Abstract
Jasmonic and salicylic acids are key hormones involved in plant responses to pests and pathogens. Existing fluorescence-based approaches to imaging plant defence hormones are constrained by the need for external illumination and by autofluorescence of plant tissues, while luminescence-based ones require exogenous substrates. Here, we use jasmonate- and salicylate-responsive promoters to engineer autoluminescent plants that report hormone signalling activity with up to a 53-fold contrast. Using consumer-grade cameras, we image reporter Arabidopsis thaliana and Nicotiana benthamiana plants throughout normal development and in response to pest and pathogen attacks, visualising local and systemic responses. Because the luminescence is self-sustained, these reporters enable non-invasive, substrate-free imaging of defence signalling over extended time courses without specialised equipment.
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Data availability
The plasmids used in this study are available for non-commercial use through Addgene [https://www.addgene.org/browse/article/28247826/]. Plant lines will be made available under an MTA upon request. Metabolomics data are available at MetaboLights repository50 under accession MTBLS13465. Raw imaging data is available at Figshare [https://doi.org/10.6084/m9.figshare.30911858]. Source data are provided with this paper.
Code availability
Python code for processing and plotting data is available at Github [https://github.com/Perfus/BL_plant_hormones].
References
Peng, Y., Yang, J., Li, X. & Zhang, Y. Salicylic acid: biosynthesis and signaling. Annu. Rev. Plant Biol. 72, 761–791 (2021).
Onkokesung, N. et al. Jasmonic acid and ethylene modulate local responses to wounding and simulated herbivory in Nicotiana attenuata leaves. Plant Physiol. 153, 785–798 (2010).
Thaler, J. S., Humphrey, P. T. & Whiteman, N. K. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci. 17, 260–270 (2012).
Gimenez-Ibanez, S., Zamarreño, A. M., García-Mina, J. M. & Solano, R. An evolutionarily ancient immune system governs the interactions between Pseudomonas syringae and an early-diverging land plant lineage. Curr. Biol. 29, 2270–2281.e4 (2019).
Xu, H.-X. et al. A salivary effector enables whitefly to feed on host plants by eliciting salicylic acid-signaling pathway. Proc. Natl. Acad. Sci. USA 116, 490–495 (2019).
Li, N., Han, X., Feng, D., Yuan, D. & Huang, L.-J. Signaling crosstalk between salicylic acid and ethylene/jasmonate in plant defense: Do we understand what they are whispering? Int. J. Mol. Sci. 20, 671 (2019).
Hou, S. & Tsuda, K. Salicylic acid and jasmonic acid crosstalk in plant immunity. Essays Biochem. 66, 647–656 (2022).
Waadt, R., Hsu, P.-K. & Schroeder, J. I. Abscisic acid and other plant hormones: methods to visualize distribution and signaling. Bioessays 37, 1338–1349 (2015).
Isoda, R. et al. Sensors for the quantification, localization and analysis of the dynamics of plant hormones. Plant J. 105, 542–557 (2021).
Engelberth, M. J. & Engelberth, J. Monitoring plant hormones during stress responses. J. Vis. Exp. 28, 1127 (2009).
Kaku, T., Sugiura, K., Entani, T., Osabe, K. & Nagai, T. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci. Rep. 11, 14994 (2021).
Kusuma, S. H., Hattori, M. & Nagai, T. Autonomous multicolor bioluminescence imaging in bacteria, mammalian, and plant hosts. Proc. Natl. Acad. Sci. USA 121, e2406358121 (2024).
Khakhar, A. et al. Building customizable auto-luminescent luciferase-based reporters in plants. eLife 9, e52786 (2020).
Gregor, C. et al. Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system. Proc. Natl. Acad. Sci. USA 116, 26491–26496 (2019).
Gregor, C., Gwosch, K. C., Sahl, S. J. & Hell, S. W. Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc. Natl. Acad. Sci. USA 115, 962–967 (2018).
Mitiouchkina, T. et al. Plants with genetically encoded autoluminescence. Nat. Biotechnol. 38, 944–946 (2020).
Close, D. M., Xu, T., Sayler, G. S. & Ripp, S. In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals. Sensors 11, 180–206 (2011).
Ge, J. et al. Integration of biological and information technologies to enhance plant autoluminescence. Plant Cell 36, 4703–4715 (2024).
Kotlobay, A. A. et al. Genetically encodable bioluminescent system from fungi. Proc. Natl. Acad. Sci. USA 115, 12728–12732 (2018).
Purtov, K. V. et al. The chemical basis of fungal bioluminescence. Angew. Chem. Int. Ed. Engl. 54, 8124–8128 (2015).
Kaskova, Z. M. et al. Mechanism and color modulation of fungal bioluminescence. Sci. Adv. 3, e1602847 (2017).
Zheng, P. et al. Metabolic engineering and mechanical investigation of enhanced plant autoluminescence. Plant Biotechnol. J. 21, 1671–1681 (2023).
Shakhova, E. S. et al. An improved pathway for autonomous bioluminescence imaging in eukaryotes. Nat. Methods 21, 406–410 (2024).
Palkina, K. A. et al. A hybrid pathway for self-sustained luminescence. Sci. Adv. 10, eadk1992 (2024).
Moreno-Giménez, E., Selma, S., Calvache, C. & Orzáez, D. GB_SynP: A modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants. ACS Synth. Biol. 11, 3037–3048 (2022).
Calvache, C., Vazquez-Vilar, M., Moreno-Giménez, E. & Orzaez, D. A quantitative autonomous bioluminescence reporter system with a wide dynamic range for plant synthetic biology. Plant Biotechnol. J. 22, 37–47 (2024).
Montiel, G., Zarei, A., Körbes, A. P. & Memelink, J. The jasmonate-responsive element from the ORCA3 promoter from Catharanthus roseus is active in Arabidopsis and is controlled by the transcription factor AtMYC2. Plant Cell Physiol. 52, 578–587 (2011).
Van Der Fits, L. & Memelink, J. The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. Plant J. 25, 43–53 (2001).
Lehmann, S. et al. Novel markers for high-throughput protoplast-based analyses of phytohormone signaling. PLoS One 15, e0234154 (2020).
Nagata, T. Hidden history of the tobacco BY-2 cell line. J. Plant Res. 136, 781–786 (2023).
Li, J., Brader, G. & Palva, E. T. The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16, 319–331 (2004).
Raman, V. et al. Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses. Sci. Rep. 9, 13503 (2019).
Glazebrook, J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227 (2005).
Uppalapati, S. R. et al. The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000. Mol. Plant Microbe Interact. 20, 955–DC3965 (2007).
Smith-Becker, J. et al. Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems1. Plant Physiol. 116, 231–238 (1998).
Gao, Y. et al. Pseudomonas syringae activates ZAT18 to inhibit salicylic acid accumulation by repressing EDS1 transcription for bacterial infection. N. Phytol. 233, 1274–1288 (2022).
Zayed, M. S., Taha, E.-K. A., Hassan, M. M. & Elnabawy, E.-S. M. Enhance systemic resistance significantly reduces the silverleaf whitefly population and increases the yield of sweet pepper, Capsicum annuum L. var. annuum. Sustain. Sci. Pract. Policy 14, 6583 (2022).
Murugan, M. & Dhandapani, N. Induced systemic resistance activates defense responses to interspecific insect infestations on tomato. J. Veg. Sci. 12, 43–62 (2007).
Ruan, J. et al. Jasmonic acid signaling pathway in plants. Int. J. Mol. Sci. 20, 2479 (2019).
Huang, W. E. et al. Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1. Plant J. 46, 1073–1083 (2006).
Lebel, E. et al. Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis. Plant J. 16, 223–233 (1998).
Sarrion-Perdigones, A. et al. GoldenBraid 2.0: A comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618–1631 (2013).
Fernandez-Moreno, J.-P. et al. A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters. Plant Biotechnol. J. 22, 1942–1956 (2024).
Alonso, J. M. & Stepanova, A. N. Arabidopsis transformation with large bacterial artificial chromosomes. Methods Mol. Biol. 1062, 271–283 (2014).
Lazo, G. R., Stein, P. A. & Ludwig, R. A. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9, 963–967 (1991).
Nagata, T., Nemoto, Y. & Hasezawa, S. Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. Int. Rev. Cytol. 132, 1–30 (1992).
Gengenbach, B. B., Opdensteinen, P. & Buyel, J. F. Robot cookies - plant cell packs as an automated high-throughput screening platform based on transient expression. Front. Bioeng. Biotechnol. 8, 393 (2020).
Clough, S. J. & Bent, A. F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Scikit-posthocs. PyPI https://pypi.org/project/scikit-posthocs/.
Yurekten, O. et al. MetaboLights: open data repository for metabolomics. Nucleic Acids Res. 52, D640–D646 (2024).
Acknowledgements
This study was partially funded by Planta LLC (https://planta.bio/) and Light Bio Inc. (https://light.bio/). We thank MiLaboratories (https://milaboratories.com/) for the access to computing and storage infrastructure. The Synthetic Biology Group (AF, KSS) is funded by the MRC Laboratory of Medical Sciences (UKRI MC-A658-5QEA0). AF and KSS were supported by UKRI Biotechnology and Biological Sciences Research Council through the International Science Partnerships Fund (grant number APP33664/UKRI249). Plasmid assembly was funded by RSF project number 25-14-00349 (https://rscf.ru/en/project/25-14-00349/; ASM). Imaging analysis was funded by RSF project number 25-76-30006 (https://rscf.ru/en/project/25-76-30006/; DAG, IVY). Experiments in N. tabacum BY-2 cell culture were funded by RSF under project number 25-44-02157 (https://rscf.ru/en/project/25-44-02157/; IVY, ESS, and TAK). Plant transformations were funded by RSF under project number 24-74-10087 (https://rscf.ru/en/project/24-74-10087/; AVB and VVM). Works in Arabidopsis were supported by the CSF project no. 21-20936J (Z.V., M.K., J.P.), Czech BioImaging project n. LM 2023050, and the HORIZON-WIDERA-2022-TALENTS-02 project no. 101090293 (M.K.). Generation of synthetic promoters was funded by NSF PAPM-EAGER 1650139 to A.N.S. and J.M.A.
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Performed experiments: T.A.K., E.S.S., K.A.P., T.Y.M., K.M., V.V.M., M.K., N.M.M., L.I.F., G.M.D., O.A.B., S.I.K., E.N.B., Z.V., K.M., T.K., J.M., N.D., J.P.F.M., and D.A.G. Planned experiments: A.V.B., T.A.K., E.S.S., K.A.P., T.Y.M., M.K., K.S.S., A.S.M., V.V.C., J.P.F.M., J.M.A., and A.N.S. Analysed data: M.M.P., A.V.B., A.F., K.S.S., A.S.M., V.V.C., J.P.F.M., J.M.A., A.N.S., and M.K. Proposed and directed the study: K.S.S., M.K., J.P., J.M.A., A.N.S., A.S.M., and I.V.Y. Reviewed the paper draft: all authors.
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A.V.B., T.A.K., T.Y.M., E.S.S., M.M.P., O.A.B., K.A.P., L.I.F., N.M.M.,. E.N.B., G.M.D., V.V.C., I.V.Y., and A.S.M. are employed by Planta LLC (https://planta.bio/). K.S.S. and I.V.Y. are employed by Light Bio Inc. (https://light.bio/). Other authors don’t claim competing interests.
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Balakireva, A.V., Karataeva, T.A., Karampelias, M. et al. Non-invasive imaging of defence responses in plants. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70075-1
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DOI: https://doi.org/10.1038/s41467-026-70075-1
