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A role for leucoanthocyanidin reductase in the extension of proanthocyanidins

Abstract

Proanthocyanidins (PAs) are the second most abundant plant polyphenolic compounds after lignin. PAs affect taste, mouth feel and astringency of many fruits, wines and beverages1,2, have been associated with reduced risks of cardiovascular disease, cancer and Alzheimer's disease35, can improve nutrition and prevent bloat in ruminant animals6 and enhance soil nitrogen retention7. PAs are oligomers and polymers of flavan-3-ols, primarily (–)-epicatechin and (+)-catechin, but the mechanism by which the monomers polymerize and become insoluble is currently unknown. Leucoanthocyanidin reductase (LAR) has been shown to convert leucocyanidin to (+)-catechin8,9. Here, we report that loss of function of LAR in the model legume Medicago truncatula leads unexpectedly to loss of soluble epicatechin-derived PAs, increased levels of insoluble PAs, and accumulation of 4β-(S-cysteinyl)-epicatechin, which provides the 4→8 linked extension units during non-enzymatic PA polymerization. LAR converts 4β-(S-cysteinyl)-epicatechin back to epicatechin, the starter unit in PAs, thereby regulating the relative proportions of starter and extension units and consequently the degree of PA oligomerization.

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Figure 1: Characterization of lar and anr mutants in M. truncatula.
Figure 2: Identification of a new substrate of LAR.
Figure 3: 4β-(S-Cysteinyl)-epicatechin is a substrate of LAR.
Figure 4: In vitro auto-polymerization between 4β-(S-cysteinyl)-epicatechin and 13C-labelled epicatechin.

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References

  1. Gonzalo-Diago, A., Dizy, M. & Fernández-Zurbano, P. Taste and mouthfeel properties of red wines proanthocyanidins and their relation to the chemical composition. J. Agric. Food Chem. 61, 8861–8870 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Lesschaeve, I. & Noble, A. C. Polyphenols: factors influencing their sensory properties and their effects on food and beverage preferences. Am. J. Clin. Nutr. 81, 330s–335s (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Bagchi, D. et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology 148, 187–197 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Cos, P. et al. Proanthocyanidins in health care: current and new trends. Curr. Med. Chem. 11, 1345–1359 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Middleton, E. Jr, Kandaswami, C. & Theoharides, T. C. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673–751 (2000).

    CAS  PubMed  Google Scholar 

  6. Lees, G. L. Condensed tannins in some forage legumes: their role in the prevention of ruminant pasture bloat. Basic Life Sci. 59, 915–934 (1992).

    CAS  PubMed  Google Scholar 

  7. Joanisse, G. D., Bradley, R. L., Preston, C. M. & Bending, G. D. Sequestration of soil nitrogen as tannin-protein complexes may improve the competitive ability of sheep laurel (Kalmia angustifolia) relative to black spruce (Picea mariana). New Phytol. 181, 187–198 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Pang, Y., Peel, G. J., Wright, E., Wang, Z. & Dixon, R. A. Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol. 145, 601–615 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tanner, G. J. et al. Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. J. Biol. Chem. 278, 31647–31656 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Devic, M. et al. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J. 19, 387–398 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Xie, D. Y., Sharma, S. B., Paiva, N. L., Ferreira, D. & Dixon, R. A. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299, 396–399 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Bogs, J. et al. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol. 139, 652–663 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liu, Y., Shi, Z., Maximova, S., Payne, M. J. & Guiltinan, M. J. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase. BMC Plant Biol. 13, 202 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tadege, M. et al. Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J. 54, 335–347 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Kiatgrajai, P., Wellons, J. D., Gollob, L. & White, J. D. Kinetics of epimerization of (+)-catechin and its rearrangement to catechinic acid. J. Org. Chem. 47, 2910–2912 (1982).

    Article  CAS  Google Scholar 

  16. Pang, Y., Peel, G. J., Sharma, S. B., Tang, Y. & Dixon, R. A. A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula. Proc. Natl Acad. Sci. USA 105, 14210–14215 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mauge, C. et al. Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera. J. Mol. Biol. 397, 1079–1091 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Liu, C., Jun, J. H. & Dixon, R. A. MYB5 and MYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula. Plant Physiol. 165, 1424–1439 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Callemien, D. & Collin, S. Use of RP-HPLC-ESI(–)MS/MS to differentiate various proanthocyanidin isomers in lager beer extracts. J. Am. S. Brewing Chem. 66, 109–115 (2008).

    Article  CAS  Google Scholar 

  20. Torres, J. L. et al. Cysteinyl-flavan-3-ol conjugates from grape procyanidins. Antioxidant and antiproliferative properties. Bioorg. Med. Chem. 10, 2497–2509 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Torres, J. L., Lozano, C. & Maher, P. Conjugation of catechins with cysteine generates antioxidant compounds with enhanced neuroprotective activity. Phytochemistry 66, 2032–2037 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Jiang, X. et al. Analysis of accumulation patterns and preliminary study on the condensation mechanism of proanthocyanidins in the tea plant [Camellia sinensis]. Sci. Rep. 5, 8742 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Dixon, R. A., Xie, D. Y. & Sharma, S. B. Proanthocyanidins—a final frontier in flavonoid research? New Phytol. 165, 9–28 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Hemingway, R. W. & Foo, L. Y. Condensed tannins: quinone methide intermediates in procyanidin synthesis. J. Chem. Soc. Chem. Commun. 1035–1036 (1983).

  25. Yan, Y., Li, Z. & Koffas, M. A. High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnol. Bioeng. 100, 126–140 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Lim, C. G. et al. Development of a recombinant Escherichia coli strain for overproduction of the plant pigment anthocyanin. Appl. Environ. Microbiol. 81, 6276–6284 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wellmann, F. et al. Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin. FEBS Lett. 580, 1642–1648 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Ferraro, K. et al. Characterization of proanthocyanidin metabolism in pea (Pisum sativum) seeds. BMC Plant Biol. 14, 1–17 (2014).

    Article  Google Scholar 

  29. Huang, Y.-F. et al. Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping. BMC Plant Biol. 12, 1–19 (2012).

    Article  CAS  Google Scholar 

  30. Akagi, T., Katayama-Ikegami, A. & Yonemori, K. Proanthocyanidin biosynthesis of persimmon (Diospyros kaki Thunb.) fruit. Sci. Hort. 130, 373–380 (2011).

    Article  CAS  Google Scholar 

  31. Liao, L. et al. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple. Frontiers Plant Sci. 6, 243 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the University of North Texas and Forage Genetics International. We thank J. Wen and X. Chen for screening for M. truncatula Tnt1 insertion mutants.

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Authors

Contributions

C.L. and R.A.D. conceived and designed the project, C.L. carried out the experiments, V.S. provided assistance for UPLC/MS analysis, X.W. carried out molecular modelling and docking analyses and C.L. and R.A.D. wrote the paper.

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Correspondence to Richard A. Dixon.

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The authors declare no competing financial interests.

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Supplementary Information

Supplementary Figures 1–23, Supplementary Methods, Supplementary References. (PDF 1336 kb)

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Liu, C., Wang, X., Shulaev, V. et al. A role for leucoanthocyanidin reductase in the extension of proanthocyanidins. Nature Plants 2, 16182 (2016). https://doi.org/10.1038/nplants.2016.182

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