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.

Non-specific activities of the major herbicide-resistance gene BAR

Abstract

Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops1,2,3,4. Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids1, indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes5,6. Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Accumulation of acetyl-aminoadipate and acetyl-tryptophan in senescent leaves of Arabidopsis carrying the BAR transgene.
Fig. 2: BAR-dependent accumulation of acetyl-aminoadipate and acetyl-tryptophan is linked to nitrogen remobilization during senescence.
Fig. 3: In vitro enzyme kinetic assays of BAR against native and non-native substrates.
Fig. 4: Structural basis for amino acid N-acetylation catalysed by BAR and structure-guided engineering of BAR with reduced non-specific activities.

References

  1. 1.

    Wehrmann, A., Van Vliet, A., Opsomer, C., Botterman, J. & Schulz, A. The similarities of BAR and PAT gene products make them equally applicable for plant engineers. Nat. Biotechnol. 14, 1274–1278 (1996).

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Duke, S. O. Taking stock of herbicide-resistant crops ten years after introduction. Pest. Manag. Sci. 61, 211–218 (2005).

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Thompson, C. J. et al. Characterization of the herbicide-resistance gene BAR from Streptomyces hygroscopicus. EMBO J. 6, 2519–2523 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Wohlleben, W. et al. Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum. Gene 70, 25–37 (1988).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Ren, Y. F. et al. A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis. J. Genet. Genomics 36, 629–639 (2009).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Brown, R. H., Raboy, V. & Bregitzer, P. Unintended consequences: high phosphinothricin acetyltransferase activity related to reduced fitness in barley. In Vitro Cell. Dev. Biol. Plant 49, 240–247 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    The National Academies of Science, Engineering and Medicine Genetically Engineered Crops: Experiences and Prospects (National Academies Press, Washington DC, 2016).

  8. 8.

    Schenk, N. et al. The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana. FEBS Lett. 581, 5517–5525 (2007).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Samson, F. et al. FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants. Nucleic Acids Res. 30, 94–97 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Sessions, A. et al. A high-throughput Arabidopsis reverse genetics system. Plant Cell 14, 2985–2994 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).

    Article  PubMed  Google Scholar 

  12. 12.

    Rosso, M. G. et al. An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol. Biol. 53, 247–259 (2003).

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Soudry, E., Ulitzur, S. & Gepstein, S. Accumulation and remobilization of amino acids during senescence of detached and attached leaves: in planta analysis of tryptophan levels by recombinant luminescent bacteria. J. Exp. Bot. 56, 695–702 (2005).

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Hörtensteiner, S. & Feller, U. Nitrogen metabolism and remobilization during senescence. J. Exp. Bot. 53, 927–937 (2002).

    Article  PubMed  Google Scholar 

  15. 15.

    Arruda, P., Kemper, E. L., Papes, F. & Leite, A. Regulation of lysine catabolism in higher plants. Trends Plant Sci. 5, 324–330 (2000).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Zhu, X., Tang, G., Granier, F., Bouchez, D. & Galili, G. A T-DNA insertion knockout of the bifunctional LYSINE-KETOGLUTARATE REDUCTASE/SACCHAROPINE DEHYDROGENASE gene elevates lysine levels in Arabidopsis seeds. Plant Physiol. 126, 1539–1545 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Vinnemeier, J., DrogeLaser, W., Pistorius, E. K. & Broer, I. Purification and partial characterization of the Streptomyces viridochromogenes Tü494 phosphinothricin-N-acetyltransferase mediating resistance to the herbicide phosphinothricin in transgenic plants. Z. Naturforsch. C. 50, 796–805 (1995).

    CAS  Google Scholar 

  18. 18.

    Dyda, F., Klein, D. C. & Hickman, A. B. GCN5-related N-acetyltransferases: a structural overview. Annu. Rev. Bioph. Biom. 29, 81–103 (2000).

    CAS  Article  Google Scholar 

  19. 19.

    Vetting, M. W. et al. Structure and functions of the GNAT superfamily of acetyltransferases. Arch. Biochem. Biophys. 433, 212–226 (2005).

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Srivastava, P. et al. Structural characterization of a GCN5-related N-acetyltransferase from Staphylococcus aureus. PLoS ONE 9 (2014).

  21. 21.

    Rojas, J. R. et al. Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide. Nature 401, 93–98 (1999).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Woolston, B. M., Edgar, S. & Stephanopoulos, G. Metabolic engineering: past and future. Annu. Rev. Chem. Biomol. Eng. 4, 259–288 (2013).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Weng, J. K. & Noel, J. P. The remarkable pliability and promiscuity of specialized metabolism. Cold Spring Harb. Symp. Quant. Biol. 77, 309–320 (2012).

    Article  PubMed  Google Scholar 

  24. 24.

    Jin, Y.-S. et al. Chemical and biologically active constituents of Salsola collina. Chem. Nat. Compd. 47, 257–260 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    Yu, P., Hegeman, A. D. & Cohen, J. D. A facile means for the identification of indolic compounds from plant tissues. Plant J. 79, 1065–1075 (2014).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Bruckhoff, V. et al. Functional characterization of CYP94-genes and identification of a novel jasmonate catabolite in flowers. PLoS ONE 11, e0159875 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Green, J. M. & Owen, M. D. Herbicide-resistant crops: utilities and limitations for herbicide-resistant weed management. J. Agric. Food Chem. 59, 5819–5829 (2011).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Herouet, C. et al. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the PAT and BAR sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants. Regul. Toxicol. Pharm. 41, 134–149 (2005).

    CAS  Article  Google Scholar 

  29. 29.

    Dan, Y. Plant Transformation Technology Revolution in Last Three Decades: Historical Technology Developments (Bentham Science Publishers, Sharjah, 2012).

  30. 30.

    Song, W. Y. & Choi, K. S. Alexis de, A., Martinoia, E. & Lee, Y. Brassica juncea plant cadmium resistance 1 protein (BjPCR1) facilitates the radial transport of calcium in the root. Proc. Natl Acad. Sci. USA 108, 19808–19813 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Foetzki, A. et al. Surveying of pollen-mediated crop-to-crop gene flow from a wheat field trial as a biosafety measure. GM Crops Food 3, 115–122 (2012).

    Article  PubMed  Google Scholar 

  32. 32.

    Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. & Scheible, W. R. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Chambers, M. C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Gowda, H. et al. Interactive XCMS Online: simplifying advanced metabolomic data processing and subsequent statistical analyses. Anal. Chem. 86, 6931–6939 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Smith, C. A. et al. METLIN: a metabolite mass spectral database. Ther. Drug Monit. 27, 747–751 (2005).

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Tropea, J. E., Cherry, S. & Waugh, D. S. Expression and purification of soluble His(6)-tagged TEV protease. Methods Mol. Biol. 498, 297–307 (2009).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271–281 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006).

    Article  PubMed  Google Scholar 

  39. 39.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  41. 41.

    Goldstein, A. L. & McCusker, J. H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553 (1999).

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Engler, C. et al. A Golden Gate modular cloning toolbox for plants. ACS Synth. Biol. (2014).

  43. 43.

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

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Schelbert, S. et al. Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21, 767–785 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Christ, B. et al. MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis. Plant Physiol. 158, 628–641 (2012).

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Guyer, L. Characterization of Dephytylation and Dechelation, Two Early Steps of Chlorophyll Breakdown in Leaves and Fruits. PhD thesis, Univ. Zurich (2015).

  47. 47.

    Christ, B. et al. Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis. Plant Cell 25, 1868–1880 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Perez-Perez, J. M. et al. Functional redundancy and divergence within the Arabidopsis RETICULATA-RELATED gene family. Plant Physiol. 162, 589–603 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Christ, B. Chlorophyll Breakdown: Modifications of Colorless Chlorophyll Catabolites. PhD thesis, Univ. Zurich (2013).

  50. 50.

    Zufferey, M. et al. The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery. J. Biol. Chem. 292, 6952–6964 (2017).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Pulido, P., Llamas, E. & Rodriguez-Concepcion, M. Both Hsp70 chaperone and Clp protease plastidial systems are required for protection against oxidative stress. Plant Signal. Behav. 12, e1290039 (2017).

    Article  PubMed  Google Scholar 

  52. 52.

    Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2 – a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D.M. Sabatini, G.R. Fink, N. Amrhein and E. Martinoia for helpful discussions. We thank J.M. Cheeseman for providing the phosphinothricin-resistant Glycine max line. We thank J. Varberg for providing the phosphinothricin-resistant B. napus line and M. Rahman for providing conventional B. napus lines. This work is based on research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6 M detector on NE-CAT 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This work was supported by the Swiss National Science Foundation (grant 31003A_149389 to S.H. and postdoctoral fellowship P2ZHP3_155258 to B.C.), the EU-funded Plant Fellows program (S.A.), the Pew Scholar Program in the Biomedical Sciences (J.K.W.) and the Searle Scholars Program (J.K.W.).

Author information

Affiliations

Authors

Contributions

B.C., S.A., S.H. and J.K.W. designed experiments; B.C., R.H., L.G., R.F. and S.A. performed experiments; B.C., R.H., L.G. and J.K.W. analysed data; B.C., S.H., S.A. and J.K.W. wrote the manuscript.

Corresponding authors

Correspondence to Stefan Hörtensteiner or Jing-Ke Weng.

Ethics declarations

Competing interests

B.C. and J.K.W. have filed a patent application on BAR and PAT mutants described in this paper that show altered acetyltransferase activity.

Additional information

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

Electronic supplementary material

Supplementary Information

Supplementary Tables 1–3, Supplementary Figures 1–17

Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Christ, B., Hochstrasser, R., Guyer, L. et al. Non-specific activities of the major herbicide-resistance gene BAR . Nature Plants 3, 937–945 (2017). https://doi.org/10.1038/s41477-017-0061-1

Download citation

Further reading

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