The Mysteries of Proanthocyanidin Transport and Polymerization

Flavonoids, including proanthocyanidins (PAs; also called condensed tannins), play a multitude of roles in plants (Winkel-Shirley, 2001). The presence of certain types of flavonoids in crops is associated with desirable and important agronomic traits; therefore, metabolic engineering of flavonoid biosynthesis has attracted considerable interest (Dixon, 2005). PAs are oligomers or polymers of flavan-3-ol units and are prominent flavonoid compounds in seed coats (where they become oxidized and confer a brownish color to the testa), leaves, fruits, flowers, and bark (Dixon et al., 2005). Characterization of a series of transparent testa (tt) and tannin-deficient seed (tds) mutants from Arabidopsis (Arabidopsis thaliana) has led to an in-depth understanding of flavonoid biosynthesis at the molecular level (Shirley et al., 1995; Winkel-Shirley, 2001; Abrahams et al., 2002; Lepiniec et al., 2006; Fig. 1). These studies clearly suggest that, in addition to structural enzymes and regulatory factors, transport proteins are also essential for flavonoid biosynthesis. Ablation of single transporter genes can result in defects in flavonoid and PA production associated with dysfunction of the central vacuole (Baxter et al., 2005; Marinova et al., 2007a, 2007b). With increasing evidence of such links between biochemistry and cell biology, transport and trafficking of plant secondary metabolites is emerging as an important, but technically challenging, research frontier (Grotewold, 2004). In this Update, we briefly review the status of our knowledge concerning PA biosynthesis and assembly, with particular emphasis on the still controversial mechanisms for transport and polymerization of the PA monomers. Genetically mapped TT genes involved in PA biosynthesis in Arabidopsis. tt mutants and their encoding enzymes are as follows: tt4, chalcone synthase; tt5, chalcone isomerase; tt6, flavanone 3-hydroxylase; tt7, flavonoid 3′ hydroxylase; tt3, dihydroflavonol 4-reductase; ban, ANR; tt18 (at the same locus as tt11), leucocyanidin dioxygenase; tt10, laccase-like polyphenol oxidase; tt12, MATE antiporter; tt15, UDP-Glc:sterol glycosyltransferase; tt19 (at the same locus as tt14), GST; aha10, P-type H+-ATPase. tt mutants encoding regulatory proteins are as follows: tt1, WIK-type zinc finger transcription factor; ttg1 (for transparent testa glabra1), WD40 repeat transcription factor; tt2, R2R3 Myb transcription factor; tt8, basic helix-loop-helix transcription factor; tt16, MADS domain transcription factor. Arabidopsis has, until recently, been the major model system for molecular studies of PA biosynthesis (Lepiniec et al., 2006). The model legume Medicago truncatula has recently emerged as another useful system, with the ultimate goal of introducing PAs into the foliar tissues of its close relative, the major forage crop alfalfa (Medicago sativa), to protect ruminant animals from pasture bloat and enhance ruminant nutrition (Xie et al., 2003; Dixon et al., 2005; Pang et al., 2007, 2008; Peel et al., 2009; Zhao and Dixon, 2009). PAs in Arabidopsis and M. truncatula consist of the same monomeric building unit, epicatechin, and have similar biological features (Abrahams et al., 2002; Lepiniec et al., 2006; Pang et al., 2007). Epicatechin shares a common biosynthetic pathway with anthocyanins from Phe to anthocyanidin (Dixon et al., 2005), from which epicatechin is formed by the action of anthocyanidin reductase (ANR; Xie et al., 2003; Fig. 1). The Medicago glycosyltransferase UGT72L1 catalyzes UDP-Glc-dependent glycosylation of epicatechin to form epicatechin 3′-O-glucoside (E3′G; Pang et al., 2008), which is the substrate for a vacuolar multidrug and toxic compound extrusion (MATE) transporter (Zhao and Dixon, 2009; Fig. 1). M. truncatula MATE1 is an ortholog of Arabidopsis TT12, and both proteins transport cyanidin 3-O-glucoside (Cy3G), although E3′G is transported with a higher affinity and velocity (Marinova et al., 2007b; Zhao and Dixon, 2009). Grapevine (Vitis vinifera), with a recently sequenced genome, has become another model plant for studying PA biosynthesis. Grapevine PAs consist of two major flavan 3-ol monomers, catechin and epicatechin. Their seed and skin PAs have average degrees of polymerization of around 10 and 30, respectively. Epicatechin, and to a lesser extent epigallocatechin, is the extension unit and catechin the terminal unit in grapevine PAs (Terrier et al., 2009). Characterization of several transcription factors that regulate grapevine PA biosynthesis has revealed both similar and distinct regulatory mechanisms from those in Arabidopsis and M. truncatula (Bogs et al., 2007; Deluc et al., 2008; Terrier et al., 2009). Two TT2-like Myb transcription factors, VvMYBPA1 and VvMYBPA2, are essential for the expression of PA biosynthetic genes and PA accumulation in grapevine hairy roots (Bogs et al., 2007; Terrier et al., 2009). VvMYBPA1, which is mainly expressed during seed development, specifically regulates PA biosynthetic genes such as ANR and LEUCOANTHOCYANIDIN REDUCTASE. It can complement the seed PA-deficient phenotype of the Arabidopsis tt2 mutant, suggesting that VvMYBPA1 is an ortholog of TT2, although it is not clear whether grapevine also possesses a TT2-TT8-TTG1-like ternary transcription complex such as controls PA biosynthesis in Arabidopsis (Lepiniec et al., 2006; Bogs et al., 2007). VvMYBPA2 is mainly expressed in berries and leaves, but constitutive expression of either VvMYBPA1 or VvMYBPA2 in hairy roots induces similar sets of genes (Terrier et al., 2009). A new player in PA biosynthesis, VvMYB5 (Deluc et al., 2008), was also found to regulate the biosynthesis of PAs and other flavonoids in grapevine, and other MYB transcription factors, DkMYB4 and PtMYB134, regulate PA biosynthesis in persimmon (Diospyros kaki) fruit and poplar (Populus spp.) leaves, respectively (Akagi et al., 2009; Mellway et al., 2009). These data suggest that MYB family transcription factors are common regulators of PA biosynthesis in different tissues. Several studies show that PAs are stored in the vacuoles of Arabidopsis seed coat endothelial cells during the early stages of seed development (Abrahams et al., 2003; Kitamura et al., 2004), and it is likely, but not yet definitively proven, that PA oligomerization/polymerization occurs in the vacuole. As biosynthesis of PA precursors is believed to occur on the cytosolic face of the endoplasmic reticulum (ER) surface, the PA starter and extension units must first be transported into the vacuole. The tt12 mutation was mapped to a MATE family transporter (Debeaujon et al., 2001), and TT12 protein was shown to localize to the vacuolar membrane. Yeast-expressed TT12 could transport Cy3G but not catechin 3-O-glucoside, which could inhibit Cy3G uptake (Marinova et al., 2007b). This was perhaps the first comprehensive characterization of a plant secondary metabolite transporter, incorporating tissue-specific expression, subcellular localization, transport activity, and metabolite profiling of loss-of-function lines. However, Cy3G is not considered to be a PA precursor. Comparative studies of MATE1 and TT12 revealed that the kinetically preferred substrate for both transporters is E3′G rather than Cy3G, which explains the seed phenotypes of the tt12 and mate1 mutants (Marinova et al., 2007b; Zhao and Dixon, 2009). The MATE transporter (CAO69962) from grapevine may carry out similar functions that remain to be determined experimentally (Terrier et al., 2009). If E3′G is universally the vacuolar transport form of epicatechin, its subsequent fate inside the vacuole is still not clear. E3′G can be detected during the early stages of seed development in M. truncatula (Pang et al., 2008), but its levels decrease to zero as PA levels increase, suggesting that it undergoes hydrolysis and subsequent incorporation into the PA polymer. However, other interpretations of the data are possible. For example, glucosylation by UGT72L1 could function in part as a detoxification mechanism to “mop up” excess epicatechin, consistent with the fact that this enzyme shows considerably higher sequence similarity to multifunctional UGTs, ascribed roles in xenobiotic detoxification, than to other flavonoid-specific UGTs (Pang et al., 2008). Alternatively, glucosylation of epicatechin could be an integral part of the PA polymerization mechanism as well as essential for monomer transport. In this respect, it is important to note that the exact nature of the starter and extension units of PAs remains to be determined. Gain- and loss-of-function experiments with UGT72L1 could give valuable information about its in vivo function. TT19, a glutathione S-transferase (GST), is an essential protein for anthocyanin and PA accumulation in Arabidopsis and has orthologs in other plants such as maize (Zea mays), petunia (Petunia hybrida), and grapevine (Marrs et al., 1995; Alfenito et al., 1998; Kitamura et al., 2004). GST is believed to function in flavonoid production through its binding to flavonoids rather than by catalyzing the conjugation of glutathione to anthocyanins (Mueller et al., 2000). GST may act as an anthocyanin-binding matrix to protect anthocyanins from oxidation, or the GST-flavonoid complex may facilitate flavonoid transport mediated either by membrane transporters (Smith et al., 2003) or via vesicle trafficking. However, the exact mechanisms underlying such processes are not understood. The tissue expression patterns of GSTs may provide additional clues to their functions in flavonoid transport. TT19 is expressed in leaves (particularly during senescence), stems, and young siliques (Kitamura et al., 2004). A TT19 promoter::GUS transgene directed high GUS activity to vascular tissues in the mid vein of Arabidopsis leaves, and a flavonoid glycosyltransferase gene exhibits a similar expression pattern (Wenzel et al., 2008). These data suggest that TT19 may be involved in long-distance transport of flavonoid-GST complexes (Fig. 2) in addition to its proposed role in anthocyanin and PA production in the seed coat, consistent with the lack of pigmentation in leaves and stems of tt19 plants (Kitamura et al., 2004). Studies on flavonoid-auxin transport interactions in vegetative axillary bud branching also support this hypothesis (Lazar and Goodman, 2006), and long-distance transport of flavonoids between cells or organs (e.g. roots and shoots) has recently been demonstrated (Buer et al., 2007). Model for PA transport and polymerization. PA regulatory transcription factors (TT factors) activate PA biosynthesis structural genes (TTs) in the nuclei of seed coat endothelial cells under appropriate conditions. PA pathway proteins are translocated to the cytosolic side of the ER for synthesis of epicatechin (white circles) and anthocyanins (red circles). These flavonoids can be compartmentalized in different ways. Epicatechin and anthocyanins are readily glycosylated, and the conjugates are transported into the vacuole by MATE (TT12) transporters. They could also be loaded into the ER membrane system or derived membrane vesicles, which are transported to the central vacuole through prevacuole compartment (PVC)-dependent vesicle trafficking, or else they could be bound to the TT19 GST, which facilitates their transport into the ER, vacuole, or other compartments. The acidic vacuolar conditions may facilitate nonenzymatic condensation of PA units, or the units may undergo enzymatic condensation catalyzed by TT10, with E3′G as a possible extension unit, or by yet unidentified proteins (red cylinders). After synthesis on the ER and modification by glycosylation in the Golgi, TT10 could be sorted and targeted within membrane vesicles that also contain epicatechin and other PA biosynthetic units. TT10 may also catalyze the condensation of PA units into oligomers (such as procyanidin B1 and B2) in the vesicles, which are transported to the vacuole, where PA chain elongation could be further catalyzed by TT10 using epicatechin glucoside and PA oligomers as substrates. PAs can also be transported through membrane vesicles or other mechanisms to the apoplastic space, where they are subjected to oxidative polymerization and further cross-linked with other cell wall components, catalyzed by apoplastic TT10-like polyphenol oxidases. Blue hexagons represent glycosylated moieties (epicatechin, membrane sterols, and TT10 protein). A Medicago GST gene is up-regulated by ectopic expression of either the Medicago Legume Anthocyanin Production1 MYB transcription factor (which controls anthocyanin biosynthesis) or the Arabidopsis TT2 MYB transcription factor (which controls PA biosynthesis; Pang et al., 2008; Peel et al., 2009). The Medicago GST gene is expressed in vegetative buds, petioles, stems, leaves, and flower tissues. Interestingly, a Medicago anthocyanin transporter, MtMATE2, shows a similar tissue-specific expression pattern to that of the GST, particularly around vascular bundles in petals (J. Zhao and R.A. Dixon, unpublished data). Therefore, both a GST and MtMATE2 may be involved in long-distance transport of anthocyanins. It is possible that transported anthocyanins could ultimately serve as precursors of PAs through deglycosylation and subsequent conversion to epicatechin, thus providing an alternative explanation for the reduced PA phenotype of the tt19 mutation. AHA10 is a putative P-type H+-ATPase, and Arabidopsis aha10 seeds show a typical tt phenotype with drastic reduction of PA levels (Baxter et al., 2005). It is not surprising that an H+-ATPase is involved in seed coat PA production, since one would be predicted to be necessary to support the activities of the TT12 and MATE1 H+ antiporters in the Arabidopsis and Medicago seed coats, respectively. Both tt12 and aha10 mutants show vacuolar morphological defects, consistent with the hypothesis that AHA10 may be tonoplast localized and provide the H+ gradient across the vacuolar membrane to support the MATE transporter's H+/flavonoid antiport function (Baxter et al., 2005). However, it is surprising that tt12 seeds do not accumulate epicatechin, whereas aha10 seeds contain higher levels of epicatechin than wild-type seeds. One possible explanation for the above paradox is that transporters in addition to TT12 may exist for PA transport. If AHA10 is located on the tonoplast like its ortholog PH5 from petunia (Verweij et al., 2008) and provides an H+ gradient for antiporters like TT12, knockout of AHA10 may not completely eliminate TT12 function because other vacuolar ATPases or vacuolar pyrophosphatases could partially support the E3′G transport function of TT12. Alternatively, AHA10 may be localized on the ER or on vesicles involved in PA transport from the cytosol to the vacuole (Fig. 2). Vesicle trafficking-mediated PA transport may require AHA10 to supply energy. Therefore, definitively determining the subcellular localization of AHA10 is critical for explaining its biological functions in PA transport. Early studies suggested that PAs are contained in cytoplasmic vesicle-like structures and that these prevacuole-like vesicles can with the central vacuole (Abrahams et al., Vesicle trafficking has also been proposed to be involved in the transport of anthocyanins from the cytosol into the central vacuole et al., 2007; et al., studies show that Arabidopsis mutants with reduced PA production morphological defects in the central vacuole of the seed coat endothelial cells (Abrahams et al., 2003; Kitamura et al., et al., for example, PAs with a pattern to prevacuole-like vesicles are found in the mutants (Abrahams et al., 2003; Kitamura et al., et al., 2005). a of vesicle from cells of Arabidopsis contain from and and flavonoids that are to the cell and 2007). In wild-type flavonoids are localized in the ER and but are in the cytosol and not associated with the in the tt12 and tt19 mutants and 2007). This the that TT12 and TT19 may be involved in the transport of flavonoids into the ER If it is not clear this is for PA transport. PAs be through ER transport vesicles to the vacuole (Fig. and TT12 be on of the involved in such trafficking, either in a or as a for the membrane protein or It is believed that PAs localize to cell in the coats of seeds. epicatechin or PA oligomers are transported out of endothelial cells across the vacuolar and remains It has been proposed that they are the vacuole as a result of cell during the seed et al., 2005). Alternatively, both vesicle trafficking and membrane transporters could be involved in the of PAs into the to a on the of and compounds in both membrane with and and membrane with are involved et al., 2009). PA oligomers could be transported to the by a similar The recently tt tt15, the UDP-Glc:sterol glycosyltransferase et al., 2005; et al., 2009). In addition to reduced PA levels in has reduced cyanidin and levels et al., reduced and levels in and and and levels in the seed coat et al., 2009). It is not clear flavonoid and PA accumulation in seeds. However, it that the mechanism membrane since and are important membrane et al., 2009). studies that is localized to the vacuolar membrane et al., et al., 2007). Therefore, of function of may vacuolar transporter activity or vesicle trafficking associated with flavonoid It be of interest to further these tt is the and TT10 a putative laccase-like polyphenol that was proposed to in the polymerization of TT10 has a predicted for into the apoplastic space, where it has been proposed to PA oligomers into the PAs of the seed coat et al., 2005). However, several remain about the seeds show a clear tt which is associated with a reduction in the levels of oxidized PAs et al., the do with metabolite profiling shows that seeds have levels of PAs and flavonoids but do not have levels of PA than wild-type seeds et al., 2005). do seeds show a tt phenotype the reduction in PA levels found in other tt additional oxidized PA that for the but that be or detected using TT10 has been proposed to catalyze the of oxidized PAs with cell wall to form an complex in the apoplastic space, studies in which epicatechin was to seeds of and wild-type Arabidopsis that the of of epicatechin by TT10 are PA oligomers such as A et al., 2005). TT10 is the either epicatechin is not the substrate or additional proteins similar to the proteins involved in et al., are necessary and do not function well in the seed the glycosylation pattern of E3′G is of flavonoids with a on the central found in are glycosylated at this at the 3′ would the of a during oxidation, and this would inhibit the of such as It remains to be determined whether E3′G can serve as a substrate for TT10 to a or higher with the typical to found in on the of seed color and PA accumulation in the vacuole show that epicatechin and PAs first accumulate in the central vacuole and during the seed (Kitamura et al., 2004). However, levels of TT10 those of the PA biosynthetic genes et al., 2005). This to be with the proposed function of TT10 in formed PAs during the stages of seed et al., 2005), the TT10 protein has a If TT10 as an catalyzing oxidative polymerization of epicatechin and PA oligomers in the apoplastic space, the mutation not epicatechin and procyanidin oligomers stored in the Kitamura et al., 2004). epicatechin or PA oligomers accumulate in the from the of seed development, the proposed functions of TT10 as proposed are to understanding of the and expression and localization of TT10 is clearly TT10 also function as a PA to catalyze PA chain elongation into higher PA TT10 was as a PA enzyme mainly on the of experiments in which seeds with epicatechin to accumulate PAs et al., 2005). However, epicatechin not the extension unit, or TT10 catalyzed PA biosynthesis under conditions found within the vacuole, the above would not be If TT10 act as the of the controversial phenotypes of the could be However, other possible polymerization such as nonenzymatic yet be out (Dixon et al., 2005). These proposed mechanisms are on an early by that of a procyanidin in the of an that could with epicatechin to the of higher Several have been proposed such an could be under conditions (Dixon et al., 2005), but these remain The in levels of epicatechin and PAs in tt mutants in expression of the and genes are and different may for of the et al., and biological factors of such between tt mutants and is of particular since it may provide clues for understanding the complex PA transport and polymerization One important the of epicatechin or PAs in seed As in these cells accumulate epicatechin and PAs in the central vacuole, and these do not until the of seed development (Kitamura et al., epicatechin or PA oligomers be inside the vacuole, and vacuolar PA oligomers can be detected in wild-type and seeds. However, the PA mutants tt12, and aha10 have an biosynthetic pathway but their seeds have levels of which could perhaps be transported to the vacuole through other or be in the Epicatechin is in Arabidopsis siliques but is in seeds (Abrahams et al., However, the tt12, tt10, and aha10 has epicatechin levels seed development et al., 2005), suggesting that TT10 is to the metabolic fate of epicatechin via PA or subsequent whether is essential for epicatechin, providing a substrate for TT12, or E3′G as an extension unit for PA polymerization is critical for our understanding of PA biosynthesis. Arabidopsis seeds do not accumulate levels of epicatechin and young M. truncatula seeds contain high levels of both (Marinova et al., 2007b; Pang et al., 2008). M. truncatula UGT72L1 is expressed in the seed coat and shows high for E3′G biosynthesis (Pang et al., 2008), but the Arabidopsis epicatechin glycosyltransferase has yet to be and of E3′G into the central vacuole may provide the building for PA additional mechanisms may exist for the transport of for PA biosynthesis, since tt12, aha10, and tt19 levels of the PAs of grapevine, and other consist of both epicatechin, as extension units, and as the starter unit, suggesting the possible for additional transporters for catechin or tt mutants from and remain to be These may represent in genes encoding proteins similar to or not involved in PA biosynthesis but PA transport or polymerization Characterization of these with of polymerization mechanisms by the nature of the that are transported to the vacuole, may the of PA transport and polymerization. understanding of these processes ultimately to PA production in crop In in of the that has been in understanding PA biosynthesis and its in the still remain which are the TT12 localized to the and do PA have to this membrane from the If PAs are in the vacuole, are they transported to the is TT19 involved in flavonoid it also involved in long-distance transport of this PA AHA10 function in PA biosynthesis, and is its subcellular glycosylation of epicatechin facilitate vacuolar or is it integral for PA TT10 involved in the polymerization of PAs in addition to of PA are the structures of PA polymers and PA can we and do our give a of their