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. 2007 May;144(1):197–205. doi: 10.1104/pp.107.097162

The FRD3-Mediated Efflux of Citrate into the Root Vasculature Is Necessary for Efficient Iron Translocation1,[OA]

Timothy P Durrett 1,2, Walter Gassmann 1, Elizabeth E Rogers 1,*
PMCID: PMC1913786  PMID: 17351051

Abstract

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; Rogers and Guerinot, 2002b). Consequently, frd3 mutant plants overaccumulate iron as well as other metals, such as manganese and zinc, in their roots. The accumulation of these additional metals is probably due to the constitutive expression of IRT1, which has previously been shown to transport other divalent metals, such as zinc and manganese (Eide et al., 1996; Korshunova et al., 1999). Accumulation of iron in frd3 shoots appears dependent on the plant's growth conditions: When grown on petri dishes under conditions of high iron availability, frd3 accumulates leaf iron levels approximately twice that of the wild type (Rogers and Guerinot, 2002a). However, when less iron is available, for example, when grown on potting soil, iron levels in frd3 leaves are 10% lower than in wild-type leaves (Lahner et al., 2003).

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). Additionally, frd3 fails to accumulate the iron storage protein ferritin, even when grown under conditions of iron sufficiency (Rogers and Guerinot, 2002a). These lower cellular iron levels in frd3 plants explain two of the characteristic frd3 phenotypes. Presumably, frd3 leaves are chlorotic because they are iron deficient. Further, because they are iron deficient, the frd3 leaf cells constantly signal the roots to take up more iron, hence the constitutive Strategy I iron uptake responses.

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). NorM from Vibrio parahaemolyticus, the first MATE family member identified, is a Na+/drug antiporter, capable of effluxing antimicrobial agents (Morita et al., 1998, 2000). Only a few of the 56 predicted Arabidopsis MATE proteins have been studied to date; their characterization, however, is consistent with them also transporting small, organic compounds. For example, the TRANSPARENT TESTA12 protein is thought to localize to the vacuolar membrane in developing seeds, where it may control the sequestration of flavonoids (Debeaujon et al., 2001). ALF5, expressed strongly in the root epidermis, is required for resistance to a number of compounds, including tetramethylammonium and an unknown contaminant of commercial agar (Diener et al., 2001). eds5 mutant plants fail to accumulate salicylic acid during pathogen attack, suggesting that EDS5 might transport salicylic acid or a precursor required for its synthesis (Nawrath et al., 2002). Lastly, DTX1 was cloned based on its ability to confer norfloxacin resistance to bacterial cells and is capable of effluxing plant-derived alkaloids such as berberine and palmatine (Li et al., 2002). The FRD3 protein localizes to the pericycle and cells internal to the pericycle cells in roots (Green and Rogers, 2004). Based on this localization, the efflux capabilities of other MATE proteins, and the frd3 mutant phenotypes, we hypothesized that FRD3 transports a small, organic iron-chelator that is necessary for the correct localization of iron throughout the plant into the xylem.

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). Analysis of xylem exudate from a number of different species reveals that iron and citrate comigrate during paper electrophoresis (Tiffin, 1966, 1970). Additionally, studies of sugar beets have demonstrated that the xylem concentration of various organic acids, including citrate, malate, and succinate, all increase under iron deficiency (Lopez-Millan et al., 2000). While these physiological experiments all demonstrate the importance of citrate in iron translocation, the means by which citrate enters the vasculature have until this point remained unknown (to our knowledge).

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.

RESULTS

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). One potential iron chelator is citrate; comparison of citrate levels revealed that frd3-1 xylem contained 40.2% less citrate than wild-type Col-0 xylem (Fig. 1A). Interestingly, xylem iron levels were also lower in frd3-1, only 49.2% of wild-type levels (Fig. 1B). This correlation between citrate and iron levels in the xylem is consistent with earlier work (Brown and Tiffin, 1965).

Figure 1.

Figure 1.

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Xylem from frd3-1 mutants contains less citrate than xylem from Col-0 wild-type plants. Xylem exudate was collected from Col-0 and frd3-1 plants and analyzed for citrate (A), iron (B), chloride (C), nitrate (D), phosphate (E), and sulfate (F). Values shown represent the mean (±sd) derived from at least five samples. Student's t test: P < 0.05, *; P < 0.01, **.

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.

Citrate Supplementation Rescues the frd3 Phenotype

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); however, addition of citrate to the growth medium reduced Fe(III) chelate reductase activity to wild-type levels (Fig. 2C). Similar results were also obtained with the frd3-3 mutant allele (data not shown). Iron accumulates in the root vasculature of frd3-1 seedlings, as indicated by Perls' stain (Green and Rogers, 2004; Fig. 2D). Yet, when grown on citrate, this overaccumulation of iron all but vanishes from the roots of frd3-1 mutant plants (Fig. 2D). Iron, however, remains present in the roots of frd3-1 seedlings treated with malate, suggesting that the effect of citrate is specific and not general to all organic acids. The ability of high levels of exogenous citrate to rescue the frd3 phenotype suggests these mutants are defective in the transport of this iron chelator, furthering the idea that FRD3 effluxes citrate into the root vasculature.

Figure 2.

Figure 2.

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Growth on citrate rescues the frd3 phenotype. A, Wild-type Col-0 and frd3-1 seedlings were grown axenically on B5 medium or B5 medium supplemented with 3 mm sodium citrate. B and C, Chlorophyll levels (B) and Fe(III) chelate reductase activity (C) were measured after 3 weeks. Values shown represent the mean (±sd) from a representative experiment. Student's t test: P < 0.05, *; P < 0.001, **. D, Perls' staining of 3-week-old Col-0 and frd3-1 seedlings grown in the absence or presence of 3 mm sodium citrate or 3 mm sodium malate.

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; Delhaize et al., 1993; Pellet et al., 1995). If FRD3 does efflux citrate, then its ectopic expression might increase the resistance of transgenic plants to aluminum. Therefore, a construct consisting of a FRD3-GFP fusion protein under the control of the 35S promoter was transformed into wild-type Col-0 plants. This 35S∷FRD3-GFP construct is capable of complementing frd3-1 mutant plants, demonstrating that the fusion protein is functional (data not shown). T3 Col-0 lines homozygous for individual insertions were analyzed for aluminum tolerance using a root growth assay. In the absence of aluminum, the root growth of most transgenic lines was similar to that of untransformed plants. However, when exposed to 75 μm and 125 μm aluminum, four of the five transgenic lines ectopically expressing FRD3 possessed an enhanced tolerance to aluminum, as evidenced by better root growth under those conditions (Fig. 3A). These results are consistent with FRD3 effluxing an organic acid. As other organic acids besides citrate, such as malate or oxaloacetate, can also confer aluminum tolerance to plant roots, root exudates were collected from these transgenic lines and citrate levels measured. Plants ectopically expressing FRD3-GFP had significantly higher amounts of citrate in their root exudates compared to untransformed control plants (Fig. 3B). Malate levels, however, were not dramatically increased in the transgenic lines compared to Col-0 wild-type plants (Fig. 3B).

Figure 3.

Figure 3.

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Transgenic plants ectopically expressing FRD3-GFP possess enhanced tolerance to aluminum due to an increased efflux of citrate. A, Seven-day-old Col-0 wild-type seedlings (black symbols) and Col-0 transgenic seedlings containing 35S∷FRD3-GFP (white symbols) were transferred to vertical plates containing growth media supplemented with 0 μm Al3+ (squares), 75 μm Al3+ (diamonds), or 125 μm Al3+ (circles). Root lengths were measured at 24-h intervals after transfer. Shown are the results from a representative experiment. Values represent the mean (±sd) of at least five roots from Col-0 wild-type and a single, representative transgenic line. B, Root exudates from 14-d-old wild-type Col-0 and transgenic 35S∷FRD3-GFP seedlings were collected, and levels of citrate (gray bars) and malate (white bars) measured. Values represent the mean (±sd) of citrate and malate levels normalized to the root mass from three replicate pools of 20 seedlings for a single, representative experiment. Student's t test: P < 0.05, *; P < 0.001, **.

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.

Figure 4.

Figure 4.

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Transport of citrate in oocytes expressing FRD3-GFP. A, Currents produced when uninjected oocytes (top) and oocytes expressing FRD3-GFP (bottom) were exposed to citrate or malate. Presence of substrate is indicated by a black bar above the current trace. Oocyte membrane potentials were clamped at −80 mV. B, Average currents induced after different times of exposure to 30 mm citrate at a membrane potential of −80 mV. Values shown represent the means (±sd) for at least 10 oocytes from four different batches of oocytes. Student's t test: P < 0.05, *; P < 0.01, **. C, Currents produced when uninjected oocytes (top) and oocytes expressing FRD3-GFP (middle) were injected with 23 nL of 1 mm citrate or oocytes expressing FRD3-GFP were injected with 23 nL of 1 mm malate (bottom), prior to clamping at a membrane potential of −80 mV.

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).

Figure 5.

Figure 5.

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Efflux of [14C]citrate via FRD3-GFP. Control oocytes (•) 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.

DISCUSSION

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). The correlation between citrate and iron levels in the xylem is consistent with earlier work that demonstrated striking parallels between the levels of iron and citrate in the xylem exudates from soybean (Glycine max) plants grown under different levels of iron deficiency (Brown and Tiffin, 1965). The concomitant lowering of iron and citrate levels in frd3 xylem provides further evidence that citrate is necessary for the efficient translocation of iron from the roots to the shoots. Based on the phenotype of frd3 plants grown on plates, which experience intracellular iron deficiency despite an overall overaccumulation of iron in their leaves, citrate also appears to be required for the efficient uptake of iron into leaf cells. We speculate that in the absence of citrate, leaf apoplastic iron precipitates onto cell walls and therefore is unavailable to the endogenous iron uptake systems. Combined with the data demonstrating that FRD3 transports citrate when expressed in Xenopus oocytes (Figs. 4 and 5), our results strongly support the hypothesis that FRD3's function in planta is to transport citrate into the xylem.

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) does not completely eliminate the function of the FRD3 protein so that it can still transport reduced amounts of citrate. This explanation is unlikely: For the most part, the phenotypes frd3-1 mutant plants are indistinguishable from other frd3 mutant alleles that all result in truncated proteins (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). Based on their sequence similarity, these paralogs are likely candidates to provide this redundant function. However, in silico analysis predicts these genes either localize to an intracellular compartment and not to the plasma membrane, or they are not expressed in the roots, suggesting that they are unlikely to be able to compensate for the loss of FRD3 (data not shown). Additionally, T-DNA insertion mutants of the closest FRD3 paralog, FRDL, possess no apparent phenotype associated with iron homeostasis: Unlike frd3 mutants, frdl mutants regulate Fe(III) chelate reductase activity normally, do not show any signs of chlorosis, and possess wild-type levels of different metals (data not shown; www.purdue.edu/dp/ionomics/). Likewise, double frd3-3 frdl mutants do not possess an enhanced phenotype relative to frd3-3 mutants (data not shown), further suggesting that FRDL is not redundant to FRD3. Therefore, it is somewhat unlikely that the higher than expected levels of citrate in frd3-1 xylem are due to the citrate transport capabilities of FRD3 paralogs. However, the possibility remains that this compensating transport of citrate could be provided by a yet unknown protein unrelated to FRD3. It is also possible for uncharged citric acid to diffuse across plant cell membranes to enter the xylem; we believe this is the most likely possibility, especially since, in the absence of functional FRD3 protein, intracellular citrate would be expected to accumulate to higher levels than in the wild type.

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). Plant cells typically use the proton gradient across their plasma membrane to power transport; it is possible that while bacterial MATE proteins are sodium antiporters, plant MATE proteins may couple efflux to proton uptake. However, given the negative plasma membrane potential of plant cells, the efflux of an anion like citrate would be energetically favorable on its own. Thus, there is no energetic requirement for coupling citrate efflux to uptake of a cation, raising the question of whether citrate efflux by FRD3 is coupled to either proton or sodium antiport. In preliminary experiments, the efflux of [14C]citrate from oocytes expressing FRD3-GFP was not dramatically affected by altering the pH or Na+ concentration of the external bathing solution, suggesting that neither of these cations is involved in the efflux of citrate by FRD3.

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; Nikolic and Romheld, 2002). This condition is reminiscent of the phenotype of frd3 mutants grown on petri dishes that are chlorotic despite overaccumulating iron in their leaves (Rogers and Guerinot, 2002a). Interestingly, plants suffering from the “iron chlorosis paradox” regreen when citrate or other weak acids are applied to their leaves (Kosegarten et al., 2001; Álvarez-Fernández et al., 2004). It is therefore tempting to speculate that there might be a connection between the role of FRD3 in providing citrate and the iron chlorosis paradox. Plants suffering from this phenomenon might possess lower levels of citrate in their xylem or apoplast due to lack of FRD3 activity. In this regard, it is interesting to note that soybean and tomato (Solanum lycopersicum) varieties resistant to iron deficiency contain more citrate and iron in xylem compared to other varieties susceptible to iron deficiency (Brown and Chaney, 1971). The further study of FRD3 orthologs in different crop species could be important to understanding this common agricultural problem.

MATERIALS AND METHODS

Arabidopsis Lines and Growth Conditions

The mutants frd3-1 and frd3-3 have been described previously (Rogers and Guerinot, 2002a) and are stocks CS6584 and CS8506 at the Arabidopsis Biological Resource Center (http://www.biosci.ohio-state.edu/∼plantbio/Facilities/abrc/abrchome.htm). Surface-sterilized seeds were plated on Gamborg's B5 medium (Caisson Laboratories) in petri dishes. Where indicated, organic acids were added to the growth medium prior to autoclaving in the form of their conjugate base sodium salt. Two weeks after germination, seedlings were transferred to fresh plates for another week, after which Fe(III) chelate reductase activity and chlorophyll levels were measured.

Fe(III) chelate reductase assays have been described previously (Yi and Guerinot, 1996) Chlorophyll was extracted by soaking approximately 20 mg of leaf tissue in 1 mL of methanol, and total chlorophyll levels quantified by measuring the absorbance at 652, 665, and 750 nm (Porra et al., 1989). Perls' stain was performed using established protocols for Arabidopsis (Arabidopsis thaliana) roots (Green and Rogers, 2004).

Xylem Collection and Analysis

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 0.25× 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 0.25× 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). Briefly, samples were analyzed using a DX-500 BioLC system (Dionex) with an IonPac AS11 analytical column and a 0.5 to 38 mm NaOH gradient at a flow rate of 2.0 mL/min. Gradient parameters were 0.5 mm NaOH for 0 to 2.5 min, 0.5 to 5 mm NaOH for 2.5 to 6 min, and 5 to 38 mm NaOH for 6 to 18 min. Anions were detected with an ED40 electrochemical detector (Dionex).

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.

Aluminum Tolerance

To obtain 35S∷FRD3-GFP, a full-length FRD3 cDNA was cloned into the yeast expression vector pFL61 (Minet et al., 1992). Homologous recombination was then used to replace the C terminus of FRD3 with sequence corresponding to the C terminus fused to GFP. The inserted sequence was confirmed with sequencing. FRD3-GFP was then cloned into 35SpBARN under the control of the 35S promoter (LeClere and Bartel, 2001). Col-0 plants were transformed with 35S∷FRD3-GFP using the floral dip method (Clough and Bent, 1998). Transgenic plants were selected by spraying T1 seedlings three times with 250 mg L−1 Basta. Lines homozygous for individual insertions were generated by analysis of segregation ratios in the T2 and T3 generations.

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). Root growth was measured by marking root lengths every 24 h. The petri plates were then digitally scanned and root growth accurately measured using NIH Image (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/).

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.

Two-Electrode Voltage Clamping and Efflux Experiments

A full-length FRD3 cDNA was subcloned into the oocyte expression vector pOO2 (Ludewig et al., 2002). The FRD3 stop codon was then replaced with the sequence of GFP. To enhance expression, the long plant 5′ untranslated region was subsequently removed to obtain pOO2.FRD3-GFPshort. For oocyte expression, pOO2.FRD3-GFPshort was linearized with MluI and capped cRNA transcribed in vitro using the SP6 mMessage mMachine kit (Ambion). The isolation of oocytes from Xenopus laevis frogs has been described previously (Osawa et al., 2006). Oocytes were injected with 23 ng of FRD3-GFP cRNA using a Nanoject II injector (Drummond Scientific) and then incubated in ND96 Ringers solution at 17°C as described previously (Osawa et al., 2006).

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.

Acknowledgments

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.

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.).

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@missouri.edu).

[OA]

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