome.
• Farnese, F.S. Menezes-Silva, P.E. Gusman, G.S. and Oliveira, J.A. 2016. When Bad Guys Become Good Ones: The Key Role of Reactive Oxygen Species and Nitric Oxide in the Plant Responses to Abiotic Stress. Frontiers in Plant Science 7(April): 471.
• Gaweł, S. Wardas, M. Niedworok, E. and Wardas, P. 2004. Malondialdehyde (MDA) as a lipid peroxidation marker. Wiadomości Lekarskie (Warsaw, Poland : 1960) 57(9–10): 453–455.
• Gill, S.S. and Tuteja, N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry 48(12): 909–930.
• Gu, C.S. Liu, L. qin Zhao, Y.H. Deng, Y. ming Zhu, X. dong and Huang, S.Z. 2014. Overexpression of Iris. lactea var. chinensis metallothionein llMT2a enhances cadmium tolerance in Arabidopsis thaliana. Ecotoxicology and Environmental Safety 10522–28.
• Guo, W.-J. Meetam, M. and Goldsbrough, P.B. 2008. Examining the Specific Contributions of Individual Arabidopsis Metallothioneins to Copper Distribution and Metal Tolerance. Plant Physiology 146(4): 1697–1706.
• Gupta, K. Sengupta, A. Chakraborty, M. and Gupta, B. 2016. Hydrogen Peroxide and Polyamines Act as Double Edged Swords in Plant Abiotic Stress Responses. Frontiers in Plant Science 7(September): 1343.
• Hernández-Hernández, H. Juárez-Maldonado, A. Benavides-Mendoza, A. Ortega-Ortiz, H. Cadenas-Pliego, G. Sánchez-Aspeytia, D. and González-Morales, S. 2018. Chitosan-PVA and Copper Nanoparticles Improve Growth and Overexpress the SOD and JA Genes in Tomato Plants under Salt Stress. Agronomy 8(9): 175.
• Ishibashi, Y. Yamaguchi, H. Yuasa, T. Iwaya-Inoue, M. Arima, S. and Zheng, S.H. 2011. Hydrogen peroxide spraying alleviates drought stress in soybean plants. Journal of Plant Physiology 168(13): 1562–1567.
• Jain, R. Verma, R. Singh, A. Chandra, A. and Solomon, S. 2015. Influence of selenium on metallothionein gene expression and physiological characteristics of sugarcane plants. Plant Growth Regulation.
• Junglee, S. Urban, L. Sallanon, H. and Lopez-lauri, F. 2014. Optimized Assay for Hydrogen Peroxide Determination in Plant Tissue Using Potassium Iodide. American Journal of Analytical Chemistry 5(August): 730–736.
• Kar, M. 2018. Determination of the expression level of stress-related genes in Cicer arietinum root cell under Cd stress and the relationship to H2O2 concentrations. Ecotoxicology 27(8): 1087–1094.
• Kim, D.-W. Shibato, J. Agrawal, G.K. Fujihara, S. Iwahashi, H. Kim, D.H. Shim, I.-S. and Rakwal, R. 2007. Gene transcription in the leaves of rice undergoing salt-induced morphological changes (Oryza sativa L.). Molecules and Cells 24(1): 45–59.
• Kumar, K.S. Dayananda, S. and Subramanyam, C. 2005. Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa. FEMS Microbiology Letters 242(1): 45–50.
• Laloi, C. Apel, K. and Danon, A. 2004. Reactive oxygen signalling: The latest news. Current Opinion in Plant Biology.
• Leblebici, Z. and Aksoy, A. 2011. Growth and Lead Accumulation Capacity of Lemna minor and Spirodela polyrhiza (Lemnaceae): Interactions with Nutrient Enrichment. Water, Air, & Soil Pollution 214(1–4): 175–184.
• Liu, J. Shi, X. Qian, M. Zheng, L. Lian, C. Xia, Y. and Shen, Z. 2015. Copper-induced hydrogen peroxide upregulation of a metallothionein gene, OsMT2c, from Oryza sativa L. confers copper tolerance in Arabidopsis thaliana. Journal of Hazardous Materials 29499–108.
• Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.) 25(4): 402–408.
• Luna, C.M. Pastori, G.M. Driscoll, S. Groten, K. Bernard, S. and Foyer, C.H. 2005. Drought controls on H2O2 accumulation, catalase (CAT) activity and CAT gene expression in wheat. Journal of Experimental Botany 56(411): 417–423.
• Mir, G. Domènech, J. Huguet, G. Guo, W.J. Goldsbrough, P. Atrian, S. and Molinas, M. 2004. A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress. Journal of Experimental Botany 55(408): 2483–2493.
• Del Río, L.A. 2015. ROS and RNS in plant physiology: An overview. Journal of Experimental Botany 66(10): 2827–2837.
• Rossatto, T. do Amaral, M.N. Benitez, L.C. Vighi, I.L. Braga, E.J.B. de Magalhaes Junior, A.M. Maia, M.A.C. and da Silva Pinto, L. 2017. Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress. Physiology and Molecular Biology of Plants : An International Journal of Functional Plant Biology 23(4): 865–875.
• Sahin, O. Taskin, M.B. Kaya, E.C. and Taskin, H. 2017. Poultry Manure Biochar Reduces Arsenic Induced Oxidative Stress and Arsenic Levels in Rice Plants Tavuk Gübresi Biyokömürünün Çeltik Bitkisi Arsenik Al ı m ı ve Arsenik Düzeyleri Üzerine Etkisi ve Oksidatif Stres İ le İ li ş kisi. Bursa Uludag Üniv. Ziraat Fak. Derg. 113103–113.
• Sewelam, N. Kazan, K. and Schenk, P.M. 2016. Global Plant Stress Signaling: Reactive Oxygen Species at the Cross-Road. Frontiers in Plant Science 7(February): 187.
• Sharma, P. Jha, A.B. Dubey, R.S. and Pessarakli, M. 2012. Reactive Oxygen Species, Oxidative Damage, and Antioxidative Defense Mechanism in Plants under Stressful Conditions. Journal of Botany 2012(1): 1,26.
• Souguir, D. El Ferjani, E. Ledoigt, G. and Goupil, P. 2013. Transcript accumulation of stress-related genes in Vicia faba roots under a short exposure to cadmium. Plant Biosystems - An International Journal Dealing with All Aspects of Plant Biology 3504(August): 1–9.
• Tamás, L. Dudíková, J. Ďurčeková, K. Halušková, L. Huttová, J. Mistrík, I. and Ollé, M. 2008. Alterations of the gene expression, lipid peroxidation, proline and thiol content along the barley root exposed to cadmium. Journal of Plant Physiology 165(11): 1193–1203.
• Tombuloglu, H. Semizoglu, N. Sakcali, S. and Kekec, G. 2012. Boron induced expression of some stress-related genes in tomato. Chemosphere 86(5): 433–438.
• Verma, S. and Dubey, R.S. 2003. Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. Plant Science 164(4): 645–655.
• Yruela, I. 2005. Copper in plants. Brazilian Journal of Plant Physiology 17(1): 145–156.