Review

Tansley review Putting the brakes on: abscisic acid as a central environmental regulator of stomatal development Author for correspondence: Julie E. Gray Tel: +44 114 2224407 Email: [email protected]

Caspar C. C. Chater, James Oliver, Stuart Casson and Julie E. Gray Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK

Received: 30 September 2013 Accepted: 13 December 2013

Contents Summary

376

VI.

A role for ABA in CO2 and carbon budget signalling

384

I.

Making holes: the developmental programme

376

VII.

ABA-mediated signals from the surface: the role of waxes and cuticle

385

II.

Controlling the valves: guard cell physiology

378 Conclusions

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III.

Ontogenetic priming to ABA and its role in stomatal development

378

Acknowledgements

387

IV.

ABA metabolism and its contribution to stomatal development

380

References

387

V.

Light-mediated changes in stomatal patterning: a role for ABA?

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VIII.

Summary New Phytologist (2014) 202: 376–391 doi: 10.1111/nph.12713

Key words: abiotic stress, abscisic acid (ABA), guard cell, stomata, stomatal development.

Stomata are produced by a controlled series of epidermal cell divisions. The molecular underpinnings of this process are becoming well understood, but mechanisms that determine plasticity of stomatal patterning to many exogenous and environmental cues remain less clear. Light quantity and quality, vapour pressure deficit, soil water content, and CO2 concentration are detected by the plant, and new leaves adapt their stomatal densities accordingly. Mature leaves detect these environmental signals and relay messages to immature leaves to tell them how to adapt and grow. Stomata on mature leaves may act as stress signal-sensing and transduction centres, locally by aperture adjustment, and at long distance by optimizing stomatal density to maximize future carbon gain while minimizing water loss. Although mechanisms of stomatal aperture responses are well characterized, the pathways by which mature stomata integrate environmental signals to control immature epidermal cell fate, and ultimately stomatal density, are not. Here we evaluate current understanding of the latter through the influence of the former. We argue that mature stomata, as key portals by which plants coordinate their carbon and water relations, are controlled by abscisic acid (ABA), both metabolically and hydraulically, and that ABA is also a core regulator of environmentally determined stomatal development.

I. Making holes: the developmental programme Stomata, formed by two specialized guard cells, are microscopic valves on the surfaces of leaves that act to maximize the influx of carbon dioxide and minimize water loss. This review concerns the 376 New Phytologist (2014) 202: 376–391 www.newphytologist.com

ways in which environmental signals, in particular drought, carbon dioxide, and light, affect stomatal development. It explores the role that stomata on mature leaves have in sensing environmental change and influencing young leaf development, and the putative integrative role of the phytohormone abscisic acid (ABA) in this Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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long-distance signalling, as illustrated in Fig. 1. It is now accepted that growth at elevated atmospheric concentrations of CO2 is, in the majority of plant species, associated with a decrease in stomatal density (Woodward, 1987). This inverse relationship is very ancient and extends back into the geological record, and is expected to affect plant growth under predicted future CO2 concentrations.

(a)

(b)

Fig. 1 Control of transpiration by mature stomata and transpirational feedback on stomatal development. (a) Model of mature to immature leaf control of stomatal development in which (1) the leaf acts as a sensory organ on which the stomata are the sensors of environmental change and (2) directs the signals from the mature leaf to (3) the developing leaf. (b) The guard cells’ response to stimuli such as photosynthetically active radiation (PAR), CO2 concentration, and humidity alters the aperture of the stomatal pore and the (1) combined actions of numerous stomata result in whole leaf transpiration change. The stomata transduce the multiple inputs from the environment, and the transpirational response determines the hydraulic and nonhydraulic signalling outputs of the stomata which convey the current conditions. (2) These signals are transmitted via the vasculature as well as by nonvascular apoplastic and symplastic routes to (3) the developing leaf which receives and decodes the signals to make cell fate decisions to determine epidermal patterning. The cellular divisions and transitions leading to stomatal development are outlined in the schematic on the right. Environmental control may act on each of these stages, resulting in altered stomatal density or index. Colour codes of stomatal precursor cell types are as follows: dark blue is the meristemoid mother cell, pink is the meristemoid, orange is the guard mother cell, and turquoise is the guard cell. Each process, from the guard cell physiological response (1) to transportation of signalling molecules (2) to the recognition of and response to signals in the developing epidermis (3) can be modulated by the phytohormone abscisic acid (ABA). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Irrespective of the CO2 concentration or light regime experienced by developing leaves, it is the concentration of CO2 or the intensity of light to which the mature leaves have been exposed that controls stomatal density in developing leaves (Lake et al., 2001). The molecular mechanisms that modulate stomatal development in response to environmental signals remain unknown but, in 2008, Lake and Woodward reported a comprehensive set of experiments demonstrating that stomatal density responses to environmental signals, including CO2 concentration and vapour pressure deficit, are correlated with changes in whole-plant transpiration and leaf ABA concentration. Thus, it is proposed that a long-distance signal that arises in the mature leaves and influences stomatal development in the young leaves is driven by mature guard cells’ modulation of transpiration (Lake & Woodward, 2008). These experiments, which demonstrate a clear link between the short-term pathways controlling stomatal aperture and the long-term pathways regulating stomatal development, also implicate ABA in the control of stomatal development. They show that the magnitude of environmentally induced reductions in stomatal density correlate well with leaf ABA concentrations. Furthermore, ABA biosynthesis mutants which exhibit greatly reduced concentrations of ABA and increased transpiration rates exhibit large increases in stomatal density (Lake et al., 2002; Xie et al., 2006; Tanaka et al., 2013), and there is limited evidence that these mutants show a reduction in stomatal density when grown at elevated CO2 (Lake et al., 2002). The question of whether the regulation of stomatal development in response to environmental signals requires a functional ABA biosynthesis pathway needs to be directly addressed. The complex process of stomatal development, from epidermal precursor cells to paired guard cells, has been the subject of several recent reviews (Lau & Bergmann, 2012; Pillitteri & Torii, 2012) and so, while critical to the discussions below, will only be explained briefly here. The process of stomatal development is best understood in the model genetic plant Arabidopsis thaliana. Immature epidermal cells enter the stomatal development pathway via an asymmetric division which produces a small cell known as a meristemoid and a larger cell which can differentiate into an epidermal pavement cell or, more often, divides asymmetrically itself to produce a secondary meristemoid (Robinson et al., 2011). Each meristemoid usually divides again asymmetrically three or four times before differentiating into a guard mother cell. The guard mother cell undergoes a single symmetric division to form the two guard cells that control the aperture of the mature stomatal pore. Each developmental transition is regulated by a specific bHLH (basic Helix Loop Helix) transcription factor. SPEECHLESS regulates the formation of meristemoids (MacAlister et al., 2007), MUTE regulates the number of meristemoid asymmetric divisions and differentiation into a guard mother cell (Pillitteri et al., 2007), and FAMA regulates the final symmetric division (Ohashi-Ito & Bergmann, 2006). The activities of these transcription factors are tightly controlled, spatially and temporally, to ensure that stomatal development does not occur inappropriately. For example, SPEECHLESS activity is restricted to individual precursor cells in the developing epidermis by a MAP kinase (mitogen-activated protein kinase) cascade involving MPK3 and MPK6 (mitogen-activated protein kinase), MKK4 and MKK5 New Phytologist (2014) 202: 376–391 www.newphytologist.com

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(mitogen-activated protein kinase kinase) and the MAP kinase kinase kinase YODA (Bergmann et al., 2004; Wang et al., 2007; Lampard et al., 2009). Phosphorylation by this kinase cascade is believed to be up- and down-regulated by a family of secreted epidermal patterning factors (EPFs; Hara et al., 2007, 2009; Hunt & Gray, 2009; Abrash & Bergmann, 2010; Abrash et al., 2011) in combination with ERECTA family receptors (Shpak et al., 2005) and the receptor-like component TOO MANY MOUTHS (TMM; Yang & Sack, 1995; Nadeau & Sack, 2002). It is not yet clear how environmental signals impact on this complex developmental signalling pathway to modulate stomatal frequency during leaf development.

II. Controlling the valves: guard cell physiology Guard cell physiology and stomatal signal transduction mechanisms are crucial to the themes outlined below and, again, current understanding of these processes can be sourced from recent reviews (Ara
ujo et al., 2011; Hauser et al., 2011; DaszkowskaGolec & Szarejko, 2013). Most plants open their stomata during the day and close their stomata at night. This diurnal response is initiated by photoreceptors via photosynthetic and nonphotosynthetic pathways (Shimazaki et al., 2007). The stomatal pores open by the increase of guard cell osmotic pressure, water influx and cell expansion, allowing greater stomatal conductance to water and CO2. The pores close by the induction of water efflux from the guard cells and the reduction in turgor pressure. This aperture response is regulated by complex signal transduction networks involving ABA, light, CO2, calcium, and reactive oxygen species (ROS), with a core of kinase and phosphatase regulation by SnRK2s (sucrose-non-fermenting-1-related kinase 2 proteins) and PP2Cs (protein phosphatase 2C proteins), respectively, which activate ion channels to modulate cell turgor, which has been extensively reviewed elsewhere (Kim et al., 2010).

III. Ontogenetic priming to ABA and its role in stomatal development Soil water availability and atmospheric vapour pressure are the principal environmental signals used by plants to respond to drought and, similarly, are the primary influences on changes in stomatal density under water deficit. ABA is the major chemical signal that regulates the physiological responses to drought, including the stomatal closure response outlined in the previous section. In the following sections, we propose that ABA also has a central role in regulating the development of stomata in young leaves, and we discuss the evidence to support this. Recently, Pantin et al. (2013b) have shown that A. thaliana stomata undergo a developmental priming of ABA sensitivity which is induced by the slight humidity gradient across the plant rosette (Fig. 2). When epidermal cell fate decisions are occurring, the very young developing leaves are often under conditions of relatively high humidity surrounded, and in some cases enclosed, by the older leaves. As mature leaves continuously direct ABA towards the developing leaves, these young shoots contain high concentrations of ABA and yet, paradoxically, their young stomata New Phytologist (2014) 202: 376–391 www.newphytologist.com

New Phytologist remain open (Jordan et al., 1975; Zeevaart, 1977; Cornish & Zeevaart, 1984). The recent observation that guard cell ABA sensitivity increases as the leaf ages explains this paradox (Pantin et al., 2013b). Elevated ABA concentrations in young leaves serve to up-regulate hydraulic and aquaporin activity to maximize the cell expansion rate to facilitate leaf growth (Parent et al., 2009; Tardieu et al., 2011; Pantin et al., 2012). Thus, ABA insensitivity must be restricted to the guard cells or epidermis of the developing leaves. Young guard cell insensitivity to ABA is believed to be induced by low vapour pressure deficit in the microenvironment around the shoot. This allows stomatal pores to open to maximize CO2 acquisition, and further promotes high transpiration rates and maintains locally high humidity (Aliniaeifard & van Meeteren, 2013; Pantin et al., 2013b). As leaves expand and their microenvironment becomes less humid, their stomatal ABA sensitivity rises (Fig. 2). Although these recent studies demonstrate that stomatal apertures of young leaves are unresponsive to ABA signals, there is much evidence that the pathways that govern stomatal development in young leaves are either directly or indirectly responsive to ABA. Therefore, in the immature leaf, some cell types must be able to respond to ABA more readily than others. The cells that give rise to epidermal cells are responsive to ABA (Tanaka et al., 2013), whereas the unprimed guard cells are unresponsive (Pantin et al., 2013a,b). Currently, however, it is not known at what leaf developmental stage ABA begins to regulate stomatal development and whether a priming event is required for these events too. ABA appears to slow leaf expansion rates under conditions of soil water deficit and low atmospheric humidity by reducing turgor pressure, cell division rate, and cell wall extensibility (Bacon et al., 1998; Cosgrove, 2005; Tardieu, 2013). This retardation of young leaf growth results in smaller leaves with lower stomatal indices (i.e. the proportion of stomata to epidermal cells is reduced). One way in which ABA may achieve this reduction in stomatal frequency is by inducing the cyclin-dependent kinase (CDK) inhibitor gene Interactor of Cdc2 Kinase 1 (ICK1) which acts on cyclin-dependent protein kinases B1,1 and B1,2 (CDKB1,1 and CDKB1,2) which are specifically required for the symmetric division that produces two guard cells, thereby blocking the G1–S cell cycle transition as well as directly inhibiting stomatal formation (Wang et al., 1998; Boudolf et al., 2004; Weinl et al., 2005; Xie et al., 2006). Disruption of these kinases and associated A2-type cyclins results in leaves with abnormal single guard cells (Lee et al., 2013). In addition, ABA controls epidermal cell fate during early leaf development by acting in a related pathway as a negative regulator of stomatal lineage entry upstream of the bHLH transcription factor SPEECHLESS, as well as an inhibitor of pavement cell expansion (Tanaka et al., 2013). As brassinosteroid biosynthesis and signalling have recently been shown to promote leaf cell division and expansion (Zhiponova et al., 2013), ABA can be seen as the brake in this mechanism. It appears that under optimal water supply ABA acts to promote maximal leaf growth by maintaining high hydraulic conductance and a high density of aquaporins, but that suboptimal water supply primes ABA-mediated stomatal closure in expanding leaves and alters the role of ABA from an indirect growth promoter to a direct growth inhibitor in these (and younger) tissues as well. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(a)

Leaf age (b)

(c)

(d)

Mature leaves sense CO2 concentrations and alter their stomatal conductances accordingly, such that at subambient atmospheric CO2 concentrations stomata are more open and at elevated concentrations stomata are more closed. The accompanying change in the rate of mature leaf transpiration, in turn, appears to regulate the degree of stomatal formation in immature leaves (Miyazawa et al., 2006; Lake & Woodward, 2008). Thus, growth at elevated atmospheric CO2 concentrations usually results in the formation of leaves with reduced stomatal density. The insensitivity of stomatal aperture responses to ABA when it is applied directly to young leaves or to mature leaves that have undergone periods of low vapour pressure deficit (Aliniaeifard & van Meeteren, 2013; Pantin et al., 2013b) suggests that the underlying mechanism behind this disruption in drought signalling may be the same as that controlling conditional responses to CO2 (Raschke, 1975; Frechilla et al., 2002; Talbott et al., 2003). We propose that stomata undergo ontogenetic priming to both ABA and CO2 concentrations that is triggered by increasing vapour pressure deficit-mediated ABA sensitivity, and that the ABA and CO2 insensitivity of developing leaves promotes maximal CO2 uptake (Fig. 2). In this scenario, the initial insensitivity of the developing leaf to drought signals and Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 2 Ontogeny and the priming of stomatal abscisic acid (ABA) sensitivity. (a) Leaf development and maturation are correlated with changes in ABA concentration and stomatal sensitivity to ABA in Arabidopsis thaliana. ABA concentrations decline as the leaf reaches maturity and older leaves export ABA to developing leaves for hydraulic- and aquaporin-controlled expansion. Arabidopsis thaliana stomata undergo a developmental priming of ABA sensitivity as they mature and expand. This increasing sensitivity of stomata to ABA may also induce sensitization to CO2 concentration, light intensity and other environmental stimuli. The red line indicates the decline in ABA concentration as the leaf matures, whereas the green line denotes the increase in guard cell ABA and CO2 sensitivity. (b) The spiral phyllotaxis of the A. thaliana plant rosette results in a spatial gradient, declining ABA concentration (red spiral) and increasing stomatal sensitivity to ABA and CO2 (green spiral). This spiral architecture provides a protective microclimate of elevated humidity to the developing leaves in the centre in which newly formed ABA-insensitive stomata remain fully open to maximize transpiration and carbon acquisition. Leaf expansion in the rosette results in a humidity gradient and the greater vapour pressure deficit experienced by the mature stomata is believed to activate, or ‘prime’, physiological responses. (c) Ontogenetic mechanisms associated with gradients in ABA concentration (red spiral), stomatal sensitivity to ABA and CO2 (green spiral), and humidity are also likely to be active within plants with multiple stems. This may result in numerous zones of ‘long-distance’ signalling from older to younger leaves within each ‘rosette’ and enable fine-tuned developmental responses based on aspect and other local environmental variation experienced across the crown, the aerial surface of the plant. (d) Extreme examples of rosette architecture in plants. Sempervivum arachnoideum, the cobweb houseleek (upper), produces dense matting which may protect its developing leaves against drought and maximize the ontogenetic humidity gradient upon expansion. Saxifraga arendsii (lower) produces numerous deep and tightly clustered rosette-forming shoots, again perhaps to increase the boundary layer and provide a greater humidity gradient. It is probable that the mechanisms governing ontogenetic control of transpiration in the rosette architecture of A. thaliana are at work in these species. Furthermore, it is likely that a similar mechanism is in place in the shoots of plants that do not form rosettes and instead protect developing leaves in other ways, including enclosure in older leaf sheaths, tight bud scales, or dense trichomes.

CO2 concentration necessitates fully primed, environmentally responsive, stomata in mature leaves to exert control over the cell fate decisions in the youngest leaves. Prolonged or subsequent exposure of developing leaves to ABA must first prime stomata, then induce their closure and then retard vegetative growth. ABA can be rapidly mobilized and transported via an elevated pH-dependent symplast-to-apoplast route to both mature and developing leaves (Bacon et al., 1998; Sharp & Davies, 2009). ABA is synthesized in and transported from the roots, the vasculature, and the guard cells themselves and it is not known if and how these distinct sources of ABA determine specific outcomes. The stomatal response to this ABA signal must be primarily mature-leaf directed, as the older leaves are more ABA-sensitive than the young leaves if they have not yet been primed by previous exposure to ABA (Pantin et al., 2012, 2013b). ABA-induced closure of mature stomata is therefore the first line of defence against excessive water loss and is accompanied by rapid de novo ABA synthesis (see the following Section IV, ABA metabolism and its contribution to stomatal development) and transportation to developing leaves (Zeevaart & Boyer, 1984). The transport of ABA and other as yet unknown drought signals to the developing leaf may therefore serve to prime young stomata in anticipation of continuing water stress and alter the course of young leaf development to one better equipped for future drought tolerance. New Phytologist (2014) 202: 376–391 www.newphytologist.com

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As well as a direct ABA induction of stomatal closure in mature leaves, ABA has recently been shown to affect stomatal water loss by an indirect hydraulic effect through a decrease in water permeability within the leaf vasculature (Pantin et al., 2013a). This vascular ABA response is proposed to provide a mechanism by which internal humidity change is sensed and translated into fine-tuned control of transpiration (Pieruschka et al., 2010) and, furthermore, could link mature leaf ABA concentration to transpiration-driven control of stomatal development (Lake & Woodward, 2008).

IV. ABA metabolism and its contribution to stomatal development Increased stomatal conductance induces rapid ABA biosynthesis in both guard cells and the vascular tissue (Bauer et al., 2013). Several mutants lacking activity of the core ABA biosynthesis enzymes have high stomatal densities in A. thaliana (Lake & Woodward, 2008; Tanaka et al., 2013), indicating that normally ABA either directly or indirectly inhibits the entry of epidermal precursor cells into the stomatal development pathway. Thus, the regulation of ABA biosynthesis, in particular the tight control of the rate-limiting enzyme 9-cis-epoxycarotenoid dioxygenase 3 (NCED3) which catalyses the cleavage of cis-xanthophylls to xanthoxin, may be critical to furthering our understanding of how stomata convert environmental signals into the long-distance endogenous signals that regulate epidermal development (Fig. 3, Table 1). The ABA biosynthesis pathway is well characterized and NCED3, and to a lesser extent other ABA biosynthetic enzymes, ABA1 (ABA DEFICIENT 1) and AAO3 (ABSCISIC ALDEHYDE OXIDASE 3), are activated by ABA in a positive feedback loop (Barrero et al., 2006). ABA2 and AAO3 proteins are localized to the vascular parenchyma between xylem and phloem vessels in plants grown in nonstressed conditions, but the NCED3 protein is undetectable until drought stress is applied (Endo et al., 2008). However, NCED3 is partially redundant with its isozyme NCED5 in the control of transpiration, and NCED5 expression does not appear to be controlled by water loss (Frey et al., 2012). NCED3 expression is controlled by several transcription factors which also up-regulate the expression levels of other genes potentially involved in both positive and negative feedback loops to modulate ABA’s regulation of abiotic stress and stomatal development. An interesting transcription factor with respect to the interplay between ABA biosynthesis, ABA signalling and stomatal development is HOMEODOMAIN GLABROUS 11 (HDG11), which directly or indirectly activates both NCED3 and ABA3 for ABA production as well as CIPK3, CAX3, ABI3 (CALCINEURIN B-LIKE-INTERACTING PROTEIN KINASE 3, CALCIUM/ PROTON EXCHANGER CAX1-LIKE, ABA INSENSITIVE 3) and ERECTA; all components known to be involved in ABA signalling or stomatal development (Yu et al., 2008, 2013). In this way, HDG11 therefore plays a role in ABA-mediated water use efficiency through both stomatal aperture control and the longdistance negative regulation of stomatal development and cell size via ABA sensitivity and ERECTA-regulated developmental fate (Yu et al., 2008, 2013). Gain-of-function and over-expression of HDG11 in A. thaliana, tobacco (Nicotiana tabacum) and rice New Phytologist (2014) 202: 376–391 www.newphytologist.com

Fig. 3 The control of abscisic acid (ABA) metabolism and developmental feedback on stomata. 9-cis-epoxycarotenoid dioxygenase (NCED) catalyses the first committed, and the rate-limiting, step in ABA biosynthesis. The control of NCED3 in particular links guard cell physiology and stomatal development via transcriptional and metabolic processes, including hydraulic and nonhydraulic regulation in the vasculature. Components of the ABA biosynthetic pathway (green) are regulated by several transcription factors (orange) principally via the tight temporal and spatial regulation of NCED gene expression and activity. There is evidence that these transcription factors also restrict the effects of ABA biosynthesis by negative feedback. ABA concentrations (red) are also down-regulated by catabolism and the balance of the free ABA pool can be altered by reversible production of the less physiologically active ABA-glucose ester (ABA-GE). Arrows pointing from transcription factors denote transcriptional regulation of their targets and arrows from signalling components denote post-translational protein modification. Solid lines denote activation, whereas dashed lines show inhibition, of processes. See Table 1 for explanation of abbreviations.

(Oryza sativa) mutants result in elevated ABA concentrations and, arguably as a consequence, reduced stomatal densities and improved drought tolerance (Yu et al., 2008, 2013). ATAF1 (ARABIDOPSIS NAC DOMAIN-CONTAINING PROTEIN 2), a guard cell-expressed NAC (NAM (No Apical Meristem), ATAF1/2 (Arabidopsis thaliana transcription factor), and CUC2 (cup-shaped cotyledon)) transcription factor, has recently been shown to interact with the NCED3 promoter to up-regulate NCED3 expression and increase ABA concentrations, as well as to activate the transcription factor DREB2A (DEHYDRATION-RESPONSE ELEMENT BINDING PROTEIN 2a), which is involved in the induction of leaf drought and heat stress responses (Fig. 3; Sakuma et al., 2006; Jensen et al., 2013). Over-expression of ATAF1 results in severely retarded plant growth (Jensen et al., 2013). Interestingly, ATAF1 transcription is up-regulated by ABA (Wu et al., 2009) and ATAF1 is co-expressed with several PP2C phosphatase-encoding genes in a putative negative feedback loop that inhibits ABA responses (Jensen et al., 2013) and is believed to be crucial in the integration of various environmental stimuli via modulation of the ABA signal (MauchMani & Flors, 2009). The transcription factor WRKY57 (WRKY Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Table 1 List of abbreviations used in Figs 3–5 Abbreviation

Expanded name

AAO3 ABA1 ABA2 ABA3 ABI TFs

ABSCISIC ALDEHYDE OXIDASE 3 ABA-DEFICIENT 1 ABA-DEFICIENT 2 ABA-DEFICIENT 3 ABSCISIC ACID-INSENSITIVE TRANSCRIPTION FACTORS ABSCISIC ACID-INSENSITIVE 4 ABA-OVERLY SENSITIVE 1/ELONGATA 2 ARABIDOPSIS HISTIDINE KINASE 1 ARABIDOPSIS NAC DOMAIN-CONTAINING PROTEIN 2 ARABIDOPSIS THALIANA TRITHORAX-LIKE 1 BODYGUARD1 BETA-GLUCOSIDASE 1,2 BRASSINOSTEROID-INSENSITIVE 2 ECERIFERUM 1 ECERIFERUM 3 ECERIFERUM 6 CONSTITUTIVE PHOTOMORPHOGENIC 1 CULLIN-4-BASED E3 LIGASE COMPLEX

ABI4 ABO/ELO2 AHK1 ATAF1 ATX1 BDG1 BG1,2 BIN2 CER1 CER3 CER6 COP1 Cul4-based E3 ligase complex CYP707A1,A3 DREB2A ERECTAS EXO FAMA FAR1 FHY3 FHL FHY1 GsWRKY20 HDG11 HIC HOS3 HXK MAPK cascade MPK6 MUTE MYB96 NCED3 NFXL2 PP2C RD22 RD29A S-1-P SHN1,2,3 SPCH T6P TPP TPS TRE VIP1 VLCFAs WRKY57 YODA

CYTOCHROME P450 707A1/A3 DEHYDRATION-RESPONSE ELEMENT BINDING PROTEIN 2A ERECTA FAMILY (ERECTA, ERL1, ERL2) EXORDIUM FAMA FAR-RED IMPAIRED RESPONSE 1 FAR-RED ELONGATED HYPOCOTYL 3 FAR-RED ELONGATED HYPOCOTYL-LIKE 1 FAR-RED ELONGATED HYPOCOTYL 1 GLYCINE SOJA WRKY 20 HOMEODOMAIN GLABROUS 11 HIGH CARBON DIOXIDE HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 3 HEXOKINASE MITOGEN-ACTIVATED PROTEIN KINASE CASCADE MITOGEN-ACTIVATED PROTEIN KINASE 6 MUTE MYELOBLASTISIS FAMILY 96 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 NUCLEAR TRANSCRIPTION FACTOR, X-BOX BINDING PROTEIN 1-LIKE 2 PROTEIN PHOSPHATASE TYPE 2C RESPONSIVE TO DESSICATION 22 RESPONSIVE TO DESSICATION 29A SPHINGOSINE-1-PHOSPHATE SHINE 1,2,3 SPEECHLESS TREHALOSE-6-PHOSPHATE TREHALOSE-6-PHOSPHATE PHOSPHATASE TREHALOSE PHOSPHATASE/SYNTHASE TREHALASE VIRE2-INTERACTING PROTEIN 1 VERY LONG CHAIN FATTY ACIDS WRKY 57 YODA

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DNA-BINDING PROTEIN 57) also binds the NCED3 promoter and results in elevated ABA concentrations, as well as the co-expression of the gene RD29A (RESPONSIVE TO DESSICATION 29A) (Fig. 3; Msanne et al., 2011; Jiang et al., 2012). Although it might be expected that the activity of these transcription factors which control ABA concentrations might also affect epidermal cell fate, this remains to be determined. An epidermally derived protein, CED1/BDG1 (9-CISEPOXYCAROTENOID DIOXYGASE DEFECTIVE 1/ BODYGUARD 1), which is involved in cutin biosynthesis, activates NCED3 and ABA biosynthesis, thereby influencing drought tolerance (Wang et al., 2011; Fig. 3). In an example of positive feedback, ABA further up-regulates BDG1 expression (Wang et al., 2011). The transcription factor NFXL2 (NUCLEAR TRANSCRIPTION FACTOR, X-BOX BINDING PROTEIN 1-LIKE 2) down-regulates BDG1 expression and may also integrate the biosynthesis and action of ABA in the control of stomatal conductance and stomatal development (Fig. 3; Lisso et al., 2012). Loss of NFXL2 produces mutants with elevated ABA concentrations, reduced stomatal apertures, altered cuticular properties and lower stomatal densities (Lisso et al., 2012). These phenotypes may in part be caused by unregulated BDG1 expression, but the authors argue that NFXL2 also dampens the effect of SHINE1, 2 and 3 transcription factors; regulators of wax biosynthesis that are known to inhibit stomatal initiation (Yang et al., 2011; Lisso et al., 2012). Expression of a rice homologue of the SHINE genes, OsWR1 (ORYZA SATIVA WAX SYNTHESIS REGULATORY GENE 1), is up-regulated by ABA and drought (Wang et al., 2012a), but stomatal phenotypes have not been investigated. The interactions between cuticular wax biosynthesis and stomatal development are discussed more fully in Section VII, ABA-mediated signals from the surface: the role of waxes and cuticle. Intriguingly, brassinosteroid has recently been implicated in the biosynthesis of ABA via nitric oxide-induced up-regulation of an NCED gene and enhanced water stress tolerance in maize (Zea mays) (Zhang et al., 2011a). Brassinosteroid has not been shown to influence NCED3 expression in A. thaliana (P
erez-P
erez et al., 2004), although ABA has been shown to tightly control the main signalling outputs of brassinosteroid (Zhang et al., 2009) and inhibit brassinosteroid responses (Divi et al., 2010) in what could be another negative feedback loop (Fig. 3). Furthermore, brassinosteroid can inhibit stomatal production by regulating the BIN2 (BRASSINOSTEROID-INSENSITIVE 2)-mediated phosphorylation of the YODA-MKK4/5-MPK3/6 MAPK cascade (Kim et al., 2012; Khan et al., 2013) and also modulates stomatal production via the activity of the transcription factor SPEECHLESS (Gudesblat et al., 2012). Just as brassinosteroid has been shown to influence MAPK signalling (Khan et al., 2013), ABA may interact with the modulation of MAPKs to prevent stomatal clustering and determine stomatal index and density. MPK6 and MPK3 have been increasingly shown to both negatively and positively regulate stomatal development and integrate a multitude of signals both in epidermal development and for mature guard cell dynamics (Pitzschke & Hirt, 2009; Brock et al., 2010; Umbrasaite et al., 2010). Putative MAPK signalling substrates with newly identified roles in stomatal development suggest further layers of New Phytologist (2014) 202: 376–391 www.newphytologist.com

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control (S€orensson et al., 2012). MPK6 plays a role in ABA biosynthesis by NCED3 and ABA2 induction in seedlings (Xing et al., 2009). MPKs 9 and 12 are also involved in ROS-mediated ABA signalling to induce stomatal closure (Jammes et al., 2009). A PP2C-type phosphatase, AP2C3 (ARABIDOPSIS PROTEIN PHOSPHATASE 2C), interacts with MPK3, MPK4 and MPK6 and modulates stomatal production as well as positively regulating stomatal closure and ABA-induced gene expression (Brock et al., 2010; Umbrasaite et al., 2010). As the MAPK network integrates brassinosteroid and ABA signalling, it could be seen as a major coordinator of epidermal development and stomatal physiology. As such, MAPKs may be instrumental in the plant’s communication and translation of environmental signals into cell fate decisions. An overview of the evidence of how control of ABA biosynthesis integrates with both stomatal aperture and developmental control is presented in Fig. 3. The findings described here in section IV are all consistent with the level of NCED3 expression controlling ABA biosynthesis, and ABA inhibiting stomatal development. Also consistent with this is the observation that an A. thaliana mutant lacking Histidine Kinase1 (AHK1) has reduced expression of NCED3 (Tran et al., 2007) accompanied by increased stomatal density and index (Fig. 3). However, the effect of AHK1 on ABA accumulation is debated (Wohlbach et al., 2008; Kumar et al., 2012). These data suggest that there may be an uncoupling of transcript and protein levels, or that ABA catabolism may be affected (Kumar et al., 2012), or that there are differences in subcellular or apoplastic partitioning of the ABA pool that may lead to developmental effects without an obvious difference in plant ABA content. Nevertheless, AHK1 seems to play an osmosensory role in the leaf vasculature and cannot yet be excluded as part of a signalling system from mature stomata to developing leaves. In addition to ABA biosynthesis, it is likely that ABA catabolism also plays an important role in the modulation of stomatal signals. ABA catabolism is catalysed by ABA 8′-hydroxylase which is encoded by genes including the humidity up-regulated CYP707A1 and CYP707A3 (CYTOCHROME P450 707A1/A3) (Okamoto et al., 2009). These hydroxylase isozymes are likely to be key players in many physiological processes (Nambara & Marion-Poll, 2005), and as such may be as important as NCED3 for altering the course of stomatal development in response to a changing environment. Interestingly, CYP707A3 expression is also up-regulated by brassinolide (Saito et al., 2004), indicating that brassinosteroids are involved in regulating ABA turnover. Recently it was shown that the bZIP (basic region/leucine zipper motif) protein VIP1 (virE2 interacting protein 1), which translocates to the nucleus under elevated turgor pressure, binds to the promoters of CYP707A1 and CYP707A3 inducing rapid catabolism of ABA (Tsugama et al., 2012). A decline in humidity and subsequent turgor loss result in the suppression of VIP1 and cessation of ABA breakdown (Tsugama et al., 2012). Not only could this regulation of ABA catabolism help to provide a mechanism by which stomata lose sensitivity to vapour pressure deficit (Aliniaeifard & van Meeteren, 2013) and by which maturing stomata become primed to the drought response, but it also provides a convincing osmosensory mechanism for stomatal and vascular systemic ABA signalling of New Phytologist (2014) 202: 376–391 www.newphytologist.com

New Phytologist humidity change in addition to the control of ABA biosynthesis (Fig. 3). If, as we have described in section III and outlined in Fig. 3, the plant ABA concentration gradient coupled with ontogenetic stomatal ABA sensitivity helps to modulate stomatal patterning, it might be expected that mutants lacking VIP1 or CYP7O7 isoforms would have low stomatal densities compared with wild type. Similarly, over-expressors of ABA biosynthetic genes such as NCED3 would have lower stomatal densities, as shown for HDG11, but unfortunately these developmental phenotypes have not been investigated. Instead, observations of reduced transpirational water loss with elevated ABA, explained mainly by reduction in stomatal apertures, pervade the literature (Thompson et al., 2000; Qin & Zeevaart, 2002; Ko et al., 2006; Yue et al., 2012; Lu et al., 2013; Zeng et al., 2013) and may not do justice to ABA’s strong effect on developmental processes.

V. Light-mediated changes in stomatal patterning: a role for ABA? Light quantity, light quality and photoperiod are powerful environmental modulators of stomatal physiology as well as stomatal development. Indeed, the differences in stomatal number observed between plants grown under differing light conditions can be far greater than the patterning differences observed between plants grown under different CO2 concentrations or water availabilities. Recent reviews have discussed the contribution of light signalling to stomatal function and development (Shimazaki et al., 2007; Chen et al., 2012; Casal, 2013), but surprisingly little is known about the cross-talk between ABA-mediated and lightmediated control of stomatal development. Long photoperiods and continuous light, like high relative humidity, have been shown to disrupt stomatal development and physiology, reducing plants’ desiccation tolerance and ABA concentrations (Arve et al., 2013), although normal responses can be maintained by growth under higher vapour pressure deficit (Aliniaeifard & van Meeteren, 2013). This suggests that ABA balance might be critical for normal stomatal responses to light. Indeed, in germinating seeds and seedlings, ABA concentrations are controlled by red-light-induced phytochrome down-regulation of expression of NCED genes combined with up-regulation of ABA 8′-hydroxylases (Sawada et al., 2008; Gu et al., 2012), but there are only limited data to suggest that a similar mechanism may be active in plant leaves. In this section we present the emerging data demonstrating that the regulation of ABA concentrations plays a role in promoting or inhibiting stomatal development under differing light intensities or qualities. These data are summarized in Fig. 4. Intriguingly, recent research suggests that the phytochrome red/ far red light receptors may be involved in both elevation of ABA sensitivity and ABA degradation (Gonz
alez et al., 2012). phyB (phytochrome B) is likely to be the key photoreceptor involved in high-light induction of stomatal differentiation, and phyB mutants have reduced stomatal density and index (Boccalandro et al., 2009; Casson et al., 2009). phyB’s effect on stomatal number is at the expense of water use efficiency as more pores cause more transpiration (Boccalandro et al., 2009); however, phyB may Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist

Fig. 4 The interaction of light and abscisic acid (ABA) signalling in stomatal aperture control and development. ABA’s modulation of light regulation (pink background) of stomatal physiology and development is likely to act via transcriptional control (orange boxes) of photosynthetic and nonphotosynthetic photoreceptor-mediated mechanisms (yellow frames). E3 ligase complexes may play a crucial role in the auto-regulation of ABA and the positive and negative regulation of ABA-mediated drought responses by light, modulating both stomatal physiology and stomatal development. Photosynthetic control (green box) feeds into carbohydrate metabolism, sugar signalling, and ABA’s interaction with carbon status sensing (blue boxes on light blue background). Arrows pointing from transcription factors denote transcriptional regulation of their targets and arrows from signalling components denote post-translational protein modification. Solid lines denote activation, whereas dashed lines show inhibition, of processes. See Table 1 for explanation of abbreviations.

counteract this effect by enhancing drought tolerance via the up-regulation of ABA signalling components and increasing ABA sensitivity (Gonz
alez et al., 2012). Under low water pressure deficit and well-watered conditions, however, phyB is involved in ABA degradation. It is clear that the effects of phyB signalling on ABA and drought require further investigation to place them in the context of the involvement of light and ABA in epidermal and stomatal differentiation. Shade and darkness severely reduce stomatal density via the expression or subcellular localization of repressors of photomorphogenesis, including CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and COP10 E3 ligases, which are involved in the degradation of positive regulator proteins including the light-labile photoreceptor phyA (Fig. 4). The stomatal clustering phenotypes and differences in stomatal density and index identified in mutants of COP1, COP10 and other components of these Cul4-based E3 ligase complexes, namely Cul4, DDB1 and DET1 (cullin 4, Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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DNA-damage binding protein 1 and de-etiolated homolog 1), as well as their substrate CDT1 (cell division control protein 10 Target 1) (Castellano et al., 2004; Delgado et al., 2012a,b), indicate that protein degradation may provide a key integration point for environmental modulation of the developing epidermis. The interplay of ABA and light in stomatal development is particularly evident in the interactions of Cul4-based E3 ligase complexes (involving DWA1/2/3 (DWD hypersensitive to ABA1/ 2/3), DDB1, DET1, COP10 and CDT1; Fig. 4) which target protein degradation and affect cell cycle, cell size, photomorphogenesis, and chloroplast compartmentation (Castellano et al., 2004; Bernhardt et al., 2006; Caspi et al., 2008; Lee et al., 2010, 2011; Carvalho et al., 2011; Delgado et al., 2012a,b). Loss-offunction mutants in many of these component genes display stomatal clustering phenotypes, light hypersensitivity, abnormal vasculature and altered stomatal transpiration. DWA1, 2, and 3 have been shown to negatively regulate ABA signalling via the possible degradation of ABI5. dwa3 mutant leaves have reduced water loss (Lee et al., 2010, 2011), suggesting a stomatal or ABArelated defect, although epidermal phenotypes have not been reported. Mutants in DDB1 have reduced ABA concentrations, believed to be caused by down-regulation of the ABA biosynthetic enzyme ABA1 (Carvalho et al., 2011; Kilambi et al., 2013). Furthermore, exogenous ABA up-regulates expression of DDB1 (Wang et al., 2008) and reverses the elevated transpiration phenotype of ddb1 mutants in tomato (Carvalho et al., 2011), suggesting that DDB1 and its associated ligase complexes (i.e. DWAs) may be involved in the auto-regulation of ABA concentrations. Additionally, transpiration phenotypes in ddb1 have been proposed to be caused by alternative compartmentalization/ sequestration of ABA in the smaller, denser chloroplasts that these mutants possess (Caspi et al., 2008; Carvalho et al., 2011). Although stomatal development is most strongly affected by red light perception, as well as the phytochromes, the blue light sensors the cryptochromes and phototropins are involved, albeit to a lesser extent, in the regulation of stomatal development (Kang et al., 2009). It has been proposed that blue light perception by cryptochromes reduces ABA concentrations, which causes increased stomatal sensitivity to light signals to promote stomatal opening as well as an increased density of stomata to be produced in young leaves (Boccalandro et al., 2012). We propose an alternative hypothesis that the elevated transpiration induced by cryptochrome’s reduction of guard cell and vascular ABA concentrations may be the cause of the observed increased stomatal density in younger leaves. The repression of photomorphogenesis and stomatal development by COP1 and possibly COP10 is inhibited by all three classes of photoreceptor. COP1 has been shown to negatively regulate stomatal development upstream of YODA, which in turn inhibits stomatal development (Fig. 4; Kang et al., 2009). ABA may feed into this process via the shade induction of brassinosteroid, which plays a crucial role in the repression of photomorphogenesis and stomatal production (Kozuka et al., 2010; Wang et al., 2012b). Brassinosteroid signalling has only recently been implicated in the control of stomatal development. Nonetheless, a framework for its involvement is emerging. BIN2, a negative regulator of brassinosteroid signalling which is itself New Phytologist (2014) 202: 376–391 www.newphytologist.com

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negatively regulated by brassinosteroid, acts to repress YODA (Kim et al., 2012) as well as SPEECHLESS (Gudesblat et al., 2012) and consequently can both inhibit and promote stomatal development. Nevertheless, brassinosteroid signalling can repress the positive regulators of stomatal signalling phyB and SPEECHLESS, and activate negative regulators such as COP1 and YODA (Liu et al., 2011; Gudesblat et al., 2012; Lau & Deng, 2012; Khan et al., 2013). Thus, the regulation of YODA and SPEECHLESS activity represents points of control where environmental signals such ABA could feed in (Zhang et al., 2009; Tanaka et al., 2013), modifying the plant’s stomatal development response to shade under drought or variable CO2 (Fig. 4). It has recently been demonstrated that FHY3 and FAR1 (FARRED ELONGATED HYPOCOTYL 3 and FAR-RED IMPAIRED RESPONSE 1), transcription factors that transduce phytochrome A-mediated events, are up-regulated by ABA and also bind to and activate the promoter of ABI5 which encodes a transcription factor associated with the regulation of ABA sensitivity, particularly during seed germination (Tang et al., 2013; Fig. 4). FHY3 also provides protection for phytochrome A against degradation by COP1 (Saijo et al., 2008). Although stomatal development phenotypes have not been reported, fhy3 and far1 single and double mutants have additively greater stomatal apertures, ABA insensitivity, and transpirational water loss (Tang et al., 2013). FHY3 and to a lesser extent FAR1 are known to have several developmental effects in plants, but understanding their cause is complicated by their effect on the regulation of chloroplast division (Ouyang et al., 2011). The activation of ABI5 by FHY3 and FAR1 counters the degradation of ABI5 by Cul4-based E3 ligase complexes, and the two processes modulate drought signalling by light. This suggests that light signalling may both positively and negatively regulate ABA signalling, stomatal physiology and the stomatal development pathway. This flexibility could provide a physiological mechanism to prevent light from having a significant positive effect on stomatal development during periods of drought. Exposure to ultraviolet (UV) light causes increased stomatal densities, and a loss-of-function mutation in a UV-B receptor, uvr8, causes reductions in stomatal density and index (Wargent et al., 2009). How UVR8 transduces the light signal to modulate the stomatal development pathway is yet to be discovered, but there is evidence that ABA and COP1 might also be involved in this response. UVR8 has a primary function in UV-B perception by plants and co-localizes and interacts directly with COP1 to protect plant tissue from UV damage (Favory et al., 2009; Rizzini et al., 2011). DDB1, probably in conjunction with COP1 and/or COP10, is also functionally involved in the plant response to UV damage (Caspi et al., 2008). ABA concentrations are up-regulated by UV-B and are involved in the plant’s defence response (Berli et al., 2010, 2011; Gil et al., 2012). Furthermore, UV-B-induced stomatal closure shares components with ABA-induced closure, although it is not clear how ABA is involved in this process (He et al., 2013).

VI. A role for ABA in CO2 and carbon budget signalling In most plants, CO2 uptake in the light results in the accumulation of photosynthates during the day. Growth under high light New Phytologist (2014) 202: 376–391 www.newphytologist.com

New Phytologist intensities normally results in higher stomatal densities and higher rates of photosynthesis and carbohydrate production. In lower light conditions, these phenotypes are reversed. Sugar signalling therefore provides a mechanism by which light quantity can modulate stomatal development. However, this mechanism cannot be simple, as growth at elevated CO2 concentrations also increases the accumulation of photosynthates but normally reduces stomatal density (Coupe et al., 2006). As elevated CO2 and low light intensities both result in stomatal closure, a more attractive explanation is that they both alter the transpiration rate which in turn affects hormone and sugar signalling via the transpiration stream to developing leaves (Pons et al., 2001). Sucrose and carbohydrate metabolism has recently been implicated in the maintenance of normal stomatal development and the prevention of stomatal clustering, as direct sugar treatment of seedlings results in defective epidermal patterning (Akita et al., 2013). Trehalose metabolism and the intermediate trehalose-6-phosphate (T6P) have also been implicated in regulating this process. Trehalose acts as a carbohydrate reserve and can induce stomatal closure (Gao et al., 2013) and, along with its intermediates, may be involved in the cross-talk between the plant’s carbon status and ABA signalling. The potential links between carbohydrate metabolism, sugar signalling, ABA, and stomatal development are discussed in this section and outlined in Fig. 4. Trehalose is a plant disaccharide synthesized in trace amounts via the conversion of UDP-glucose to T6P by T6P synthase (TPS), and dephosphorylation by T6P phosphatase (TPP; Ponnu et al., 2011). T6P is an indicator of high carbon availability and acts as a signal to modulate carbon utilization in plants (Wingler et al., 2012; O’Hara et al., 2013). Interestingly, disruption of the enzyme TPS6 results in changes in epidermal morphology and stomatal density (Chary et al., 2008). Manipulation of TPS expression alters sugar responses. TPS1 expression is induced by ABA and induces expression of ABA-responsive genes including ABI4 (Leonhardt et al., 2004; Kim et al., 2005; G
omez et al., 2010; Li et al., 2011). TPS1 over-expression results in glucose and ABA insensitivity, while weak nonlethal mutations cause stomatal ABA hypersensitivity, correlating with T6P concentrations and reduced epidermal cell size (Avonce et al., 2004; G
omez et al., 2010). Similarly, there is a correlation between T6P concentrations and ABA sensitivity (Vandesteene et al., 2012). T6P inhibits hexokinase activity in yeast, to negatively regulate the entry of glucose and fructose into glycolysis (Bl
azquez et al., 1993), and a similar process might occur in plants. Hexokinase phosphorylates sucrose and has recently been shown to play a role in ABA-mediated stomatal closure (Kelly et al., 2013). Thus, it appears possible that, when the photosynthetic rate is increased through elevated CO2 concentrations or light intensities, T6P production by TPS1 signals the change in sugar status to developing leaves so that they adjust their stomatal density accordingly. As described above, T6P metabolism will be affected by ABA concentrations and the transpiration rate, which may account for the opposing effects of light and CO2 signals on stomatal development. This sugar sensing pathway may also provide a mechanism for the ABA priming effect outlined in Section IV, ABA metabolism and its contribution to stomatal development. Elevated CO2 and light can induce sucrose Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist accumulation under stress conditions and therefore T6P may also act to prime developing tissue for growth once the stress has lifted, via SnRK1 kinase activity and induction of gene expression (Nunes et al., 2013). TPS1 may thus be involved in the priming of maturing stomata to ABA, thereby inducing greater transpiration control. In this way, T6P may play an essential role in the regulation of leaf development by the coordination of long-distance ABA and sugar signalling. TPP genes exhibit specific spatio-temporal expression patterns, indicating tight local control of T6P turnover. It has recently been shown that the TPPG transcript is restricted to trichomes and stomata, and is involved in stomatal ABA responses, as outlined in Fig. 4. The loss-of-function tppg mutation causes stomatal insensitivity to ABA, and over-expression of TPPG results in stomatal ABA hypersensitivity (Vandesteene et al., 2012). Thus, this enzyme may link guard cell sensing of carbon homeostasis to various aspects of growth and development (Vandesteene et al., 2012). Trehalase too has recently been shown to control the transpiration rate by acting both in the modulation of ABA-induced stomatal closure and in the control of stomatal development (Van Houtte et al., 2013). Loss-of-function mutants in the trehalase gene TRE1 result in elevated trehalose concentrations and an impaired drought response, whereas over-expression causes ABA-hypersensitive stomata, reduced transpiration, and a reduction in stomatal index (Van Houtte et al., 2013). An apoplastic brassinosteroid-responsive protein EXO (EXORDIUM) provides further links between extra- and intracellular carbon status, leaf growth, trehalose and ABA signalling (Lisso et al., 2013). EXO is induced at night, by low CO2 concentrations and carbon limitation (Schr€oder et al., 2011). Loss of EXO function results in smaller leaves with smaller cells, ABA accumulation, and down-regulation of TRE1 and trehalase activity which is reversed upon treatment with trehalose (Lisso et al., 2013). The effects of EXO on stomatal density and index have not been reported, and further work is required to determine how EXO interacts with the trehalose biosynthesis pathway and T6P signalling. There are likely to be many other intersection points between the guard cell signalling and stomatal development pathways where CO2 and carbon status sensing mechanisms interact with ABA. For example, glucose induces ABA biosynthesis (Hassibi et al., 2011) and plays a direct role in ABA signalling as a moiety of the inactive ABA-glucose ester (ABA-GE) in the apoplast (Sauter et al., 2002) and guard cells may have access to ABA via cleavage of the ABAglucose ester by b-glucosidase 1 (BG1; Lee et al., 2006; Bauer et al., 2013; see Fig. 3). Recently, another b-glucosidase, BG2, has been shown to be strongly activated by drought stress, and bg2 lossof-function mutants have limited transpirational control because of impaired ABA production (Xu et al., 2012). It has been proposed that ABA-GE may be more suitable as a long-distance regulator of stomatal development than ABA itself, as a consequence of its low membrane permeability and weak ABA-like activity which is probably attributable to conversion to ABA itself (Jiang & Hartung, 2008; Kato-Noguchi & Tanaka, 2009; Kepka et al., 2011). An ABA-GE transporter has recently been identified (Burla et al., 2013) and may pave the way for future studies of ABA-GE’s role in plant development. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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b-carbonic anhydrases have recently been shown to influence both guard cell physiology and stomatal development (Hu et al., 2011) and may feed into plants’ carbon status sensing. bca1bca4 double mutants show altered stomatal responses to CO2 and have increased stomatal density in a seemingly light- and ABAindependent manner, and ABA and drought have been shown to induce the activity of b-carbonic anhydrases in barley (Hordeum vulgare) (Popova et al., 1996). In concert with stomatal conductance, internal conductance to CO2 can change rapidly via ABA-mediated induction of the activities of aquaporins and carbonic anhydrases (Warren, 2008). The roles of b-carbonic anhydrases in the regulation of transpiration and in long-distance regulation of stomatal development are only beginning to be understood. Similarly, the EPF peptides, which regulate stomatal differentiation and inhibit clustering, may play a role in the developmental response to CO2 (Doheny-Adams et al., 2012; Richardson & Torii, 2013) as the manipulation of their expression under elevated CO2 conditions results in variable stomatal density responses, but a direct mechanism remains unknown. The effect on transpiration of CO2-mediated stomatal density changes clearly has consequences for drought tolerance and therefore ABA regulation, but it remains to be seen where and how ABA and CO2 signalling converge in the regulation of these processes.

VII. ABA-mediated signals from the surface: the role of waxes and cuticle The waxy cuticle of the stoma is the threshold between the guard cell and the external environment, its first point of contact with intercepted light, CO2 and humidity and thus the first line of defence for the epidermis. Epicuticular wax properties determine epidermal wettability and protection from and absorbance of light, as well as responses to CO2 and pathogens (Gray et al., 2000; Pf€undel et al., 2007; Koch et al., 2008). There is a strong link between cuticle formation and epidermal patterning which suggests that there is cross-talk between cuticle composition and epidermal cell fate which is highly responsive to the environment (Bird & Gray, 2003; see Fig. 4). Although the identity of the cuticular signals and the mechanism of their perception remain unknown, ABA again is implicated. An increase in ABA concentrations, triggered by changes in relative humidity, affects the properties of the waxy cuticle by reducing cuticle permeability and altering the cell wall composition (Curvers et al., 2010). The ABAresponsive R2R3-type MYB transcription factor MYB96 promotes drought resistance by activating cuticular wax biosynthesis and affecting stomatal aperture, but no effect on stomatal density was observed (Seo et al., 2009, 2011). Recently, a drought- and ABAactivated transcription factor in soybean (Glycine max), GsWRKY20, has been shown to up-regulate the expression of a suite of ABA signalling and wax biosynthesis genes, and its overexpression results in more glaucous leaves with thicker cuticles which have fewer, but ABA-hypersensitive, stomata (Fig. 5; Luo et al., 2013). As described above in section IV, the activity of SHINE transcription factors, and in addition many lipid metabolism enzymes involved in wax biosynthesis, affects stomatal New Phytologist (2014) 202: 376–391 www.newphytologist.com

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2009). The disruption of HOS3 results in ABA-hypersensitive stomata and lower rates of water loss, suggesting that HOS3 acts as a negative regulator of stomatal ABA signalling by controlling production of sphingosine-1-phosphate (S-1-P) by sphingosine kinase (Ng et al., 2001; Coursol et al., 2003; Quist et al., 2009). Interestingly, S-1-P is known to induce stomatal closure, thus regulating plant transpiration efficiency; physiologically by affecting guard cell sensitivity to CO2, ABA and UV-B, and developmentally by controlling epidermal cell size and stomatal development (Nilson & Assmann, 2010; Zhang et al., 2011b; He et al., 2013). ABA therefore has a far-reaching influence on stomatal development by cuticular modification via the control of transcription factor activity, as well as the control of VLCFA, wax and cutin biosynthesis.

VIII. Conclusions

Fig. 5 Cuticle and wax biosynthesis signalling cross-talk with stomatal ABA signalling and development. Cuticular modification is modulated by ABA and affects ABA signalling and biosynthesis, influencing stomatal physiology and stomatal development via the control of transcription factor activity (orange boxes) and components of very long chain fatty acid (VLCFA), wax and cutin biosynthesis (grey boxes). Arrows pointing from transcription factors denote transcriptional regulation of their targets and arrows from signalling components denote post-translational protein modification. Solid lines denote activation, whereas dashed lines show inhibition, of processes. See Table 1 for explanation of abbreviations.

development. The loss of function of a sterol-desaturase-like protein, CER3 (ECERIFERUM 3), results in reduced stomatal density and index, as well as increased cuticle permeability (Chen et al., 2003). Disruption of 3-ketoacyl coenzyme A synthase genes involved in the synthesis of very long chain fatty acids (VLCFAs), including CER1, CER6 and HIC (High Carbon dioxide), results in increased stomatal indices (Fig. 5; Gray et al., 2000). CER6 transcription is up-regulated by light and drought and the CER6 promoter contains consensus light- and ABA-responsive element sequences (Hooker et al., 2002), indicating a mechanism for environmental regulation of wax composition. Interestingly, the hic stomatal phenotype is apparent only when plants are grown under elevated CO2, suggesting that HIC is involved in regulating wax composition and stomatal development in response to changes in CO2 concentration (Gray et al., 2000). Other VLCFA biosynthesis proteins have also been shown to influence stomatal signalling. A mutant of ABO1/ELO2 (ABA-OVERLY INSENSITIVE 1/ELONGATA 2), a holo-elongator subunit, has increased ABA sensitivity, reduced stomatal density as a result of arrested guard cell development, smaller stomatal apertures and improved drought tolerance (Chen et al., 2006). A similar elongase protein, HOS3 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 3), is involved in the synthesis of VLCFAs for the production of sphingolipids such as ceramide (Quist et al., New Phytologist (2014) 202: 376–391 www.newphytologist.com

The ontogenetic priming of stomata to ABA and the mature guard cell response to ABA-mediated hydraulic and nonhydraulic signalling demonstrated by Pantin et al. (2012, 2013a,b) support a fundamental role of ABA in shaping plant development in a changing environment. Furthermore, they provide a mechanism by which the stomata of mature expanded leaves, no longer sheltered and instead exposed to the elements, can modulate the epidermal growth programme to maximize future gas exchange. Transpiration is key to this process (Lake & Woodward, 2008). We review the evidence which demonstrates that a plant’s ability to sense, respond to, and regulate the rate of transpiration, principally through ABA, is fundamental to this process and that mature stomata are the control centres of transpiration. Their transepidermal location allows them to receive direct environmental signals to and across the guard cell cuticle and also messages from the surrounding epidermal cells, as well as ABA, humidity and other signals from below. Here we have outlined several aspects of ABA control to highlight processes in which ABA may act as a conduit for environmental information to immature leaves. We have focussed on core components that may have dual functions in mature guard cell physiological control and the control of stomatal development in an attempt to convey the considerable overlap of these seemingly distinct stages. We conclude that recent findings with regard to ABA’s biosynthesis, catabolism, control of gene expression, and interactions with brassinosteroid and photoreceptor signalling are strong indicators of its central role in uniting these stages. We believe that its role as the plant’s brake system for transpiration puts ABA in a prime position to be a long-distance brake on epidermal development. Further research is needed to identify the missing links in this system. Only very recently have we begun to understand the mechanisms of ABA and ABA-GE transport and ABA’s recognition by receptors (Park et al., 2009; Kang et al., 2010; Kanno et al., 2012; Burla et al., 2013). There is evidence that variation in spatiotemporal control of different ABA receptor classes helps to determine local ABA sensitivity and accumulation in specific cell types (Boursiac et al., 2013). Humidity, light, and/or a combination of environmental signals could trigger this variation and thus be responsible for ontogenetic priming of the guard cell apparatus. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist Acknowledgements The authors would like to thank Ian Woodward for helpful comments during the writing of the manuscript.

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