The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Abstract

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.

Figure 1.

Simplified Scheme of the Biosynthetic Pathway in Arabidopsis Leading to PAs, Flavonols, and Anthocyanins, Including Proposed Vacuolar Transport Steps. Dihydrokaempferol is synthesized from 4-coumaroyl CoA and three molecules of malonyl CoA by the sequential action of chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H). Enzymes are represented in uppercase bold letters. Corresponding genetic mutations are in lowercase italic letters. Regulatory mutants are indicated by parentheses. AHA10, P-type H⁺-ATPase; ANR, anthocyanidin reductase; CE, condensing enzyme; DFR, dihydroflavonol reductase; F3′H, flavonol 3′-hydroxylase; FLS, flavonol synthase; (F)GT, (flavonol) glycosyltransferase; LDOX, leucoanthocyanidin dioxygenase.

Figure 2.

Flavonoid Composition of Mature Seeds. (A) Flavonol composition of seeds from the tt12 mutant, the wild type, TT12 overexpressor (TT12), and GFP5:TT12 overexpressor. Values represent the average and se of six independent measurements. Q-di-R, quercetin-3,7-di-O-rhamnoside; G, glucoside; H, hexoside; I, isorhamnetin; K, kaempferol; Q, quercetin; R, rhamnoside. (B) Detection of epicatechin and soluble PAs (dimers to heptamers) by LC-MS. EC, epicatechin. (C) Analysis of soluble and insoluble PAs measured after acid-catalyzed hydrolysis. For (B) and (C), values represent the average and se of three independent measurements.

Figure 3.

Activity of the TT12 Promoter in Wild-Type Developing Seeds of Arabidopsis. The expression of the ProTT12:uidA cassette was exclusively observed in developing seeds at 0 DAF (A), 2 DAF (B), 3 DAF ([C] and [G]), 4 DAF (D), 5 DAF (E), and 6 DAF (F). GUS activity was observed with Nomarski optics on whole mounts for (A) to (F) and on section for (G). The ii1 layer is also called endothelium. c, chalaza; e, embryo; ii, inner integument; m, micropyle; oi, outer integument; ps, pigment strand (chalazal tissue); t, testa. Bar = 25 μm in (A), 40 μm in (B), 65 μm in (C), 70 μm in (D), 80 μm in (E) and (F), and 33 μm in (G).

Figure 4.

Subcellular Localization of the TT12 Protein after Transient and Stable Expression. (A) to (C) Mesophyll protoplasts isolated after transient expression of cTT12-GFP ([A] and [B]) or the tonoplast control cTPK1-GFP (C) by infiltration of N. benthamiana leaves with agrobacteria carrying the corresponding constructs. DIC, differential interference contrast. Bars = 5 μm. (A) GFP and chlorophyll autofluorescence were false-colored in green and red, respectively. GFP fluorescence surrounds the plastids (see merged image). (B) A vacuole released by gentle lysis exhibits GFP fluorescence on the tonoplast. (C) Transient cTPK1-GFP expression is similar to cTT12-GFP, resulting in GFP fluorescence surrounding plastids and the nucleus (asterisk). In (B) and (C), the green GFP and the red chlorophyll autofluorescence channels are merged. (D) to (G) Confocal microscopy of Arabidopsis roots ([D] and [E]), mesophyll protoplasts (F), and isolated vacuoles (G) after stable transformation of GFP-cTT12 in the tt12 background. (D) GFP fluorescence (green) of a root tip of a 7-d-old seedling. Bar = 20 μm. (E) Merged image of GFP-cTT12 fluorescence (green) after propidium iodide counterstaining of cell walls (red). A single root cell possessing two large vacuoles exhibiting GFP-cTT12 fluorescence exclusively on the tonoplast membranes is shown. Bar = 5 μm. (F) GFP-cTT12 fluorescence (green) is visible on an intracellular membrane surrounding the plastids exhibiting red chlorophyll autofluorescence (asterisks in the merged image) that delimits the vacuole(s) (DIC). Bar = 5 μm. (G) Isolated leaf mesophyll vacuole prepared from stably transformed GFP-cTT12/tt12 plants exhibits GFP fluorescence on the tonoplast. Bar = 5 μm.

Figure 5.

TT12-Mediated Transport of C3G in Yeast Microsomal Vesicles. (A) and (B) Absorption scans of C3G washed off from filters after the transport experiment representing the vesicle-associated amount of C3G. (A) Result of a transport experiment in the presence of MgATP. (B) Corresponding control in the absence of ATP. Transport was stopped by filtration after 30 (dotted lines) or 90 s of vesicular uptake (solid lines). Black and gray lines represent uptake experiments performed with vesicles isolated from cTT12- or empty vector control (NEV)–transformed yeasts, respectively. Dashed line in corresponds to authentic C3G standard. (C) HPLC profile of authentic C3G standard. (D) HPLC analysis of the uptake of C3G into microsomal vesicles prepared from yeasts transformed with TT12. Vesicles isolated from TT12-transformed yeasts have taken up C3G after 90 s (black solid line) compared with 30 s (black dotted line) of incubation. Corresponding control vesicles (empty vector; gray lines) do not display any time-dependent increase in C3G. (E) Time-dependent uptake of C3G into vesicles isolated from yeasts transformed either with TT12 (closed symbols) or the empty vector (open symbols) in the presence of MgATP. (F) Quantification of uptake activity. Averages ± se of 20 transport experiments performed with independent vesicle preparations are presented. Black and white bars stand for yeasts transformed with TT12 (pNEV-TT12) and empty vector (pNEV), respectively.

Figure 6.

C3G Specifically Inhibits TT12-Mediated C3G Transport. (A) Inhibition of the uptake of 1 mM C3G by the flavonols Q3G, Q3R, the nonglucosylated flavan-3-ols epicatechin and catechin, and by cat3G. All competitors were added at a final concentration of 2 mM. Experiments were performed in the presence of MgATP. The C3G transport activity with vesicles isolated from TT12-transformed yeasts in the absence of competitors was set to 100%. Transport rates were calculated after subtraction of unspecific binding. (B) Dose-dependent inhibition of C3G uptake by cat3G. All experiments were performed in triplicate with two independent vesicle preparations.

Figure 7.

Anthocyanin Deposition in Immature Seeds of ban and the Double Mutant ban tt12. (A) and (B) The characteristic red color observed in the endothelial layer of ban seed caused by anthocyanins is reduced and more diffuse in immature ban tt12 seeds as seen in whole siliques ([A]; bar = 1 mm) or individual immature seeds ([B]; bar = 250 μm). (C) Entire siliques (left panel) or immature seeds (right panel) of identical developmental stages were extracted in acidic methanol, and the anthocyanin content was determined photometrically at 525 nm. ban tt12 siliques or seeds contain significantly less anthocyanins compared with ban (Student's t test, two-tailed distribution, P < 0.01).