Oryza sativa Osmyb4 geni aktarılmış transgenik patateste Osmyb4 gen ifadesinin tuzluluk toleransına etkisi

Year 2020, Volume: 35 Issue: 1, 115 - 123, 14.02.2020

Gülsüm Aydın

https://doi.org/10.7161/omuanajas.624749

Cited By: 1

Abstract

Bu çalışmada Osmyb4
geni ile transforme edilmiş patateste, MYB4’ün tuz toleransına olan potansiyel
etkileri araştırılmıştır. Daha önce yapmış olduğumuz bir çalışmada Osmyb4
geni aktarılmış transgenik patates bitkilerinin yüksek konsantrasyonda tuz
içeren ortamda, gen aktarılmamış bitkilerden daha iyi fizyolojik gelişim
gösterdiği tespit edilmiştir. Bu noktadan hareketle MYB4’ün patateste tuz toleransına
etkisini araştırmak amacıyla, transgenik bitkiler (TR) ve gen aktarılmamış
bitkiler (WT) 300 mM tuz stresine tabi tutularak elektrolit salınımı,
malondialdehit (MDA) miktarı, nispi su içeriği (RWC), hidrojen peroksit (H₂O₂)
konsantrasyonu, klorofil içeriği ve prolin miktarı belirlenmiştir. Bunların
yanı sıra literatürde tuz stresi ile ilişkili olduğu belirtilen NAC072,
NAC024, CDPK4 ve P5CS genlerinin ekspresyonu gerçek zamanlı
kantitatif PCR (qRT PCR) aracılığı ile incelenmiştir. Biyokimyasal analizler
sonucunda elde edilen veriler elektrolit salınımı, prolin miktarı ve H₂O₂
miktarı bakımından WT bitkiler ile TR bitkiler arasında anlamlı bir farklılık
olmadığını ortaya koymuştur. RWC transgenik S2 hattında transgenik olmayan
bitkilere kıyasla anlamlı oranda (P < 0.05) yüksek bulunmuştur. MDA miktarı
her iki transgenik hatta (S2 ve M48) WT bitkilere kıyasla anlamlı oranda düşük
bulunmuştur. qPCR analizi sonucunda elde edilen relatif ekspresyon değerleri,
S2’de NAC072 geninin ekspresyon seviyesinin WT ile karşılaştırıldığında
anlamlı olarak (P < 0.05) yüksek olduğunu göstermiştir. NAC024 ve P5CS
genlerinin ekspresyon seviyeleri de transgenik bitkilerde transgenik
olmayanlara oranla yüksek bulunmuştur. CDPK4 geninin ekspresyon seviyesi
ise S2 bitkileri ile WT bitkilerde birbirine yakın seviyede tespit edilmiştir.
Elde edilen veriler bir bütün olarak değerlendirildiğinde MYB4 transkripsiyon
faktörünün RWC, MDA miktarı ve stresle ilişkili çeşitli genlerin ekspresyon
seviyesini etkilemek suretiyle tuz stresi tolerans mekanizmasında rol
oynayabileceğine işaret etmiştir. 

Keywords

Abiyotik stres, MYB4, patates, transgenik, tuzluluk

Supporting Institution

Selçuk Üniversitesi

Project Number

16401046

Thanks

Bu çalışma Selçuk Üniversitesi Bilimsel Araştırmalar Koordinatörlüğü tarafından 16401046 nolu proje ile desteklenmiştir.

Effect of Osmyb4 gene expression on salinity tolerance of potato transformed with Oryza sativa Osmyb4 gene

Abstract

In this study, potential involvement of MYB4 in
salt tolerance of potato trasformed with Osmyb4 was investigated. In our
previous studies transgenic potato plants heterologously expressing Osmyb4
gene displayed better growth compared to non-transgenic plants upon exposure to
high salt concentrations. These results have led to a detailed analysis of
potential involvement of MYB4 in salt tolerance of potato. For this purpose
transgenic (TR) and non-transgenic (WT) plants were subjected to 300 mM salt
concentrations and electrolyte leakage, malonedialdehyde level (MDA), relative
water content (RWC), the amount of hydrogen peroxide (H₂O₂),
chlorophyll content and the level of proline was identified. The expression of
certain genes reported to be involved in salinity tolerance, namely, NAC072,
NAC024, CDPK4 and P5CS was also investigated by
quantitative real time PCR (qRT-PCR). The data obtained through biochemical
tests showed that there were no significant difference between WT and TR plants
with respect to electrolyte leakage, proline content and H₂O₂
content. RWC was significantly higher (P <0.05) in the transgenic line S2
compared to TR plants. MDA content in both transgenic lines (S2 and M48) were
found to be significantly lower compared to WT plants. Relative expression data
obtained by qPCR analysis revealed that expression of NAC072 was
significantly higher in S2 compared to WT (P < 0.05). Expression level of NAC024
and P5CS genes were also higher in transgenic plants compared to
non-transgenic WT. On the other hand expression level of CDPK4 was
similar in WT and transgenic lines. The results have indicated that MYB4
transcription factor may regulate salt stress tolerance mechanism in potato by
affecting RWC, MDA content and expression of certain stress related genes.

Keywords

Abiotic stress, MYB4, Potato, Salinity, Transgenic

Project Number

16401046

References

• Aydin, G., Yucel, M., Chan, M.-T., Oktem, H.A., 2014. Evaluation of abiotic stress tolerance and physiological characteristics of potato (Solanum tuberosum L. cv. Kennebec) that heterologously expresses the rice Osmyb4 gene. Plant Biotechnology Reports, 8(3): 295-304.
• Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil, 39: s. 205–207.
• Bernt, E., Bergmeyer, H.U., 1974. Inorganic Peroxides, In: Bergmeyer, H.U. (Eds). Methods of enzymatic analysis. Academic Press. pp. 2246-2248.
• Bhattacharya, R. C., Maheswari, M., Dineshkumar, V., Kirti, P. B., Bhat, S. R., Chopra, V. L., 2004. Transformation of Brassica oleracea var. capitata with bacterial betA gene enhances tolerance to salt stress. Scientia Horticulturae, 100: 215–227.
• Bray, E. A., Bailey-Serres, J., Wewrwtilnyk, E., 2000. Responses to abiotic stresses. In: Buchanan, B.B., Gruissem, W., Jones, R.L. (Eds.). Biochemistry & molecular biology of plants. Rockville, Md.: American Society of Plant Physiologists. pp. 1158-1249.
• Burssens, S., Himanen, K., Cotte, B.V., Beeckman, T., Montagu, M.V., Inze, D. Verbruggen, N., 2000. Expression of Cell Cycle Regulatory Genes and Morphological Alterations in Response to Salt Stress in Arabidopsis thaliana. Planta, 211: 632-640.
• Chen, R. M., Ni, Z. F., Nie, X. L., Qin, Y. X., Dong, G. Q., Sun, Q. X., 2005. Isolation and characterization of genes encoding Myb transcription factor in wheat (Triticum aestivem L.). Plant Science, 169(6): 1146-1154.
• Çulha, Ş., Çakırlar, H., 2011. Tuzluluğun Bitkiler Üzerine Etkileri ve Tuz Tolerans Mekanizmaları. Afyon Kocatepe Üniversitesi Fen Bilimleri Dergisi, 11: 11-34.
• Dajic, Z., 2006. Salt Stress, Physiology and Molecular Biology of Stress Tolerance in Plants. 345p, Dordrecht, The Netherlands.
• de-Lacerda, C.F., Cambraia, J., Oliva, M.A., Ruiz, H.A., Prisco, J.T., 2003. Solute accumulation and distribution during shoot and leaf development in two sorghum genotypes under salt stress. Environ Exp Bot, 49:107–120.
• Du, H., Zhang, L., Liu, L., Tang, X. F., Yang, W. J., Wu, Y. M., Huang, Y. B., Tang, Y. X., 2009. Biochemical and molecular characterization of plant MYB transcription factor family. Biochemistry-Moscow, 74(1): 1-11.
• Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., Lepiniec, L., 2010. MYB transcription factors in Arabidopsis. Trends Plant Sci, 15: 573–581.
• Farquhar, G.D., S.C. Wong, J.R. Evans, K.T. Hubic, 1989. Photosynthesis and gas exchange. In: H.G. Jones, T.J. Flowers & M.B. Jones (Eds.), Plant under Stress. pp 47–69. Cambridge University Press, Cambridge.
• Golldack, D., Li, C., Mohan, H., Probst, N., 2014. Tolerance to drought and salt stress in plants: Unraveling the signaling Networks. Front. Plant Sci, 5: 151.
• Gomaa, A. M., Raldugina, G. N., Burmistrova, N. A., Radionov, N. V., & Kuznetsov, V. V., 2011. Response of transgenic rape plants bearing the Osmyb4 gene from rice encoding a trans-factor to low above-zero temperature. Russian Journal of Plant Physiology, 59(1): 105–114.
• Hayat, S., Hayat, Q., Alyemeni, M.N., Wani, A.S., Pichtel, J., Ahmad, A., 2012. Role of proline under changing environments. Plant Signaling & Behavior, 7(11): 1456–1466.
• Hemm, M.R., Herrmann, K.M., Chapple, C., 2001. AtMYB4: a transcription factor general in the battle against UV. Trends in Plant Science, 6(4): 135-136.
• Hernandez, M., Fernandez-Garcia, N., Diaz-Vivancos, P., Olmos, E., 2010. A different role for hydrogen peroxide and the antioxidative system under short and long salt stress in Brassica oleracea roots. J Exp Bot, 61: 521–535.
• Hettenhausen, C., Yang, D.-H., Baldwin, I. T., Wu, J., 2013. Calcium-dependent protein kinases, CDPK4 and CDPK5, affect early steps of jasmonic acid biosynthesis in Nicotiana attenuata. Plant Signaling & Behavior, 8(1): e22784.
• Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular, 347: 1-32.
• Hu, Y., Schmidhalter, U., 2005. Drought and Salinity: A Comparison of Their Effects on Mineral Nutrition of Plants. Journal of Plant Nutrient and Soil Science, 168: 541-549.
• Jin, H., Cominelli, E., Bailey, P., Parr, A., Mehrtens, F., Jones, J., Tonelli, C., Weisshaar, B., Martin, C., 2000. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J, 19(22): 6150-6161.
• Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., & Shinozaki, K., 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnology, 17(3): 287-291.
• Laura, M., Consonni, R., Locatelli, F., Fumagalli, E., Allavena, A., Coraggio, I., Mattana, M., 2010. Metabolic response to cold and freezing of Osteospermum ecklonis overexpressing Osmyb4. Plant Physiol Biochem, 48(9): 764-771.
• Li, C., Ng, C.K.-Y., Fan, L.-M., 2015. MYB transcription factors, active players in abiotic stres signaling. Environ. Exp. Bot., 114: 80–91.
• Lichtenthaler, H. K., 1987. Chlorophyll and carotenoids: Pigments of photosynthetic biomembranes. Meth. Enzym., 148: 331-382.
• Lilley, J.M., Ludlow, M.M., 1996. Expression of osmotic adjustment and dehydration tolerance in diverse rice lines. Field Crop Res, 48: 185–197.
• Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., Shinozaki, K., 1998. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10(8): 1391-1406.
• Lugojan,C., Ciulca, S., 2011. Evaluation of relative water content in winter wheat. J. Hortic. Fores. Biotechnol., 15: 173–177
• Lutts, S., Majerus, V., Kinet, J-M. 1999. NaCl effects on proline metabolism in rice ( Oryza sativa ) seedlings. Physiol Plant, 105:450–458.
• Madhava Rao, K.V., Srestry, T.V., 2000. Antioxidative parameters in the seedlings of pigeon pea in response to zinc and nickel stress. Plant Science, 157: 113-128.
• Mahajan, S., Tuteja, N., 2005. Cold, salinity and drought stresses: An overview. Archives of Biochemistry and Biophysics, 444(2): 139-158.
• Mattana, M., Biazzi, E., Consonni, R., Locatelli, F., Vannini, C., Provera, S., Coraggio, I., 2005. Overexpression of Osmyb4 enhances compatible solute accumulation and increases stress tolerance of Arabidopsis thaliana. Physiologia Plantarum, 125(2): 212-223.
• Mohammad, M., Shibli, R., Ajlouni, M. Nimri, L., 1998. Tomato Root and Shoot Responses to Salt Stress Under Different Levels of Phosphorus Nutrition. Journal of Plant Nutrition, 21(8): 1667-1680.
• Munns, R. Tester, M., 2008. Mechanisms of Salinity Tolerance. Annual Review of Plant Biology, 59: 651-681.Munns, R., 2002. Salinity, Growth and Phytohormones, Salinity: Environment-Plants-Molecules. Kluwer Academic Publishers, 522p, Dordrecht, The Netherlands.
• Munns, R., James, R.A., 2003. Screening method for salinity tolerance: A case study with tetraploid wheat. Plant Soil, 253: 201-218.
• Nanjo, T., Kobayashi, M., Yoshiba, Y., Kakubari, Y., Yamaguchi-Shinozaki, K., Shinozaki, K., 1999. Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett, 461: 205–210.
• Nicot, N., Hausman, J.F., Hoffmann, L., Evers, D., 2005. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany, 56(421): 2907-2914.
• Pasquali, G., Biricolti, S., Locatelli, F., Baldoni, E., Mattana, M., 2008. Osmyb4 expression improves adaptive responses to drought and cold stress in transgenic apples. Plant Cell Rep, 27(10): 1677-1686.
• Pérez-Arellano, I., Carmona-Álvarez, F., Martínez, A. I., Rodríguez-Díaz, J., Cervera, J., 2010. Pyrroline-5-carboxylate synthase and proline biosynthesis: From osmotolerance to rare metabolic disease. Protein Science, 19: 372-382.
• Poljakoff- Mayber, A., Gale, J., 1975. Plant in Salin Environments. Springer-Verlag, 213p, Berlin.
• Quan, L.J., Zhang, B., Shi, W.W., Li, H.Y., 2008. Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. Journal of Integrative Plant Biology, 50: 2–18.
• Raldugina, G. N., Maree, M., Mattana, M., Shumkova, G., Mapelli, S., Kholodova, V. P., … Kuznetsov, V. V., 2018. Expression of rice OsMyb4 transcription factor improves tolerance to copper or zinc in canola plants. Biologia Plantarum, 62(3): 511–520.
• Reddy, M.P., Iyengar, E.R.R., 1999. Crop Responses to Salt Stress: Seawater Application and Prospects, Handbook of Plant Crop Stress, 1198p, New York.
• Sharma, S., Villamor, J.G., Verslues, P.E., 2011. Essential role of tissuespecific proline synthesis and catabolism in growth and redox balance at low water potential. Plant Physiology, 157: 292–304.
• Singh, A.K., Sharma, V., Pal, A.K., Acharya, V., Ahuja, P.S., 2013. Genome-wide organization and expression profiling of the NAC transcription factor family in potato (Solanum tuberosum L.). DNA Research, 20: 403–423.
• Smart, R.E., Bingham, G.E., 1974. Rapid estimates of relative water content. Plant Physiol, 53: 258–260.
• Szabados, L., Savoure´, A., 2010. Proline: a multifunctional aminoacid. Trends Plant Sciences, 15:89–97.
• Thipyapong, P., Melkonian, J., Wolf, D.W., Steffens, J.C., 2004. Suppression of polyphenol oxidases increases stress tolerance in tomato. Plant Sci., 167: 693-703.
• Tran, L.S., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K., 2004. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell, 16: 2481–2498.
• Vannini, C., Campa, M., Iriti, M., Genga, A., Faoro, F., Carravieri, S., Rotino, G. L., Rossoni, M., Spinardi, A., Bracale, M., 2007. Evaluation of transgenic tomato plants ectopically expressing the rice Osmyb4 gene. Plant Science, 173(2): 231-239.
• Vannini, C., Iriti, M., Bracale, M., Locatelli, F., Faoro, F., Croce, P., Pirona, R., Di Maro, A., Coraggio, I., Genga, A., 2006. The ectopic expression of the rice Osmyb4 gene in Arabidopsis increases tolerance to abiotic, environmental and biotic stresses. Physiological and Molecular Plant Pathology, 69(1-3): 26-42.
• Vannini, C., Locatelli, F., Bracale, M., Magnani, E., Marsoni, M., Osnato, M., Mattana, M., Baldoni, E., Coraggio, I., 2004. Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants. Plant Journal, 37(1): 115-127.
• Wang, W. X., Vinocur, B., Altman, A., 2003. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 218(1): 1-14.
• Wolf, O., Munns, R., Tonnet, M., Jeschke, W. D., 1991. The Role of the Stem in the Partitioning of Na+ and K+ in Salt-Treated Barley. J. of Exp. Bot., 42: 278-282.
• Yang, A., Dai, X., Zhang, W.-H., 2012. A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. Journal of Experimental Botany, 63(7): 2541–2556.
• Zhao, G.Q., Ma, B.L., Ren, C.Z., 2007. Growth, gas exchange, chlorophyll fluorescence and ion content of naked oat in response to salinity. Crop Sci., 47: 123-131.