The Arabidopsis ATP-binding cassette protein AtMRP5/AtABCC5 is a high affinity inositol hexakisphosphate transporter involved in guard cell signaling and phytate storage

Abstract

Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule.

FIGURE 1.

Ablation of AtMRP5 disturbs inositol hexakisphosphate, phosphate, and phosphorus levels in seeds. The conductivity profiles of seed extracts is shown. A, traces for Col0 (red line), mrp5-2 (black line), and ipk1-1 (blue line). B, traces for Ws-2 (red line) and mrp5-1 (black line). Individual traces are offset on the y axis to aid visualization. C, seed InsP₆. D, inorganic phosphate (Pi). E, total phosphorus (P) content of two independent AtMRP5 mutants and their corresponding wild types. The data represent the means and standard deviations of four to eight independent measurements.

FIGURE 2.

Analysis of cation reserves in seeds of Arabidopsis. Seed content of cations that differ significantly in the two independent AtMRP5 mutants (mrp5-1 and mrp5-2) as compared with their corresponding wild types. The data represent the means and standard deviations of four independent measurements. Na, sodium; Mg, magnesium; K, potassium; Ca, calcium.

FIGURE 3.

AtMRP5 is a high affinity inositol hexakisphosphate transporter. A, inositol hexakisphosphate uptake into microsomes isolated from yeast ycf1 mutant cells transformed with an empty vector (pNEV) or with vector harboring AtMRP5 cDNA (pNEV-MRP5). For non-ATP-dependent uptake, the reaction mix lacked ATP. Transport under all conditions was performed with the same vesicle preparation. Open square, pNEV-ATP; filled square, pNEV+ATP; open circle, pNEV-AtMRP5-ATP; filled circles, pNEV-AtMRP5+ATP. The InsP₆ concentration used for the time-dependent uptake was 80 nm. B, partisphere SAX HPLC analysis of the ³³P recovered from filtered and washed microsomes showing the integrity of inositol hexakisphosphate after transport. C, inositol hexakisphosphate uptake by microsomes isolated from yeast cells harboring AtMRP5. Uptake velocities were measured at different inositol hexakisphosphate concentrations, as indicated. D, a double reciprocal plot was used to determine the K_m value of AtMRP5.

FIGURE 4.

Profiles of inositol phosphates from [³H]inositol-labeled seedlings. A, traces for Ws-2 labeled with low phosphate (black line) and high phosphate (red line). B, traces for mrp5-1 labeled with low phosphate (black line) and high phosphate (red line) and for ipk1-l labeled with high phosphate (blue line). Note that the retention time of inositol phosphates can vary; InsP₆ was confirmed for all genotypes by spiking parallel samples with an InsP₆ standard. The traces for mrp5-1 and Ws-2 labeled with high phosphate and the ipk1-1 trace have been offset on the y axis to aid visualization.

FIGURE 5.

Guard cell-targeted expression of AtMRP5 restores stomatal phenotype in AtMRP5 mutant. A and B, the MYB60 promoter targets GUS exclusively to guard cells. C, the mrp5-1 mutant is insensitive to ABA (ABA inhibits opening of stomata in response to light). The MYB60::AtMRP5 transgene restores ABA sensitivity to mrp5-1. The results for Ws-2, mrp5-1, and three independent transgenic T3 lines are shown. At least 200 stomates of abaxial epidermal strips were measured for each genotype. Two independent experiments were performed, and three additional T3 lines of the transgenic gave the same results (data not shown). The error bars represent the S.E. D, the MYB60::AtMRP5 transgene does not complement the seed inositol hexakisphosphate content of mrp5-1. The data represent the means and standard deviations of three independent measurements. For C, the p value indicates the confidence of significance between light treatment and light+ABA treatment within the same genotype. For D, the p value indicates the confidence of significance between wild type and the four other genotypes.

FIGURE 6.

Hypothetical model that links AtMRP5, InsP₆ signaling and guard cell movements. In wild type plants, inositol hexakisphosphate stimulates the release of Ca²⁺ by specific channels and inhibits the K⁺ inward channel. To avoid continuous InsP₆ signaling, inositol hexakisphosphate has to be transported into the vacuole by AtMRP5. AtMRP5 mutant plants are impaired in the export of InsP₆ into the vacuole. Increased concentrations of cytosolic InsP₆ might complex divalent cations or induce continuous Ca²⁺ release, thus disturbing Ca²⁺-dependent signaling pathways. Furthermore, during the light period, increased InsP₆ levels may reduce K⁺ uptake into guard cells by inhibiting K⁺ inward rectifying channels.