Expression pattern of promoters driving eGFP expression in Arabidopsis thaliana hairy roots

Nga T. P. Mai

doi:10.7324/JABB.2025.238739

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Abstract

Plants are widely used expression system for the production of recombinant proteins. The expression systems mostly rely on promoters. In our study, hairy roots (HRs) of Arabidopsis thaliana were chosen to express the model protein enhanced green fluorescent protein (eGFP). To enhance recombinant protein expression, a strongly expressed promoter in the HRs of A. thaliana from microarray data – MT promoter – was used to control eGFP expression. We obtained different transformation HR lines with equal or lower eGFP production compared with the strong line controlled by the 35S promoter. In rhizocalli, which was induced by growing HRs in the medium supplemented with 2.4-D hormone, much lower eGFP content was quantified. The histological analysis showed that, under the control of MT promoter, eGFP was only expressed in the context, but not in the style or calli-like structure of roots which resulted in lower eGFP production. The structural interaction between the MT promoter and eGFP gene may be responsible for this low eGFP production. Further studies need to be generated to understand the different expression patterns of MT promoters toward heterologous protein eGFP. The discussion was raised when choosing promoters for heterologous protein production to get high productivity.

Keyword: Arabidopsis thaliana Hairy roots Heterologous protein Promoter Rhizocallis

Citation:

Mai NTP. Expression pattern of promoters driving eGFP expression in Arabidopsis thaliana hairy roots. J App Biol Biotech. 2025. Online First. http://doi.org/10.7324/JABB.2025.238739

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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13. Mai NT, Boitel-Conti M, Guerineau F. Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell Tissue Organ Cult 2016;127:489-96. https://doi.org/10.1007/s11240-016-1073-7
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18. García-Tomsig NI, Guedes-García SK, Jiménez-Zurdo JI. A Workflow for the functional characterization of noncoding rnas in legume symbiotic bacteria. Methods Mol Biol 2024;2751:179-203. https://doi.org/10.1007/978-1-0716-3617-6_12
19. Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 1992;122:383-4. https://doi.org/10.1016/0378-1119(92)90232-E
20. Jones HD, Doherty A, Wu H. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 2005;1:5. https://doi.org/10.1186/1746-4811-1-5
21. Guerineau F, Mai NT, Boitel-Conti M. Arabidopsis hairy roots producing high level of active human gastric lipase. Mol Biotechnol 2020;623:168-76. https://doi.org/10.1007/s12033-019-00233-y
22. Düzenli ÖF, Okay S. Promoter engineering for the recombinant protein production in prokaryotic systems. AIMS Bioeng 2020;7:62-81. https://doi.org/10.3934/bioeng.2020007
23. Xu N, Zhu J, Zhu Q, Xing Y, Cai M, Jiang T, et al. Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris. Yeast 2018;35:379-85. https://doi.org/10.1002/yea.3301
24. Stadlmayr G, Mecklenbräuker A, Rothmüller M, Maurer M, Sauer M, Mattanovich D, et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 2010;150:519-29. https://doi.org/10.1016/j.jbiotec.2010.09.957
25. Tang H, Wu Y, Deng J, Chen N, Zheng Z, Wei Y, et al. Promoter architecture and promoter engineering in Saccharomyces cerevisiae. Metabolites 2020;10:320. https://doi.org/10.3390/metabo10080320
26. Streatfield SJ. Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 2007;5:2-15. https://doi.org/10.1111/j.1467-7652.2006.00216.x
27. Chen X, Dong Y, Huang Y, Fan J, Yang M, Zhang J. Whole-genome resequencing using next-generation and Nanopore sequencing for molecular characterization of T-DNA integration in transgenic poplar 741. BMC Genomics 2021;221:329. https://doi.org/10.1186/s12864-021-07625-y
28. Gong W, Zhou Y, Wang R, Wei X, Zhang L, Dai Y, et al. Analysis of T-DNA integration events in transgenic rice. J Plant Physiol 2021;266:153527. https://doi.org/10.1016/j.jplph.2021.153527

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1. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs 2014;28:145-59. https://doi.org/10.1007/s40259-013-0062-1

2. Wani KI, Aftab T. Tools and techniques used in plant molecular farming. In: Plant Molecular Farming. Cham: Springer; 2022. p. m11-30. https://doi.org/10.1007/978-3-031-12794-6_2

3. Roy A. Hairy root culture an alternative for bioactive compound production from medicinal plants. Curr Pharm Biotechnol 2021;22:136-49. https://doi.org/10.2174/18734316MTEyfNzcD0

4. Georgiev MI, Agostini E, Ludwig-Müller J, Xu J. Genetically transformed roots: From plant disease to biotechnological resource. Trends Biotechnol 2012;30:528-37. https://doi.org/10.1016/j.tibtech.2012.07.001

5. Khan SA, Siddiqui MH, Osama K. Bioreactors for hairy roots culture: A review. Curr Biotechnol 2019;7:417-27. https://doi.org/10.2174/2211550108666190114143824

6. Gutierrez-Valdes N, Häkkinen ST, Lemasson C, Guillet M, Oksman- Caldentey KM, Ritala A, et al. Hairy root cultures-a versatile tool with multiple applications. Front Plant Sci 2020;11:33. https://doi.org/10.3389/fpls.2020.00033

7. Serganova I, Blasberg RG. Molecular imaging with reporter genes: Has its promise been delivered? J Nucl Med 2019;60:1665-81. https://doi.org/10.2967/jnumed.118.220004

8. Hu GY, Ma JY, Li F, Zhao JR, Xu FC, Yang WW, et al. Optimizing the protein fluorescence reporting system for somatic embryogenesis regeneration screening and visual labeling of functional genes in cotton. Front Plant Sci 2022;12:825212. https://doi.org/10.3389/fpls.2021.825212

9. MacGilvary NJ, Tan S. Fluorescent Mycobacterium tuberculosis reporters: Illuminating host-pathogen interactions. Pathog Dis 2018;76:fty017. https://doi.org/10.1093/femspd/fty017

10. Yilmazer I, Abt MR, Liang Y, Seung D, Zeeman SC, Sharma M. Determining protein-protein interaction with GFP-trap beads. Methods Mol Biol 2022;2564:317-23. https://doi.org/10.1007/978-1-0716-2667-2_17

11. Kummari D, Palakolanu SR, Kishor PB, Bhatnagar-Mathur P, Singam P, Vadez V, et al. An update and perspectives on the use of promoters in plant genetic engineering. J Biosci 2020;45:1-24. https://doi.org/10.1007/s12038-020-00087-6

12. Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho MJ, et al. Morphogenic regulators baby boom and wuschel improve monocot transformation. Plant Cell 2016;28:1998-2015. https://doi.org/10.1105/tpc.16.00124

13. Mai NT, Boitel-Conti M, Guerineau F. Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell Tissue Organ Cult 2016;127:489-96. https://doi.org/10.1007/s11240-016-1073-7

14. Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 1968;50:151-8. https://doi.org/10.1016/0014-4827(68)90403-5

15. Azizi P, Rafii MY, Mahmood M, Abdullah SN, Hanafi MM, Latif MA, et al. Evaluation of RNA extraction methods in rice and their application in expression analysis of resistance genes against Magnaporthe oryzae. Biotechnol Biotechnol Equip 2017;31:75-84. https://doi.org/10.1080/13102818.2016.1259015

16. Lee YS, Chen CH, Tsai CN, Tsai CL, Chao A, Wang TH. Microarray labeling extension values: Laboratory signatures for Affymetrix GeneChips. Nucleic Acids Res 2009;37:e61. https://doi.org/10.1093/nar/gkp168

17. Helliwell EE, Vega-Arreguín J, Shi Z, Bailey B, Xiao S, Maximova SN, et al. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins. Plant Biotechnol J 2016;14:875-86. https://doi.org/10.1111/pbi.12436

18. García-Tomsig NI, Guedes-García SK, Jiménez-Zurdo JI. A Workflow for the functional characterization of noncoding rnas in legume symbiotic bacteria. Methods Mol Biol 2024;2751:179-203. https://doi.org/10.1007/978-1-0716-3617-6_12

19. Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 1992;122:383-4. https://doi.org/10.1016/0378-1119(92)90232-E

20. Jones HD, Doherty A, Wu H. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 2005;1:5. https://doi.org/10.1186/1746-4811-1-5

21. Guerineau F, Mai NT, Boitel-Conti M. Arabidopsis hairy roots producing high level of active human gastric lipase. Mol Biotechnol 2020;623:168-76. https://doi.org/10.1007/s12033-019-00233-y

22. Düzenli ÖF, Okay S. Promoter engineering for the recombinant protein production in prokaryotic systems. AIMS Bioeng 2020;7:62-81. https://doi.org/10.3934/bioeng.2020007

23. Xu N, Zhu J, Zhu Q, Xing Y, Cai M, Jiang T, et al. Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris. Yeast 2018;35:379-85. https://doi.org/10.1002/yea.3301

24. Stadlmayr G, Mecklenbräuker A, Rothmüller M, Maurer M, Sauer M, Mattanovich D, et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 2010;150:519-29. https://doi.org/10.1016/j.jbiotec.2010.09.957

25. Tang H, Wu Y, Deng J, Chen N, Zheng Z, Wei Y, et al. Promoter architecture and promoter engineering in Saccharomyces cerevisiae. Metabolites 2020;10:320. https://doi.org/10.3390/metabo10080320

26. Streatfield SJ. Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 2007;5:2-15. https://doi.org/10.1111/j.1467-7652.2006.00216.x

27. Chen X, Dong Y, Huang Y, Fan J, Yang M, Zhang J. Whole-genome resequencing using next-generation and Nanopore sequencing for molecular characterization of T-DNA integration in transgenic poplar 741. BMC Genomics 2021;221:329. https://doi.org/10.1186/s12864-021-07625-y

28. Gong W, Zhou Y, Wang R, Wei X, Zhang L, Dai Y, et al. Analysis of T-DNA integration events in transgenic rice. J Plant Physiol 2021;266:153527. https://doi.org/10.1016/j.jplph.2021.153527