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.
Highlights
Surround a central pore, the stoma, through which gas exchange occurs
Because AtMRP5 is strongly expressed in seeds, we were interested whether knock-out mutants of this ABC transporter exhibit the same low inositol hexakisphosphate phenotype
Because we have shown that inositol hexakisphosphate is a physiological regulator of vacuolar (Ca2ϩ-permeable) and plasma membrane conductances [13, 14], we sought to determine a role for AtMRP5 in guard cell function
Summary
Chemical Analysis of AtMRP5 Loss-of-function Mutants— Fifty milligrams of dried seed material was incinerated for 8 h at 550 °C, and subsequently the ash was solubilized in 2 ml of 6 N HCl, briefly heated up to 100 °C, purified through Whatman No 40 ashless filter paper, and transferred to double-distilled water to a total volume of 50 ml. Inositol hexakisphosphate uptake was terminated by the transfer of three aliquots, equivalent to 18.9 nCi (700 Bq) of starting material, onto 0.45-m-diameter pore size nitrocellulose filters. Under these conditions, the rate of net transport was linear with time during the first 5.5 min. After 8 min, Ins(1,[33P]2,3,4,5,6)P6 was added to the reaction mix, and inositol hexakisphosphate uptake was determined as above. Partisphere SAX HPLC—Extracts of radiolabeled Arabidopsis seedlings, products of transport assays, and preparations of Ins(1,[33P]2,3,4,5,6)P6 were all analyzed by anion exchange HPLC on a 23.5-cm ϫ 4.6-mm internal diameter Partisphere SAX WVS cartridge with guard cartridge [18]. Arabidopsis plants (Ws-2, mrp, and MYB60::MRP5, independent homozygous transformed lines from the T3 generation) were grown in soil in a phytotron
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