Revisiting the Basal Role of ABA – Roles Outside of Stress

Trends in Plant Science

Review

Revisiting the Basal Role of ABA – Roles Outside of Stress Takuya Yoshida,1,4,* Alexander Christmann,2 Kazuko Yamaguchi-Shinozaki,3 Erwin Grill,2 and Alisdair R. Fernie1,4 The physiological roles of abscisic acid (ABA) as a stress hormone in plant responses to water shortage, including stomatal regulation and gene expression, have been well documented. However, less attention has been paid to the function of basal ABA synthesized under well-watered conditions in recent studies. In this review, we summarize progress in the understanding of how low concentrations of ABA are perceived at the molecular level and how its signaling affects plant metabolism and growth under nonstressed conditions. We also discuss the dual nature of ABA in acting as an inhibitor and activator of plant growth and development.

Highlights

A Plant Hormone with Many Faces

Basal ABA levels support plant growth and development via a beneficial effect on plants’ water status, which comprises proper adjustment of stomatal aperture, stimulation of tissue hydraulic conductivity, and a positive regulatory role in xylem development.

Structural and biological evidence suggests that ABA receptor complexes operate at basal, nonstress ABA levels. Basal ABA balances primary metabolism and leaf growth in arabidopsis. Low levels of ABA play opposite roles in different tissues, inhibitory effects on leaf emergence, and promotion of root growth.

Abscisic acid (ABA; see Glossary) is an isoprenoid-derived phytohormone that accumulates in response to water shortage and seed maturation. ABA controls stomatal aperture and stressresponsive genes, thereby reducing the entry of CO2 into the leaf, which limits photosynthesis and enhances tolerance to unfavorable conditions [1–3]. Some seeds, buds, and fruits contain high levels of ABA and ABA metabolites [4,5], which appear to be associated with maintenance of dormancy and seed development. Although ABA is also involved in plant pathogen responses, such aspects of ABA are well summarized in other reviews [6,7], and are out of the scope of this article. ABA is one of the best-characterized signaling molecules in terms of its biosynthesis and catabolism, transport, and signal transduction. Currently, ABA is known to be synthesized at several sites, such as root and leaf vascular cells, and translocated to its site of action, which includes guard cells [8]. Given that plants have multiple ABA transporters, ABA transport is dynamically regulated within a plant under various growth conditions. ABA signal transduction is, at the first glance, a three-step signal relay, from perception by a binary receptor complex via protein kinases acting as response mediators, to the targets, such as ABA-responsive transcription factors and membrane channels (Figure 1, Key Figure). ABA is perceived by receptors, designated as REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR), PYRABACTIN RESISTANCE1 (PYR1), or PYR1-LIKE (PYL), and clade A protein phosphatases of type 2C (PP2C), which function as coreceptors [9,10]. ABA binding stabilizes the receptor complex, thereby the active site of the phosphatase is blocked and an associated protein kinase is released from PP2C inhibition [11]. This protein kinase belongs to a small subfamily of sucrose non-fermenting-1 (SNF1)-related protein kinase 2s (SnRK2s), of which three have been tightly associated with ABA responses [12–14]. Here we will summarize recent progress in the elucidation of the precise molecular mechanism underlying the ABA receptor complex formation and the dissection of the regulation of metabolism by ABA signaling under nonstress conditions, combining this information with an up-todate signaling model. Based on this synthesis, we will discuss how ABA signaling maintains plant growth, metabolism, and development under well-watered conditions. Trends in Plant Science, July 2019, Vol. 24, No. 7

1

Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 PotsdamGolm, Germany 2 Lehrstuhl für Botanik, Technische Universität München, D-85354 Freising, Germany 3 Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 113-8657 Tokyo, Japan 4 https://www.mpimp-golm.mpg.de

*Correspondence: [email protected] (T. Yoshida).

https://doi.org/10.1016/j.tplants.2019.04.008 © 2019 Elsevier Ltd. All rights reserved.

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Key Figure

Glossary

The Core ABA Signaling Components and Post-translational Regulation

Abscisic acid (ABA): abscisic acid is a weak-acidic plant hormone, named from ‘abscission’, which is currently known to be its indirect target. Ethylene: ethylene (C2H4) is a gaseous plant hormone in growth and development, popularly known to be in ripening. Hydrotropism: the tropic response in roots toward water. Stomata: pores on leaves surrounded by a pair of guard cells, responsible for transpiration and gas exchange. Xylem: the lignified water-transporting tissue in vascular plants.

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Figure 1. The three-step signal relay from perception by a binary receptor complex (RCAR-ABA-PP2C) via proteins kinases (SnRK2) acting as response mediators to the target, such as ABA-responsive transcription factors and membrane channels. The three core ABA signaling components are post-translationally regulated. RCARs are phosphorylated by AELs, CARK1, and/or TOR kinase, which is phosphorylated by SnRK2s. The SnRK2s are phosphorylated and dephosphorylated by BIN2 and EGR2, respectively. Protein abundance of the core components is controlled by multiple E3 ligases and/or adaptor proteins, including DDA1 for RCARs, PUB12/13 and RGLG1/5 for PP2Cs, and PP2-B11 for SnRK2s. RCARs are also degraded via RSL1, an E3 ligase localized to plasma membrane, and a component of the endosomal sorting complex, FYVE1. Intracellular localization of RCARs and PP2Cs are controlled by a small lipid-binding protein (CAR) or a receptorlike kinase (RDK1), respectively. Binding affinity between RCARs and PP2Cs is regulated via GEF and ROP. RCARs are possibly inactivated via Tyr nitration caused by nitric acids. EAR1 and DOG1, proteins of unknown function, appear to regulate activities of PP2Cs. Abbreviations: ABA, abscisic acid; AEL, Arabidopsis EARLY FLOWERING 1 (EL1)-like; BIN2, BRASSINOSTEROID INSENSITIVE 2; CAR, C2-DOMAIN ABA-RELATED; CARK1, CYTOSOLIC ABA RECEPTOR KINASE 1; DDA1, DET1-, DDB1-ASSOCIATED1; DOG1, DELAY OF GERMINATION1; EAR1, ENHANCER OF ABA CORECEPTOR1; EGR2, CLADE-E GROWTH-REGULATING 2; FYVE1, named after proteins harboring a phosphatidylinositol-3-phosphate binding motif: Fab1b, YOTB, Vac1p, and EEA1; GEF, guanine nucleotide exchange factor; PP2-B11, phloem protein 2-B11; PP2C, protein phosphatases of type 2C; PUB, plant U-box type E3 ligase; RCAR, REGULATORY COMPONENTS OF ABA RECEPTOR; PYR1, PYRABACTIN RESISTANCE1; PYL, PYR1-LIKE; RDK1, RECEPTOR DEAD KINASE1; RGLG, RING DOMAIN LIGASE; ROP, plant Rho GTPase; RSL1, RING FINGER OF SEED LONGEVITY1; SnRK2, sucrose non-fermenting-1 (SNF1)-related protein kinase 2; TOR, TARGET OF RAPAMYCIN.

Post-translational Regulation of the Core ABA Signaling Components Recent studies have reported that the three ABA signaling core components are posttranslationally regulated by a number of mechanisms (Figure 1 and Table 1). Phosphorylation/dephosphorylation is a central protein modification regulating protein activities. A reciprocal regulation by phosphorylation has been suggested among RCAR/PYR/PYL receptors, the SnRK2 kinases, and TARGET OF RAPAMYCIN (TOR) kinase [15]. EARLY FLOWERING 1 (EL1)-like casein kinases were also shown to inactivate RCAR/PYR/PYLs via phosphorylation [16]. By contrast, CYTOSOLIC ABA RECEPTOR KINASE 1 (CARK1) was found to phosphorylate RCAR/ PYR/PYLs and enhance the inhibitory effect on the PP2Cs, resulting in activation of ABA signaling [17,18]. Whilst the SnRK2s are thought to be autoactivated by releasing from the PP2Cs in response to ABA [11], a glycogen synthase kinase 3 (GSK3)-like kinase, BRASSINOSTEROID 626

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Table 1. The Upstream Regulators of the Core ABA Signaling Componentsa Regulation/Function

Regulators

Targetsc

References

RCAR/PYR/PYLb

Phosphorylation

Subfamily I

Subfamily II

Subfamily III

PP2C

SnRK2

Inhibition

TOR

RCAR1–4

RCAR8–10

RCAR11–14

–

–

[15]

Destabilization

AEL1/3/4

RCAR2/4

RCAR8–10

RCAR11–14

–

–

[16]

Activation

CARK1

RCAR3

–

RCAR11–14

–

–

[17,18]

Activation

BIN2

–

–

–

–

SRK2D/I

[19]

Dephosphorylation

Inhibition

EGR2

–

–

–

–

SRK2D/E/I

[20]

Ubiquitylation

Degradation

DDA1

RCAR3

–

–

–

–

[21]

Triggering endocytosis

RSL1

–

RCAR10

RCAR11

–

–

[22,23]

Degradation

PUB12/13

–

–

–

ABI1

–

[24]

Degradation

RGLG1/5

–

–

–

ABI2, HAB2, PP2CA

–

[25]

Degradation

PP2-B11

–

–

–

–

SRK2I

[26]

Endosomal trafficking

FYVE1

–

RCAR10

RCAR11

–

–

[23]

Mediating intracellular localization

CAR1/4

RCAR3

RCAR9/10

RCAR11/12

–

–

[27]

Plasma membrane localization

RDK1

–

–

–

ABI1, ABI2, HAB1

–

[28]

Competitive binding

Activation

GEFs–ROP

–

–

–

ABI1, ABI2

–

[29,30]

Tyr nitration

Inhibition

–

RCAR3

–

RCAR11

–

–

[32]

Unknown

Activation

EAR1

–

–

–

ABI1, ABI2, AHG1, HAB1, HAB2, PP2CA

–

[31]

Inhibition

DOG1

–

–

–

AHG1, PP2CA

–

[33,34]

Physical interaction

a

Abbreviations: AEL, Arabidopsis EARLY FLOWERING 1 (EL1)-like; CAR, C2-DOMAIN ABA-RELATED; FYVE1, named after proteins harboring a phosphatidylinositol-3phosphate binding motif: Fab1b, YOTB, Vac1p, and EEA1; PP2-B11, phloem protein 2-B11; PUB, plant U-box type E3 ligase; RDK1, RECEPTOR DEAD KINASE1; RGLG, RING DOMAIN LIGASE; –, not applicable. b RCAR/PYR/PYLs are classified into three subfamilies as in Figure 2: subfamily I (RCAR1/PYL9, RCAR2/PYL7, RCAR3/PYL8, and RCAR4/PYL10), subfamily II (RCAR5/ PYL11, RCAR6/PYL12, RCAR7/PYL13, RCAR8/PYL5, RCAR9/PYL6, and RCAR10/PYL4), and subfamily III (RCAR11/PYR1, RCAR12/PYL1, RCAR13/PYL3, and RCAR14/PYL2). c In addition to the phosphorylation, dephosphorylation, or ubiquitylation targets analyzed in detail, possible targets based on biochemical experiments, such as in vitro phosphorylation or ubiquitylation assays, are listed. Proteins that showed only interaction with the regulators are excluded. For others, possible targets supported by in planta analyses are listed.

INSENSITIVE 2 (BIN2), has been shown to activate the SnRK2s [19]. Besides clade A PP2C phosphatases, a plasma membrane-localized clade E PP2C, CLADE-E GROWTHREGULATING 2 (EGR2), has been proposed to inhibit the SnRK2s [20]. Ubiquitylation is another important protein modification, and protein abundance of core components has been shown to be controlled via multiple ubiquitin ligases. Degradation of several RCAR/PYR/PYL receptors is regulated by at least two types of machineries. Whilst DET1-, DDB1-ASSOCIATED1 (DDA1) functions as a substrate adaptor of the ubiquitin ligase [21], RING FINGER OF SEED LONGEVITY1 (RSL1) is the E3 ligase localized to plasma membrane and mediates trafficking of RCAR10/PYL4 to the vacuole via a component of the endosomal sorting complex [22,23]. Likewise, abundance of clade A PP2Cs, including ABA INSENSITIVE 1 (ABI1) and PP2CA/ABAHYPERSENSITIVE GERMINATION 3 (AHG3), are controlled by different types of ubiquitin ligases, the U-box E3 ligases and the REALLY INTERESTING NEW GENE (RING)-type E3 ligases [24,25]. Interestingly, among the three core SnRK2s, the SRK2I/SnRK2.3 protein seems to be specifically Trends in Plant Science, July 2019, Vol. 24, No. 7

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degraded via interaction with the F-box protein in a ubiquitin ligase complex [26]. Moreover, the intracellular localization of RCAR/PYR/PYLs and PP2Cs are apparently controlled by a small lipidbinding protein [27] or a receptor-like kinase [28]. It is noteworthy to mention other reported regulatory components, including guanine nucleotide exchange factors (GEFs) and plant Rho GTPase (ROP) [29,30], and an uncharacterized protein, ENHANCER OF ABA CO-RECEPTOR1 (EAR1) [31], in activation of PP2Cs, nitric acids in inactivation of RCAR/PYR/PYLs via Tyr nitration [32], and DELAY OF GERMINATION1 (DOG1), a protein of unknown function, in inhibition of ABAHYPERSENSITIVE GERMINATION 1 (AHG1) in the control of seed dormancy [33,34]. Each signaling component is represented by a small gene family, and further studies are thus necessary to assess whether these emerging features of post-translational regulation are specific to certain proteins in each of the core component families or are a general feature of the family in question.

ABA Perception at Nanomolar Levels Similar to receptor complexes for other plant hormones, ABA is perceived by a heterodimeric protein complex: RCAR/PYR/PYL receptor proteins and clade A PP2Cs [9,10]. Given that there are 14 RCAR/PYR/PYLs and nine PP2Cs of clade A in arabidopsis (Arabidopsis thaliana), functional analyses suggest that plants are able to respond to a wide range of ABA concentrations in different cell types and tissues, possibly by dozens of combinations of the two types of proteins [9,10,35]. This idea has been substantiated in a recent study examining the ABA affinity and interaction between RCAR/PYR/PYLs and PP2Cs by assessing ABA-responsive gene expression in protoplasts [36]. There are three subgroups of ABA receptors which are conserved in higher plants [9,37–40]. Arabidopsis subfamily I members readily interact with the PP2C coreceptors (Figure 2) and this complex formation is thought to generate the high-affinity interaction at basal levels of ABA. The preponderance of subfamily I receptors for binding to PP2Cs without ligand have been shown by protein interaction analyses in yeast (Saccharomyces cerevisiae) [9,41] and seems to be a prerequisite for regulation of the ABA response at nanomolar levels. Analysis of ABA responsiveness of receptors in poplar (Populus × canescens) [38], soybean (Glycine max) [42], wheat (Triticum aestivum) [40], and maize (Zea mays) [43], reveal the high regulatory capacity of this subclass. Consistent with the PP2C-mediated ligand coordination [44], ABA responsiveness is

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Figure 2. Comparative ABA Responsiveness of ABA Receptor Complexes. (A) The protein phosphatases of type 2C (PP2C)-mediated inhibition of abscisic acid (ABA)-responsive gene regulation was alleviated by ectopic expression of ABA receptors at resting ABA concentration of arabidopsis protoplasts of approximately 20 nM. The data present the fraction of the REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR)-mediated ABA response at basal ABA levels compared to the mean induction value of the respective RCAR among PP2Cs without the outlier ABA-HYPERSENSITIVE GERMINATION 1 (AHG1) at 10 μM exogenous ABA. (B) Induction of the ABA response by specific RCARs as a mean value of PP2Cs without the outlier AHG1 at 10 μM ABA. The RCARs from RCAR1 to RCAR14 (R1–R14) are color-coded as shown in (B) and the corresponding PYR1/PYL (P1–P13) nomenclature is given. The data are modified with permission from Tischer et al. (2017) [36].

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affected by PP2C coreceptors, among which HYPERSENSITIVE TO ABA 1 (HAB1) and PP2CA provide highly ABA-sensitive regulation (Figure 2). The different ternary ABA-RCAR-PP2C complexes allow for a dynamic response over a wide range of ABA concentrations, ranging from basal levels to induced levels under stress. The most ABA-sensitive RCAR appears to be RCAR4/PYL10, RCAR1/PYL9, and RCAR3/ PYL8 (Figure 2), for which the low ABA level present in aba2-1 protoplasts (2 nM) was sufficient to activate the ABA response [36]. Roots sensitively respond to ABA under nonstress conditions. In roots, RCAR3, RCAR8/PYL5, RCAR10, and RCAR11/PYR1 are prominently expressed, as well as the PP2Cs PP2CA, ABI1, and HAI1 (named from highly ABA-induced PP2C genes) [45]. RCAR1 and RCAR3 promote ABA-induced lateral root growth by augmenting auxin signaling [46,47]. ABA stabilizes RCAR3 in root cells and mediates the receptor accumulation in the nucleus and in neighboring cells [48,49]. Hence, not only ABA [50,51] seems to act cell nonautonomously and provide feedback mechanisms to modulate the ABA response by regulating signal abundance and sensitivity of ABA perception.

The Role of ABA in Water Acquisition and Control of Water Status at Basal Hormone Levels A major effort for plants is balancing water uptake with water vapor loss according to photosynthetic demand, ambient humidity, and temperature at nonstress conditions. ABA plays a key role by sensitively regulating stomatal aperture and whole plant hydraulics [52–55]. This regulation requires fast and long distance-coordinated homeostasis of water relation involving sensitive readouts of changes in water pressures and potentials [56]. Regulation of aquaporin activity [57,58], xylem differentiation [59], and suberization [60] by ABA signaling contribute to adjusting the water flux into and out of the plant. Stomatal control involves ABA-dependent reduction of stomatal aperture in response to high CO2 and low ambient humidity, resulting in high vapor pressure deficits [61,62]. Notably, recent studies on a couple of ABA-biosynthesis and ABA-signaling mutants revealed that the stomatal closing responses to higher CO2 and lower air humidity are not solely caused by altered ABA levels [51,63], though basal ABA is suggested to be essential to facilitate the stomatal response to elevated CO2 [63]. The receptors RCAR2/PYL7, RCAR8, and RCAR10, and ABI1 and PP2CA are predominantly expressed in stomata [64]. The ABA sensitivity of these RCAR–PP2C pairings (Figure 2) would

Box 1. Precise Absolute Quantification of ABA The reported resting ABA levels of plant leaves are highly variable, yet for arabidopsis, concentrations were found to be in the range of 6 to 32 nM [36,65,97–101]. Quantification of ABA is generally performed by immunological detection using antibodies against ABA, gas chromatography–tandem mass spectrometry (GC–MS/MS), or liquid chromatography– electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Although the immunoassay is used in many laboratories thanks to commercially available kits, it is not appropriate for precise quantification and high-throughput analyses due to the limitation of antibody specificities against ABA and ABA metabolites, its high cost, and its relatively narrow working range. By contrast, a large number of samples are rapidly analyzed by GC–MS/MS [99,102]. Although several plant hormones can be measured by GC–MS/MS at high sensitivity [99], several steps of extraction, purification, and derivatization are required, and derivatization procedures need to be optimized for each plant hormone. By contrast, LC–ESI– MS/MS is suitable for simultaneous measurement of several plant hormones and their metabolites with high selectivity and sensitivity [103,104]. To date, 101 plant hormone-related compounds can be measured from small amounts of materials via LC–MS/MS-based profiling [105]. Regardless of separation methods, that is, GC or LC, the accuracy of quantification is assured by isotope labeled internal standards, such as [2H6]-ABA. It is, however, important to note that even when using highly optimized methods [105], some inaccuracies remain in multihormone methods, with a 9.1% inconsistency with known theoretical amounts being reported to ABA.

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suggest that closing of stomata requires ABA levels above resting levels. Higher ABA levels in guard cells compared to the surrounding tissue might be responsible for the observed ABAdependent fine-tuning of stomatal aperture at nonstress conditions [62,65,66]. The reported ABA levels represent leaf mean concentrations (Box 1), and levels in guard cells could differ from these given the guard cell-specific roles of certain ABA catabolic enzymes [67], transporters [68], and the mobility of ABA [51]. To directly address this question requires that we overcome the challenge of measuring ABA concentrations with cellular resolution by using genetically encoded fluorescence resonance energy transfer (FRET) sensors [69,70] without interference of the endogenous ABA pool (Box 1). Water uptake is fostered by the ABA-mediated hydrotropism of roots [48,71], which allows the root system to efficiently explore water resources of the soil. Roots of ABA-deficient and ABA-insensitive mutants showed a reduced hydrotropic response while ABA-hypersensitive mutants were enhanced in this response [72] (Figure 3). The requirement of basal ABA levels for proper root growth is evident from the stimulation of root extension by nM exogenous ABA in wild type [71,73,74] and the complementation of impaired root growth in the ABA-deficient aba2-1 mutant, possibly via redox homeostasis [75].

Altered ABA Signaling Resulted in Physiological and Metabolic Changes In agreement with the structural and molecular biological studies on the ABA receptor complexes, arabidopsis overexpressing single RCAR/PYR/PYLs showed altered growth even under wellwatered conditions [76]. Similarly, multiple knockout mutants of rice (Oryza sativa) RCAR/PYR/ PYLs displayed improved growth and yield [77]. These observations suggest basal levels of ABA are essential for maintaining plant growth and development. Indeed, ABA affects leaf and

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Figure 3. Physiological Roles of Basal ABA. Basal abscisic acid (ABA) is involved in water homeostasis, growth, and development. In roots, ABA is involved in processes stimulating water uptake, including hydrotropism and xylem formation. Deposition of hydrophobic suberin layers and primary root growth are promoted by ABA. In the shoot, transpiration is controlled by ABA. Leaf initiation is inhibited by ABA signaling and metabolically via ABA control on the tricarboxylic acid (TCA) cycle, the metabolic pathway essential for energy and amino acid production. ABA also promotes leaf growth via attenuation of ethylene biosynthesis. Created with BioRender.

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shoot growth via ethylene (Figure 3). The analyses of ABA-deficient arabidopsis and tomato (Solanum lycopersicum) plants, which showed stunted growth under high humidity conditions under which the leaf water potentials were comparable to that in control plants, suggested a role of ABA in promoting shoot growth via inhibition of ethylene production [78,79]. Hypocotyl elongation in the dark is also known to be triggered by ABA. The growth stimulatory effect of ABA in the dark was initially reported in mesocotyl in monocots, such as rice, oat (Avena sativa), and maize [80,81]. Later, in eudicots, shorter hypocotyls were observed in ABA-deficient arabidopsis and tomato in the dark [82,83]. Endogenous ABA under nonstress conditions is also important for proper stomatal development [84]. In addition, as evidenced by mutant analyses of the PP2Cs and the SnRK2s [85,86], basal ABA is involved in floral transition through yet-to-be-elucidated complex mechanisms (reviewed in [87]). Despite these reports, the idea of ABA in maintenance of plant growth had until recently not been supported by biochemical evidence. In arabidopsis, the ABA receptor–phosphatase complexes directly control three subclass III SnRK2s (SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1, and SRK2I/SnRK2.3) that are key positive regulators of ABA signaling. Indeed, the triple knockout mutant of the kinases, srk2d srk2e srk2i (also referred to as srk2d/e/i or snrk2.2/2.3/2.6), shows extreme insensitivity to ABA in seeds and seedlings [12–14]. In addition, the knockout seedlings show further abnormalities, including an increased number of leaves under nonstress conditions [13], and the leaf number phenotype was also observed in an ABA-biosynthesis mutant, aba2-1 [88]. The rates of leaf emergence were increased in the srk2d srk2e srk2i and aba2-1 mutants, whilst the quadruple mutant of ABA-RESPONSIVE ELEMENT (ABRE)-BINDING proteins (AREBs)/ABRE-BINDING FACTORS (ABFs) transcription factors, which regulate a large proportion of the stress-responsive and/or ABA-responsive genes controlled by the SnRK2s, was similar to wild-type plants in terms of leaf numbers and leaf emergence rates. These results suggest that besides its roles in stress-responsive gene expression, ABA signaling mediated by the SnRK2s is important for leaf initiation under nonstress conditions. But, how is the leaf phenotype linked with primary metabolism that drives plant growth and development? ABA biosynthesis plays a key role in a rapid metabolic adjustment in response to dehydration [89]. Moreover, the transcriptional regulation by the SnRK2s in ABA signaling is important for starch degradation in response to osmotic stress [90]. However, roles of ABA in carbon metabolism under nonstress conditions are just emerging. Profiling of primary metabolism examined by gas chromatography–mass spectrometry (GC–MS) revealed that the metabolite profile of the srk2d srk2e srk2i mutant was similar to that of the aba2-1 mutant, but distinct from that of wild-type plants [88]. In particular, citrate and trehalose were more highly accumulated in these mutants, whilst Pro and galactinol accumulated to a lesser extent. A pioneering metabolite profiling of the ABA-biosynthesis mutant, nced3, reported a number of dehydration-induced amino acids and organic acids [89], however, except for Pro, these metabolites were unaltered in the srk2d srk2e srk2i or aba2-1 mutants [88]. Further, isotope labeling experiments revealed that estimated 14CO2 emission through the tricarboxylic acid (TCA) cycle was increased in the srk2d srk2e srk2i mutant, and that label accumulation into citrate and several amino acids increased. Collectively, in addition to its roles in the metabolic response under abiotic stress [89,90], the ABA signaling mediated by the SnRK2s was shown to be essential to fine-tune the TCA cycle for proper metabolism and leaf initiation under nonstress conditions (Figure 3). In animals, plants, and fungi, TOR kinase plays essential roles in cellular homeostasis by regulating metabolism, nutrient sensing, and growth (reviewed in [91]). Indeed, TOR kinase has been

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proposed to maintain growth under nonstress conditions by inhibiting stress responses via phosphorylation of RCAR/PYR/PYL ABA receptors [15]. RCAR/PYR/PYLs were inhibited by TOR kinase via phosphorylation of a conserved Ser residue in the absence of ABA. Furthermore, in the presence of ABA, the SnRK2s inversely phosphorylated and inhibited REGULATORY ASSOCIATED PROTEIN OF TOR (RAPTOR) 1B, a key scaffold protein recruiting substrates of TOR kinase (Figure 1). These results suggest a reciprocal regulation between TOR kinase and the SnRK2 kinases, and raise the hypothesis that the metabolic changes in the srk2d srk2e srk2i mutant under nonstress conditions [88] is due to activation of TOR kinase. This hypothesis should be carefully tested in future analyses, since the growth characteristics of TOR kinase mutants are not easily explained. In an estradiol-induced TOR RNAi line, some raptor1b T-DNA knockouts showed enhanced ABA-sensitivity in young seedlings, increased activity of the SnRK2s, and elevated expression of stress-responsive genes [15]. However, other T-DNA knockout lines of RAPTOR1B showed decreased ABA contents and ABAdeficiency symptoms under vegetative growth, including increased stomatal conductance, which were partially rescued by ABA application [92]. Given that endogenous ABA levels were reported to be unchanged with respect to altered ABA sensitivities in the multiple mutants of PP2Cs and SnRK2s [13,93], it follows that the ABA-deficient phenotypes in the raptor1b mutants [92] might not be a direct consequence of activated ABA signaling [15]. TOR kinase may affect ABA responses at several levels, including ABA biosynthesis, catabolism, and/or signaling. In conclusion, whilst evidence suggests a link between ABA signaling and TOR kinase signaling, further validation is necessary to unveil how these pathways are mechanistically connected with one another.

Growth Promotion in Roots By contrast to the suggested roles of ABA in maintaining leaf initiation, ABA promotes root growth, which is crucially associated with water uptake, in several ways (Figure 3). In addition to xylem formation via the microRNA and its target transcription factors [59], hydrotropism based on asymmetric expansion of cortical cells is regulated by basal ABA [48,71]. ABA was also reported to be a crucial signal in establishing hydrophobic suberin barriers to prevent water and nutrients flows [60]. Suberin coats covering root endodermis are plastically deposited or removed, depending on nutrient availability and stresses. Deficiencies of Iron (Fe), Manganese (Mn), or Zinc (Zn) induce ethylene signaling to inhibit suberization, whilst deficiencies of Potassium (K) or sulfur (S) and salt stress activate ABA signaling to enhance suberin deposition. Notably, suberin deposition was decreased in the abi1 mutant background under control conditions, indicating that basal ABA supports transport of xylem-derived nutrients as well as water to the shoot. Primary root growth is also promoted by ABA, possibly via attenuation of ethylene biosynthesis. Root meristems of arabidopsis have been well characterized, and the quiescent center (QC) plays a crucial role in root development. Ethylene promotes QC cell division and the ethylene-overproducing mutant shows stunted roots [94], whilst ABA inhibits its division [95]. At molecular levels, low concentrations of ABA induces expression of the transcription factors, which in turn represses expression of the genes in ethylene biosynthesis, resulting in promotion of primary root growth [96]. Collectively, the suggested roles of basal ABA in cellular and tissue development vary among cell and tissue types and are dependent on growth conditions. Our lack of understanding of why this is the case might be bridged by comprehensive analyses. For example, tissue-specific metabolite and hormone profiling of ABA-related mutants followed by integration of the data to simulate metabolic fluxes at the whole plant level will likely lead to a better understanding of the role of ABA in plant growth and development. 632

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Concluding Remarks and Future Perspectives

Outstanding Questions

Since the identification of cytosolic receptors of ABA a decade ago [9,10], our knowledge of ABA signaling in plants has greatly improved. First, multiple post-translational regulators have been identified, indicating that ABA signaling is finely controlled by higher order mechanisms for proper outputs under fluctuating environments. Secondly, a number of recent studies suggest that ABA is not a simple stress hormone, but is also associated with several metabolic and cell biological processes. Quantification and visualization of local ABA levels at cellular and tissue resolution under various environmental conditions will be necessary for a better understanding of the physiological role of ABA, however, it is still challenging (see Outstanding Questions). Similarly, little is known about variations in protein abundances of 14 RCAR/PYR/PYLs, nine PP2Cs, and three SnRK2s at cellular and tissue levels. Given that ABA signaling components are controlled by multiple protein modifications, it is also important to dissect cellular and tissue specificities of the regulations by monitoring readouts of ABA signaling, including gene expression, stomatal closure, and metabolic changes. Management of water status is essential for all organisms. In plants, transpiration through stomata and uptake from root is tightly controlled via endogenous hydraulics, ABA, and environmental cues, such as CO2 and H2O. Revealing the molecular links between ABA signaling and sensing of CO2 as well as the water status in order to fully understand the growth and water management system in plants will be a challenge over the next decade.

How can ABA levels be measured at cellular resolution? Which proteins of RCAR/PYR/PYLs, PP2Cs, and SnRK2s are predominant in various cell and tissue types? Are the regulatory mechanisms specific to certain proteins of the core ABA signaling components and to certain tissues? How is ABA signaling connected with environmental cues, such as CO2 and H2O?

Acknowledgments The preparation of this review was supported by the Max-Planck Society (to A.R.F.).

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