Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis

CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)–related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.     

Environmental Nitrate Stimulates Abscisic Acid Accumulation in Arabidopsis Root Tips by Releasing It from Inactive Stores

Abscisic acid (ABA) signaling plays a major role in root system development, regulating growth and root architecture. However, the precise localization of ABA remains undetermined. Here, we present a mechanism in which nitrate signaling stimulates the release of bioactive ABA from the inactive storage form, ABA-glucose ester (ABA-GE). We found that ABA accumulated in the endodermis and quiescent center of Arabidopsis thaliana root tips, mimicking the pattern of SCARECROW expression, and (to lower levels) in the vascular cylinder. Nitrate treatment increased ABA levels in root tips; this stimulation requires the activity of the endoplasmic reticulum-localized, ABA-GE-deconjugating enzyme β-GLUCOSIDASE1, but not de novo ABA biosynthesis. Immunogold labeling demonstrated that ABA is associated with cytoplasmic structures near, but not within, the endoplasmic reticulum. These findings demonstrate a mechanism for nitrate-regulated root growth via regulation of ABA accumulation in the root tip, providing insight into the environmental regulation of root growth.     

Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.     

Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases

Abscisic acid (ABA) induces stomatal closure and inhibits light-induced stomatal opening. The mechanisms in these two processes are not necessarily the same. It has been postulated that the ABA receptors involved in opening inhibition are different from those involved in closure induction. Here, we provide evidence that four recently identified ABA receptors (PYRABACTIN RESISTANCE1 [PYR1], PYRABACTIN RESISTANCE-LIKE1 [PYL1], PYL2, and PYL4) are not sufficient for opening inhibition in Arabidopsis (Arabidopsis thaliana). ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant. ABA inhibition of the opening of the mutant’s stomata remained intact. ABA did not induce either the production of reactive oxygen species and nitric oxide or the alkalization of the cytosol in the quadruple mutant, in accordance with the closure phenotype. Whole cell patch-clamp analysis of inward-rectifying K⁺ current in guard cells showed a partial inhibition by ABA, indicating that the ABA sensitivity of the mutant was not fully impaired. ABA substantially inhibited blue light-induced phosphorylation of H⁺-ATPase in guard cells in both the mutant and the wild type. On the other hand, in a knockout mutant of the SNF1-related protein kinase, srk2e, stomatal opening and closure, reactive oxygen species and nitric oxide production, cytosolic alkalization, inward-rectifying K⁺ current inactivation, and H⁺-ATPase phosphorylation were not sensitive to ABA.The phytohormone abscisic acid (ABA), which is synthesized in response to abiotic stresses, plays a key role in the drought hardiness of plants. Reducing transpirational water loss through stomatal pores is a major ABA response (Schroeder et al., 2001). ABA promotes the closure of open stomata and inhibits the opening of closed stomata. These effects are not simply the reverse of one another (Allen et al., 1999; Wang et al., 2001; Mishra et al., 2006).A class of receptors of ABA was identified (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009; Nishimura et al., 2010). The sensitivity of stomata to ABA was strongly decreased in quadruple and sextuple mutants of the ABA receptor genes PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE-LIKE/REGULATORY COMPONENT OF ABSCISIC ACID RECEPTOR (PYR/PYL/RCAR; Nishimura et al., 2010; Gonzalez-Guzman et al., 2012). The PYR/PYL/RCAR receptors are involved in the early ABA signaling events, in which a sequence of interactions of the receptors with PROTEIN PHOSPHATASE 2Cs (PP2Cs) and subfamily 2 SNF1-RELATED PROTEIN KINASES (SnRK2s) leads to the activation of downstream ABA signaling targets in guard cells (Cutler et al., 2010; Kim et al., 2010; Weiner et al., 2010). Studies of Commelina communis and Vicia faba suggested that the ABA receptors involved in stomatal opening are not the same as the ABA receptors involved in stomatal closure (Allan et al., 1994; Anderson et al., 1994; Assmann, 1994; Schwartz et al., 1994). The roles of PYR/PYL/RCAR in either stomatal opening or closure remained to be elucidated.Blue light induces stomatal opening through the activation of plasma membrane H⁺-ATPase in guard cells that generates an inside-negative electrochemical gradient across the plasma membrane and drives K⁺ uptake through voltage-dependent inward-rectifying K⁺ channels (Assmann et al., 1985; Shimazaki et al., 1986; Blatt, 1987; Schroeder et al., 1987; Thiel et al., 1992). Phosphorylation of the penultimate Thr of the plasma membrane H⁺-ATPase is a prerequisite for blue light-induced activation of the H⁺-ATPase (Kinoshita and Shimazaki, 1999, 2002). ABA inhibits H⁺-ATPase activity through dephosphorylation of the penultimate Thr in the C terminus of the H⁺-ATPase in guard cells, resulting in prevention of the opening (Goh et al., 1996; Zhang et al., 2004; Hayashi et al., 2011). Inward-rectifying K⁺ currents (I_Kin) of guard cells are negatively regulated by ABA in addition to through the decline of the H⁺ pump-driven membrane potential difference (Schroeder and Hagiwara, 1989; Blatt, 1990; McAinsh et al., 1990; Schwartz et al., 1994; Grabov and Blatt, 1999; Saito et al., 2008). This down-regulation of ion transporters by ABA is essential for the inhibition of stomatal opening.A series of second messengers has been shown to mediate ABA-induced stomatal closure. Reactive oxygen species (ROS) produced by NADPH oxidases play a crucial role in ABA signaling in guard cells (Pei et al., 2000; Zhang et al., 2001; Kwak et al., 2003; Sirichandra et al., 2009; Jannat et al., 2011). Nitric oxide (NO) is an essential signaling component in ABA-induced stomatal closure (Desikan et al., 2002; Guo et al., 2003; Garcia-Mata and Lamattina, 2007; Neill et al., 2008). Alkalization of cytosolic pH in guard cells is postulated to mediate ABA-induced stomatal closure in Arabidopsis (Arabidopsis thaliana) and Pisum sativum and Paphiopedilum species (Irving et al., 1992; Gehring et al., 1997; Grabov and Blatt, 1997; Suhita et al., 2004; Gonugunta et al., 2008). These second messengers transduce environmental signals to ion channels and ion transporters that create the driving force for stomatal movements (Ward et al., 1995; MacRobbie, 1998; Garcia-Mata et al., 2003).In this study, we examined the mobilization of second messengers, the inactivation of I_Kin, and the suppression of H⁺-ATPase phosphorylation evoked by ABA in Arabidopsis mutants to clarify the downstream signaling events of ABA signaling in guard cells. The mutants included a quadruple mutant of PYR/PYL/RCARs, pyr1/pyl1/pyl2/pyl4, and a mutant of a SnRK2 kinase, srk2e.     

Abscisic Acid Mediates a Divergence in the Drought Response of Two Conifers

During water stress, stomatal closure occurs as water tension and levels of abscisic acid (ABA) increase in the leaf, but the interaction between these two drivers of stomatal aperture is poorly understood. We investigate the dynamics of water potential, ABA, and stomatal conductance during the imposition of water stress on two drought-tolerant conifer species with contrasting stomatal behavior. Rapid rehydration of excised shoots was used as a means of differentiating the direct influences of ABA and water potential on stomatal closure. Pinus radiata (Pinaceae) was found to exhibit ABA-driven stomatal closure during water stress, resulting in strongly isohydric regulation of water loss. By contrast, stomatal closure in Callitris rhomboidea (Cupressaceae) was initiated by elevated foliar ABA, but sustained water stress saw a marked decline in ABA levels and a shift to water potential-driven stomatal closure. The transition from ABA to water potential as the primary driver of stomatal aperture allowed C. rhomboidea to rapidly recover gas exchange after water-stressed plants were rewatered, and was associated with a strongly anisohydric regulation of water loss. These two contrasting mechanisms of stomatal regulation function in combination with xylem vulnerability to produce highly divergent strategies of water management. Species-specific ABA dynamics are proposed as a central component of drought survival and ecology.By guarding the interface between plant and atmosphere, the stomata of land plants occupy a uniquely important role that connects diverse aspects of plant biology with atmospheric processes. Capitalizing upon the potential for stomata to be used to modify plant growth and survival, or as a tool for interpreting environmental change, requires a mechanistic understanding of how these tiny valves operate. Yet, an integrated understanding of stomatal control remains elusive. Foremost in this uncertainty is an explanation for how complex signals from the environment are translated into guard cell movement. A particularly challenging feature of stomatal behavior is the fact that environmental perturbation induces both physical and chemical responses within the plant and that turgor-regulated stomata are responsive to both signals. Disentangling these distinct contributions to stomatal conductance (g_s) has been made more complicated by the limited communication between molecular-scaled disciplines of mutant characterization and membrane transport biology and researchers at the larger scale of plant water relations and xylem transport. As a result, two contrasting views of stomatal control exist. Molecular biologists view stomata as osmotically regulated valves uniquely responsive to plant hormone levels and the resultant movement of ions across the guard cell membranes (Schroeder et al., 2001; Roelfsema and Hedrich, 2005). By contrast, most process-based models assume a direct influence of soil water content on stomatal aperture (Buckley, 2005; Damour et al., 2010).The phytohormone abscisic acid (ABA) is seen as a cornerstone of stomatal function because it has been shown to trigger responses in guard cell membrane channels and transporters that cause a reduction in guard cell turgor, thereby closing stomata. ABA-mediated stomatal closure in seed plants (but not in ferns and lycophytes; Brodribb and McAdam, 2011) is broadly accepted as the explanation for stomatal closure during water stress (Zhang and Davies, 1989; Bauer et al., 2013); yet, there are very few studies that show a good correlation between the level of ABA and g_s during water stress in the field. The traditional explanation for this lack of a strong relationship suggests that ABA is a root-derived hormone that is delivered to the leaf in the transpiration stream (Zhang et al., 1987; Davies and Zhang, 1991) and hence that the xylem ABA flux, rather than the leaf level of ABA, should dictate the intensity of the stomatal response to soil drying (Tardieu et al., 1992; Tardieu and Davies, 1993). The flux-based model for ABA action in the leaf remains the most widely used interpretation of how stomata sense and respond to drying soil, despite the fact that there is mounting evidence for significant ABA synthesis in the leaf and guard cells, and short term responses to ABA that cannot be explained by xylem transport (Christmann et al., 2005; Lee et al., 2006; Georgopoulou and Milborrow, 2012). Furthermore, the ABA flux approach has never been successfully applied to explain variation in transpiration in trees (Sperry, 2000; Cochard et al., 2002), suggesting that there may be some benefit in reexamining some of the principles and assumptions used to link water stress, ABA, and transpiration.Here, we examine the dynamics of stomatal closure, leaf ABA levels, and xylem tension during the gradual imposition of water stress upon two conifer species, Pinus radiata and Callitris rhomboidea, known for having contrasting stomatal responses to desiccation. Our primary aim is to separate the interacting effects of ABA and water tension on guard cell turgor pressure and stomatal diffusive conductance and hence to reveal the relative importance of water tension and ABA levels during drought as effectors of stomatal closure. Conifers are particularly suitable for identifying different closing signals because they do not appear to produce hydropassive stomatal movements (McAdam and Brodribb, 2012). This makes them ideal for examining the direct effects of ABA and water tension without the mechanical interactions between subsidiary cells and guard cells (Franks and Farquhar, 2007) that greatly complicate the mechanics of angiosperm stomatal movements. Both conifer species examined grow naturally in low rainfall habitats, but P. radiata is strongly isohydric (meaning that stomata close in a very narrow range of leaf hydration), while C. rhomboidea is anisohydric (meaning that stomata have a relatively low sensitivity to leaf hydration).     

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.Plant vacuoles are vital organelles for maintaining cell volume and cell turgor, regulating ion homeostasis and pH, disposing toxic materials, and storing and degrading unwanted proteins (Marty, 1999). To perform these diverse functions, vacuoles require an array of different and complex proteins. These proteins are synthesized at the endoplasmic reticulum (ER) and are transported to the vacuole through the vacuolar trafficking pathway. Perturbation of the vacuolar trafficking machinery affects many cellular processes, including tropisms, responses to pathogens, cytokinesis, hormone transport, and signal transduction (Surpin and Raikhel, 2004). The vacuolar trafficking system is comprised of several compartments: the ER, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), and the vacuole. Vacuolar proteins synthesized at the ER are transported to the cis-Golgi via coat protein complex II (COPII) vesicles and are then transported to the TGN through the Golgi apparatus. In the TGN, proteins are sorted for delivery to their respective locations according to their targeting signal. Vacuolar proteins carrying a vacuolar sorting signal are thought to be recognized by vacuolar sorting receptors (VSRs), which are mainly located in the PVC, although sorting of vacuolar proteins may also occur at the ER and VSRs can be recycled from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). Multiple studies suggest that plant VSRs serve as sorting receptors both for lytic vacuole proteins (daSilva et al., 2005; Foresti et al., 2006; Kim et al., 2010) and for storage vacuole proteins (Shimada et al., 2003; Fuji et al., 2007; Zouhar et al., 2010).Osmotic stress is commonly associated with many environmental stresses, including drought, cold, and high soil salinity, that have a severe impact on the productivity of agricultural plants worldwide. Therefore, understanding how plants perceive and respond to osmotic stress is critical for improving plant resistance to abiotic stresses (Zhu, 2002; Fujita et al., 2013). It has long been recognized that osmotic stress can activate several signaling pathways that lead to changes in gene expression and metabolism. One important regulator of these signaling pathways is the phytohormone abscisic acid (ABA), which accumulates in response to osmotic stress. ABA regulates many critical processes, such as seed dormancy, stomatal movement, and adaptation to environmental stress (Finkelstein and Gibson, 2002; Xiong and Zhu, 2003; Cutler et al., 2010). De novo synthesis of ABA is of primary importance for increasing ABA levels in response to abiotic stress. ABA is synthesized through the cleavage of a C40 carotenoid originating from the 2-C-methyl-d-erythritol-4-phosphate pathway, followed by a conversion from zeaxanthin to violaxanthin catalyzed by the zeaxanthin epoxidase ABA1 and then to neoxanthin catalyzed by the neoxanthin synthase ABA4. Subsequently, a 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves the violaxanthin and neoxanthin to xanthoxin. Xanthoxin, in turn, is oxidized by a short-chain alcohol dehydrogenase (ABA2) to abscisic aldehyde, which is converted to ABA by abscisic acid aldehyde oxidase3 (AAO3) using a molybdenum cofactor activated by the molybdenum cofactor sulfurase (ABA3; Nambara and Marion-Poll, 2005). In this pathway, it is generally thought that the cleavage step catalyzed by NCED is the rate-limiting step (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). In Arabidopsis (Arabidopsis thaliana), five members of the NCED family (NCED2, NCED3, NCED5, NCED6, and NCED9) have been characterized (Tan et al., 2003). Of those, NCED3 has been suggested to play a crucial role in ABA biosynthesis, and its expression is induced by dehydration and osmotic stress (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). Thus, understanding how the NCED3 gene is activated in response to osmotic stress is important for the elucidation of the mechanisms that govern plant acclimation to abiotic stress.We have used the firefly luciferase reporter gene driven by the stress-responsive NCED3 promoter to enable the genetic dissection of plant responses to osmotic stress (Wang et al., 2011). Here, we report the characterization of a unique regulator of ABA biosynthesis, 9-cis Epoxycarotenoid Dioxygenase Defective2 (CED2). The ced2 mutants are impaired in osmotic stress tolerance and are defective in the expression of genes required for ABA synthesis and consequently osmotic stress-induced ABA accumulation. The CED2 gene encodes VSR1, previously known to be involved in vacuolar trafficking but not known to be critical for osmotic stress induction of ABA biosynthesis and osmotic stress tolerance. Our study further suggests that intracellular pH changes might act as an early stress response signal triggering osmotic stress-activated ABA biosynthesis.     

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.