Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system

Autoři: Yuichi Furuhata aff001;  Ayako Sakai aff001;  Tomi Murakami aff001;  Akira Nagasaki aff001;  Yoshio Kato aff001
Působiště autorů: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227477


Bioluminescent detection has become a powerful method that is used extensively in numerous areas in life science research. Given that fluorescence detection in plant cells is difficult owing to the autofluorescence of chlorophyll, the use of a luciferin–luciferase system should be effective in plant biology. However, the suitable optical window for a luminescence system in plants remains unexplored. In this study, we sought to determine the optical window and optimal luciferase reporter system for terrestrial plant analyses using Arabidopsis thaliana as a model organism. We compared six different luciferase systems and found the green enhanced Nano-lantern (GeNL)–furimazine combination to be the optimal luciferase reporter. Spectral measurements of GeNL–furimazine showed that its luminescence peak falls within the range of optical transparency for chlorophyll and, therefore, enables greater penetration through a layer of cultured A. thaliana cells. Moreover, A. thaliana plants expressing GeNL with furimazine emitted strong luminescence, which could be detected even with the naked eye. Thus, the GeNL–furimazine combination should facilitate biological analyses of genes and cellular functions in A. thaliana and all other terrestrial plants.

Klíčová slova:

Agrobacteria – Arabidopsis thaliana – Cell cultures – Fluorescence – Chlorophyll – Luciferase – Luminescence – Plant cells


1. Greer LF, Szalay AA. Imaging of light emission from the expression of luciferases in living cells and organisms: A review. Luminescence. 2002;17: 43–47. doi: 10.1002/bio.676 11816060

2. Fan F, Wood KV. Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol. 2007;5: 127–136. doi: 10.1089/adt.2006.053 17355205

3. Shimomura O. Bioluminescence: Chemical principles and methods, revised edition [Internet]. Bioluminescence: Chemical Principles and Methods, Revised Edition 5 Toh Tuck Link, Singapore: World Scientific Publishing Co. Pte. Ltd.; 2012. doi: 10.1142/8239

4. Fleiss A, Sarkisyan KS. A brief review of bioluminescent systems (2019). Curr Genet. 2019;65: 877–882. doi: 10.1007/s00294-019-00951-5 30850867

5. Kaskova ZM, Tsarkova AS, Yampolsky IV. 1001 lights: Luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine. Chem Soc Rev. 2016;45: 6048–6077. doi: 10.1039/c6cs00296j 27711774

6. Markova SV, Vysotski ES. Coelenterazine-dependent luciferases. Biochem. 2015;80: 714–732. doi: 10.1134/S0006297915060073 26531017

7. Tomabechi Y, Hosoya T, Ehara H, Sekine SI, Shirouzu M, Inouye S. Crystal structure of nanoKAZ: The mutated 19 kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine. Biochem Biophys Res Commun. 2016;470: 88–93. doi: 10.1016/j.bbrc.2015.12.123 26746005

8. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012;7: 1848–1857. doi: 10.1021/cb3002478 22894855

9. Ow DW, Wood KV, Deluca M, De Wet JR, Helinski DR, Howell SH. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science. 1986;234: 856–859. doi: 10.1126/science.234.4778.856 17758108

10. Horecker BL. The absorption spectra of hemoglobin and its derivatives in the visible and near infra-red regions. J Biol Chem. 1943;148: 173–183. Available:

11. Taiz L, Zeiger E, Møller IM, Murphy A. Plant physiology and development. 6th ed. Sinauer Associates. Massachusetts; 2015.

12. Kuchimaru T, Iwano S, Kiyama M, Mitsumata S, Kadonosono T, Niwa H, et al. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging. Nat Commun. 2016;7: 11856. doi: 10.1038/ncomms11856 27297211

13. Shakhmin A, Hall MP, Machleidt T, Walker JR, Wood KV, Kirkland TA. Coelenterazine analogues emit red-shifted bioluminescence with NanoLuc. Org Biomol Chem. 2017;15: 8559–8567. doi: 10.1039/c7ob01985h 28972606

14. Nakajima Y, Kimura T, Sugata K, Enomoto T, Asakawa A, Kubota H, et al. Multicolor luciferase assay system: One-step monitoring of multiple gene expressions with a single substrate. Biotechniques. 2005;38: 891–894. doi: 10.2144/05386ST03 16018550

15. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, et al. Structural basis for the spectral difference in luciferase bioluminescence. Nature. 2006;440: 372–376. doi: 10.1038/nature04542 16541080

16. Saito K, Chang YF, Horikawa K, Hatsugai N, Higuchi Y, Hashida M, et al. Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun. 2012;3: 1262. doi: 10.1038/ncomms2248 23232392

17. Hoshino H, Nakajima Y, Ohmiya Y. Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging. Nat Methods. 2007;4: 637–639. doi: 10.1038/nmeth1069 17618293

18. Takai A, Nakano M, Saito K, Haruno R, Watanabe TM, Ohyanagi T, et al. Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging. Proc Natl Acad Sci U S A. 2015;112: 4352–4356. doi: 10.1073/pnas.1418468112 25831507

19. Suzuki K, Kimura T, Shinoda H, Bai G, Daniels MJ, Arai Y, et al. Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nat Commun. 2016;7: 13718. doi: 10.1038/ncomms13718 27966527

20. Mitiouchkina T, Mishin AS, Somermeyer LG, Markina NM, Chepurnyh TV., Guglya EB, et al. Plants with self-sustained luminescence. BioRxiv [Preprint]. 2019 bioRxiv 809376 [posted 2019 Oct 18; cited 2019 Nov 22]: [17 p.]. Available from:

21. Khakhar A, Starker C, Chamness J, Lee N, Stokke S, Wang C, et al. Building customizable auto-luminescent luciferase-based reporters in plants. BioRxiv [Preprint]. 2019 bioRxiv 809533 [posted 2019 Oct 17; cited 2019 Nov 22]: [22 p.]. Available from:

22. Kotlobay AA, Sarkisyan KS, Mokrushina YA, Marcet-Houben M, Serebrovskaya EO, Markina NM, et al. Genetically encodable bioluminescent system from fungi. Proc Natl Acad Sci U S A. 2018;115: 12728–12732. doi: 10.1073/pnas.1803615115 30478037

23. Furuhata Y, Sakai A, Murakami T, Morikawa M, Nakamura C, Yoshizumi T, et al. A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall. Sci Rep. 2019;9: 2163. doi: 10.1038/s41598-018-38119-9 30770845

24. French CS, Young VK. The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and chlorophyll. J Gen Physiol. 1952;35: 873–890. doi: 10.1085/jgp.35.6.873 14938526

25. Berkaloff C, Caron L, Rousseau B. Subunit organization of PSI particles from brown algae and diatoms: polypeptide and pigment analysis. Photosynth Res. 1990;23: 181–193. doi: 10.1007/BF00035009 24421060

26. Furuhata Y, Sakai A, Kato Y. Protein electroporation of Cre recombinase into cultured Arabidopsis cells with an intact cell wall. Protoc Exch. 2019; doi: 10.1038/protex.2019.027

27. Urquiza-García U, Millar AJ. Expanding the bioluminescent reporter toolkit for plant science with NanoLUC. Plant Methods. 2019;15: 68. doi: 10.1186/s13007-019-0454-4 31316580

Článek vyšel v časopise


2020 Číslo 1