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Plant Cell. Aug 2009; 21(8): 2323–2340.
PMCID: PMC2751950

MATE Transporters Facilitate Vacuolar Uptake of Epicatechin 3′-O-Glucoside for Proanthocyanidin Biosynthesis in Medicago truncatula and Arabidopsis[C][W]

Abstract

Expression of the Arabidopsis thaliana MYB transcription factor TRANSPARENT TESTA 2 (TT2) in Medicago trunculata hairy roots induces both proanthocyanidin accumulation and the ATP-dependent vacuolar/vesicular uptake of epicatechin 3′-O-glucoside; neither process is active in control roots that do, however, possess anthocyanidin 3-O-glucoside vacuolar uptake activity. A vacuolar membrane-localized multidrug and toxic compound extrusion (MATE) transporter, Medicago MATE1, was identified at the molecular level and shown to preferentially transport epicatechin 3′-O-glucoside. Genetic evidence has implicated TT12, a tonoplastic MATE transporter from Arabidopsis, in the transport of precursors for proanthocyanidin biosynthesis in the seed coat. However, although Arabidopsis TT12 facilitates the transport of cyanidin 3-O-glucoside into membrane vesicles when expressed in yeast, there is no evidence that cyanidin 3-O-glucoside is converted to proanthocyanidins after transport into the vacuole. Here, we show that Arabidopsis TT12, like Medicago MATE1, functions to transport epicatechin 3′-O-glucoside as a precursor for proanthocyanidin biosynthesis, and Medicago MATE1 complements the seed proanthocyanidin phenotype of the Arabidopsis tt12 mutant both quantitatively and qualitatively. On the basis of biochemical properties, tissue-specific expression pattern, and genetic loss-of-function analysis, we conclude that MATE1 is an essential membrane transporter for proanthocyanidin biosynthesis in the Medicago seed coat. Implications of these findings for the assembly of oligomeric proanthocyanidins are discussed.

INTRODUCTION

Proanthocyanidins (PAs; also called condensed tannins) are oligomers of flavan-3-ol units and are found as prominent compounds in seed coats, leaves, fruits, flowers, and bark of many plant species (Ariga et al., 1981; Gabetta et al., 2000; Gu et al., 2004; Dixon et al., 2005). They are derived from the flavonoid/anthocyanin branch of the phenylpropanoid pathway. PAs and their presumed monomeric building blocks, such as catechin and epicatechin, are potent antioxidants with beneficial effects on cardiac health, immunity, and longevity (Santos-Buelga and Scalbert, 2000; Serafini et al., 2003). The presence of PAs in forage plants is regarded as a positive trait that protects ruminant animals from pasture bloat and enhances ruminant nutrition (Lees, 1992). It is therefore important to understand PA biosynthesis as a basis for metabolic engineering of PA production in plants.

In Arabidopsis thaliana and the model legume barrel medic (Medicago truncatula), the PAs found in the seed coat consist essentially of epicatechin units (Abrahams et al., 2002; Lepiniec et al., 2006; Pang et al., 2007) (Figure 1). Epicatechin and anthocyanins share a common biosynthetic pathway from Phe to anthocyanidin (Dixon et al., 2005), which is then converted to epicatechin by anthocyanidin reductase (ANR) (Xie et al., 2003). Characterization of a series of Arabidopsis mutants with pale-colored seeds (termed transparent testa [tt] or tannin-deficient seeds [tds]) has revealed many genes that are essential for PA and anthocyanin accumulation in seeds (Shirley et al., 1995; Abrahams et al., 2002; Lepiniec et al., 2006). The genes defined by the tt and tds mutations encode biosynthetic enzymes, such as ANR, regulatory transcription factors, and proteins involved in transport of intermediates in the PA and anthocyanin biosynthesis pathways (Lepiniec et al., 2006).

Figure 1.
Diagrammatic Representation of Key Reactions for PA Precursor Synthesis and Transport in Arabidopsis.

Anthocyanins and PAs accumulate in the vacuole, where polymerization of PA precursors is believed to occur followed by conversion to brown oxidation products (Lepiniec et al., 2006). However, the nature of the intermediates that are transported to the vacuole, and then polymerize to give PA oligomers, is not fully understood. ANR is a soluble cytoplasmic enzyme (Pang et al., 2007), and epicatechin or its derivatives destined for PA biosynthesis therefore likely have an extravacuolar origin. Blocking flavonoid transport from the cytosol into the central vacuole reduces anthocyanin and PA production, and ATP binding cassette (ABC) and multidrug and toxin extrusion (MATE) transporter proteins have been shown genetically to be involved in both anthocyanin and PA precursor transport (Debeaujon et al., 2001; Abrahams et al., 2003; Goodman et al., 2004). The Arabidopsis TT12 gene was identified as encoding a MATE transporter that could transport potential PA precursor(s) into the vacuole (Debeaujon et al., 2001). Expression of TT12 is regulated by the transcription factors TT2, TTG1, and TT8 in the Arabidopsis seed coat (Lepiniec et al., 2006). Membrane vesicles from yeast expressing Arabidopsis TT12 could transport cyanidin 3-O glucoside (Cy3G) (Figure 1) but not catechin 3-O-glucoside (C3G), and C3G strongly inhibited TT12-mediated Cy3G uptake (Marinova et al., 2007). This result, however, does not confirm the nature of the transported species or explain why the tt12 mutant has reduced PA accumulation, since catechin is not a component of Arabidopsis seed coat PAs. In this regard, Marinova et al. (2007) suggested that TT12 might transport a glucoside of epicatechin, but the exact nature of this compound was not described.

We have recently identified a Medicago uridine diphosphate glucosyltransferase, UGT72L1, with high specificity for production of epicatechin 3′-O-glucoside (E3′G) (Pang et al., 2008), in which the sugar unit is attached to the 3′ hydroxyl group of the B-ring of the flavonoid instead of at the 3-O-position of the central heterocyclic C ring (Figure 1). UGT72L1 is preferentially expressed in the seed coat and is transcriptionally activated by the PA regulatory transcription factor Arabidopsis TT2 when this gene is expressed in Medicago hairy roots. The expression pattern of UGT72L1 in the seed coat correlates with the transient appearance of an epicatechin glucoside, levels of which decrease as oligomeric PA levels increase (Pang et al., 2008). These results are consistent with E3′G being a precursor of PAs in Medicago and, therefore, a likely substrate for transport to the vacuole. This series of experiments was therefore designed to identify the putative E3′G transporter in Medicago. Vacuole-enriched membrane vesicles from Arabidopsis TT2-expressing Medicago hairy roots take up E3′G in an ATP-dependent manner, and Medicago MATE1 was identified as a vacuolar E3′G transporter with a strong preference for E3′G over Cy3G. Arabidopsis TT12 was also shown to be an E3′G transporter. Medicago MATE1 complements the tt12 mutation in Arabidopsis, and genetic loss-of-function studies confirmed a role for MATE1 in PA biosynthesis in Medicago, further supporting the critical role of E3′G as a PA precursor.

RESULTS

Arabidopsis TT2 Induces Vacuolar Transport of E3′G in Medicago Hairy Roots

Ectopic expression of Arabidopsis TT2 strongly activates transcription of ANR (for production of epicatechin) and UGT72L1 in Medicago hairy roots, associated with transient production of E3′G and accumulation of PAs, whereas control Medicago hairy roots accumulate anthocyanin but not PAs (Pang et al., 2008). To determine whether TT2-mediated induction of PA biosynthesis is associated with enhanced transport of E3′G in the hairy roots, we first generated E3′G by incubation of (−)-epicatechin and uridine diphosphate glucose in the presence of recombinant UGT72L1 (Pang et al., 2008). The glucosylated product was isolated and purified, and its structure confirmed by nuclear magnetic resonance (NMR) analysis (Pang et al., 2008). We then isolated vacuole-enriched membrane vesicles from TT2-expressing and control hairy root tissues by differential centrifugation and sucrose gradient purification. Protein gel blot analysis of marker enzymes showed that the 20% sucrose and 20/30% sucrose interface fractions were enriched in vacuolar membranes (as seen by the higher level of the vacuolar H+-ATPase marker, Figure 2A). The membrane vesicles from control hairy roots took up both Cy3G and daidzin, the 7-O-glucoside of the isoflavone daidzein, a known vacuolar component in Medicago roots (Naoumkina et al., 2007), in an ATP-dependent manner (Figures 2B and 2D; see Supplemental Figure 1 online) but did not take up E3′G (Figure 2C). However, vesicles from TT2-expressing hairy roots did exhibit ATP-dependent E3′G uptake as well as increased uptake of both Cy3G and daidzin (Figures 2B to 2D; see Supplemental Figure 1 online).

Figure 2.
Uptake of Cy3G, E3′G, and Daidzin (D7) by Vacuole-Enriched Membrane Vesicles from M. truncatula Hairy Roots.

Concentration dependence studies of Cy3G and E3′G uptake into vacuolar membrane preparations from Arabidopsis TT2-expressing Medicago hairy roots indicated that E3′G and Cy3G uptake was saturable (Figures 2E and 2F). Double reciprocal plot analysis of initial rate data gave a Km of 16.10 μM and Vmax of 0.34 nmol/mg protein/min for E3′G uptake, with corresponding values of 49.10 μM and 4.23 nmol/mg protein/min for Cy3G. Uptake of Cy3G into vacuolar vesicles from vector control hairy roots was characterized by a Km of 34 μM and Vmax of 0.75 nmol/mg protein/min. Kinetic constants for daidzin uptake were similar (Km 27.10 mM and Vmax 0.83 nmol/min/mg protein). Overall, these results suggest that Arabidopsis TT2 expression upregulates a high-affinity E3′G transporter and a lower affinity but higher capacity Cy3G transporter. The data also suggest that the transporter responsible for E3′G uptake is a different protein from the constitutively expressed Cy3G transporter(s).

Various inhibitors were used to further characterize the properties of the transporters responsible for uptake of Cy3G, E3′G, and daidzin in the Medicago hairy root membranes. When applied at appropriate concentrations in the uptake reaction, bafilomycin A1, a vacuole-type ATPase inhibitor (Drose and Altendorf, 1997), NH4Cl, which dissipates the vacuolar membrane pH gradient (Rodrigues et al., 1999), and gramicidin D, a monovalent selective ionophore that dissipates the membrane potential and pH gradient (Luvisetto and Azzone, 1989), significantly inhibited Cy3G and E3′G uptake, whereas vanadate, a commonly used inhibitor of ABC transporters (Pezza et al., 2002), had almost no effect (Figure 2G). These data suggest that uptake of both Cy3G and E3′G into vacuoles is mediated by pH gradient-dependent H+/antiporters. The observation that uptake of daidzin, but not of Cy3G or E3′G, was partially inhibited by vanadate (Figure 2G), suggests that daidzin uptake occurs by a mechanism distinct from that of Cy3G and E3′G uptake and may possibly involve an ABC transporter in addition to a pH gradient-dependent H+/antiporter.

Competition assays showed that E3′G uptake into vacuolar membrane-enriched vesicles was slightly inhibited by Cy3G but not by various flavonoid aglycones, including free epicatechin and catechin (see Supplemental Figure 2 online). Likewise, Cy3G uptake was inhibited by E3′G but not by flavonoid aglycones or by isoflavone glucosides such as daidzin. Uptake of daidzin was, predictably, inhibited by genistin (genistein 7-O-glucoside), which differs from daidzin by the presence of a single hydroxyl group on the A-ring of the isoflavone moiety (see Supplemental Figure 2 online). Neither free epicatechin nor catechin was taken up by the membrane vesicles.

Identification of Medicago MATE1

The above-described uptake studies in Medicago root membrane vesicles predict the presence of a MATE transporter for vacuolar E3′G uptake in the model legume. The gene encoding this protein could be critical for engineering PA accumulation in forage legumes. No candidate gene was represented on the Medicago Affymetrix array previously used to study the Arabidopsis TT2-induced Medicago hairy root transcriptome (Pang et al., 2008). We therefore mined for Medicago MATE sequences using the Arabidopsis TT12 protein sequence to BLAST against the M. truncatula annotated genome protein database (http://biodecypher.noble.org/decypher/algo-tera-blast/tera-blastp_aa.shtml). Similar to Arabidopsis, which has 56 putative MATE transporters encoded in its genome, the M. truncatula genome sequence available to date contains >40 putative MATE-type transporters (see Supplemental Figure 3 and Supplemental Data Set 1 online). CT485797_21.5 (hereafter referred to as MATE1) is located on chromosome 5 and shares 70% identity and 80% similarity with Arabidopsis TT12 at the amino acid level (see Supplemental Figure 4A online). Phylogenetic analysis on available databases suggests that Medicago MATE1 is grouped into the same clade as Arabidopsis TT12 and TT12-like MATE transporters from several other species (Figure 3A). Most of these species, such as grapevine (Vitis vinifera) and poplar (Populus trichocarpa), produce high levels of PAs. Arabidopsis TT12 has been previously characterized as a Cy3G transporter but implicated in PA biosynthesis (Marinova et al., 2007), whereas CAO69962 from V. vinifera was suggested to be a putative PA precursor transporter based on microarray data from V. vinifera plants overexpressing the Arabidopsis TT2-like Myb transcription factors Vv MybPA1 and Vv MybPA2 (Terrier et al., 2009). CAO69962 transcripts (GSVIVP00018839001) were induced by these Myb transcription factors, coincidentally with other PA biosynthetic genes (Terrier et al., 2009). However, biochemical functions consistent with roles for Arabidopsis TT12 and the V. vinifera MATE transporter CAO69662 in PA biosynthesis have yet to be directly demonstrated. This group of MATE transporters related to PA accumulation is clearly distinct from the three known anthocyanin MATE transporters anthoMATE1 (AM1) and AM3 from V. vinifera and MTP77 from maize (Zea mays), as well as a nicotine MATE transporter from tobacco (Nicotiana tabacum; Figure 3A; see Supplemental Data Set 2 online) (Gomez et al., 2009; Morita et al., 2009; Shoji et al., 2009).

Figure 3.
Phylogeny and Expression Pattern of Medicago MATE1.

Protein structure prediction indicates that Medicago MATE1 and Arabidopsis TT12 have very similar secondary structures, each with 12 putative transmembrane domains (see Supplemental Figure 4B online).

Because Medicago MATE1 probe sets were not present on the Affymetrix chip used for the previous analyses of Arabidopsis TT2-induced transcripts in Medicago hairy roots (Pang et al., 2008), quantitative RT-PCR (qRT-PCR) was used to examine Medicago MATE1 expression. We used two transgenic hairy root lines that highly expressed Arabidopsis TT2 and accumulated PAs and the corresponding vector control lines (Pang et al., 2008). MATE1 expression was strongly induced in the two TT2-expressing lines but not in the vector control lines, in a similar manner to ANR expression (Figure 3B). However, another Medicago MATE transporter gene, AC121237_16.5, was not dramatically induced by AtTT2. qRT-PCR indicated that MATE1 is predominantly expressed in flowers, young pods, and seed coats, with very low expression level in leaves, roots, petioles, stems, and vegetative buds (Figure 3C). MATE1 expression increased in seedpods during the first 12 d after flowering and then decreased dramatically. Its transcript levels were low in mature seed coats. This expression pattern, which is similar to that of TT12 in Arabidopsis siliques, parallels the expression patterns of previously identified Medicago PA biosynthesis-related genes, such as ANR and UGT72L1 (Pang et al., 2008).

To examine the biochemical properties of MATE1, its open reading frame (ORF) was cloned from a cDNA library of young M. trucatula seedpods and expressed in yeast, both as the wild-type protein and as a fusion with the N terminus of the green fluorescent protein (GFP) ORF. Confocal microscopy indicated that the MATE1-GFP fusion protein localized to vesicle-like structures within the yeast cells (see Supplemental Figure 5 online). Uptake assays were then performed with microsomal fractions isolated from yeast cells expressing wild-type Medicago MATE1, MATE1-GFP fusion protein, or the empty vector (pYES) control. Time course and concentration dependence studies indicated that wild-type MATE1 transported E3′G at a higher velocity than Cy3′G (Figures 4A to 4D), and analysis of initial rate data at various substrate concentrations (Figures 4E and 4F) gave a Km of 36.60 μM and Vmax of 0.99 nmol/mg per min for transport of E3′G. Corresponding kinetic constants for Cy3G were 103.80 μM and 0.16 nmol/mg per min, respectively. The MATE1-GFP fusion protein was also functionally active (see Supplemental Figure 6A online).

Figure 4.
Uptake of Cy3G and E3′G by Yeast Microsomal Vesicles Expressing Medicago MATE1.

Arabidopsis TT12 does not transport flavonol glycosides, such as quercetin or kaempferol 3-O-glucosides (Marinova et al., 2007). The same was true for Medicago MATE 1 when expressed in yeast (see Supplemental Figure 5C online); the yeast vesicles also failed to transport daidzein 7-O-glucoside or free epicatechin, catechin, or cyanidin (see Supplemental Figure 5C online). The uptake of E3′G by these vesicles was dependent upon the H+ gradient, as determined by the inhibition of uptake by gramicidin D, NH4Cl, and bafilomycin A1 (see Supplemental Figure 5B online).

Medicago MATE1 Is Localized to the Tonoplast Membrane

To determine the subcellular localization of the MATE1 protein, the 35S promoter-driven functionally active MATE1-GFP fusion was bombarded into tobacco leaf epidermal cells. A construct for transient expression of free GFP was used as control. MATE1-GFP fluorescence was detected only around the periphery of tobacco epidermal cells (Figures 5A, 5B, and 5G), quite different from the localization of free GFP, which shows strong cytosolic and plasma membrane signal, in addition to labeling of the nucleus (Figure 5D). MATE1-GFP could be visualized in a distinct line around the nucleus and in many small, fine vesicle-like structures that could potentially be prevacuolar (Figures 5A and 5G; see Supplemental Figures 7A to 7E online). A comparison with the localization of free GFP shows the clear lack of nuclear labeling with MATE1-GFP and the presence of the tonoplast membrane internal to the location of chloroplasts (Figures 5G to 5I). These features distinguish MATE1-GFP localization from that of plasma membrane–localized proteins (Lefebvre et al., 2004; Sutter et al., 2006).

Figure 5.
Subcellular Localization of MATE1-GFP.

To further confirm this observation, we examined the localization of the plasma membrane in the same cell types, using fluorescent dyes that are endocytosed into the membrane (Emans et al., 2002). Plasma membrane labeled with the green fluorescent dye FM1-43 appeared as a highly uniform, sharp signal, with no evidence of labeling around the nucleus or on prevacuolar bodies (cf. Figures 5C and 5A). Furthermore, the membrane labeled with FM1-43 was outside the red fluorescent chloroplasts, as opposed to within them as observed for MATE1-GFP (cf. Figures 5G to 5I and 5J to 5L). Finally, we performed a comparative localization of MATE1-GFP fluorescence with the plasma membrane by labeling bombarded cells with the fluorescent marker FM4-64. A single cell expressing MATE1-GFP showed GFP signals in the tonoplast, around the nuclear membrane (long arrow), and in some of the prevacuole-like membrane vesicles (arrowheads) (Figure 5M); by contrast, FM4-64 label on the plasma membrane (red fluorescence in Figure 5N) only partially overlapped with that of MATE1-GFP on the inner side of the plasma membrane but not around the nucleus (long arrow) or on the prevacuole-like membrane vesicles (Figure 5O). These images suggest that MATE1-GFP is localized to the vacuolar membrane and are consistent with previous confocal images of tonoplast-targeted proteins, including TIPs and TT12-GFP (Hunter et al., 2007; Marinova et al., 2007).

Genetic Analysis of MATE1 Function

A Medicago line harboring a Tnt1 retrotransposon insertion in the MATE1 gene was isolated from the Noble Foundation's M. truncatula core Tnt1 mutant collection (Tadege et al., 2008). PCR with Tnt1- and MATE1-specific primers confirmed the insertion (in the 2nd exon of the gene) and the loss of MATE1 transcripts in the plant (Figure 6A; see Supplemental Figure 8 online). F2 generation seeds of a homozygous line showed a clear tt phenotype, as indicated by the pale-colored seeds (Figure 6B). Staining whole seeds with dimethylaminocinnamaldehyde (DMACA), a reagent specific for PAs and their flavan 3-ol precursors (Li et al., 1996), confirmed that mate1-1 seeds contain lower PA levels than wild-type seeds (Figure 6C). Analysis of cross sections through seeds indicated that DMACA-reactive material was virtually absent from the endothelial layer of seeds from the NF2629 line from the Noble Foundation's Medicago transposon insertion mutant collection (Figures 6D to 6H). Lack of PAs was confirmed by extraction and quantitative analysis of PAs from wild-type (R108) and mutant seeds (Figure 6I). Analysis of extracted soluble PAs by normal phase HPLC indicated that wild-type M. truncatula R108 contained a range of PA oligomers and polymers, whereas these were essentially lacking from the mate1 mutant (Figures 6K and 6L). Prevention of E3′G transport in this line did not result in accumulation of epicatechin or E3′G (Figure 6L). Tnt1 insertion lines lacking MATE1 expression showed normal expression of transcripts encoding the PA-specific gene ANR and the anthocyanin pathway genes dihydroflavonol reductase and anthocyanidin synthase (see Supplemental Figure 7 online), suggesting that the tt phenotype was due to a lack of precursor transport rather than to a lesion elsewhere in the pathway.

Figure 6.
Loss-of-Function Analysis of MATE1 in M. truncatula.

Arabidopsis TT12 Transports E3′G More Efficiently Than Cyanidin 3-O-Glucoside

Previous studies have shown that, when expressed in yeast, TT12 facilitates the vacuolar uptake of Cy3G and that this uptake is inhibited by C3G, which is not itself a substrate for transport (Marinova et al., 2007). Since Arabidopsis PAs are composed of epicatechin-type units like those of M. truncatula, and because we have recently demonstrated the formation of E3′G in M. truncatula (Pang et al., 2008) and shown that Medicago MATE1 is a transporter of E3′G, it was important to determine whether E3′G is also a preferred substrate for transport by Arabidopsis TT12. Using purified E3′G and commercial Cy3G as substrates, we conducted uptake assays with membrane vesicles isolated from yeast cells expressing either TT12 or empty vector control. Membrane vesicles from TT12-expressing yeast took up both E3′G and Cy3G when the compounds were present at 100 μM in the uptake mixture, whereas membrane vesicles from the vector control showed no uptake activity (Figures 7A and 7B). The initial rate of E3′G uptake was higher than that of Cy3G. Kinetic analyses revealed a Km for E3′G uptake of 50.2 μM and a Vmax of 0.73 nmol/mg protein/min; uptake of Cy3G by TT12 was less efficient, with a Km of 293.6 μM and a Vmax of 0.40 nmol/mg protein/min (calculated from Figures 7C and 7D). Cy3G inhibited E3′G uptake, but only at high concentrations relative to that of E3′G (Figure 7E), whereas E3′G inhibited Cy3G uptake at lower concentrations (see Supplemental Figure 9 online). In both cases, the competition was competitive.

Figure 7.
Uptake of Cy3G and E3′G by Yeast Microsomal Vesicles Expressing Arabidopsis TT12.

As previously shown for Cy3G uptake (Marinova et al., 2007), TT12-mediated uptake of E3′G was sensitive to the vacuolar H+ gradient dissipaters Bafilomycin A1 and NH4Cl but not to the ABC transporter inhibitor vanadate, suggesting that an H+/antiport mechanism is involved in the uptake (Figure 7F). Taken together, our results are consistent with E3′G being the preferred substrate for transport by TT12, a finding that explains the tt phenotype of the TT12 knockout mutation (Debeaujon et al., 2001).

Complementation of the tt12 Mutation by Medicago MATE1

To confirm that Medicago MATE1 is a true functional ortholog of Arabidopsis TT12, we complemented the Arabidopsis tt12 mutant by genetic transformation with a construct harboring the complete MATE1 ORF. PCR screening identified >20 transgenic tt12/MATE1 lines; three lines expressing MATE1 transcripts were analyzed (Figure 8A). Comparison of seed coat phenotypes indicated restoration of seed coat color and DMACA staining as a result of expression of MATE1 in the tt12 background (Figure 8B), and levels of soluble and insoluble PAs were restored to wild-type levels through complementation with MATE1 (Figure 8C). MATE1 complemented the loss of PA phenotype in the tt12 background (Figures 8C and 8E) quantitatively (Figure 8C), but there were slight differences in size distribution between the PAs in wild-type Wassilewskija (Ws) and tt12/MATE1 (Figures 8D and 8F) that were reproducible in the three independent lines. HPLC analysis of the PAs after acid-butanol hydrolysis confirmed that the extension units were derived wholly from (epi)catechin units in wild-type Ws and tt12/MATE1 lines (see Supplemental Figure 8 online).

Figure 8.
Complementation of the Arabidopsis tt12 Mutation by Medicago MATE1.

DISCUSSION

Medicago MATE1 and Arabidopsis TT12 Are E3′G Transporters

Previous studies have identified one Arabidopsis tt mutant, tt12, as defining a lesion in a MATE transporter involved in PA biosynthesis, and tt12 plants show a defect in vacuolar PA accumulation in immature seeds (Debeaujon et al., 2001; Marinova et al., 2007). It was further shown that TT12 is localized to the tonoplast and can function as a transporter of Cy3G (Marinova et al., 2007). While the color of the seeds in Arabidopsis is attributed to accumulation of both anthocyanins and PAs (Routaboul et al., 2006), mature tt12 seeds contain significantly lower levels of PAs and flavonols, such as quercetin-3-rhamnoside, but normal levels of anthocyanins (Marinova et al., 2007). Such phenotypes suggest that TT12 could function to specifically transport PA precursors into the vacuole (Marinova et al., 2007). However, biochemical studies in yeast showed that Arabidopsis TT12 can transport Cy3G, but not C3G or flavonol glucosides (Marinova et al., 2007), which does not explain why the tt12 mutant has reduced PA accumulation. It was suggested that a glucoside of epicatechin might be the substrate transported by TT12 in vivo (Marinova et al., 2007).

We recently identified a seed coat–expressed UDP-glucosyltransferase acting specifically on epicatechin to produce the corresponding 3′-O-glucoside (Pang et al., 2008). Using the recombinant enzyme to produce this compound as a substrate for in vitro uptake studies, we have now shown that E3′G is efficiently transported by TT12 (Figure 7). An M. truncatula transporter protein (MATE1) sharing high sequence identity with TT12 was identified and shown to also transport E3′G and Cy3G. Both TT12 and MATE1 transport E3′G with higher affinity and velocity than Cy3G. MATE1 has a higher affinity and uptake velocity for transport of E3′G than does TT12, and the greater preference for E3′G compared with Cy3G is more apparent for MATE1 than for TT12.

Generally, flavonoids are transported in the form of glycosides. In the case of anthocyanidins, the first position to be glycosylated biosynthetically is the 3-O-position of the heterocyclic C-ring, and this stabilizes the highly reactive anthocyanidin molecule. It is interesting that TT12 and MATE1 transport both Cy3G and E3′G, although the latter is the kinetically preferred substrate (Figures 4 and and7).7). Clearly, the position of glycosylation is not critical for transport. One reason that C3G cannot be transported by TT12 (Marinova et al., 2007) may be that the trans-stereochemistry of catechin gives it a very different shape from that of 2,3-cis-epicatechin or the planar cyanidin. It is also possible that transport of Cy3G by TT12 and MATE1 simply reflects a relaxed specificity for binding of structurally related flavonoids that accommodates the planar anthocyanin molecule and that other transport systems, such as TT19 (Kitamura et al., 2004), or the endoplasmic reticulum-to-vacuole protein sorting route in Arabidopsis (Poustka et al., 2007) may represent the true physiological transporters for movement of anthocyanins to the vacuole. On the other hand, it is still not clear whether C3G occurs in planta, since so far there has, to the best of our knowledge, been no report of its isolation from plants. A biotransformation study showed that tobacco cells can transform both epicatechin and catechin into their 3′-O-glucosides (~38 to 46%), 5-O-glucosides (~7 to 10%), and 7-O-glucosides (~15 to 17%) (Shimoda et al., 2007). The study by Shimoda et al. (2007), together with our previous report that UTG72L1 catalyzes 3′-O-glycosylation of epicatechin (Pang et al., 2008), suggests that plant enzyme systems may preferentially glycosylate flavan-3-ols at the 3′-O position rather than the 3-O position favored for anthocyanin conjugation.

Neither epicatechin nor E3′G appear to accumulate in seeds of the Arabidopsis tt12 mutant (Marinova et al., 2007) or in mate1 (Figure 6). The reason for this is not clear at present, but the observation suggests that either these compounds are metabolized to other (non-PA) products in these mutants or that there is some feedback loop between transport and synthesis that blocks their formation. It is interesting that epicatechin does accumulate in the aha10 mutant of Arabidopsis, which is also compromised in PA precursor transport (Baxter et al., 2005).

TT2 Induces Flavonoid Transporters in Medicago Hairy Roots

Hairy roots of M. truncatula contain significant levels of anthocyanins and isoflavone glycosides, but no PAs. However, massive accumulation of PAs is induced in these roots by expression of the Arabidopsis MYB transcription factor TT2 (Pang et al., 2008). Consistent with these findings, vacuole-enriched membrane vesicle preparations from control hairy roots exhibit ATP-dependent uptake of both Cy3G and daidzein 7-O-glucoside, but not of E3′G. Vacuole-enriched vesicles from TT2 expressing roots take up all three compounds, with increased uptake of both Cy3G and D7G. Inhibitor studies suggest that the mechanism of uptake of D7G is different from that of Cy3G and E3′G, and most likely requires an ABC-type transporter, whereas the latter compounds are taken up by MATE-type transporters. ABC transporters have been identified as an important class of transporters for plant secondary metabolites (Yazaki, 2005; Kitamura, 2006) and are involved, for example, in the secretion of the isoflavone genistein from soybean (Glycine max) roots (Sugiyama et al., 2007). Our results indicate that vacuolar E3′G uptake is dependent on expression of a PA regulatory transcription factor and that there is more than one transporter for Cy3G, as suggested above. One Cy3G transporter is constitutively expressed in the hairy roots, and another is induced by Arabidopsis TT2. The Cy3G transport induced by TT2 may result from the activity of MATE1, which nevertheless shows a kinetic preference for the uptake of E3′G.

Modification of flavonoids and other compounds by processes such as glycosylation and acylation is often a necessary prerequisite for their transport into the vacuole by MATE or ABC transporters (Klein et al., 1996; Bartholomew et al., 2002; Marinova et al., 2007; Gomez et al., 2009), and flavonoid aglycones are generally not suitable substrates for MATE transporters. Arabidopsis TT12 transports Cy3G but not C3G, flavonoid aglycones, or flavonol glucosides, such as quercetin-3-O-glucoside (Marinova et al., 2007). Grape anthoMATE1 transports acylated anthocyanins, but not nonacylated anthocyanins, into the vacuole (Gomez et al., 2009). However, a MATE transporter from tobacco can transport nonmodified nicotine directly into the vacuole (Morita et al., 2009; Shoji et al., 2009). Medicago MATE1 preferentially transports E3′G over Cy3G and does not transport daidzin (an isoflavone glucoside) or flavonoid aglycones (see Supplemental Figure 6 online). Overall, these studies indicate that some MATE transporters exhibit a degree of substrate specificity. Such specificity may have important physiological consequences during seed development and PA biosynthesis. A low level of epicatechin glucoside has been detected in Arabidopsis seeds (Marinova et al., 2007), and developing Medicago seeds accumulate E3′G up to 0.4 nmol per mg fresh weight of seed coat at 12 d after pollination (Pang et al., 2008). E3′G is therefore likely to be preferentially transported into the vacuole at the expense of Cy3G transport by TT12 or MATE-1 during PA accumulation.

MATE1 Is Required for PA Biosynthesis in Medicago

Reverse genetic screening led to the identification of one M. truncatula line with a Tnt-1 retrotransposon insertion in the MATE1 gene. Seeds of this line exhibited a tt phenotype similar to that of Arabidopsis tt12 (Debeaujon et al., 2001), and metabolite analysis confirmed that mate1 seeds have drastically reduced PA levels. Other genes of PA biosynthesis were not downregulated in mate1, suggesting that it is the insertion in the MATE1 gene that confers the loss of PA phenotype. The in vivo function of MATE1 was confirmed by complementation of the Arabidopsis tt12 mutant. Interestingly, PA production was restored quantitatively, but there were small differences in PA quality as regards polymer size distribution on comparing wild-type Arabidopsis Ws and tt12 expressing Medicago MATE1 (Figures 8D and 8F) The reason for this is not clear at present. Clearly though, MATE1 is a functional ortholog of TT12. Recently, several different MATE-type transporters have been reported to participate in vacuolar sequestration of various secondary metabolites (Yazaki, 2005; Marinova et al., 2007; Morita et al., 2009; Shoji et al., 2009), suggesting that this class of transporters may be generally involved in transport of plant secondary metabolites into the vacuole.

As a plant secondary transporter, Medicago MATE1 requires an electrochemical H+ gradient across the vacuolar membrane for transport of E3′G or C3G, with the downhill force of the H+ gradient between the vacuole and the cytosol. Although it is well known that V-ATPase and pyrophosphatase generally provide the H+ gradient across the vacuolar membrane (Gaxiola et al., 2002), there is no report so far that these proteins are involved in PA biosynthesis. By contrast, the Arabidopsis H+ ATPase 10 (AHA10), an annotated plasma membrane protein, has been shown to be necessary for PA production in Arabidopsis seeds (Baxter et al., 2005). It has been suggested that AHA10 may either be involved in vacuolar acidification to energize TT12 as a PA precursor transporter or in transport of PA precursors through vesicle trafficking, since the localization of AHA10 has not been resolved (Baxter et al., 2005; Marinova et al., 2007). Interestingly, a recent study shows that hybrid petunia (Petunia × hybrida) PH5, a putative ortholog of AHA10, is involved in flower color determination and PA production in seeds (Verweij et al., 2008); moreover, PH5 is localized to the tonoplast and believed to acidify the vacuole to enhance anthocyanin coloration and, probably, PA precursor loading into the vacuole (Verweij et al., 2008). Therefore, it is possible that AHA10 is also localized to the vacuolar membrane.

At least 40 other MATE transporters are present in the Medicago genome. Because the PAs of both Medicago and Arabidopsis are somewhat unusual in containing only epicatechin units (Lepiniec et al., 2006; Pang et al., 2007), Medicago MATE1 and Arabidopsis TT12 may be the only transporters necessary for PA biosynthesis in these species, unless the epicatechin-terminal and extension units in PAs have different biosynthetic origins (see below). Our studies with M. truncatula are ultimately targeted toward improvement of alfalfa (Medicago sativa), and this species has been reported to possess seed coat PAs with catechin-terminal units and epicatechin (major) and epigallocatechin (minor) extension units (Koupai-Abyazani et al., 1993). Studies to determine whether Medicago MATE1 or related transporters from alfalfa can facilitate uptake of catechin and epigallocatechin glucosides are in progress.

Unresolved Questions Concerning PA Monomer Transport

The results in this article define the biochemical functions of Arabidopsis TT12 and its putative ortholog from M. truncatula. E3′G, formed in Medicago by glycosylation of the product of the ANR reaction by UGT72L1, is transported to the vacuole for subsequent use in PA biosynthesis. However, what happens next is still far from clear because the nature of the condensing units in PA biosynthesis has yet to be determined. It is generally believed that the terminal catechin or epicatechin units of PAs originate from free catechin or epicatechin (Marles et al., 2003), and conversion of E3′G back to the flavanol aglycone could easily be accomplished by the activity of a vacuolar glucosidase. Indeed, a glucosidase is induced over threefold by TT2 in M. truncatula hairy roots (Pang et al., 2008), but its intracellular localization and biochemical activity remain to be determined. The nature of the condensing units is more problematical. The long-held model based on nonenzymatic chemical synthesis of PAs at low pH places leucoanthocyanidin as the condensing unit (Marles et al., 2003), but this seems unlikely since this compound has 2,3-trans stereochemistry and cannot therefore account for epicatechin-type extension units (Marles et al., 2003; Xie and Dixon, 2005; He et al., 2008). Alternative models have been proposed in which epicatechin, or some derivative of epicatechin, is oxidized to yield a corresponding quinone methiode or carbocation, which then attacks the 8-position of the A-ring of the (epi)catechin-terminal unit to extend the chain (Xie and Dixon, 2005; He et al., 2008). Such a model would only require transport of epicatechin units to the vacuole. However, similar reactive intermediates could be generated from achiral cyanidin derivaties (Marles et al., 2003; Xie and Dixon, 2005), consistent with a role for an additional anthocyanin transporter, such as TT19, which appears to function in both PA precursor and anthocyanin transport in Arabidopsis (Kitamura et al., 2004). It seems unlikely that TT12 or MATE1 would transport both Cy3G and E3′G into seed coat vacuoles in parallel because high concentrations of E3′G would inhibit transport of Cy3G (see Supplemental Figure 9 online), and an inverse relationship between expression of ANR and anthocyanidin 3-O-glucosyltransferase has been demonstrated and proposed to result in selective direction of cyanidin into either PA or anthocyanin biosynthesis (Lee et al., 2005).

Finally, our studies, although establishing the involvement of a MATE transporter in PA monomer transport, do not exclude the possibility that membrane vesicle-mediated transport of flavonoids (Grotewold, 2004; Poustka et al., 2007) may also be involved in PA biosynthesis and accumulation. Further experimentation will be necessary to confirm whether the vesicle-like structures that label with MATE1-GFP (see Supplemental Figure 7 online) are indeed destined for the vacuole.

METHODS

Plant Materials, Medicago truncatula Tnt1 Mutant Screening, and Growth of Hairy Roots

Seeds of Medicago truncatula line R108 were scarified with concentrated sulfuric acid, rinsed, sterilized with 2% sodium hypochlorite, and vernalized at 4°C for 3 d on moist, sterile filter paper. Germinated seedlings were transplanted to pots containing soil and placed in a greenhouse set to the following conditions: 16-h/8-h light/dark regime, 200 μE m−2 s−1 light irradiance, 24°C, and 40% relative humidity. Reverse genetic screening of M. truncatula Tnt1 mutants in the R108 background was performed as described previously (Peel et al., 2009). For identification of the Tnt1 insertion mutant for Medicago MATE1, PCR amplification was performed using a combination of MATE1-specific primers and Tnt1-specific primers (see Supplemental Table 1 online), and the PCR products were fully sequenced.

Growth of Arabidopsis thaliana TT2-expressing M. truncatula hairy roots was as described previously (Pang et al., 2008). Both TT2-expressing and empty vector control hairy root lines were used for isolation of microsomal fractions and preparation of vacuolar membrane-enriched vesicles.

Complementation of the Arabidopsis tt12 Mutant

The ORF of Medicago MATE1 in the binary vector pB2GW7 was driven by the 35S cauliflower mosaic virus promoter and terminated by the NOS terminator. Both pB2GW7-MtMATE1 and empty pB2GW7 vector (control) were transformed into Agrobacterium tumefaciens strain LBA4404 using the electroporation method. Transformed colonies were grown on YEP medium with selection at 28°C for transformation of Arabidopsis tt12 flowers using the floral dip infiltration method (Clough and Bent, 1998). Arabidopsis tt12 mutant in the Ws background (kindly provided by Isabelle Debeaujon, Jean-Pierre Bourgin Institute, Institut National de la Recherche Agronomique, France) and wild-type Ws plants were grown in a growth chamber at 20 to ~22°C, 100 μE m−2 s−1 light intensity provided by cold fluorescent tubes with a 18/6-h light/dark period. The F1 transgenic plants were selected with phosphinothricin (7.5 μg/mL) and kanamycin (20 μg/mL) on one-half Murashige and Skoog plates. F2 generation plants with confirmed MATE1 expression were used for analysis of seed phenotype and PA levels in seeds.

Gene Cloning and Vector Construction

Arabidopsis TT12 cDNA was cloned from a cDNA library constructed from young wild-type (Columbia-0) Arabidopsis siliques (4-week-old plants, 1 to 5 d after flowering) with a forward primer and a reverse primer that enable PCR fragments to be directionally cloned into pENTR vector (see Supplemental Table 1 online). MATE1 was cloned from cDNA constructed from young pods (9 to 12 d after flowering) of M. truncatula wild-type (R108) plants. Total RNA was extracted from Arabidopsis siliques or M. truncatula seedpods using the RNeasy plant mini kit procedure (Qiagen). First-strand cDNA was synthesized from total RNA with the SuperScript III first-strand synthesis system (Invitrogen) according to the manufacturer's protocol. The MATE1 ORF was amplified with MtMATE1-5′ forward primer and MtMATE1-3′ reverse primer (see Supplemental Table 1 online). The PCR products were cloned into the pENTR/D-TOPO vector (Invitrogen) for sequencing, and the confirmed clone was subcloned into Gateway destination vectors, the yeast expression vector pYES (uracil selection marker), or the plant binary vector pB2GW7 by recombination using Gateway LR Clonase (Invitrogen).

The MATE1-GFP fusion was constructed by cloning the MATE1 ORF in frame with the N terminus of GFP. The MATE1 ORF was amplified with a pair of primers, MtMATE1-GFP-5′ and MtMATE1-GFP-3′ (see Supplemental Table 1 online). PCR products were subcloned into a yeast GFP cassette shuttle vector fusion in frame to the N terminus of GFP. The construct pHGDP-MtMATE1-GFP was transformed into yeast cells for testing MATE1-GFP expression. A 2.1-kb MATE1-GFP coding sequence was then amplified with the primer pair MtMATE1 SFI-5′ and MtMATE1 SFI-3′ (see Supplemental Table 1 online) and subcloned into the pENTR/D-TOPO vector, followed by subcloning into the plant binary vector pB2GW7 by recombination with Gateway LR Clonase. The 35S:MATE1-GFP construct was used for localization studies. All clones were confirmed by sequencing.

Chemicals and Reagents

Flavonoid aglycones, cyanidin 3-O-glucoside, and daizein 7-O-glucoside (daidzin) were obtained from Indofine Chemical Company. E3′G was purified from incubation of epicatechin with recombinant UGT71G1 as described previously and its structure confirmed by NMR spectroscopy (Pang et al., 2008).

Yeast Culture, Transformation, and Microsomal Fraction Preparation

Saccharomyces cerevisiae strain W303A (Matα ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100) (kindly provided by Kendal Hirschi at Baylor College of Medicine) was used to express Arabidopsis TT12 and Medicago MATE1 transporter. The Gateway yeast expression vectors pYESDEST (Invitrogen) and pYES harboring TT12 or MATE1 were transformed into W303A by the polyethylene glycol/lithium acetate method. Transformants were grown in YNB (−uracil) + galactose medium overnight at 30°C and then transferred to 500 mL YPD medium at 30°C for overnight growth. Yeast cells were collected for microsomal extraction as described previously (Nakanishi et al., 2001). Briefly, cells were collected by centrifugation at 3000g and washed once with 0.1 M Tris-HCl, pH 9.4, 50 mM 2- mercaptoethanol, and 0.1 M glucose. Cells were then digested with zymolyase medium (50 mM Tris-MES, pH 7.6, 0.9 M sorbitol, 0.1 M glucose, 5 mM DTT, and Zymolyase [Seikagaku Corporation]) at 30°C for 2 h with gentle agitation. Spheroplasts were collected by centrifugation at 3000g for 10 min and washed with 1 M sorbitol. The spheroplasts were resuspended in 50 mM Tris-MES, pH 7.6, 1.1 M glycerol, 1.5% polyvinylpyrrolidone, 5 mM EGTA-Tris, 1 mM DTT, 0.2% BSA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mg/liter leupeptin and then homogenized with a motor-driven Teflon homogenizer. After centrifugation at 2000g for 10 min, the precipitate was washed with the same buffer and centrifuged again. All of the supernatant fractions were pooled and centrifuged at 120,000g for 30 min. The pellets were suspended in 5 mM Tris-MES, pH 7.6, 0.3 M sorbitol, 1 mM DTT, 0.1 M KCl, and protease inhibitor cocktail (10 μg/mL leupeptin, 2 μg/mL aprotinin, 2 μg/mL pepstatin, 1 mM PMSF, and 1 mM EDTA). The suspension was stored at −80°C until use.

Preparation of Membrane Vesicles from Hairy Roots

Vacuolar membrane vesicles were prepared from hairy roots of M. truncatula essentially according to the method described previously (Zhao et al., 2009). All procedures were performed on ice or at 4°C unless otherwise stated. Hairy roots were homogenized in ice-cold homogenization buffer containing 100 mM Tris-HCl, pH 7.6, 10% (v/v) glycerol, 0.5% (w/v) polyvinylpolypyrrolidone, 5 mM EDTA, 150 mM KCl, 5 mM DTT, and 2 mM PMSF. The homogenate was strained through Miracloth (Calbiochem) and centrifuged at 10,000g for 10 min. The resulting supernatant was centrifuged at 100,000g for 40 min. The pellet was resuspended in buffer containing 20 mM Tris-HCl, pH 7.6, 10% (v/v) glycerol, 1 mM DTT, and protease inhibitor cocktail (Sigma-Aldrich). The suspension was layered over a 20% to 30% to 40% to 50% (w/v, in 20 mM Tris-HCl buffer, pH 7.6, 1 mM DTT, and 1 mM EDTA) discontinuous sucrose gradient in a 13-mL tube. The tubes were subjected to centrifugation at 100,000g for 180 min. Fractions recovered from the interfaces were resuspended in ~35 mL of resuspension buffer and centrifuged at 100,000g for 40 min. Each pellet was resuspended in resuspension buffer supplemented with protease inhibitor cocktail and stored at −80°C until use. Vacuole-enriched membrane vesicles were present in the 20% and 20/30% interface fractions, as determined by protein gel blot analysis with antibodies against marker proteins.

Transport Activity Assays

The measurement of uptake by vacuolar membrane vesicles was performed at 25°C by a method modified from that of Bartholomew et al. (2002). The 600- to 1000-μL assay mixtures contained 25 mM Tris-MES, pH 8.0, 0.4 M sorbitol, 50 mM KCl, 5 mM Mg-ATP, 0.1% (w/v) BSA, and the indicated concentrations of transport substrate. ATP was omitted from the nonenergized controls. Assays were initiated by the addition of membrane vesicles (~50 μg of protein) and brief agitation. Batches of the reaction mixture (100 μL) were removed at various times, and their reactions terminated with 1.0 mL of ice-cold washing solution (25 mM Tris-MES, pH 8.0, containing 0.4 M sorbitol). The mixtures were then subjected to vacuum filtration through prewetted GV Durapore polyvinylidene difluoride membrane filters (0.2-μm pore diameter; Millipore). The dried filters were transferred to 20-mL glass vials containing 1.0 mL of 50% (v/v) methanol. The vials were capped and the filters were extracted for 1 h at room temperature in an orbital shaker. The eluate was analyzed by HPLC.

For inhibitor assays, transport inhibitors were incubated with the membrane vesicles at the following final concentrations: 1 mM vanadate, 5 mM gramicidin D, 0.1 mM bafilomycin A1, or 5 mM NH4Cl. The inhibitors were added to the reaction mixtures 2 min prior to initiation of uptake reactions by addition of substrate. Transport inhibitors were prepared in stock solutions of 1 M vanadate in water, 5 mM bafilomycin A1 in DMSO, 5 mM gramicidin D (in DMSO), and 1 M NH4Cl in water.

For competition assays with hairy root membrane vesicles, 10 mM stock solutions of epicatechin, catechin, genistein, daidzin, daidzein, E3′G, and Cy3G were prepared in 50% DMSO or methanol. Flavonoid aglycones or glucosides were added to the reaction mixture at a final concentration of 250 μM, while the uptake substrate concentration was set at 50 μM. For competition assays with yeast membrane vesicles expressing Arabidopsis TT12, various concentrations of Cy3G were used with E3′G as substrate at 100 μM. After incubation at 25°C for 10 min, transported E3′G was measured by HPLC. Reverse-phase HPLC analyses were performed on an Agilent HP1100 HPLC using a gradient mobile phase: solvent A (1% phosphoric acid) and B (acetonitrile) at 1 mL/min flow rate: 0 to 5 min, 5% B; 5 to 10 min, 5 to 10% B; 10 to 25 min, 10 to 17% B; 25 to 30 min, 17 to 23% B; 30 to 65 min, 23 to 50% B; 65–79 min, 50 to 100% B; 79 to 80 min, 100 to 5% B. Data were collected at 206 and 530 nm for epicatechin and cyanidin derivatives, respectively. Identifications were based on chromatographic behavior and UV spectra compared with those of authentic standards.

Determination of Transport Kinetics

Transport assays were performed with different concentrations of E3′G, Cy3G, and daidzin (from 25 to 200 μM) and 5 mM MgATP. After incubation at 25°C for 10 min, transported glycosides were measured by HPLC as described above. Lineweaver-Burk plots were used to calculate Km and Vmax values.

Protein Gel Blot Analysis

For immunoblotting, ~10 μg of vacuolar membrane vesicle proteins were denatured in protein loading buffer at 98°C for 5 min, subjected to SDS/7 to 12% PAGE (Bio-Rad), and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk, incubated with primary antibodies and secondary horseradish peroxidase–conjugated anti-rabbit IgG antibodies, and then subjected to immunodetection with a WesternBreeze Chemiluminescent Immunodetection Kit (Invitrogen) according to standard procedures. Antibodies used for immunodetection were against plasma membrane H+-ATPase and endoplasmic reticulum marker SEC12 (Sar1p exchange factor 12) (kindly provided by Rujin Chen, Noble Foundation) and vacuolar V-type H+-ATPase (subunit E) from Arabidopsis (Agrisera AB; used at 1:1000 dilution).

Light and Confocal Microscopy

Seeds of M. truncatula wild-type R108 and Tnt1 insertion mutant mate1-1, as well as Arabidopsis Ws, tt12, and tt12/MtMATE1, were imaged before and after DMACA staining. Seeds were stained with 2% (w/v) DMACA in 3 M HCl/50% methanol (v/v) for 4 d and washed three times with 70% ethanol for 2 d (Abrahams et al., 2002). An Olympus SZX12 fluorescence microscope with a Spot RT color camera run by Spot Basic and Spot Advanced software was used to visualize seeds. For visualizing DMACA-stained seed cross sections, seeds were quickly frozen in liquid nitrogen and processed with a Cryostat Leica CM 1850 Microtome into 20-μm sections for observation under a Nikon SMZ1500 fluorescence microscope installed with a Nikon DXM 1200 run by ACT-1 software.

Five micrograms of plasmid DNA containing 35S:MATE1-GFP or 35S:GFP was mixed with 20 μL of an aqueous suspension containing 1.0-μm gold particles. After washing, the gold was spread onto plastic carrier discs for biolistic bombardment of tobacco epidermal cells using a Bio-Rad 1000/HE particle delivery system. After 16 to 48 h, epidermal cells of tobacco leaves were viewed directly with a Leica TCS-SP2 AOBS confocal laser scanning microscope (Leica Microsystems) to examine the localization patterns of MATE1-GFP. GFP was excited using the 488-nm line of the argon laser and emission was detected at 520 nm.

To compare the difference between MATE1-GFP–labeled tonoplast and the plasma membrane, a styryl FM dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide) was used to label the plasma membrane (Emans et al., 2002). Tobacco leaf strips were incubated in a 10 μM aqueous solution of FM1-43 for 10 min and then observed under the confocal microscope. FM1-43 was excited at 488 nm and emission was detected at 560 nm. Chloroplasts were visualized at 560 to 610 nm under excitation at 543 nm.

To further distinguish MATE1-GFP signal from the plasma membrane signal, tobacco leaf epidermal cells expressing MATE1-GFP were stained with a 10 μM aqueous solution of the dye FM4-64 for 10 min before being observed under the confocal microscope. FM4-64–labeled plasma membrane was excited at 543 nm with the argon laser and emission was detected from 620 to 680 nm.

All images were acquired at a resolution of 512 × 512 pixels using a ×60/1.20 water-immersion objective and analyzed with Leica LAS AF software.

qRT-PCR

For testing expression of MATE1 and ANR in hairy roots, total RNA was isolated from M. truncatula hairy roots expressing Arabidopsis TT2 or β-glucuronidase (control) using the RNeasy plant mini kit (Qiagen) for cDNA synthesis. Equal amounts of total RNA were treated with DNaseI (Invitrogen) and were subsequently heat inactivated. cDNAs were synthesized with the SuperScript III first-strand synthesis system (Invitrogen). Diluted fractions were used for PCR (Promega; Mixgo polymerase). Medicago MATE1, ANR, and ACTIN were amplified in 25 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 30 min). Primers are shown in Supplemental Table 1 online.

For q-RT-PCR, cDNA from M. truncatula tissues was prepared as previously described (Benedito et al., 2008). Pods were collected at different times from 3 to 36 d after fertilization for cDNA synthesis. Seed coat RNA was isolated from seeds at 12 d after pollination (Pang et al., 2008). qPCR was performed with an ABI PRISM 7900 HT sequence detection system (Applied Biosystems). Reactions contained 2.5 μL of SYBR Green Master Mix reagent (Applied Biosystems), 0.5 μL of cDNA, and 200 nM of each gene-specific primer in a final volume of 5 μL and underwent 40 cycles of amplification. Data were analyzed using the SDS 2.2.1 software (Applied Biosystems). PCR efficiency (E) was estimated using the LinRegPCR software (Ramakers et al., 2003), and the transcript levels were determined by relative quantification (Pfaffl, 2001) using the Medicago ACTIN gene as a reference.

Quantitative Analysis of PAs

Extraction and subsequent analysis of total soluble and insoluble PAs were performed as previously described (Pang et al., 2008). Soluble PA levels were determined by microplate assay using DMACA reagent, and the components of the soluble PAs analyzed using normal phase HPLC with postcolumn DMACA derivatization (Peel and Dixon, 2007). Levels of insoluble PAs in dried residues were determined by reaction with acid-butanol, and the butanol extracts were then separated by HPLC to determine the nature of the resulting anthocyanin units (Pang et al., 2008).

Phylogenetic and Topological Analysis

Alignment of protein sequences was done with ClustalW (http://www.ebi.ac.uk/clustalw/) using the default parameters. Formatting of aligned sequences was done with box shade program (http://www.ch.embnet.org/software/BOX_form.html). Sequence alignments were analyzed with Mesquite software, and the nonrooted neighbor-joining tree was generated by PAUP 4.0 BETA programs. All nodes are supported by at least 1000 bootstrap replicates. Topological analysis for transmembrane domains was performed using the TMHMM2 program in SMART (http://smart.embl-heidelberg.de).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: M. truncatula MATE1, FJ858726; Arabidopsis TT12, NP_191462; Vitis vinifera AM1, FJ264202; V. vinifera AM3, FJ264203; Zea mays (maize) MTP77, AAQ55183; Nicotiana tabacum (tobacco) nicotine transporter, CAQ51477; V. vinifera predicted MATE transporters, XP_002282932 and CAO69962; Populus trichocarpa predicted MATE transporter, XP_002307572; Brassica rapa predicted MATE transporter, ACJ36213.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. ATP-Dependent Flavonoid Glucoside Uptake into Vacuole-Enriched Vesicles from M. truncatula Hairy Roots Expressing Arabidopsis TT2.
  • Supplemental Figure 2. Competition of Uptake of E3′G, Cy3G, and D7G into Vacuole-Enriched Vesicles by Various Flavonoid Glucosides and Aglycones.
  • Supplemental Figure 3. Phylogenetic Analysis of Plant MATE Transporters.
  • Supplemental Figure 4. Amino Acid Alignments of Arabidopsis TT12 and Medicago MATE1.
  • Supplememtal Figure 5. Confocal Microscopy Image of MATE1-GFP Expressed in Yeast.
  • Supplemental Figure 6. Transport Properties of Medicago MATE1 Expressed in Yeast Cells.
  • Supplemental Figure 7. Subcellular Localization of MATE1-GFP Transiently Expressed in Tobacco Leaf Epidermal Cells, Viewed by Confocal Microscopy.
  • Supplemental Figure 8. PCR Analysis of PA Pathway Gene Transcripts in Wild-Type R108 and Seven Homozygous Progeny of mate1.
  • Supplemental Figure 9. Competition of the Uptake of Cy3G by E3′G in Yeast Microsomes Expressing Arabidopsis TT12.
  • Supplemental Figure 10. HPLC Chromatograph of Anthocyanins Released by Hydrolysis of Insoluble PAs in Butanol-HCl.
  • Supplemental Table 1. Primers Used in This Study.
  • Supplemental Data Set 1. Protein Sequences Used in Phylogenetic Analysis in Supplemental Figure 3.
  • Supplemental Data Set 2. Protein Sequences Used in Phylogenetic Analysis in Figure 3A.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Elison Blancaflor (Noble Foundation) for assistance with confocal microscopy, Parastoo Azadi (Complex Carbohydrate Research Center, University of Georgia at Athens) for NMR analysis, Markus Klein (University of Zurich) for provision of the Arabidopsis TT12 cDNA, Isabelle Debeaujon (INRA, Versailles, France) for provision of seeds of the Arabidopsis tt12 mutant, Carla Welch (Noble Foundation) for assistance with plant growth, Vagner Benedito (Noble Foundation) for help with qRT-PCR, and Vagner Benedito and Stephen Temple (Forage Genetics International) for critical reading of the manuscript. The M. truncatula plants used in this research project, which are jointly owned by the Centre National de la Recherche Scientifique, were created through research funded, in part, by Grant 703285 from the National Science Foundation. NMR analysis was supported in part by the Department of Energy-funded (DE-FG09-93R-20097) Center for Plant and Microbial Complex Carbohydrates. This work was supported by Forage Genetics International and the Samuel Roberts Noble Foundation.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Richard A. Dixon (gro.elbon@noxidar).

[C]Some figures in this article are displayed in color online but in black and white in the print edition.

[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.109.067819

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