Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean

  • Kim, Hye Jeong (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University) ;
  • Cho, Hyun Suk (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University) ;
  • Pak, Jung Hun (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University) ;
  • Kwon, Tackmin (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University) ;
  • Lee, Jai-Heon (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University) ;
  • Kim, Doh-Hoon (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University) ;
  • Lee, Dong Hee (Genomine Advanced Biotechnology Research Institute, Genomine Inc.) ;
  • Kim, Chang-Gi (Bio-Evaluation Center, KRIBB) ;
  • Chung, Young-Soo (Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University)
  • Received : 2017.10.12
  • Accepted : 2018.03.07
  • Published : 2018.05.31
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Abstract

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from $T_2$ seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than ${\underline{n}}on-{\underline{t}}ransgenic$ (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants ($T_2$) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

Keywords

References

  1. Abdeen, A., Schnell, J., and Miki, B. (2010). Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3. BMC Genomics 11, 69. https://doi.org/10.1186/1471-2164-11-69
  2. Bruce, W.B., Edmeades, G.O., and Barker, T.C. (2002). Molecular and physiological approaches to maize improvement for drought tolerance. J. Exp. Bot. 53, 13-25. https://doi.org/10.1093/jexbot/53.366.13
  3. Choi, H.I., Hong, J.H., Ha, J.O., Kang, J.Y., and Kim, S.Y. (2000). ABFs, a family of ABA-responsive element binding factors. J Biol Chem. 275, 1723-1730. https://doi.org/10.1074/jbc.275.3.1723
  4. Choi, Y.S., Kim, Y.M., Hwang, O.J., Han, Y.j., Kim, S.Y., and Kim, J.I. (2013). Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass. Plant Biotechnol. Rep. 7, 165-173. https://doi.org/10.1007/s11816-012-0245-0
  5. Dan, Y. (2008). Biological functions of antioxidants in plant transformation. In Vitro Cell. Dev. Biol. Plant 44, 149-161. https://doi.org/10.1007/s11627-008-9110-9
  6. Di, R., Purcell, V., Collins, G.B., and Ghabrial, S.A. (1996). Production of transgenic lines expressing the bean pod mottle virus coat protein precursor gene. Plant Cell Rep. 15, 746-750. https://doi.org/10.1007/BF00232220
  7. Fan, L., Zheng, S., and Wang, X. (1997). Antisense suppression of phospholipase $D{\alpha}$ retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell 9, 2183-2196.
  8. Finkelstein, R., Gampala, S.S.L., Lynch, T.J., Thomas, T.L., and Rock, C.D. (2005). Redundant and distinct functions of the ABA response loci ABA-INSENSITIVE(ABI)5 and ABRE-BINDING FACTOR (ABF)3. Plant Mol. Biol. 59, 253-267. https://doi.org/10.1007/s11103-005-8767-2
  9. Fu, Y., Li, H., and Yang, Z. (2002). The ROP2 GTPase controls the formation of cortical fine F-Actin and the early phase of directional cell expansion during Arabidopsis organogenesis. Plant Cell 14, 777-794. https://doi.org/10.1105/tpc.001537
  10. Gao, S.Q., Chen, M., Xu, Z.S., Zhang, C.P., Li, L., Xu, H.J., Tang, Y.M., Zhao, X., and Ma, Y.Z. (2011). The soybean GmbZIP1 transcription factor enhances multiple abiotic stress tolerances in transgenic plants. Plant Mol. Biol. 75, 537-553. https://doi.org/10.1007/s11103-011-9738-4
  11. Hansen, G., and Wright, M.S. (1999). Recent advances in the transformation of plants. Trends Plant Sci. 4, 226-231. https://doi.org/10.1016/S1360-1385(99)01412-0
  12. Hinchee, M.A.W., Connor-Ward, D.V., Newell, C.A., McDonnell, R.E., Sato, S.J., Gasser CS, Fischhoff, D.A., Re, D.B., Fraley, R.T., and Horsch, R.B. (1988). Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Nat. Biotechnol. 6, 915-922. https://doi.org/10.1038/nbt0888-915
  13. Hossain, M.A., Cho, J.I., Han, M.H, Ahn, C.H., Jeon, J.S., An, G.H., and Park, P.B. (2010). The ABRE-binding bZIP transcription factor OsABF2 is a positive regulator of abiotic stress and ABA signaling in rice. J. Plant Physiol. 167, 1512-1520. https://doi.org/10.1016/j.jplph.2010.05.008
  14. Hu, R., Fan, C., Li, H. Zheng, Q., and Fu, Y.F. (2009). Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol. Biol. 10, 93. https://doi.org/10.1186/1471-2199-10-93
  15. Huang, X.Y., Chao, D.Y., Gao, J.P., Zhu, M.Z., Shi, M., and Lin, H.X. (2009). A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control. Genes Dev. 23, 1805-1817. https://doi.org/10.1101/gad.1812409
  16. Jia, H., Wang, C., Wang, F., Liu, S., Li, G., and Guo, X. (2015). GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana. PLoS ONE 10, e0120646. https://doi.org/10.1371/journal.pone.0120646
  17. Kang, J.Y., Choi, H.I., Im, M.Y., Kim, and S.Y. (2002). Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell 14, 343-357. https://doi.org/10.1105/tpc.010362
  18. Karimi, M., Inze, D., and Depicker, A. (2002). $Gateway^{TM}$ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193-195. https://doi.org/10.1016/S1360-1385(02)02251-3
  19. Kim, J.B. Kang, J.Y., and Kim, S.Y. (2004). Over-expression of a transcription factor regulating ABA-responsive gene expression confers multiple stress tolerance. Plant Biotechnol. J. 2, 459-466. https://doi.org/10.1111/j.1467-7652.2004.00090.x
  20. Kim, M.J., Kim, H.J., Pak, J.H., Cho, H.S., Choi, H.K., Jung, H.W., Lee, D.H., and Chung Y.S. (2017). Overexpression of AtSZF2 from Arabidopsis Showed Enhanced Tolerance to Salt Stress in Soybean. Plant Breed. Biotech. 5, 1-15. https://doi.org/10.9787/PBB.2017.5.1.1
  21. Kim, M.J., Kim, J.K., Kim, H.J., Pak, J.H., Lee, J.H., Kim, D.H., Choi, H.K., Jung, H.W., Lee, J.D., Chung, Y.S., et al. (2012). Genetic modification of the soybean to enhance the ${\beta}$-carotene content through seed-specific expression. PLoS ONE 7, e48287. https://doi.org/10.1371/journal.pone.0048287
  22. Kim, H.J., Kim, M.J., Pak, J.H., Jung, H.W., Choi, H.K., Lee, Y.H., Baek, I.Y., Ko, J.M., Jeong, S.C., Pack, I.S., et al. (2013). Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi. Plant Biotechnol Rep 7, 425-433. https://doi.org/10.1007/s11816-013-0279-y
  23. Lee, K.J., Seo, J.K., Lee, H.Y., Jeon, E.H., Shin, S.H., Lee, J.H., Kim, D.H., Ko, J.M., Hahn, W.Y., Baek, I.Y., et al. (2006). Optimization of genetic transformation conditions for Korean soybean cultivars. Kor. J. Life Sci. 16, 289-296. https://doi.org/10.5352/JLS.2006.16.2.289
  24. Lee, S.C., and Luan, S. (2012). ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Environ. 35, 53-60. https://doi.org/10.1111/j.1365-3040.2011.02426.x
  25. Li, S., Cong, Y., Liu, Y., Wang, T., Shuai, Q., Chen, N., Gai, J., and Li, Y. (2017). Optimization of Agrobacterium-mediated transformation in soybean. Front. Plant Sci. 8, 246.
  26. Luan, S. (2002). Signalling drought in guard cells. Plant Cell Environ 25, 229-237. https://doi.org/10.1046/j.1365-3040.2002.00758.x
  27. Manavalan, L.P., Guttikonda, S.K., Tran, L.S.P., and Nguyen, H.T. (2009). Physiological and molecular approaches to improve drought resistance in soybean. Plant Cell Physiol. 50, 1260-1276. https://doi.org/10.1093/pcp/pcp082
  28. Meurer, C.A., Dinkins, R.D., and Collins, G.B. (1998). Factors affecting soybean cotyledonary node transformation. Plant Cell Rep. 18, 180-186. https://doi.org/10.1007/s002990050553
  29. Nakashima, K., and Yamaguchi-Shinozaki, K. (2013). ABA signaling in stress-response and seed development. Plant Cell Rep. 32, 959-970. https://doi.org/10.1007/s00299-013-1418-1
  30. Oh, S.J., Song, S.I., Kim, Y.S., Jang, H.J., Kim, S.Y., Kim, M.J., Kim, Y.K., Nahm, B.H., and Kim, J.K. (2005). Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiol. 138, 341-351. https://doi.org/10.1104/pp.104.059147
  31. Olhoft, P.M., Flagel, L.E., Donovan, C.M., and Somers, D.A. (2003). Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216, 723-735.
  32. Pathan, S., Nguyen, H.T., Sharp, R.E., and Shannon, J.G. (2010). Soybean improvement for drought, salt and Flooding tolerance. Kor J. Breed. Sci. 42, 329-338.
  33. Paz, M.M., Martinez, J.C., Kalvig, A.B., Fonger, T.M., and Wang, K. (2006). Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacteriummediated soybean transformation. Plant Cell Rep. 25, 206-213. https://doi.org/10.1007/s00299-005-0048-7
  34. Schroeder, J.I., Kwak, J.M., and Allen, G.J. (2001). Guard cell abscisic acid signalling and engineering drought hardiness in plants. Nature 410, 327-330. https://doi.org/10.1038/35066500
  35. Sinclair, T.R., Purcell, L.C., and Sneller, C.H. (2004). Crop transformation and the challenge to increase yield potential. Trends Plant Sci. 9, 2.
  36. Specht, J.E., Hume, D.J., and Kumudini, S.V. (1999). Soybean yield potential - a genetic and physiological perspective. Crop Sci. 39, 1560-1570. https://doi.org/10.2135/cropsci1999.3961560x
  37. Vanjildorj, E., Bae, T.W., Riu, K.Z., Kim, S.Y., and Lee, H.Y. (2005). Overexpression of Arabidopsis ABF3 gene enhances tolerance to drought and cold in transgenic lettuce (Lactuca sativa). Plant Cell Tissue Org 83, 41-50. https://doi.org/10.1007/s11240-005-3800-3
  38. Wang, R.S., Pandey, S., Li, S., Gookin, T.E., Zhao, Z., Albert, R., and Assmann, S.M. (2011). Common and unique elements of the ABAregulated transcriptome of Arabidopsis guard cells. BMC Genomics 12, 216. https://doi.org/10.1186/1471-2164-12-216
  39. Wang, H., Wang, H., Shao, H., and Tang, X. (2016). Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology. Front. Plant Sci. 7, 67.
  40. Wu, C., Niu, Z., Tang, Q., and Huang, W. (2008). Estimating chlorophyll content from hyperspectral vegetation indices: modeling and validation. Agr. Forest Meteorol. 148, 1230-1241. https://doi.org/10.1016/j.agrformet.2008.03.005
  41. Xoconostle-Cazares, B., Ramirez-Ortega, FA., Flores-Elenes, L., and Ruiz-Medrano, R. (2010). Drought tolerance in crop plants. Am. J. Plant Physiol. 5, 241-256. https://doi.org/10.3923/ajpp.2010.241.256
  42. Yang, W.Y., Liu, X.D., Chi, X.J., Wu, C.A., Li, Y.Z., Song, L.L., Liu, X.M., Wang, Y.F., Wang, F.W., Zhang, C., et al. (2011). Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways. Planta 233, 219-229. https://doi.org/10.1007/s00425-010-1279-6
  43. Zeevaart, J.A.D., and Creelman, R.A. (1988). Metabolism and physiology of abscisic acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 439-473. https://doi.org/10.1146/annurev.pp.39.060188.002255
  44. Zhang, H., Liu, W., Wan, L., Li, F., Dai, L., Li, D., Zhang, Z., and Huang, R. (2010). Functional analyses of ethylene response factor JERF3 with the aim of improving tolerance to drought and osmotic stress in transgenic rice. Transgenic Res 19, 809-818. https://doi.org/10.1007/s11248-009-9357-x
  45. Zhu, J.K. (2001). Plant salt tolerance. Trends Plant Sci. 6, 2. https://doi.org/10.1016/S1360-1385(00)01795-7
  46. Zhu, J.K. (2002). Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53, 247-273. https://doi.org/10.1146/annurev.arplant.53.091401.143329