Proanthocyanidins (PAs) are primarily composed of the flavan-3-ol subunits (-)-epicatechin and/or (+)-catechin, but the basis for their different starter and extension unit compositions remains unclear. Genetic and biochemical analyses show that, in the model legume Medicago truncatula, two 2-oxoglutarate-dependent dioxygenases, anthocyanidin synthase (ANS) and its homologue leucoanthocyanidin dioxygenase (LDOX), are involved in parallel pathways to generate, respectively, the (-)-epicatechin extension and starter units of PAs, with (+) catechin being an intermediate in the formation of the (-)-epicatechin starter unit. The presence/absence of the LDOX pathway accounts for natural differences in PA compositions across species, and engineering loss of function of ANS or LDOX provides a means to obtain PAs with different compositions and degrees of polymerization for use in food and feed.
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Porter, L. J. in The flavonoids: Advances in research since 1986 (ed. Harborne, J. B.) 23–53 (Chapman & Hall, London, 1994).
Prior, R. L. & Gu, L. Occurrence and biological significance of proanthocyanidins in the American diet. Phytochemistry 66, 2264–2280 (2005).
Donaldson, J. R., Stevens, M. T., Barnhill, H. R. & Lindroth, R. L. Age-related shifts in leaf chemistry of clonal aspen (Populus tremuloides). J. Chem. Ecol. 32, 1415–1429 (2006).
Balentine, D. A., Wiseman, S. A. & Bouwens, L. C. The chemistry of tea flavonoids. Crit. Rev. Food Sci. 37, 693–704 (1997).
Debeaujon, I., Leon-Kloosterziel, K. M. & Koornneef, M. Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant Physiol. 122, 403–414 (2000).
Cos, P. et al. Proanthocyanidins in health care: current and new trends. Curr. Med. Chem. 11, 1345–1359 (2004).
Bagchi, D. et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology 148, 187–197 (2000).
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).
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).
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).
Jorgensen, E. M., Marin, A. B. & Kennedy, J. A. Analysis of the oxidative degradation of proanthocyanidins under basic conditions. J. Agr. Food Chem. 52, 2292–2296 (2004).
Ma, W. et al. A review on astringency and bitterness perception of tannins in wine. Trends Food Sci. Tech. 40, 6–19 (2014).
Min, B. R., Pinchak, W. E., Fulford, J. D. & Puchala, R. Effect of feed additives on in vitro and in vivo rumen characteristics and frothy bloat dynamics in steers grazing wheat pasture. Anim. Feed Sci. Tech. 124, 615–629 (2005).
Dixon, R. A., Xie, D. Y. & Sharma, S. B. Proanthocyanidins—a final frontier in flavonoid research? New Phytol. 165, 9–28 (2005).
Dixon, R. A., Liu, C. G. & Jun, J. H. Metabolic engineering of anthocyanins and condensed tannins in plants. Curr. Opin. Biotech. 24, 329–335 (2013).
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).
Jun, J. H., Liu, C. G., Xiao, X. R. & Dixon, R. A. The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula. Plant Cell 27, 2860–2879 (2015).
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).
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).
Pang, Y. Z., Peel, G. J., Sharma, S. B., Tang, Y. H. & 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).
Zhao, J. & Dixon, R. A. MATE transporters facilitate vacuolar uptake of epicatechin 3′-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatula and Arabidopsis. Plant Cell 21, 2323–2340 (2009).
Xie, D. Y. & Dixon, R. A. Proanthocyanidin biosynthesis—still more questions than answers? Phytochemistry 66, 2127–2144 (2005).
Gonzalez-Centeno, M. R. et al. Proanthocyanidin composition and antioxidant potential of the stem winemaking byproducts from 10 different grape varieties (Vitis vinifera L.). J. Agric. Food Chem. 60, 11850–11858 (2012).
Pang, Y. Z., Peel, G. J., Wright, E., Wang, Z. Y. & Dixon, R. A. Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol. 145, 601–615 (2007).
Ito, C. et al. Characterisation of proanthocyanidins from black soybeans: isolation and characterisation of proanthocyanidin oligomers from black soybean seed coats. Food Chem. 141, 2507–2512 (2013).
Saito, K., Kobayashi, M., Gong, Z., Tanaka, Y. & Yamazaki, M. Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma of Perilla frutescens. Plant J. 17, 181–189 (1999).
Abrahams, S. et al. The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development. Plant J. 35, 624–636 (2003).
Liu, C., Wang, X., Shulaev, V. & Dixon, R. A. A role for leucoanthocyanidin reductase in the extension of proanthocyanidins. Nat. Plants 2, 16182 (2016).
Turnbull, J. J. et al. Are anthocyanidins the immediate products of anthocyanidin synthase?. Chem. Commun. 24, 2473–2474 (2000).
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).
Peel, G. J., Pang, Y. Z., Modolo, L. V. & Dixon, R. A. The LAP1 MYB transcription factor orchestrates anthocyanidin biosynthesis and glycosylation in Medicago. Plant J. 59, 136–149 (2009).
Wilmouth, R. C. et al. Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. Structure 10, 93–103 (2002).
Creasy, L. L. & Swain, T. Structure of condensed tannins. Nature 208, 151–153 (1965).
Pang, Y. et al. Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering. Plant Physiol. 161, 1103–1116 (2013).
Jacques, D., Opie, C. T., Porter, L. J. & Haslam, E. Plant proanthocyanidins, Part 4. Biosynthesis of procyanidins and observations on the metabolism of cyanidin in plants. J. Chem. Soc. Perkin I 14, 1637–1643 (1977).
Wang, H. L. et al. Gene transcript accumulation, tissue and subcellular localization of anthocyanidin synthase (ANS) in developing grape berries. Plant Sci. 179, 103–113 (2010).
Winkel-Shirley, B. Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiol. Plantarum 107, 142–149 (1999).
Fujino, N. et al. Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species. Plant J. 94, 372–392 (2018).
Perkins, J. R., Diboun, I., Dessailly, B. H., Lees, J. G. & Orengo, C. Transient protein‒protein interactions: structural, functional, and network properties. Structure 18, 1233–1243 (2010).
Gou, M., Ran, X., Martin, D. W. & Liu, C. J. The scaffold proteins of lignin biosynthetic cytochrome P450 enzymes. Nat. Plants 4, 299–310 (2018).
Tadege, M. et al. Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J. 54, 335–347 (2008).
Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).
Rao, X. et al. A deep transcriptomic analysis of pod development in the vanilla orchid (Vanilla planifolia). BMC Genomics 15, 964 (2014).
Rao, X. et al. Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages. J. Exp. Bot. 67, 1649–1662 (2016).
Haas, B. J. et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512 (2013).
Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009).
We thank S. Temple for critical reading of the manuscript and V. Shulaev for assistance with high mass accuracy LCMS. This work was supported by Forage Genetics International Inc. and the University of North Texas.
R.A.D. and J.H.J. are inventors on a United States provisional patent application filed by the University of North Texas that describes methods for engineering PA composition.
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Jun, J.H., Xiao, X., Rao, X. et al. Proanthocyanidin subunit composition determined by functionally diverged dioxygenases. Nature Plants 4, 1034–1043 (2018). https://doi.org/10.1038/s41477-018-0292-9
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