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:

Role of bifunctional ammonia-lyase in grass cell wall biosynthesis

Nature Plants volume 2, Article number: 16050 (2016) Cite this article

Subjects

Abstract

L-Phenylalanine ammonia-lyase (PAL) is the first enzyme in the biosynthesis of phenylpropanoid-derived plant compounds such as flavonoids, coumarins and the cell wall polymer lignin. The cell walls of grasses possess higher proportions of syringyl (S)-rich lignins and high levels of esterified coumaric acid compared with those of dicotyledonous plants, and PAL from grasses can also possess tyrosine ammonia-lyase (TAL) activity, the reason for which has remained unclear. Using phylogenetic, transcriptomic and in vitro biochemical analyses, we identified a single homotetrameric bifunctional ammonia-lyase (PTAL) among eight BdPAL enzymes in the model grass species Brachypodium distachyon. 13C isotope labelling experiments along with BdPTAL1-downregulation in transgenic plants showed that the TAL activity of BdPTAL1 can provide nearly half of the total lignin deposited in Brachypodium, with a preference for S-lignin and wall-bound coumarate biosynthesis, indicating that PTAL function is linked to the characteristic features of grass cell walls. Furthermore, isotope dilution experiments suggest that the pathways to lignin from L-phenylalanine and L-tyrosine are distinct beyond the formation of 4-coumarate, supporting the organization of lignin synthesis enzymes in one or more metabolons.

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

Figure 1: Phylogeny, gene expression and enzymatic activity of Brachypodium BdPALs.
Figure 2: Purification of the native PTAL enzyme from Brachypodium stem tissue.
Figure 3: Labelling of Brachypodium seedlings with 13C9-L-Phe and 13C9-L-Tyr.
Figure 4: Phenotype and lignin characteristics of Brachypodium PTAL1 RNAi lines.
Figure 5: Model for the early steps of lignin synthesis including both PAL and TAL pathways in monocot plants.

Similar content being viewed by others

References

  1. Kenrick, P. & Crane, P. R. The origin and early evolution of plants on land. Nature 389, 33–39 (1997).

    Article  CAS  Google Scholar 

  2. Bateman, R. M. et al. Early evolution of land plants: phylogeny, physiology, and ecology of the primary terrestrial radiation. Annu. Rev. Ecol. Syst. 29, 263–292 (1998).

    Article  Google Scholar 

  3. Dixon, R. A. et al. The phenylpropanoid pathway and plant defence –a genomics perspective. Mol. Plant Pathol. 3, 371–390 (2002).

    Article  CAS  Google Scholar 

  4. Maeda, H. & Dudareva, N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu. Rev. Plant Biol. 63, 73–105 (2012).

    Article  CAS  Google Scholar 

  5. Vanholme, R. et al. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341, 1103–1106 (2013).

    Article  CAS  Google Scholar 

  6. Appert, C., Logemann, E., Hahlbrock, K., Schmid, J. & Amrhein, N. Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.). Eur. J. Biochem. 225, 491–499 (1994).

    Article  CAS  Google Scholar 

  7. Kyndt, J. A., Meyer, T. E., Cusanovich, M. A. & Van Beeumen, J. J. Characterization of a bacterial tyrosine ammonia-lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Lett. 512, 240–244 (2002).

    Article  CAS  Google Scholar 

  8. Cochrane, F. C., Davin, L. B. & Lewis, N. G. The Arabidopsis phenylalanine ammonia-lyase gene family: kinetic characterization of the four PAL isoforms. Phytochemistry 65, 1557–1564 (2004).

    Article  CAS  Google Scholar 

  9. Havir, E. A., Reid, P. D. & Marsh, H. V. L-phenylalanine ammonia-lyase (maize) evidence for a common catalytic site for L-phenylalanine and L-tyrosine. Plant Physiol. 48, 130–136 (1971).

    Article  CAS  Google Scholar 

  10. Rosler, J., Krekel, F., Amrhein, N. & Schmid, J. Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiol. 113, 175–179 (1997).

    Article  CAS  Google Scholar 

  11. Jangaard, N. O. The characterization of phenylalanine ammonia-lyase from several plant species. Phytochemistry 13, 1765–1768 (1974).

    Article  CAS  Google Scholar 

  12. Hsieh, L., Ma, G., Yang, C. & Lee, P. Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii. Phytochemistry 71, 1999–2009 (2010).

    Article  CAS  Google Scholar 

  13. Brown, S. A., Wright, D. & Neish, A. C. Studies of lignin biosynthesis using isotopic carbon: VII.: The role of p-hydroxyphenylpyruvic acid. Can. J. Biochem. Physiol. 37, 25–34 (1959).

    Article  CAS  Google Scholar 

  14. Higuchi, T., Ito, Y. & Kawamura, I. p-Hydroxyphenylpropane component of grass lignin and role of tyrosine-ammonia lyase in its formation. Phytochemistry 6, 875–881 (1967).

    Article  CAS  Google Scholar 

  15. Louie, G. V. et al. Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonia-lyases. Chem. Biol. 13, 1327–1338 (2006).

    Article  CAS  Google Scholar 

  16. Watts, K. T., Mijts, B. N., Lee, P. C., Manning, A. J. & Schmidt-Dannert, C. Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family. Chem. Biol. 13, 1317–1326 (2006).

    Article  CAS  Google Scholar 

  17. Rétey, J. Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group. Biochim. Biophys. Acta 1647, 179–184 (2003).

    Article  Google Scholar 

  18. MacDonald, M. J. & D'Cunha, G. B. A modern view of phenylalanine ammonia-lyase. Biochem. Cell Biol. 85, 273–282 (2007).

    Article  CAS  Google Scholar 

  19. Cass, C. et al. Effects of phenylalanine ammonia lyase (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium. J. Exp. Bot. 66, 4317–4335 (2015).

    Article  CAS  Google Scholar 

  20. Reichert, A., He, X. & Dixon, R. A. Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes. Biochem. J. 424, 233–242 (2009).

    Article  CAS  Google Scholar 

  21. Hyun, M. W., Yun, Y. H., Kim, J. Y. & Kim, S. H. Fungal and plant phenylalanine ammonia-lyase. Mycobiology 39, 257–265 (2011).

    Article  CAS  Google Scholar 

  22. Vogel, J. Unique aspects of the grass cell wall. Curr. Opin. Plant Biol. 11, 301–307 (2008).

    Article  CAS  Google Scholar 

  23. Harris, P. J. & Hartley, R. D. Detection of bound ferulic acid in cell walls of the Gramineae by ultraviolet fluorescence microscopy. Nature 259, 508–510 (1976).

    Article  CAS  Google Scholar 

  24. Mann, D. G. J. et al. Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species. Plant Biotechnol. J. 10, 226–236 (2012).

    Article  CAS  Google Scholar 

  25. Rasmussen, S. & Dixon, R. A. Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11, 1537–1551 (1999).

    Article  CAS  Google Scholar 

  26. Achnine, L., Blancaflor, E. B., Rasmussen, S. & Dixon, R. A. Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16, 3098–3109 (2004).

    Article  CAS  Google Scholar 

  27. Bassard, J.-E. et al. Protein–protein and protein–membrane associations in the lignin pathway. Plant Cell 24, 4465–4482 (2012).

    Article  CAS  Google Scholar 

  28. Winkel, B. S. J. Metabolic channeling in plants. Annu. Rev. Plant Biol. 55, 85–107 (2004).

    Article  CAS  Google Scholar 

  29. Zhao, Q., & Dixon, R. A. Altering the cell wall and its impact on plant disease: from forage to bioenergy. Annu. Rev. Phytopathol. 52, 62–91 (2014).

    Article  Google Scholar 

  30. Yamamoto, K., Nobayashi, N., Yoshitama, K., Teramoto, S. & Komamine, A. Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora. Plant Cell Physiol. 42, 969–975 (2001).

    Article  CAS  Google Scholar 

  31. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003).

    Article  Google Scholar 

  32. Nuin, P. A. S., Wang, Z. & Tillier, E. R. M. The accuracy of several multiple sequence alignment programs for proteins. BMC Bioinform. 7, 471 (2006).

    Article  Google Scholar 

  33. Scott, D. A., Hammond, P. M., Brearley, G. M. & Price, C. P. Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase. J. Chromatogr. B 573, 309–312 (1992).

    Article  CAS  Google Scholar 

  34. Shevchenko, A., Tomas, H., Havli, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols 1, 2856–2860 (2006).

    Article  CAS  Google Scholar 

  35. Rolando, C., Monties, B. & Lapierre, C. in Methods in Lignin Chemistry (eds Lin, S. Y. & Dence, C. W. ) 334–340 (Springer, 1992).

    Book  Google Scholar 

  36. Broeckling, C. D. et al. Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J. Exp. Bot. 56, 323–336 (2005).

    Article  CAS  Google Scholar 

  37. Rohde, A. et al. Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism. Plant Cell 16, 2749–2771 (2004).

    Article  CAS  Google Scholar 

  38. Franke, R. et al. The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J. 30, 33–45 (2002).

    Article  CAS  Google Scholar 

  39. Zuo, Y., Wang, C. & Zhan, J. Separation, characterization, and quantitation of benzoic and phenolic antioxidants in American cranberry fruit by GC–MS. J. Agricult. Food Chem. 50, 3789–3794 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Huhman and the Noble Foundation Analytical Chemistry Facility for the analysis of soluble phenolic compounds, Dr X. Wang for assistance with protein purification, and Dr L. Gallego-Giraldo for critical reading of the manuscript. This work was supported by a Barrie Foundation Fellowship (to J.B.), The University of North Texas and the BioEnergy Science Center (Oak Ridge National Laboratory), a US Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.

Author information

Authors and Affiliations

  1. BioDiscovery Institute, University of North Texas, Denton, 76203, Texas, USA

    Jaime Barros, Juan C. Serrani-Yarce, Fang Chen, David Baxter, Barney J. Venables & Richard A. Dixon

  2. Department of Biological Sciences, University of North Texas, Denton, 76203, Texas, USA

    Jaime Barros, Juan C. Serrani-Yarce, Fang Chen & Richard A. Dixon

  3. BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, 37831, Tennessee, USA

    Fang Chen & Richard A. Dixon

Authors
  1. Jaime Barros
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan C. Serrani-Yarce
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Baxter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Barney J. Venables
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard A. Dixon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.A.D. and J.B. conceived the study; J.B., J.S.-Y., F.C. and R.A.D. designed the experiments; J.B, J.S.-Y. and D.B. performed experimental work; J.B., J.S.-Y., D.B., B.V., F.C. and R.A.D. discussed and interpreted results; J.B. and R.A.D. wrote the paper.

Corresponding author

Correspondence to Richard A. Dixon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Barros, J., Serrani-Yarce, J., Chen, F. et al. Role of bifunctional ammonia-lyase in grass cell wall biosynthesis. Nature Plants 2, 16050 (2016). https://doi.org/10.1038/nplants.2016.50

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nplants.2016.50

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