Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR–Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.
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Plant Methods Open Access 28 July 2021
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Raw and associated data generated with plate-reader-, RT-qPCR- and microscope-specific software that support the findings of this study are available from the corresponding author upon request. The plasmids used in all experiments are available at AddGene, and the plasmid maps at the public repository JBEI-ICE (https://public-registry.jbei.org/folders/577). Source data are provided with this paper.
The numerical integration, fitting process and identifiability analysis with the profile likelihood method were performed in MATLAB using the freely available Data2Dynamics software. Details relative to the equations used can be found in the Supplementary Note. Source data are provided with this paper.
Deisseroth, K. & Hegemann, P. The form and function of channelrhodopsin. Science 357, eaan5544 (2017).
Alberio, L. et al. A light-gated potassium channel for sustained neuronal inhibition. Nat. Methods 15, 969–976 (2018).
Ye, H., Baba, M. D.-E., Peng, R.-W. & Fussenegger, M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, 1565 (2011).
Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–384 (2012).
Shin, Y. et al. Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets. Cell 168, 159–171.e14 (2017).
van Bergeijk, P., Adrian, M., Hoogenraad, C. C. & Kapitein, L. C. Optogenetic control of organelle transport and positioning. Nature 518, 111–114 (2015).
Kolar, K., Knobloch, C., Stork, H., Žnidarič, M. & Weber, W. OptoBase: a web platform for molecular optogenetics. ACS Synth. Biol. 7, 1825–1828 (2018).
Müller, K. et al. A red light-controlled synthetic gene expression switch for plant systems. Mol. Biosyst. 10, 1679–1688 (2014).
Chatelle, C. et al. A green-light-responsive system for the control of transgene expression in mammalian and plant cells. ACS Synth. Biol. 7, 1349–1358 (2018).
Ochoa-Fernandez, R. et al. in Optogenetics: Methods and Protocols (ed. Kianianmomeni, A.) 125–139 (Humana Press, 2016).
Nash, A. I. et al. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Comput. Biol. 108, 9449–9945 (2011).
Motta-Mena, L. B. et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat. Chem. Biol. 10, 196–202 (2014).
Moosmann, P., Georgiev, O., Thiesen, H., Hagmann, M. & Schaffner, W. Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Krüppel-type zinc finger factor. Biol. Chem. 378, 669–677 (1997).
Baaske, J. et al. Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells. Sci. Rep. 8, 15024 (2018).
Ikeda, M. & Ohme-Takagi, M. A novel group of transcriptional repressors in Arabidopsis. Plant Cell Physiol. 50, 970–975 (2009).
Kelly, J. M. & Lagarias, J. C. Photochemistry of 124-kilodalton Avena phytochrome under constant illumination in vitro. Biochemistry 24, 6003–6010 (1985).
Müller, K. et al. Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 (2013).
Li, Z. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nat. Plants 3, 930–936 (2017).
Selma, S. et al. Strong gene activation in plants with genome-wide specificity using a new orthogonal CRISPR/Cas9-based programmable transcriptional activator. Plant Biotechnol. J. 17, 1703–1705 (2019).
Simon, R., Igeño, M. I. & Coupland, G. Activation of floral meristem identity genes in Arabidopsis. Nature 384, 59–62 (1996).
de Felipe, P. et al. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol. 24, 68–75 (2006).
Zipfel, C. et al. Perception of the Bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749–760 (2006).
Kunze, G. et al. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16, 3496–3507 (2004).
Lacombe, S. et al. Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nat. Biotechnol. 28, 365 (2010).
Suzuki, N. et al. Respiratory burst oxidases: the engines of ROS signaling. Curr. Opin. Plant Biol. 14, 691–699 (2011).
Gaupels, F., Durner, J. & Kogel, K.-H. Production, amplification and systemic propagation of redox messengers in plants? The phloem can do it all! N. Phytol. 214, 554–560 (2017).
Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133 (2009).
Gulati, S. et al. Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor. Nat. Commun. 9, 1996 (2018).
Schornack, S. et al. Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60, 744–754 (2009).
Yu, D. et al. Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins. Nat. Methods 16, 1095–1100 (2019).
Moore, I., Samalova, M., Kurup, S., For, T. & Analysis, M. Transactivated and chemically inducible gene expression in plants. Plant J. 45, 651–683 (2006).
Zuo, J. & Chua, N. H. Chemical-inducible systems for regulated expression of plant genes. Curr. Opin. Biotechnol. 11, 146–151 (2000).
Andres, J., Blomeier, T. & Zurbriggen, M. D. Synthetic switches and regulatory circuits in plants. Plant Physiol. 179, 862–884 (2019).
Cosentino, C. et al. Engineering of a light-gated potassium channel. Science 348, 707–710 (2015).
Papanatsiou, M. et al. Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth. Science 363, 1456–1459 (2019).
Martin-Arevalillo, R. & Vernoux, T. Shining light on plant hormones with genetically encoded biosensors. Biol. Chem. 400, 477–486 (2018).
Kolar, K. & Weber, W. Synthetic biological approaches to optogenetically control cell signaling. Curr. Opin. Biotechnol. 47, 112–119 (2017).
Levskaya, A., Weiner, O. D., Lim, W. A. & Voigt, C. A. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009).
Toettcher, J. E., Weiner, O. D. & Lim, W. A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013).
Renicke, C., Schuster, D., Usherenko, S., Essen, L. O. & Taxis, C. A LOV2 domain-based optogenetic tool to control protein degradation and cellular function. Chem. Biol. 20, 619–626 (2013).
Bonger, K. M., Rakhit, R., Payumo, A. Y., Chen, J. K. & Wandless, T. J. General method for regulating protein stability with light. ACS Chem. Biol. 9, 111–115 (2014).
Niopek, D. et al. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat. Commun. 5, 4404 (2014).
Yumerefendi, H. et al. Control of protein activity and cell fate specification via light-mediated nuclear translocation. PLoS One 10, e0128443 (2015).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343 (2009).
Beyer, H. M. et al. AQUA Cloning: a versatile and simple enzyme-free cloning approach. PLoS One 10, e0137652 (2015).
Sarrion-Perdigones, A. et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618 (2013).
Binder, A. et al. A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants. PLoS One 9, e88218 (2014).
Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, e16765 (2011).
Müller, K., Zurbriggen, M. D. & Weber, W. Control of gene expression using a red- and far-red light–responsive bi-stable toggle switch. Nat. Protoc. 9, 622 (2014).
Sellaro, R. et al. Cryptochrome as a sensor of the blue/green ratio of natural radiation in Arabidopsis. Plant Physiol. 154, 401 (2010).
Bauer, P. Regulation of iron acquisition responses in plant roots by a transcription factor: regulation of iron acquisition responses. Biochem. Mol. Biol. Educ. 44, 438–449 (2016).
Vazquez-Vilar, M. et al. GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. Nucleic Acids Res. 45, 2196–2209 (2017).
Naranjo-Arcos, M. A. et al. Dissection of iron signaling and iron accumulation by overexpression of subgroup Ib bHLH039 protein. Sci. Rep. 7, 10911 (2017).
Trujillo, M. in Environmental Responses in Plants: Methods and Protcools (ed. Duque, P.) 323–329 (Humana Press, 2016).
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
This study was supported in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (CEPLAS—EXC-1028 project no. 194465578 to R.S. and M.D.Z., EXC-2048/1—project no. 390686111 to R.S. and M.D.Z., CIBSS – EXC-2189—project no. 390939984 to T.O., J.T. and W.W., and BIOSS – EXC-294 to J.T. and W.W.), the iGRAD Plant (IRTG 1525 to R.O.F., J.S., R.S. and M.D.Z.), and the Collaborative Research Centers SFB1208 (project no. 267205415; project A13 to M.D.Z.) and SFB924 (INST 95/1126-2; project B4 to T.O.), the European Commission – Research Executive Agency (H2020 Future and Emerging Technologies FET-Open project no. 801041 CyGenTig to M.D.Z.). J.B.M. is supported by a fellowship from the Eastern Academic Research Consortium. We thank D. Orzaez (Polytechnic University of Valencia) and K. Gardner (City University of New York) for kindly providing the GoldenBraid and EL222 plasmids, respectively, T. Brumbarova (University of Düsseldorf) for aid with quantitative reverse-transcription PCR experiments, R. Wurm and M. Gerads (University of Düsseldorf) for technical assistance, and J. Schmidt (Technical Workshop Biology, University of Freiburg) for designing and constructing the light boxes used in this work. We are indebted to J. Casal (University of Buenos Aires), D. Nusinow (Danforth Center), S. Romero, H. Beyer and U. Urquiza (University of Düsseldorf) for careful reading and their suggestions to improve the manuscript.
The authors declare no competing interests.
Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Model-based functional characterization, and prediction and validation of PULSE function.
a,b, Quantitative characterization of On-Off PULSE kinetics and reversibility. PULSE-driven FLuc expression assays in Arabidopsis protoplasts. (a) FLuc/RLuc ratios for protein expression kinetics, n = 6 protoplast samples. (b) Normalized starting quantity (SQ) of FLuc transcript to the SQ geometric mean of EF and TIP41L transcripts (internal normalization controls), n = 2 technical replicates for each transcript (b). Protoplasts were transformed and kept in the dark, 12 h for protein (a) and 16 h for mRNA (b) determination, followed by illumination with either 10 µmol m−2 s−1 of red or blue light, or kept in darkness. Arrows indicate the time point where the samples were split into different illumination conditions, for example, red to dark, red to blue (On-Off), red to blue to red (On-Off-On). The curves are the fits to the ODE-based model. The shaded areas represent the error bands as calculated in 95% confidence intervals with a constant Gaussian error model using the profile likelihood method. c, Model aided prediction of PULSE-controlled protein expression levels as a function of red light intensities and illumination times. The calibrated model yields estimated FLuc/RLuc expression ranges (heatmap). d, Experimental validation of the model predictions of the operating range of PULSE. Selected model simulated expression levels at different red light intensities and illumination times as indicated in (c) were experimentally tested and the resulting FLuc/RLuc ratios (means and 2xSEM are plotted, n = 6 protoplast samples for each condition, black circles) were compared to the predicted values (grey squares). RLU = Relative Luminescence Units.
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Ochoa-Fernandez, R., Abel, N.B., Wieland, FG. et al. Optogenetic control of gene expression in plants in the presence of ambient white light. Nat Methods 17, 717–725 (2020). https://doi.org/10.1038/s41592-020-0868-y
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