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First published online March 9, 2007; 10.1104/pp.107.097162 Plant Physiology 144:197-205 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The FRD3-Mediated Efflux of Citrate into the Root Vasculature Is Necessary for Efficient Iron Translocation1,[OA]Department of Biochemistry (T.P.D., E.E.R.), Division of Plant Sciences (W.G.), Department of Nutritional Sciences (E.E.R.), and C.S. Bond Life Sciences Center (T.P.D., W.G., E.E.R.), University of Missouri, Columbia, Missouri 65211
Iron, despite being an essential micronutrient, becomes toxic if present at high levels. As a result, plants possess carefully regulated mechanisms to acquire iron from the soil. The ferric reductase defective3 (frd3) mutant of Arabidopsis (Arabidopsis thaliana) is chlorotic and exhibits constitutive expression of its iron uptake responses. Consequently, frd3 mutants overaccumulate iron; yet, paradoxically, the frd3 phenotypes are due to a reduction in the amount of iron present inside frd3 leaf cells. The FRD3 protein belongs to the multidrug and toxin efflux family, members of which are known to export low-Mr organic molecules. We therefore hypothesized that FRD3 loads an iron chelator necessary for the correct distribution of iron throughout the plant into the xylem. One such potential chelator is citrate. Xylem exudate from frd3 plants contains significantly less citrate and iron than the exudate from wild-type plants. Additionally, supplementation of growth media with citrate rescues the frd3 phenotypes. The ectopic expression of FRD3-GFP results in enhanced tolerance to aluminum in Arabidopsis roots, a hallmark of organic acid exudation. Consistent with this result, approximately 3 times more citrate was detected in root exudate from plants ectopically expressing FRD3-GFP. Finally, heterologous studies in Xenopus laevis oocytes reveal that FRD3 mediates the transport of citrate. These results all strongly support the hypothesis that FRD3 effluxes citrate into the root vasculature, a process important for the translocation of iron to the leaves, as well as confirm previous reports suggesting that iron moves through the xylem as a ferric-citrate complex. Our results provide additional answers to long-standing questions about iron chelation in the vasculature and organic acid transport.
Plants, like most other organisms, require iron for essential everyday processes. Iron's usefulness is primarily derived from its ability to adopt two different ionic states; consequently, iron is present in many enzymes that catalyze redox reactions or are involved in electron transfer. Iron is abundant in most soils, yet exists mostly as Fe(III) hydroxides, which are sparingly soluble at neutral pH. Plants use two different strategies to extract iron under these conditions. One approach, called Strategy I and utilized by nongraminaceous species, involves the coordinate up-regulation of three biochemical activities in the roots of iron-deficient plants (Marschner, 1995  ). The rhizosphere is acidified by the release of protons from the roots, increasing the solubility of iron present. A membrane-bound Fe(III) reductase, encoded by FRO2 in Arabidopsis (Arabidopsis thaliana), reduces Fe(III) to Fe(II) (Robinson et al., 1999). Once in its reduced form, iron can then be transported into the root epidermal cells via the IRT1 transporter (Eide et al., 1996; Vert et al., 2002). While the components of iron uptake into the roots have for the most part been identified, very little is known about how iron is translocated from the roots to the shoots, where most iron is ultimately localized.
The Arabidopsis ferric reductase defective3 (frd3) mutants are chlorotic and constitutively express all three of their Strategy I iron uptake responses, even when grown under iron-sufficient conditions (Yi and Guerinot, 1996
Iron localization is dramatically altered in frd3 mutant plants. Protoplasts isolated from frd3 leaves contain about one-half the iron levels of wild-type protoplasts, despite the fact that the frd3 leaves used for the protoplast isolation accumulated twice as much iron as corresponding wild-type leaves (Green and Rogers, 2004
FRD3 is a member of the multidrug and toxin efflux (MATE) family, a group of proteins with 12 to 14 transmembrane domains capable of transporting small, organic compounds (Brown et al., 1999
The most likely candidate for FRD3's substrate is citrate, which is predicted to chelate 99.5% of iron present in xylem exudate (White et al., 1981 Here, we present data demonstrating that FRD3 loads citrate into the vasculature, a process necessary for the correct localization of iron throughout the plant. First, xylem from frd3 plants contains less citrate and iron than wild-type xylem. Additionally, growing frd3 plants on citrate rescues their characteristic mutant phenotypes. The ectopic expression of FRD3 confers tolerance to aluminum, consistent with the observed efflux of citrate. Finally, expression of FRD3 in Xenopus laevis oocytes shows that FRD3 mediates the transport of citrate.
frd3-1 Xylem Exudate Contains Less Citrate Than Columbia-0 Wild-Type Exudate
To identify FRD3's substrate, xylem fluid was collected from wild-type Columbia (Col)-0 and frd3-1 mutant plants grown to maximize their vegetative growth. Under these conditions, the frd3-1 phenotype was similar to soil-grown plants at the same developmental stage: frd3-1 plants were slightly more chlorotic than wild-type plants and overaccumulated zinc and manganese in their leaves (data not shown). Leaf iron levels were 3% lower in frd3-1 than in wild-type plants (data not shown), consistent with previous analysis of soil-grown frd3-1 plants (Lahner et al., 2003
To confirm that the differences in the concentrations of citrate and xylem were not the result of indirect factors like chlorosis or reduced size of frd3-1, levels of other anions present in xylem were also measured. No significant differences in the concentrations of chloride and phosphate ions were noted (Fig. 1, C and E). Additionally, nitrate and sulfate ion concentrations were actually higher in frd3-1 xylem (Fig. 1, D and F), suggesting that the lower levels of citrate and iron in frd3-1 xylem exudate are caused by the elimination of FRD3 function. The increased xylem nitrate and sulfate may be caused by reduced transpiration in the frd3 mutant due to its smaller size.
To determine if exogenously supplying citrate could compensate for reduced xylem citrate levels, frd3-1 seedlings were germinated on plates with citrate added to the growth medium. These seedlings were noticeably larger and greener than those grown on unsupplemented media (Fig. 2A
). This regreening is reflected in the significantly higher chlorophyll levels of frd3-1 seedlings grown on citrate, which approach those of Col-0 wild-type plants grown under identical conditions (Fig. 2B). Growth on media supplemented with other organic acids, such as malate and succinate, had no effect on the frd3-1 phenotype (Fig. 2B; data not shown). Other characteristic frd3 phenotypes were also rescued by growth on citrate-containing media. The frd3-1 mutant possesses constitutive Fe(III) chelate reductase activity, even when grown under iron-sufficient conditions (Rogers and Guerinot, 2002a
Ectopically Overexpressing FRD3 Confers Increased Tolerance to Aluminum
To protect themselves from the toxic effects of aluminum, the roots of a number of different plant species release organic acids into the rhizosphere (Miyasaka et al., 1991
Expression of FRD3 in Xenopus Oocytes Mediates Transport of Citrate To further confirm that FRD3 transports citrate and to investigate the specificity of this transport, the characteristics of FRD3 were examined in oocytes using two-electrode voltage clamping. Oocytes injected with FRD3-GFP cRNA produced inward currents when the bath solution contained citrate but not malate (Fig. 4A ). These currents were specific for the transport of citrate by FRD3: Removal of citrate from the bath solution quickly returned the current to resting levels. Additionally, the magnitude of these currents was dependent on the concentration of citrate. There was a delay observed after the addition of citrate and before the observation of strong inward currents. While this long of a delay is not typical, it may be explained by the need for uncharged citric acid to diffuse inside the oocyte, where it can dissociate to citrate and be effluxed by FRD3. Under these experimental conditions, the concentration of citrate molecules that are uncharged and therefore able to diffuse across the plasma membrane of the oocytes is approximately 160 µM, a concentration that is about 2-fold higher than that found in xylem.
In contrast to FRD3-GFP injected oocytes, uninjected oocytes responded to neither citrate nor malate. Figure 4B summarizes the response of a number of oocytes from different batches, demonstrating that the response to citrate was significantly larger in FRD3-GFP injected oocytes compared to uninjected oocytes. By convention, these inward currents are indicative of the influx of net positive charge or the efflux of net negative charge. To study the efflux of citrate more directly, oocytes were injected with citrate immediately prior to voltage clamping. In these experiments, uninjected oocytes experienced an outward current (Fig. 4C). These currents may be the result of the membrane resealing after being disrupted by the citrate injection and by the subsequent penetration for voltage clamping. However, FRD3-GFP oocytes experienced a strong inward current when injected with citrate before voltage clamping (Fig. 4C). These inward currents are indicative of the flow of anions out of the oocytes and are therefore consistent with the efflux of citrate by FRD3. Such inward currents were not observed with FRD3-GFP oocytes injected with malate; instead, outward currents resembling those from uninjected oocytes were observed (Fig. 4C). To further study the efflux of citrate from oocytes expressing FRD3-GFP, oocytes were loaded with either [14C]citrate or a mixture of [14C]citrate and 55Fe (as FeCl3) and the efflux of radioactivity measured. Oocytes expressing FRD3-GFP effluxed almost twice as much 14C compared to uninjected oocytes (Fig. 5A ), confirming that FRD3 can mediate the efflux of citrate. The same oocytes did not efflux more 55Fe than did uninjected oocytes (Fig. 5B); this is consistent with our hypothesis that FRD3 does not participate in loading iron into the xylem. The efflux of [14C]citrate was not affected by the presence or absence of iron in the loading solution (data not shown).
FRD3 was previously hypothesized to transport a low-Mr compound, most probably an iron chelator, into the xylem (Green and Rogers, 2004 ). FRD3 function is required for the correct localization of iron throughout the plant; in the absence of functional FRD3 protein, leaf cells contain reduced amounts of iron, resulting in chlorosis and the constitutive activation of the root iron uptake responses. Here, we demonstrate that FRD3 is capable of effluxing citrate, a molecule long hypothesized to chelate iron in the xylem (Tiffin, 1966; White et al., 1981). This suggests that FRD3 is responsible for the loading of citrate into the xylem to chelate iron and allows the efficient translocation of iron to aerial portions of the plant.
The first indication that citrate was a substrate for FRD3 was analysis of xylem exudates that revealed that frd3-1 mutants contain less citrate in their xylem compared to Col-0 wild-type plants (Fig. 1A). Interestingly, iron levels in frd3-1 xylem were also lower than those of wild-type plants (Fig. 1B). These lower levels of iron in the xylem could be caused by one of two possibilities. First, FRD3 could efflux both iron and citrate, possibly as a ferric citrate complex; however, the data in Figure 5B tend to disprove this hypothesis. Alternatively, iron may be less soluble in the xylem stream of frd3-1 plants because of the lowered citrate levels. This second possibility agrees well with the dramatic accumulation of iron observed in the frd3 root vasculature by Perls' staining (Fig. 2D; Green and Rogers, 2004
Surprisingly, xylem citrate levels were only reduced by 40.2% in the frd3-1 mutant. If FRD3 is the only way citrate can enter the root vasculature, one might expect xylem citrate levels to be more dramatically reduced in the absence of FRD3. A number of explanations have been considered to explain this discrepancy. One possibility is that the Ala-to-Asp change caused by the frd3-1 mutation (Rogers and Guerinot, 2002a
Another probable explanation for the surprisingly high frd3-1 xylem citrate levels could be the presence of other citrate efflux transporters capable of compensating for the loss of FRD3 function. In a dendrogram of MATE family members, FRD3 falls into a discrete cluster with four other Arabidopsis genes (Rogers and Guerinot, 2002a There are several possible mechanisms through which the citrate provided in the growth media could enter the vasculature to rescue the frd3 phenotype (Fig. 2). It is possible for exogenous citrate to enter the root vasculature through the root apical meristem. This portion of the root does not contain a Casparian strip, a layer of cells with suberin-containing walls, which acts as a barrier to the radial movement of water and ions in the roots. The Casparian strip would block the entry of exogenous citrate from ultimately entering the root vasculature apoplastically throughout much of the root length. However, the tip of the root could be the entry point for exogenous citrate to enter the root vasculature and thereby rescue the frd3 mutant phenotypes. It is also possible for citrate to enter through non-FRD3-dependent routes: Low affinity transporters could efflux citrate when exposed to these high substrate levels. Alternatively, uncharged citrate could diffuse across membranes.
The bacterial MATE protein NorM uses the antiport of sodium ions to energize the efflux of toxins from the cell (Morita et al., 2000 A number of lines of evidence suggest that FRD3 specifically transports citrate and not other organic acids. First, transgenic lines ectopically expressing FRD3-GFP exhibited an enhanced release of citrate but not malate from their roots (Fig. 3B), suggesting that FRD3 transports citrate preferentially over malate. Additionally, oocytes expressing FRD3-GFP experienced inward currents only when exposed to citrate, and not when exposed to comparable levels of malate (Fig. 4A). Similarly, experiments involving the direct injection of malate failed to produce inward currents in oocytes expressing FRD3-GFP (Fig. 4C). Consistent with these results, only citrate appears capable of rescuing the frd3 phenotype (Fig. 2). The supplementation of media with other organic acids such as malate and succinate failed to restore a wild-type phenotype to frd3-1 mutant plants (Fig. 2; data not shown).
Transport of citrate via FRD3 into the root vasculature is important for the translocation of iron to the shoots and subsequent uptake by leaf cells. This role of FRD3 might be especially relevant when considering the so-called "iron chlorosis paradox," a phenomenon where the leaves of plants grown on calcareous soils become chlorotic despite possessing normal levels of iron in their leaves (Morales et al., 1994
Arabidopsis Lines and Growth Conditions
The mutants frd3-1 and frd3-3 have been described previously (Rogers and Guerinot, 2002a
Fe(III) chelate reductase assays have been described previously (Yi and Guerinot, 1996
Wild-type Col-0 and frd3-1 mutant seeds were grown axenically on B5 medium for 2 weeks, after which they were transferred to Rockwool (Worms Way) soaked in 0.25x Gamborg's B5 medium without Suc. Seedlings were grown under an 8-h-day/16-h-night light cycle and were watered once a week with 0.25x Gamborg's B5 medium. After 7 weeks, the shoots were excised from the roots at the hypocotyl with a sharp razor blade. To reduce cellular contamination, the first drop of xylem exuded from the decapitated roots was always discarded. Xylem was collected for no more than 2 h after the shoots were initially removed.
Anion and organic acid quantification of xylem exudate was performed by HPLC as described previously (Dionex Corporation, 2004 Iron levels were measured by adding the xylem to a solution of 0.3 M ascorbate and 1 mM ferrozine and incubating in the dark overnight. Iron concentrations were then determined by comparing absorption readings at 562 nm with a standard curve.
To obtain 35S::FRD3-GFP, a full-length FRD3 cDNA was cloned into the yeast expression vector pFL61 (Minet et al., 1992
Seedlings were grown on vertical plates in the absence of Al3+ for 7 d, after which they were transferred to fresh vertical plates containing different concentrations of AlCl3. The medium used in these vertical plates, including its supplementation with AlCl3, has been described previously (Sivaguru et al., 2003 To measure citrate efflux, 14-d-old seedlings grown on B5 media were carefully washed and their roots placed in 3 mL of water for 24 h. This root-bathing solution was lyophilized and resuspended in 150 µL of water. Citrate levels were determined according to the instructions of a citrate analysis kit (Roche Molecular Biochemicals): NADH consumption was measured in a solution containing 100 mM glycyl-glycine, pH 7.9, 0.2 mM ZnCl2, 0.56 mM NADH, 12 units/mL malate dehydrogenase, 24 units/mL lactate dehydrogenase, and 1.6 units/mL citrate lyase. Malate levels were measured by measuring the production of NADH in a solution containing 50 mM 2-amino-2-methylpropanol, pH 9.9, 40 mM Glu, 2 mM NAD+, 3.5 units/mL malate dehydrogenase, and 0.9 units/mL Asp transaminase. Enzymes, NAD+, and NADH were obtained from Sigma.
A full-length FRD3 cDNA was subcloned into the oocyte expression vector pOO2 (Ludewig et al., 2002 Oocytes were voltage clamped 4 d after cRNA injection in a bath solution containing 5 mM MES-Tris, pH 5.0, 1 mM MgCl2, and 1.8 mM CaCl2, with osmolality adjusted to 240 to 260 mosmol kg–1 with D-sorbitol. Voltage clamping was controlled and currents were recorded with a TEV-200A amplifier (Dagan) and Axotape 2.0 software (Axon Instruments). To study organic acid efflux directly, oocytes were injected with 23 nL of 1 mM citrate or malate using glass micropipettes. The injected oocytes were allowed to recover for 1 min before clamping at a membrane potential of –80 mV. For [14C]citrate and 55FeCl3 efflux, six oocytes (either uninjected or injected 4 d previously with FRD3-GFP cRNA) were injected with either 23 nL of 2 mM [14C]citrate (4.6 nCi/oocyte) or 23 nL of 2 mM [14C]citrate (4.6 nCi/oocyte) and 50 µM 55FeCl3 (8.5 nCi/oocyte) in ND96 buffer using fine-tipped glass micropipettes. The oocytes were incubated for 1 min in ice-cold ND96 buffer, pH 5.0, and then transferred into 750 µL of ND96, pH 5.0, at room temperature. At indicated time points, 650 µL of the buffer was removed for measuring radioactivity and replaced with fresh buffer. At the end of each experiment, the oocytes were dissolved in 10% SDS. Radioactivity of the efflux buffer at the various time points and remaining in the oocytes was counted using full-spectrum DPM counting in a Perkin-Elmer Tri-Carb 2800TR liquid scintillation analyzer. Radioactivity effluxed was expressed as a percentage of total radioactivity injected.
We thank Hiroki Osawa and Sharon Pike for assistance with two-electrode voltage clamping, George Kracke and Brenda Peculis for providing oocytes, and Mary Lou Guerinot for critical reading of the manuscript. We are especially grateful to Tom Mawhinney and Joe Leykam for analyzing xylem anion levels, and to David Salt and Bret Lahner for frdl and frd3-3 frdl elemental analysis. We would also like to thank Mayandi Sivaguru for advice about aluminum tolerance assays. Sarene Alsharif provided excellent technical assistance. Received February 1, 2007; accepted February 26, 2007; published March 9, 2007.
1 This work was supported by the MU Interdisciplinary Plant Group and Monsanto (predoctoral fellowship to T.P.D.), and by the U.S. Department of Agriculture (grant nos. 2002–35100–12331 and 2005–35100–16060 to E.E.R.). 
2 Present address: Department of Plant Biology, Michigan State University, East Lansing, MI 48824. 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.plantphysiol.org) is: Elizabeth E. Rogers (rogersee{at}missouri.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.097162 * Corresponding author; e-mail rogersee{at}missouri.edu; fax 573–884–9676.
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) and oocytes expressing FRD3-GFP (
) were injected with [14C]citrate and 55FeCl3 and the effluxed radioactivity measured at the indicated time points. A, 14C counts; B, 55Fe counts. Values are expressed as the percentages of the total radioactivity injected and represent the mean (±SD) of at least six samples of five or six oocytes each from a single, representative experiment.
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