Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Synthetic memory circuits for stable cell reprogramming in plants

Nature Biotechnology volume 40, pages 1862–1872 (2022)Cite this article

Subjects

Abstract

Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the potential to integrate multiple customizable input signals through a processing unit constructed from biological parts to produce a predictable and programmable output. Here we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Specifically, using recombinases and plant control elements, we activated transgenes in YES, OR and AND gates and repressed them in NOT, NOR and NAND gates; we also implemented the A NIMPLY B gate that combines activation and repression. Through use of genetic recombination, these circuits create stable long-term changes in expression and recording of past stimuli. This highly compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealized traits into plants.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Recombination-based gene circuits built with plant-compatible parts can control plant gene expression.
Fig. 2: Identification of additional functional recombinases for the construction of complex gene circuits.
Fig. 3: AND and OR gate construction using recombination-based gene circuits in plants.
Fig. 4: Negation (NOT) function implementation in plant cells by output gene CDS or promoter excision.
Fig. 5: Construction of AND and NAND gates using split-recombinases.

Similar content being viewed by others

Data availability

All raw values of data presented in this study, output of statistical tests and summaries (including central tendency and variation), list of primer sequences and the sequences of the plasmids used in this study are available in a Zenodo repository64 with the identifier 10.5281/zenodo.6381286.

Code availability

R code used to perform statistical tests and generate plots is available in a Zenodo repository64 with the identifier 10.5281/zenodo.6381286.

References

  1. Thompson, A. J. et al. Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J. 23, 363–374 (2000).

    Article  CAS  Google Scholar 

  2. Iuchi, S. et al. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27, 325–333 (2001).

    Article  CAS  Google Scholar 

  3. Feeney, M., Frigerio, L., Cui, Y. & Menassa, R. Following vegetative to embryonic cellular changes in leaves of Arabidopsis overexpressing LEAFY COTYLEDON2. Plant Physiol. 162, 1881–1896 (2013).

    Article  CAS  Google Scholar 

  4. Vanhercke, T. et al. Step changes in leaf oil accumulation via iterative metabolic engineering. Metab. Eng. 39, 237–246 (2017).

    Article  CAS  Google Scholar 

  5. He, R. et al. Overexpression of 9-cis-epoxycarotenoid dioxygenase cisgene in grapevine increases drought tolerance and results in pleiotropic effects. Front. Plant Sci. 9, 970 (2018).

    Article  Google Scholar 

  6. Brophy, J. A. N. Toward synthetic plant development. Plant Physiol. 188, 738–748 (2021).

  7. Brophy, J. A. N., Magallon, K. J., Kniazev, K. & Dinneny, J. R. Synthetic genetic circuits enable reprogramming of plant roots. Preprint at https://www.biorxiv.org/content/10.1101/2022.02.02.478917v1 (2022).

  8. Pires, N. D. et al. Recruitment and remodeling of an ancient gene regulatory network during land plant evolution. Proc. Natl Acad. Sci. USA 110, 9571–9576 (2013).

    Article  CAS  Google Scholar 

  9. Madrid, E., Chandler, J. W. & Coupland, G. Gene regulatory networks controlled by FLOWERING LOCUS C that confer variation in seasonal flowering and life history. J. Exp. Bot. 72, 4–14 (2021).

    Article  CAS  Google Scholar 

  10. Setty, Y., Mayo, A. E., Surette, M. G. & Alon, U. Detailed map of a cis-regulatory input function. Proc. Natl Acad. Sci. USA 100, 7702–7707 (2003).

    Article  CAS  Google Scholar 

  11. Krakauer, D. C., Müller, L., Prohaska, S. J. & Stadler, P. F. Design specifications for cellular regulation. Theory Biosci. 135, 231–240 (2016).

    Article  CAS  Google Scholar 

  12. Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    Article  CAS  Google Scholar 

  13. Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).

    Article  CAS  Google Scholar 

  14. Lohmueller, J. J., Armel, T. Z. & Silver, P. A. A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Nucleic Acids Res. 40, 5180–5187 (2012).

    Article  CAS  Google Scholar 

  15. Nevozhay, D., Zal, T. & Balázsi, G. Transferring a synthetic gene circuit from yeast to mammalian cells. Nat. Commun. 4, 1451 (2013).

    Article  Google Scholar 

  16. Siuti, P., Yazbek, J. & Lu, T. K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).

    Article  CAS  Google Scholar 

  17. Gaber, R. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat. Chem. Biol. 10, 203–208 (2014).

    Article  CAS  Google Scholar 

  18. Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S. & Lu, T. K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016).

    Article  Google Scholar 

  19. Weinberg, B. H. et al. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat. Biotechnol. 35, 453–462 (2017).

    Article  CAS  Google Scholar 

  20. Müller, M. et al. Designed cell consortia as fragrance-programmable analog-to-digital converters. Nat. Chem. Biol. 13, 309–316 (2017).

    Article  Google Scholar 

  21. Guiziou, S., Mayonove, P. & Bonnet, J. Hierarchical composition of reliable recombinase logic devices. Nat. Commun. 10, 456 (2019).

    Article  CAS  Google Scholar 

  22. Zúñiga, A. et al. Rational programming of history-dependent logic in cellular populations. Nat. Commun. 11, 4758 (2020).

    Article  Google Scholar 

  23. Bowyer, J. E., Ding, C., Weinberg, B. H., Wong, W. W. & Bates, D. G. A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits. PLoS Comput. Biol. 16, e1007849 (2020).

    Article  CAS  Google Scholar 

  24. Schreiber, T., Prange, A. & Tissier, A. F. Split-TALE—a TALE-based two-component system for synthetic biology applications in planta. Plant Physiol. 179, 1001–1012 (2019).

  25. Bernabé-Orts, J. M. et al. A memory switch for plant synthetic biology based on the phage ϕC31 integration system. Nucleic Acids Res. 48, 3379–3394 (2020).

    Article  Google Scholar 

  26. Lloyd, J. P. B. & Lister, R. Epigenome plasticity in plants. Nat. Rev. Genet. 23, 55–68 (2022).

  27. Jones, J. M. & Gellert, M. The taming of a transposon: V(D)J recombination and the immune system. Immunol. Rev. 200, 233–248 (2004).

    Article  CAS  Google Scholar 

  28. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  Google Scholar 

  29. Engler, C. et al. A golden gate modular cloning toolbox for plants. ACS Synth. Biol. 3, 839–843 (2014).

    Article  CAS  Google Scholar 

  30. Diamos, A. G. & Mason, H. S. Chimeric 3′ flanking regions strongly enhance gene expression in plants. Plant Biotechnol. J. 16, 1971–1982 (2018).

    Article  CAS  Google Scholar 

  31. Andreou, A. I., Nirkko, J., Ochoa-Villarreal, M. & Nakayama, N. Mobius Assembly for Plant Systems highlights promoter–terminator interaction in gene regulation. Preprint at https://www.biorxiv.org/content/10.1101/2021.03.31.437819v1 (2021).

  32. Efroni, I. et al. Root regeneration triggers an embryo-like sequence guided by hormonal interactions. Cell 165, 1721–1733 (2016).

    Article  CAS  Google Scholar 

  33. Wu, F.-H. et al. Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009).

    Article  Google Scholar 

  34. Schaumberg, K. A. et al. Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nat. Methods 13, 94–100 (2016).

    Article  CAS  Google Scholar 

  35. Padidam, M. & Cao, Y. Elimination of transcriptional interference between tandem genes in plant cells. Biotechniques 31, 328–330, 332–334 (2001).

    Article  Google Scholar 

  36. Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol. 51, 328–332 (2010).

    Article  CAS  Google Scholar 

  37. Rayson, S. et al. A role for nonsense-mediated mRNA decay in plants: pathogen responses are induced in Arabidopsis thaliana NMD mutants. PLoS ONE 7, e31917 (2012).

    Article  CAS  Google Scholar 

  38. Lloyd, J. P. B. & Davies, B. SMG1 is an ancient nonsense-mediated mRNA decay effector. Plant J. 76, 800–810 (2013).

    Article  CAS  Google Scholar 

  39. Causier, B., Hopes, T., McKay, M., Paling, Z. & Davies, B. Plants utilise ancient conserved peptide upstream open reading frames in stress-responsive translational regulation. Plant Cell Environ. 45, 1229–1241 (2022).

  40. Sanfaçon, H. & Hohn, T. Proximity to the promoter inhibits recognition of cauliflower mosaic virus polyadenylation signal. Nature 346, 81–84 (1990).

    Article  Google Scholar 

  41. Han, Y.-J., Kim, Y.-M., Hwang, O.-J. & Kim, J.-I. Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation. Plant Cell Rep. 34, 265–275 (2015).

    Article  CAS  Google Scholar 

  42. 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).

    Article  CAS  Google Scholar 

  43. Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S. & Somerville, C. R. Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl Acad. Sci. USA 97, 3718–3723 (2000).

    Article  CAS  Google Scholar 

  44. Heidstra, R., Welch, D. & Scheres, B. Mosaic analyses using marked activation and deletion clones dissect Arabidopsis SCARECROW action in asymmetric cell division. Genes Dev. 18, 1964–1969 (2004).

    Article  CAS  Google Scholar 

  45. Vergunst, A. C., Jansen, L. E. & Hooykaas, P. J. Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Res. 26, 2729–2734 (1998).

    Article  CAS  Google Scholar 

  46. Vergunst, A. C. & Hooykaas, P. J. Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre. Plant Mol. Biol. 38, 393–406 (1998).

    Article  CAS  Google Scholar 

  47. Sieburth, L. E., Drews, G. N. & Meyerowitz, E. M. Non-autonomy of AGAMOUS function in flower development: use of a Cre/loxP method for mosaic analysis in Arabidopsis. Development 125, 4303–4312 (1998).

    Article  CAS  Google Scholar 

  48. Marquès-Bueno, M. D. M. et al. A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis. Plant J. 85, 320–333 (2016).

    Article  Google Scholar 

  49. Craft, J. et al. New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis. Plant J. 41, 899–918 (2005).

    Article  CAS  Google Scholar 

  50. Weinberg, B. H. et al. High-performance chemical- and light-inducible recombinases in mammalian cells and mice. Nat. Commun. 10, 4845 (2019).

    Article  Google Scholar 

  51. Odell, J., Caimi, P., Sauer, B. & Russell, S. Site-directed recombination in the genome of transgenic tobacco. Mol. Gen. Genet. 223, 369–378 (1990).

    Article  CAS  Google Scholar 

  52. Russell, S. H., Hoopes, J. L. & Odell, J. T. Directed excision of a transgene from the plant genome. Mol. Gen. Genet. 234, 49–59 (1992).

    Article  CAS  Google Scholar 

  53. Schürholz, A.-K. et al. A comprehensive toolkit for inducible, cell type-specific gene expression in Arabidopsis. Plant Physiol. 178, 40–53 (2018).

    Article  Google Scholar 

  54. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  55. Logemann, E., Birkenbihl, R. P., Ülker, B. & Somssich, I. E. An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods 2, 16 (2006).

    Article  Google Scholar 

  56. Shimada, T. L., Shimada, T. & Hara-Nishimura, I. A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61, 519–528 (2010).

    Article  CAS  Google Scholar 

  57. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden Gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4, e5553 (2009).

    Article  Google Scholar 

  58. Patron, N. J. et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol. 208, 13–19 (2015).

    Article  CAS  Google Scholar 

  59. Libiakova, G., Jørgensen, B., Palmgren, G., Ulvskov, P. & Johansen, E. Efficacy of an intron-containing kanamycin resistance gene as a selectable marker in plant transformation. Plant Cell Rep. 20, 610–615 (2001).

    Article  CAS  Google Scholar 

  60. Wick, R. R., Judd, L. M., Gorrie, C. L. & Holt, K. E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13, e1005595 (2017).

    Article  Google Scholar 

  61. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  62. Naseer, S. et al. Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin. Proc. Natl Acad. Sci. USA 109, 10101–10106 (2012).

    Article  CAS  Google Scholar 

  63. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2016).

  64. Lloyd, J. P. B. et al. Synthetic memory circuits for programmable cell reconfiguration in plants. https://doi.org/10.5281/zenodo.6381286 (2022).

Download references

Acknowledgements

We would like to thank B. Johnston for advice on R code; M. Oliva and D. Collings for help with confocal image analysis; I. Roux for suggestion of the in vitro recombinase assay; Y.-H. Chooi for the kind donation of purified recombinant Cre enzyme; B. Crawford for the kind donation of a pOp6 promoter-containing plasmid; C. Helliwell for graciously providing the LhGR seeds used in this study; and W. Wong for generous donation of mammalian BLADE plasmids. The plasmid sequencing data were generated on instrumentation supported by the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics and Genomics WA. This work was supported by the following grants to R.L.: Australian Research Council (ARC) Centre of Excellence in Plant Energy Biology (CE140100008), ARC DP210103954, NHMRC Investigator Grant GNT1178460, Silvia and Charles Viertel Senior Medical Research Fellowship and Howard Hughes Medical Institute International Research Scholarship. T.S. was supported by the Hackett Postgraduate Research Scholarship. M.A.K. was supported by an International Postgraduate Research Scholarship. B.K. was supported by the CSIRO Synthetic Biology Future Science Platform.

Author information

Authors and Affiliations

  1. Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, Australia

    James P. B. Lloyd, Florence Ly, Patrick Gong, Christian Pflueger, Elliott Fourie, Muhammad Adil Khan, Brendan N. Kidd & Ryan Lister

  2. Harry Perkins Institute of Medical Research, The University of Western Australia, Perth, WA, Australia

    Jahnvi Pflueger, Tessa Swain, Christian Pflueger & Ryan Lister

Authors
  1. James P. B. Lloyd
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Florence Ly
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patrick Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jahnvi Pflueger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tessa Swain
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christian Pflueger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elliott Fourie
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Muhammad Adil Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Brendan N. Kidd
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ryan Lister
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.P.B.L. and R.L. conceived of the project and wrote the manuscript. J.P.B.L., with F.L. and P.G., conducted the experiments, with assistance from B.K. and E.F. J.P., T.S. and C.P. conducted the plasmid sequencing. M.A.K. developed the protoplast transformation protocol. M.A.K. and B.K. provided technical assistance with the protoplast assay, luciferase assay and cloning. All authors approved of and contributed to the final version of the manuscript.

Corresponding author

Correspondence to Ryan Lister.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Biotechnology thanks Diego Orzaez, Anna Stepanova and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and uncropped gels for Supplementary Fig. 2

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lloyd, J.P.B., Ly, F., Gong, P. et al. Synthetic memory circuits for stable cell reprogramming in plants. Nat Biotechnol 40, 1862–1872 (2022). https://doi.org/10.1038/s41587-022-01383-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-022-01383-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing