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.Guard cells form pairs of cells, which are conjoined at their ends, in the epidermis of the aerial tissues of plants. The cells surround a central pore, the stoma, through which gas exchange occurs. The principal gases exchanged are CO 2 and water vapor, and the function of the stomatal complex may be considered as the maximization of CO 2 assimilation by photosynthesis for the minimization of water loss.Guard cells and hence the aperture of the central pore are sensitive to environmental factors including light, temperature, CO 2 , and ozone (1). Stomatal closure is initiated by the drought stress hormone abscisic acid (ABA).3 The closure of stomata is a result of a loss of turgor of the delimiting guard cells as a consequence of ion efflux, predominantly Cl Ϫ and K ϩ , and metabolic conversion of organic acids into starch (2).Although the molecular identity of genes encoding the outward and inward K ϩ conductances are known for Arabidopsis thaliana (3), it remains to be demonstrated whether the recently identified SLAC protein (4, 5) encodes the S-type anion channel or is a subunit thereof.The ATP-binding cassette family of membrane proteins is among the most ubiquitous and variable group of membrane proteins and is most commonly associated with membrane transport phenomena. The substrates transported are especially diverse, and consequently a major obstacle to the interpretation of ABC transporter function, particularly pertinent in the context of guard cell function, is the identificatio...

Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters

“…Members of this family have also been shown to transport GSH conjugates and to confer cadmium resistance in plants and humans (23)(24)(25)(26). In addition, for many ABCC proteins in Arabidopsis, vacuolar localization has been demonstrated or proposed on the basis of data obtained using either Western blotting of membrane fractions, GFP fusion proteins, or vacuolar proteomics approaches (27)(28)(29)(30). We have therefore isolated homozygous knockout lines for all 15 Arabidopsis ABCC genes and have grown these mutants in the presence of arsenic and arseniccontaining herbicides.…”

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

“…However, AtABCC2 was previously shown to exhibit a higher transport activity for several substrates compared with AtABCC1 when expressed in the same pYES3 vector (Lu et al, 1998). To validate that MgATPactivated uptake of ABA-GE into yeast vesicles expressing AtABCC2 has the characteristics of ABC transportermediated transport, we tested the effects of the ABC transporter inhibitors orthovanadate and probenecid (Nagy et al, 2009) on AtABCC2-expressing yeast vesicles. The presence of 1 mM orthovanadate and 1 mM probenecid strongly inhibited the MgATP-enhanced ABA-GE uptake by 92% and 90%, respectively, which corresponds to the uptake activity in the absence of MgATP (Table II).…”

Ion Transport at the Vacuole during Stomatal Movements

“…In patch-clamp experiments, it was found that mrp5 mutant guard cells displayed reduced ABA activation of plasma membrane Ca 2+ and S-type anion currents as well as reduced activation of anion currents by cytoplasmic Ca 2+ (Suh et al, 2007). While it was initially thought that the transporter localizes to the plasma membrane, it was later reasoned that it is vacuole localized (Nagy et al, 2009). To explain the observed mutant phenotypes, the authors hypothesized that excess cytosolic IP 6 in the mrp5 mutant might complex cytosolic Ca 2+ or increase its release from the vacuole through the stimulation of putative IP 6 -sensitive vacuolar Ca 2+ channels.…”

“…It is a guard cell-expressed ABC-type transporter that mediates high-affinity uptake of IP 6 into vacuoles (Nagy et al, 2009). Although AtMRP5/AtABCC5 also is expressed in other cell types, mrp5 knockout mutant plants show strong guard cell mutant phenotypes (Klein et al, 2003).…”

“…(1) Nagy et al (2009); (2) Klein et al (2003) Arabidopsis guard cell Mal 22 concentration increases during stomatal opening 2-to 3-fold and generally correlates with stomatal aperture (Monda et al, 2011;Ding et al, 2014;Takahashi et al, 2015;Medeiros et al, 2016). We have gained good understanding of the importance of individual transport processes at the vacuole.…”