ABA transport factors found in Arabidopsis ABC transporters

Abstract

Abscisic acid (ABA) is a phytohormone that plays an important role in responses to environmental stresses as well as seed maturation and germination. Intracellular signaling by ABA has been rigorously investigated in relation to stomatal guard-cell regulation, seed germination and abiotic stress responses. However, intercellular regulation of ABA, including the molecular basis of ABA transport systems, has hardly been examined in any plant species. Based on genetic and biochemical analyses, we present evidence that one of the ATP-binding cassette (ABC) transporter genes, AtABCG25, encodes a protein that functions as an ABA exporter through the plasma membrane and is involved in the intercellular ABA signaling pathway. The ABC-type transporter is conserved in model species from E. coli to humans and is reported to transport various metabolites or signaling molecules in an ATP-dependent manner. At same time, another ABC transporter in Arabidopsis, AtABCG40, was independently reported to function as an ABA importer in plant cells. These findings strongly suggest the active control of ABA transport between plant cells, and they provide a novel impetus for examining ABA intercellular regulation.

Functional Analysis of the AtABCG25 Gene

We present evidence that one of the ATP-binding cassette (ABC) transporter genes, AtABCG25, encodes a protein that is responsible for ABA transport and responses in Arabidopsis. We originally isolated atabcg25 mutants by genetic screening of ABA hypersensitive mutants. The corresponding gene, AtABCG25, is a member of the ABCG subfamily of putative ABC transporters in the Arabidopsis genome (Fig. 1).^1,2 ABC transporters are broadly conserved from prokaryotes to higher eukaryotes.³ In particular, plants have larger ABC families in their genomes, implying that some ABC transporters have plant-specific and important functions in development or responses.^4–8 As ABC transporters are localized to various membranes in vivo, we investigated the subcellular localization of AtABCG25 in plant cells. The fluorescent protein-fused AtABCG25 was localized at the plasma membrane. By using the reporter gene to detect the promoter activity, we found AtABCG25 was expressed mainly in vascular tissues. Interestingly, ABA is predominantly biosynthesized in vascular parenchyma cells.^9–11 These data led us to hypothesize that AtABCG25 is an ABA transporter that excretes ABA, and atabcg25 mutants are ABA-hypersensitive under ABA treatment because they cannot prevent entry of excess ABA into the cells. Therefore, we studied whether AtABCG25 had ABA efflux activity. However, it was very difficult to obtain stable ABA molecules inside cells to detect the efflux activity in vivo. Hence, we used a vesicle transport assay to detect the intrinsic export function through uptake of labeled molecules.^12,13 We were able to practically assess the involvement of AtABCG25 after addition of ATP, which the ABC transporter requires for operation.³ These results provide strong evidence that AtABCG25 is an ABA exporter in plant cells.

Figure 1.

ABCG subfamily of ABC transporters in Arabidopsis. ABCG subfamily is the largest one in Arabidopsis ABC transporters, composed of the half-size type (from 1 to 28) and the full-size type (since 29).¹ AtABCG25 is a member of half-size type ABC transporters, previously called AtWBC26; AtABCG40 is a member of full-size type ABC transporters, previously called AtPDR 12. The phylogenetic relationship in this figure is referred to the report by Sanchez-Fernandez et al.²

Phenotypes of AtABCG25-Overexpressing Plants

We further characterized the AtABCG25-overexpressing transgenic plants. At the seedling stage, ABA inhibition of post-germinative growth was significantly reduced in AtABCG25-overexpressing transgenic plants, i.e., AtABCG25 overexpression led to ABA-insensitive phenotypes.¹⁴ This overexpression phenotype was totally opposite to the knockout mutant phenotype, supporting the conjecture that AtABCG25 functions as an efflux factor of ABA. On the other hand, in the adult stage, AtABCG25-overexpressing plants had less transpiration from rosette leaves, indicating ABA sensitivity.¹⁴ We propose two possible interpretations for this difference in apparent ABA sensitivity between seedlings and adult leaves of overexpressing plants. First, the 35S promoter does not work well in guard cells, and insufficient ABA would be excreted from the guard cells.¹⁵ Second, even when ABA is readily excreted from guard cells in overexpressing plants, the guard cells are still more sensitive to ABA than other cells such as mesophyll cells. In latter case, more stomata would be closed in AtABCG25 overexpressing plants than in wild-type plants (even under well-watered conditions) because excreted ABA could be delivered into the transpiration stream to guard cells, resulting in stomatal closure. Interestingly, this is compatible with a recent study on AtABCG40 (Fig. 1) by Kang et al. who reported that AtABCG40 mediated ABA uptake for cellular influx in Arabidopsis and also showed that AtABCG40 was highly expressed in guard cells.¹⁶ Hence, we can propose a simple model: ABA is exported from ABA-biosynthesizing cells to the apoplastic area by AtABCG25; then, ABA is imported from the apoplast to the inside of guard cells by AtABCG40 (Fig. 2). Thus, stomatal closure through AtABCG25 overexpression was enhanced, possibly through the influence of AtABCG40 ABA influx activity in guard cells.

Figure 2.

Schematic view of hypothetical ABA intercellular transmission. This diagram is an Arabidopsis leaf section showing two distinct cell types: vascular tissues including vascular parenchyma cells and guard cells on the leaf epidermis. AtABCG25 could function as an ABA exporter from ABA-biosynthesizing cells; ABA would diffuse into apoplastic areas. AtABCG40 could function as an ABA importer from outside to inside guard cells to facilitate stomatal closure.

Active Control of ABA Transport in Plant Cells

From analyses of AtABCG25 and AtABCG40, active control of ABA intercellular transport can be proposed (Fig. 2). We can infer the necessity of ABA transporters across plasma membranes from the viewpoint of an anion trap.^17,18 Because ABA is a weak acid (pKa 4.7), it cannot passively pass through the lipid of plasma membranes, as it is mostly in the ionized form in the cytosol, where the pH is around neutral.^16–18 AtABCG25 would be necessary for ABA diffusion from inside to outside cells over the anion trap. Similarly, stress conditions elevate the apoplastic pH, so AtABCG40 would be necessary for cellular uptake of ionized ABA at guard cells, particularly under stress conditions.¹⁶ In addition, this is consistent with recent reports that some ABA receptors that trigger ABA signaling in cells are localized in the cytosol.^19–22 Interestingly, the Km saturation kinetics of ATP-dependent ABA transport are 260 nM and 1 µM for AtABCG25 and AtABCG40, respectively, although different assay systems were used to calculate activity.¹⁶

Auxin is another major phytohormone that coordinates plant development.²³ Recently, the auxin transport system was found to include both cellular efflux and influx carriers.^24,25 In current models, auxin basically migrates to adjacent cells through polar transport to regulate disproportionate cell growth.^26,27 However, the stress hormone ABA is required for rapid signaling, especially under stress conditions.²⁸ For example, under drought, ABA promotes stomatal closure in guard cells to prevent transpiration.^14,22,28 It may be that ABA spreads rapidly to peripheral cells to cope with environmental changes. We consider that the ABA transport system developed for rapid transmission of ABA molecules and effective distribution of stress signaling between plant cells.

Addendum to: Kuromori T, Miyaji T, Yabuuchi H, Shimizu H, Sugimoto E, Kamiya A, et al. ABC transporter AtABCG25 is involved in abscisic acid transport and responses. Proc Natl Acad Sci USA. 2010 doi: 10.1073/pnas.0912516107.