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Science China Life Sciences

, Volume 62, Issue 8, pp 1070–1077 | Cite as

A transient expression system in soybean mesophyll protoplasts reveals the formation of cytoplasmic GmCRY1 photobody-like structures

  • Lu Xiong
  • Cong Li
  • Hongyu Li
  • Xiangguang Lyu
  • Tao Zhao
  • Jun Liu
  • Zecheng ZuoEmail author
  • Bin LiuEmail author
Research Paper
  • 121 Downloads

Abstract

Soybean (Glycine max (L.) Merr.), grown for its plant oils and proteins, is one of the most important crops throughout the world. Generating stable and heritable transgenic soybeans is relatively inefficient; therefore, there is an urgent need for a simple and high-efficient transient transformation method by which to enable the investigation of gene functions in soybeans, which will facilitate the elucidation and improvement of the molecular mechanisms regulating the associated agronomic traits. We established a system of transient expression in soybean mesophyll protoplasts and obtained a high level of protoplast transfection efficiency (up to 83.5%). The subcellular activity of the protoplasts was well preserved, as demonstrated by the dynamic formation of GmCRY nucleus photobodies (NPs) and/or cytoplasmic photobody-like structures (CPs) in response to blue light. In addition, we showed that GmCRY1b CPs colocalized with GmCOP1b, a co-ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which provided new insight into the potential roles of GmCRY1s in the cytoplasm.

Keywords

soybean protoplast transfection subcellular localization cryptochrome photobodies 

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Notes

Acknowledgements

This work was supported by the National Key Research and Development Plan (2016YFD0101005), the National Natural Science Foundation of China (31871705 and 31422041), and the Central Public-Interest Scientific Institution Basal Research Fund (Y2016JC13).

Supplementary material

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Supplementary material, approximately 2.61 MB.

References

  1. Assmann, S.M., Simoncini, L., and Schroeder, J.I. (1985). Blue light activates electrogenic ion pumping in guard cell protoplasts of Vicia faba. Nature 318, 285–287.CrossRefGoogle Scholar
  2. Balcerowicz, M., Kerner, K., Schenkel, C., and Hoecker, U. (2017). SPA proteins affect the subcellular localization of COP1 in the COP1/SPA ubiquitin ligase complex during photomorphogenesis. Plant Physiol 174, 1314–1321.CrossRefGoogle Scholar
  3. Chen, P., Du, Q., Liu, X., Zhou, L., Hussain, S., Lei, L., Song, C., Wang, X., Liu, W., Yang, F., et al. (2017). Effects of reduced nitrogen inputs on crop yield and nitrogen use efficiency in a long-term maize-soybean relay strip intercropping system. PLoS ONE 12, e0184503.CrossRefGoogle Scholar
  4. Christou, P., Murphy, J.E., and Swain, W.F. (1987). Stable transformation of soybean by electroporation and root formation from transformed callus. Proc Natl Acad Sci USA 84, 3962–3966.CrossRefGoogle Scholar
  5. Chu, S., Wang, J., Zhu, Y., Liu, S., Zhou, X., Zhang, H., Wang, C.E., Yang, W., Tian, Z., Cheng, H., et al. (2017). An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean. PLoS Genet 13, e1006770.CrossRefGoogle Scholar
  6. Cocking, E.C. (1960). A method for the isolation of plant protoplasts and vacuoles. Nature 187, 962–963.CrossRefGoogle Scholar
  7. Frearson, E.M., Power, J.B., and Cocking, E.C. (1973). The isolation, culture and regeneration of Petunia leaf protoplasts. Dev Biol 33, 130–137.CrossRefGoogle Scholar
  8. Fujikawa, Y., and Kato, N. (2007). TECHNICAL ADVANCE: Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. Plant J 52, 185–195.CrossRefGoogle Scholar
  9. Jia, N., Zhu, Y., and Xie, F. (2018). An efficient protocol for model legume root protoplast isolation and transformation. Front Plant Sci 9, 670.CrossRefGoogle Scholar
  10. Kereszt, A., Li, D., Indrasumunar, A., Nguyen, C.D.T., Nontachaiyapoom, S., Kinkema, M., and Gresshoff, P.M. (2007). Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat Protoc 2, 948–952.CrossRefGoogle Scholar
  11. Kulkarni, K.P., Patil, G., Valliyodan, B., Vuong, T.D., Shannon, J.G., Nguyen, H.T., and Lee, J.D. (2018). Comparative genome analysis to identify SNPs associated with high oleic acid and elevated protein content in soybean. Genome 61, 217–222.CrossRefGoogle Scholar
  12. Li, J.F., Chung, H.S., Niu, Y., Bush, J., McCormack, M., and Sheen, J. (2013a). Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25, 1507–1522.CrossRefGoogle Scholar
  13. Li, J.F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G.M., and Sheen, J. (2013b). Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31, 688–691.CrossRefGoogle Scholar
  14. 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.Google Scholar
  15. Li, Y., Zhou, G., Ma, J., Jiang, W., Jin, L., Zhang, Z., Guo, Y., Zhang, J., Sui, Y., Zheng, L., et al. (2014). De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits. Nat Biotechnol 32, 1045–1052.CrossRefGoogle Scholar
  16. Lin, W. (1983). Isolation of mesophyll protoplasts from mature leaves of soybeans. Plant Physiol 73, 1067–1069.CrossRefGoogle Scholar
  17. Liu, B., Liu, H., Zhong, D., and Lin, C. (2010). Searching for a photocycle of the cryptochrome photoreceptors. Curr Opin Plant Biol 13, 578–586.CrossRefGoogle Scholar
  18. Lu, S., Zhao, X., Hu, Y., Liu, S., Nan, H., Li, X., Fang, C., Cao, D., Shi, X., Kong, L., et al. (2017). Natural variation at the soybean J locus improves adaptation to the tropics and enhances yield. Nat Genet 49, 773–779.CrossRefGoogle Scholar
  19. Matsumura, H., Kitajima, H., Akada, S., Abe, J., Minaka, N., and Takahashi, R. (2009). Molecular Cloning and linkage mapping of cryptochrome multigene family in soybean. Plant Genome J 2, 271–281.CrossRefGoogle Scholar
  20. Meng, Y., Li, H., Wang, Q., Liu, B., and Lin, C. (2013). Blue light-dependent interaction between Cryptochrome 2 and CIB1 regulates transcription and leaf senescence in Soybean. Plant Cell 25, 4405–4420.CrossRefGoogle Scholar
  21. Miao, Y., and Jiang, L. (2007). Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc 2, 2348–2353.CrossRefGoogle Scholar
  22. Miao, Y., Li, K.Y., Li, H.Y., Yao, X., and Jiang, L. (2008). The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells. Plant J 56, 824–839.CrossRefGoogle Scholar
  23. Rasmussen, J.O., and Rasmussen, O.S. (1993). PEG mediated DNA uptake and transient GUS expression in carrot, rapeseed and soybean protoplasts. Plant Sci 89, 199–207.CrossRefGoogle Scholar
  24. Rizzo, G., and Baroni, L. (2018). Soy, soy foods and their role in vegetarian diets. Nutrients 10, 43.CrossRefGoogle Scholar
  25. Schmutz, J., Cannon, S.B., Schlueter, J., Ma, J., Mitros, T., Nelson, W., Hyten, D.L., Song, Q., Thelen, J.J., Cheng, J., et al. (2010). Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183.CrossRefGoogle Scholar
  26. Sheen, J. (2001). Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127, 1466–1475.CrossRefGoogle Scholar
  27. Shen, J., Fu, J., Ma, J., Wang, X., Gao, C., Zhuang, C., Wan, J., and Jiang, L. (2014). Isolation, culture, and transient transformation of plant protoplasts. Curr Protoc Cell Biol 63, 2. 8. 1–17.Google Scholar
  28. Shen, Y., Liu, J., Geng, H., Zhang, J., Liu, Y., Zhang, H., Xing, S., Du, J., Ma, S., and Tian, Z. (2018). De novo assembly of a Chinese soybean genome. Sci China Life Sci 61, 871–884.CrossRefGoogle Scholar
  29. van Buskirk, E.K., Decker, P.V., and Chen M.(2011). Photobodies in light signaling. Plant Physiol 158, 52–60.CrossRefGoogle Scholar
  30. von Arnim, A.G., and Deng, X.W. (1994). Light inactivation of Arabidopsis photomorphogenic repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning. Cell 79, 1035–1045.CrossRefGoogle Scholar
  31. Wang, Q., Zuo, Z., Wang, X., Gu, L., Yoshizumi, T., Yang, Z., Yang, L., Liu, Q., Liu, W., Han, Y.J., et al. (2016). Photoactivation and inactivation of Arabidopsis cryptochrome 2. Science 354, 343–347.CrossRefGoogle Scholar
  32. Wu, F., and Hanzawa, Y. (2018). A simple method for isolation of soybean protoplasts and application to transient gene expression analyses. J Vis Exp 131.Google Scholar
  33. Wu, G., and Spalding, E.P. (2007). Separate functions for nuclear and cytoplasmic cryptochrome 1 during photomorphogenesis of Arabidopsis seedlings. Proc Natl Acad Sci USA 104, 18813–18818.CrossRefGoogle Scholar
  34. Wu, J.Z., Liu, Q., Geng, X.S., Li, K.M., Luo, L.J., and Liu, J.P. (2017). Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz). BMC Biotech 17, 29.CrossRefGoogle Scholar
  35. Yang, D.G., Zhao, W., Meng, Y.Y., Li, H.Y., and Liu, B. (2015). A CIB1-LIKE transcription factor GmCIL10 from soybean positively regulates plant flowering. Sci China Life Sci 58, 261–269.CrossRefGoogle Scholar
  36. Yoo, S.D., Cho, Y.H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat Protoc 2, 1565–1572.CrossRefGoogle Scholar
  37. Zhang, Q., Li, H., Li, R., Hu, R., Fan, C., Chen, F., Wang, Z., Liu, X., Fu, Y., and Lin, C. (2008). Association of the circadian rhythmic expression of GmCRY1a with a latitudinal cline in photoperiodic flowering of soybean. Proc Natl Acad Sci USA 105, 21028–21033.CrossRefGoogle Scholar
  38. Zhang, S.R., Wang, H., Wang, Z., Ren, Y., Niu, L., Liu, J., and Liu, B. (2017). Photoperiodism dynamics during the domestication and improvement of soybean. Sci China Life Sci 60, 1416–1427.CrossRefGoogle Scholar
  39. Zhou, Z., Jiang, Y., Wang, Z., Gou, Z., Lyu, J., Li, W., Yu, Y., Shu, L., Zhao, Y., Ma, Y., et al. (2015). Resequencing 302 wild and cultivated accessions identifies genes related to domestication and improvement in soybean. Nat Biotechnol 33, 408–414.CrossRefGoogle Scholar
  40. Zuo, Z.C., Meng, Y.Y., Yu, X.H., Zhang, Z.L., Feng, D.S., Sun, S.F., Liu, B., and Lin, C.T. (2012). A study of the blue-light-dependent phosphorylation, degradation, and photobody formation of Arabidopsis CRY2. Mol Plant 5, 726–733.CrossRefGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijingChina
  2. 2.Jilin Province Engineering Laboratory of Plant Genetic Improvement, College of Plant ScienceJilin UniversityChangchunChina

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