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

Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar β-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar K_m values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites.Abscisic acid (ABA) is a major plant hormone involved in various physiological and developmental processes. ABA signaling is fundamental in plant responses to abiotic stresses, including drought, cold, osmotic, and salt stress (Cutler et al., 2010). The best-characterized function of ABA is the regulation of stomatal aperture in response to environmental signals, such as soil and air humidity, temperature, and CO₂ concentration (Nilson and Assmann, 2007; Kim et al., 2010). However, ABA also has important functions in seed development, dormancy, and germination (Holdsworth et al., 2008), lateral root formation (Galvan-Ampudia and Testerink, 2011), and leaf senescence (Lim et al., 2007). Besides, ABA is not restricted only to plants; it was also identified to have functions in species from all kingdoms, including humans, and may even have universal functions (e.g. in UV-B stress response; Tossi et al., 2012).ABA is synthesized de novo from the carotenoid zeaxanthin, whereby the first ABA-specific biosynthetic step occurs in the plastid and the final two steps take place in the cytosol (Nambara and Marion-Poll, 2005). The catabolism of ABA is mediated via oxidative and Glc conjugation pathways (Nambara and Marion-Poll, 2005). The ABA 8′-hydroxylation catalyzed by P450 cytochromes of the CYP707A subfamily represents the predominant catabolic pathway of ABA and has been demonstrated to be a key regulatory step in ABA action (Kushiro et al., 2004). The major oxidative ABA catabolites, phaseic acid (PA) and dihydroxyphaseic acid (DPA), exhibit lower and no biological activity, respectively (Sharkey and Raschke, 1980; Kepka et al., 2011). The conjugation of ABA and its oxidative catabolites PA and DPA with Glc catalyzed by UDP-glucosyltransferases represents the other mechanism of ABA inactivation. Abscisic acid glucosyl ester (ABA-GE) appears to be the major conjugate, which was found in various organs of different plant species (Piotrowska and Bajguz, 2011). In contrast to the oxidative pathway, the inactivation of ABA by Glc conjugation is reversible, and hydrolysis of ABA-GE catalyzed by β-glucosidases results in free ABA (Dietz et al., 2000; Lee et al., 2006; Xu et al., 2012). ABA-GE levels were shown to substantially increase during dehydration and specific seed developmental and germination stages (Boyer and Zeevaart, 1982; Hocher et al., 1991; Chiwocha et al., 2003). Furthermore, ABA-GE is present in the xylem sap, where it was shown to increase under drought, salt, and osmotic stress (Sauter et al., 2002). Apoplastic ABA β-glucosidases in leaves have been suggested to mediate the release of free ABA from xylem-borne ABA-GE (Dietz et al., 2000). Therefore, ABA-GE was proposed to be a root-to-shoot signaling molecule. However, under drought stress, ABA-mediated stomatal closure occurs independently of root ABA biosynthesis (Christmann et al., 2007). Thus, the involvement of ABA-GE in root-to-shoot signaling of water stress conditions remains to be revealed (Goodger and Schachtman, 2010).The intracellular compartmentalization of ABA and its catabolites is important for ABA homeostasis (Xu et al., 2013). Free ABA, PA, and DPA mainly occur in the extravacuolar compartments. In contrast to these oxidative ABA catabolites, ABA-GE has been reported to accumulate in vacuoles (Bray and Zeevaart, 1985; Lehmann and Glund, 1986). Since the sequestered ABA-GE can instantaneously provide ABA via a one-step hydrolysis, this conjugate and its compartmentalization may be of importance in the maintenance of ABA homeostasis. The identification of the endoplasmic reticulum (ER)-localized β-glucosidase AtBG1 that specifically hydrolyzes ABA-GE suggests that ABA-GE is also present in the ER (Lee et al., 2006). Plants lacking functional AtBG1 exhibit pronounced ABA-deficiency phenotypes, including sensitivity to dehydration, impaired stomatal closure, earlier germination, and lower ABA levels. Hydrolysis of ER-localized ABA-GE, therefore, represents an alternative pathway for the generation of free cytosolic ABA (Lee et al., 2006; Bauer et al., 2013). This finding raised the question of whether vacuolar ABA-GE also has an important function as an ABA reservoir. This hypothesis was supported by recent identifications of two vacuolar β-glucosidases that hydrolyze vacuolar ABA-GE (Wang et al., 2011; Xu et al., 2013). The vacuolar AtBG1 homolog AtBG2 forms high molecular weight complexes, which are present at low levels under normal conditions but significantly accumulate under dehydration stress. AtBG2 knockout plants displayed a similar, although less pronounced, phenotype to AtBG1 mutants: elevated sensitivity to drought and salt stress, while overexpression of AtBG2 resulted in exactly the opposite effect (i.e. increased drought tolerance). The other identified vacuolar ABA-GE glucosidase, BGLU10, exhibits comparable mutant phenotypes to AtBG2 (Wang et al., 2011). This redundancy may explain the less pronounced mutant phenotypes of vacuolar ABA-GE glucosidases compared with the ER-localized AtBG1. Moreover, the fact that overexpression of the vacuolar AtBG2 is able to phenotypically complement AtBG1 deletion mutants indicates an important role of vacuolar ABA-GE as a pool for free ABA during the abiotic stress response (Xu et al., 2012).The described accumulation and functions of vacuolar ABA-GE raise the question of by which mechanisms ABA-GE is sequestered into the vacuoles. To answer this question, we synthesized radiolabeled ABA-GE and characterized the ABA-GE transport into isolated mesophyll vacuoles. We showed that the vacuole comprises two distinct transport systems involved in the accumulation of ABA-GE: proton gradient-dependent and directly energized ATP-binding cassette (ABC)-type transport. In a targeted approach, we furthermore show that the Arabidopsis (Arabidopsis thaliana) ABC transporters AtABCC1 and AtABCC2 exhibit ABA-GE transport activity in vitro.