Determination of Expression Levels of Fatty Acid Desaturase-2 Genes in Safflower (Carthamus tinctorius L.) Varieties Exposed to Copper

Yıl 2022, Cilt: 38 Sayı: 2, 401 - 415, 23.08.2022

Ekrem Bölükbaşı , Sumer Aras

Öz

Heavy metal pollution is an important environmental problem all over the world. It is known that high concentrations of heavy metals in soils and waters cause genotoxicity in living things and damage most of the functional biomolecules. For instance, while low concentration of copper, is essential for all organisms, high concentration of copper is toxic element that negatively affect every living organism from plants to humans. Safflower is an agricultural plant with high economic value grown for its seed oil. Safflower oil is source of Omega-9 and Omega-6 used in many food and industrial applications. In this study, it investigated the expression levels of CtFAD2 (FAD2-6, FAD2-7, FAD2-11) genes responsible for conversion of Omega-9 to Omega-6 at root, cotyledon and leaf tissues of four different safflower varieties subjected to copper heavy metals stress by qRT-PCR. RNA isolation, cDNA synthesis and qRT-PCR analysis were performed in root, cotyledon and leaf tissues exposed to copper stress for 24 hours after cultivation for 21 days. The increases were observed at concentrations of 40 and 80 mg L-1. It was determined that the expression levels of FAD2 genes decreased at increasing copper concentrations and increased again after 160 and 320 mg L-1. The decrease firstly in the expression of FAD2 genes at increasing copper concentrations and their re-increase after 160 and 320 mg L-1, which can be considered as critical points, are accepted as an indication that the defense mechanism against stress is activated and FAD2 genes play a role in the defense against stress.
In conclusion, it has been determined that FAD2 genes, which are responsible for fatty acid desaturation in safflower cultivars exposed to copper stress, are also associated with the stress mechanism and play a role in defense.

Anahtar Kelimeler

Safflower, Copper, FAD2 Gene, Real Time PCR

Destekleyen Kurum

Ankara University Scientific Research Unit

Proje Numarası

16L0430009

Teşekkür

The authors gratefully acknowledge the financial support of this work by Ankara University Scientific Research Unit (Project No: 16L0430009).

Bakır Stresine Maruz Kalan Aspir (Carthamus tinctorius L.) Çeşitlerinde Yağ Asitleri Desaturaz-2 Genlerinin İfade Düzeylerinin Belirlenmesi

Öz

Ağır metal kirliliği tüm dünyada önemli bir çevre sorunudur. Ağır metallerin toprak ve sulardaki yüksek konsantrasyonlarının canlılarda genotoksisiteye neden olduğu ve fonksiyonel biyo-moleküllerin çoğuna zarar verdiği bilinmektedir. Örneğin, düşük bakır konsantrasyonu tüm organizmalar için gerekliyken, yüksek konsantrasyon bakır, bitkilerden insanlara kadar her canlı organizmayı olumsuz yönde etkileyen toksik bir elementtir. Aspir, tohum yağı için yetiştirilen, ekonomik değeri yüksek bir tarım bitkisidir. Aspir yağı, pek çok gıda ve endüstriyel uygulamalarda kullanılan Omega-9 ve Omega-6 kaynağıdır. Bu çalışmada, bakır ağır metal stresine maruz kalan dört farklı aspir çeşidinin kök, kotiledon ve yaprak dokularında Omega-9'un Omega-6'ya dönüştürülmesinden sorumlu CtFAD2 (FAD2-6, FAD2-7, FAD2-11) genlerinin qRT-PCR ile ekspresyon düzeyleri araştırılmıştır. 21 gün yetiştirildikten sonra, 24 saat boyunca bakır stresine maruz bırakılan kök, kotiledon ve yaprak dokularında RNA izolasyonu, cDNA sentezi ve qRT-PCR analizi yapılmıştır. 40 ve 80 mg L-1 konsantrasyonlarında artış gözlemlenmiştir. Artan bakır konsantrasyonlarında ise FAD2 genlerinin ekspresyon düzeylerinin azaldığı, 160 ve 320 mg L-1 'den sonra tekrar arttığı tespit edilmiştir. FAD2 genlerinin artan bakır konsantrasyonlarında ekspresyonunun önce azalması ve kritik nokta olarak kabul edilen 160 ve 320 mg L-1 sonrasında yeniden artması, strese karşı savunma mekanizmasının devreye girdiğinin ve FAD2 genlerinin strese karşı savunmada rol oynadığının bir göstergesi olarak kabul edilmiştir.
Sonuç olarak, bakır stresine maruz kalan aspir çeşitlerinde yağ desatürasyonundan sorumlu olan FAD2 genlerinin aynı zamanda stres mekanizmasıyla da ilişkili olduğu ve savunmada rol oynadığı tespit edilmiştir.

Anahtar Kelimeler

Aspir, Bakır, FAD2 Gen, Real Time PCR

Proje Numarası

16L0430009

Kaynakça

• Hapke, H.J. 1991. Effects of metals on domestic animals. VCH Verlagsgesellschaft mbH.
• Yarsan, E., Bilgili, A., Türel, I. 2000. Heavy metal levels in mussels (Unio stevenianus Krynicki) obtained from Van Lake. Turk J. Vet. Animal Science, 24, 93-96.
• Lichtenhaler, H.K. 1996. Vegetation stress: An introduction to the stress concept in plants. J. Plant Physiol, 148, 4-14.
• Buyuk, I., Bolukbasi, E., Aras, E.S. 2016. Expression of CtFAD2 gene for early selection in safflower oleic linoleic oil content. Journal of Animal and Plant Sciences, 26(5); 1383-1388. https://www.thejaps.org.pk/docs/v-26-05/27.pdf
• Levitt, J. 1972. Responses of plants to environmental Stresses. London: Academic Press, 697, New York.
• Kirbag, F. and Munzuroglu, O. 2006. Toxic effects of cadmium (Cd++) on metabolism of sunflower (Helianthus annuus L.) seedlings. Acta Agric Scand, Section B-Soil and Plant Sci, 3(56); 224-229.
• Yu, M.H. 2005. Environmental toxicology: Biological and health effects of pollutants. CRC press, Boca Raton.
• Okçu, M., Tozlu, E., Kumlay, A.M., Pehluvan, M. 2009. Ağır metallerin bitkiler üzerine etkileri. Alınteri, 17, 14-26.
• Bolukbasi, E., Aras, E.S. 2016. Determination of DNA Methylation Levels with CRED-RA Technique in the Genome of Sunflower Seedlings (Helianthus annuus L.) Subjected to Zinc Stress. International Journal of Environment, Agriculture and Biotechnology. 1(3), 438-444.
• Duffus, J.H. 2002. Heavy metals a meaningless term (IUPAC Technical Report). Pure and Applied Chemistry, 74, 793-807.
• Hall, J.L. 2002. Cellular mechanisms for heavy metal detoxification and tolerance. Journal of Experimental Botany, 53, 1-11.
• Burzynski, M., Klobus, G. 2004. Changes of photosynthetic parameters in cucumber leaves under Cu, Cd and Pb stress. Photosynth, 42(4); 505-510.
• Mithofer, A., Schulze, B., Boland, W. 2004. Biotic and heavy metal stress response in plants: Evidence for Common Signals. FEBS Lett, 566, 1-5. doi: 10.1016/j.febslet.2004.04.011
• Bolukbasi, E. 2021. Expression analysis of some stress-related genes induced by cadmium on tomato (Solanum lycopersicum L.) plants. Hittite Journal of Science and Engineering, 8(4), 339-345. doi.org/10.17350/HJSE19030000247
• Belitz, H.D., Grosch, W. 1987. Food Chemistry. Springer Verlag Berlin, Heidelberg, New York, Paris, Londra, Tokyo.
• Allan, R. 1997. Introduction: mining and metals in the environment. J. Geochem. Expl, 58, 95-100.
• Reddy, A.M., Kumar, S.G., Jyothsnakumari, G., Thimmanaik, S., Sudhakar, C. 2005. Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemosphere, 60, 97-104.
• Upadhyay, R.K., Panda, S.K. 2009. Copper-induced growth inhibition, oxidative stress and ultrastructural alterations in freshly grown water lettuce (Pistia stratiotes L.). C.R. Biol. 332, 623-632.
• Nzengue, Y., Candeias, S.M., Sauvaigo, S., Douki, T., Favier, A., Rachidi, W. and P. Guiraud. 2011. The toxicity redox mechanisms of cadmium alone or together with copper and zinc homeostasis alteration: its redox biomarkers. J. Trace Elem. Med. Biol, 25, 171-180.
• Gautam, S., Anjani, K., Srivastava, N. 2016. In vitro evaluation of excess copper affecting seedlings and their biochemical characteristics in Carthamus tinctorius L. (variety PBNS-12). Physiol Mol Biol Plants, 22(1); 121-129.
• Li, S., Zhang, G., Gao, W., Zhao, X., Deng, C., Lu, L. 2015. Plant Growth, Development and Change in Gsh Level in Safflower (Carthamus tinctorius L.) Exposed to Copper and Lead. Arch. Biol. Sci, 67(2); 385-396.
• Babaoglu, M. 2005. Safflower cultivation (Carthamus tinctorius L.). Trakya Agricultural Research Institute, p. 7, Edirne.
• Davis, P.H. 1975. Flora of Turkey and the East Aegeans Islands. Vol: 5, The University Press. Edinburg, England.
• Singh, V., Nimbkar, N. 2006. Safflower (Carthamus tinctorius L.). Chap. 6. In: Singh RJ (ed) Genetic resource, chromosome engineering, and crop improvement, Vol 4, 167-194, New York.
• Babaoglu, M. 2007. Safflower and its cultivation. Trakya Agricultural Research Institute, Edirne.
• Anonymous. 2013. Web sitesi: https://www.agroscope.admin.ch/agroscope/en/home/topics/plantproduction/fieldcrops/kulturarten/alternative-kulturpflanzen/saflor.html, Erişim tarihi: 26.06.2018.
• Bayrak, A. 1997. Investigation of fatty acid composition of summer-winter Safflower (Carthamus tinctorius L.) cultivars and lines tested in Ankara and Şanlıurfa. Journal of Food, 22(4); 269-277.
• Ahlawat, I.P.S. 2008. Agronomy-Rabi Crops Safflower. Division of Agronomy Indian Agricultural Research Institute, 10, New Delhi/India.
• Er, C., Başalma, D., Ekiz, H., Sancak, C. 2011. Field Crops-II, Anadolu University Publication No: 2254, I. Edition, 235, Eskişehir.
• Macartney, A., Maresca, B., Cossins, A.R. 1994. Acyl-CoA desaturases and the adaptive regulation of membrane lipid composition, in: A.R. Cossins (Ed.), Temperature Adaptation of Biological Membranes, Portland Press, pp. 129-139, London.
• Murata, N., Wada, H. 1995. Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria. Biochem. J, 308, 1-8. doi: 10.1042/bj3080001
• Delshad, E., Yousefi, M., Sasannezhad, P., Rakhshandeh, H., Ayati, Z. 2018. Medical uses of Carthamus tinctorius L. (Safflower): A comprehensive review from traditional medicine to modern medicine. Electronic Physician, 10(9); 6672-6681. doi: 10.19082/6672
• Sahin, G., Tasligil, N. 2016. Safflower (Carthamus tinctorius L.): An industrial plant with increasing strategical importance. Turkish Journal of Geography, 66, 51-62. doi: 10.17211/tcd.48394
• Karabaş, H. 2013. Ülkemiz ıslahçı çeşitlerinden Remzibey-05 Aspir (Carthamus tinctorius L.) tohumlarından üretilen biyodizelin yakıt özelliklerinin incelenmesi. Uludağ Üniversitesi Ziraat Fakültesi Dergisi, 27(1); 9-17.
• Tortopoğlu, A.İ. 2011. Benzin ve motorinde kullanılacak biyoyakıt üretimi için gerekli eşdeğer tarım arazisi miktarı. Hasad Aylık Tarım Dergisi, 27(319); 17-32.
• Gilbert, J. 2008. International safflower production-An Overview. 7. International Safflower Conference. Australian Oilseeds Federation. Wagga Wagga, Australia.
• Mastin, B.J., Rodgers, J.H. 2014. Toxicity and bioavailability of copper herbicides (Clearigate, Cutrine-Plus, and Copper Sulfate) to freshwater animals. Environmental Contamination and Toxicology, 39(4), 445-51.
• Guan, L.L., Wang, Y.B., Shen, H., Hou, K., Xu, Y. 2012a. Molecular cloning and expression analysis of genes encoding two microsomal oleate desaturases (FAD2) from safflower (Carthamus tinctorius L.). Plant mol. Boil. Rep, 30, 139-148. doi: 10.1007/s11105-011-0322-5
• Guan, L.L., Xu, Y.W., Wang, Y.B., Chen, L., Shao, J. 2012b. Isolation and characterization of temperature-regulated microsomal oleate desaturase gene (FAD2) from normal type and high oleic safflower (Carthamus tinctorius L.). Plant Mol. Biol. Rep. 30, 391-402. doi: 10.1007/s11105-011-0349-7
• Cao, S., Zhou, X.R., Wood, C.C., Green, A.G., Singh, S.P., Liu, L., Liu, Q. 2013. A large and functionally diverse family of FAD2 genes in safflower (Carthamus tinctorius L.). BMC Plant Biology, 13(5); 1-18.
• Livak, J.K., Schmittgen, D.T. 2001. Analysis of relative gene expression data using Real- Time Quantitative PCR and the 2-ΔΔCt method. Methods, 25, 402-408. doi: 10.1006/meth.2001.1262
• Dunnett, C.W. 1955. A multiple comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association, 50, 1096-1121. https://www.jstor.org/stable/pdf/2281208.pdf
• Roscoe, J.T. 1975. Fundemental research statistics for the behavioral sciences. New York: Holt, Rinehart and Winston, Inc. 63.
• Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jonak, J. 2006. The real-time polymerase chain reaction. Mol. Aspects Med., 2(3); 95-125. doi: 10.1016/j.mam.2005.12.007
• Bolukbasi, E. 2022. Analysis of Genetic and Epigenetic Effects of Sunflower (Helianthus Annuus L.) Seedlings in Response to Copper Stress. Fresenius Environmental Bulletin, 31(4), 4596-4602.
• Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E. 1994. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 6, 147-158. doi: 10.1105/tpc.6.1.147
• Zhang, M., Barg, R., Yin, M., Gueta-Dahan, Y., Leikin-Frenkel, A., Salts, Y., Shabtai, S., Ben-Hayyim, G. 2005. Modulated fatty acid desaturation via overexpression of two distinct ω -3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants. Plant J., 44, 361-371.
• Tang, G.Q., Novitzky, W.P., Griffin, H.C., Huber, S.C., Dewey, R.E. 2005. Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. The Plant Journal, 44, 433-446. doi: 10.1111/j.1365-313X.2005.02535.x
• Wang, H.S., Yu, C., Tang, X.F., Zhu, Z.J., Ma, N.N. 2014. A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress. Plant Cell Rep., 33, 131-142. doi: 10.1007/s00299-013-1517-z
• Khodakovskaya, M., McAvoy, R., Peters, J., Wu, H., Li, Y. 2006. Enhanced cold tolerance in transgenic tobacco expressing a chloroplast omega-3 fatty acid desaturase gene under the control of a cold-inducible promoter. Planta, 223, 1090-1100. doi: 10.1007/s00425-005-0161-4
• Rodríguez-Vargas, S,, Sánchez-García, A., Martínez-Rivas, J.M., Prieto, J.A., Randez-Gil, F. 2007. Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt. Applied and Environmental Microbiology, 73, 110-116. doi: 10.1128/AEM.01360-06
• Zhang, D.Y., Pirtle, I.L., Park, S.J., Nampaisansuk, M., Neogi, P., Wanjie, S.W., Pirtle, R.M., Chapman, K.D. 2009. Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. Plant Physiol Biochem, 47, 462-471.
• Zhang, J., Liu, H., Sun, J., Li, B., Zhu, Q., Chen, S., Zhang, H. 2012. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth. PloS one, 7(1); 303-313. doi: 10.1371/journal.pone.0030355
• Zhang, Q.Y., Yu, R., Xie, L.H., Rahman, M.M., Kilaru, A., Niu, L.X., Zhang, Y.L. 2018. Fatty acid and associated gene expression analyses of three tree peony species reveal key genes for α-linolenic acid synthesis in seeds. Frontiers in plant science, 9, 106-117. doi: 10.3389/fpls.2018.00106
• Yuan, S., Wu, X., Liu, Z., Luo, H., Huang, R. 2012. Abiotic stresses and phytohormones regulate expression of FAD2 gene in Arabidopsis thaliana. Journal of Integrative Agriculture, 11(1); 62-72. doi: 10.1016/S1671-2927(12)60783-4
• Kargiotidou, A., Deli, D., Galanopoulou, D., Tsaftaris, A., Farmaki, T. 2008. Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum). The Journal of Experimental Botany, 59, 2043-2056.
• Maeda, H., Sage, T.L., Isaac, G., Welti, R., Dellapenna, D. 2008. Tocopherols modulate extraplastidic polyunsaturated fatty acid metabolism in Arabidopsis at low temperature. The Plant Cell, 20, 452-70.
• Vrinten, P., Hu, Z., Munchinsky, M.A., Rowland, G., Qiu, X. 2005. Two FAD3 desaturase genes control the level of linolenic acid in flax seed. Plant Physiol., 139, 79-87.
• Feng, J., Dong, Y., Liu, W., He, Q., Daud, M.K. 2017. Genome wide identification of membrane-bound fatty acid desaturase genes in Gossypium hirsutum and their expressions during abiotic stress. Scientific Reports, 7, 1-12. doi: 10.1038/srep45711
• Wang, J.W., Ming, F., Pittman, J., Han, Y., Hu, J. 2006. Characterization of a rice (Oryza sativa L.) gene encoding a temperature-dependent chloroplast omega-3 fatty acid desaturase. Biochem. Biophys. Res. Commun., 340, 1209-1216. doi: 10.1016/j.bbrc.2005.12.126
• Li, L.Y., Wang, X.L., Gai, J.Y., Yu, D.Y. 2007. Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. J Plant Physiol, 164, 1516-1526. doi: 10.1016/j.jplph.2006.08.007
• Maksymiec, W. 2007. Signaling responses in plants to heavy metal stress. Acta Physiol Plant, 29, 177-187.
• Li, D., Hu, B., Wang, Q., Liu, H., Pan, F. 2015. Identification and evaluation of reference genes for accurate transcription normalization in safflower under different experimental conditions. PloS One, 10(10); 1-16. doi: 10.1371/journal.pone.0140218
• Gautam, S., Anjani, K. , Srivastava, N. 2016. In vitro evaluation of excess copper affecting seedlings and their biochemical characteristics in Carthamus tinctorius L. (variety PBNS-12). Physiol Mol Biol Plants, 22(1); 121-129.
• Yang, Q., Fan, C., Guo, Z., Qin, J., Wu, J. 2012. IdentiWcation of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents. Theor. Appl. Genet, 125, 715-729. doi: 10.1007/s00122-012-1863-1
• Xue, Y., Yin, N., Chen, B., Liao, F., Win, A.N. 2017. Molecular cloning and expression analysis of two FAD2 genes from chia (Salvia hispanica). Acta physiologiae plantarum, 39(4); 82-95. doi: 10.1007/s11738-017-2390-0