Update on Stomatal Evolution

Evolution of the Stomatal Regulation of PlantWater Content[OPEN]

Timothy J. Brodribb* and Scott A. M. McAdam

School of Biological Sciences, University of Tasmania, Hobart TAS 7001, Australia

ORCID IDs: 0000-0002-4964-6107 (T.J.B.); 0000-0002-9625-6750 (S.A.M.M.).

Terrestrial productivity today is regulated by stomatalmovements, but this has only been the case since “stoma-tophytes” became dominant on the land 390 million yearsago. In this review,we examine evidence for the functionofearly stomata, foundonor near the reproductive structuresof bryophyte-like fossils, and consider how this functionmay have changed through time as vascular plantsevolved and diversified. We explore the controversial in-sights that have come from observing natural variation,rather than genetic manipulation, as a primary tool forunderstanding the function of the stomatal valve system.

WHY IS STOMATAL EVOLUTION IMPORTANT?

The variation in stomatal behavior among extant plantgroups has stimulated great interest recently across diversefields of science. Behavioral differences in the responses ofstomata to water stress within plant communities havebeen recognized as an important axis of variation in eco-logical strategy (Martínez-Vilalta and Garcia-Forner, 2016;Meinzer et al., 2016), while systematic variation in stomatalsize, density, and response characteristics (Santelia andLawson, 2016) among plant clades have important impli-cations for interpreting plant-atmosphere interactions(Franks et al., 2012; Franks e Buckley, 2017).Understandinghow stomatal behavior feeds into processes of plant selec-tion is a fundamental goal in plant science, and muchprogress over thepast threedecadeshasbeenmade towardthis goal by applying the tools of genetic manipulation tothemodel angiosperm,Arabidopsis thaliana (Schroeder et al.,2001; Hetherington andWoodward, 2003; Kim et al., 2010;Hedrich, 2012; Eisenach and De Angeli, 2017; Jezek andBlatt, 2017). A very different but potentially complemen-tary approach, especially as we approach a postgenomicsera, is the use of natural variation as a lens through whichthe function of stomata canbeviewed fromaperspective ofplant selection over evolutionary time. This novel evolu-tionary approach to understanding stomatal function pre-sents huge potential for enhancing our understanding ofnot only the influence of stomatal behavior on the coloni-zation of land by plants, but also the key processes thatgovern stomatal responses in modern species.

Stomatal evolution has become a focus of some de-bate in recent years (Brodribb et al., 2009; Brodribb andMcAdam, 2011; Chater et al., 2011; Ruszala et al., 2011;Franks and Britton-Harper, 2016), largely initiated by ev-idence that although the stomata of ancient lineages ofvascular plants open similarly to angiosperms in responseto light and CO2, they close differently. In particular, theobservation that stomatal conductance and transpirationof ferns and lycophytes does not significantly decline inresponse to abscisic acid (ABA; Brodribb and McAdam,2011) led to the theory that stomatal closure during waterstress originated in early vascular plants as a passive re-sponse of guard cells to dehydration, and that the “active”closure mechanism, mediated by ABA, evolved muchlater in the earliest seed plants. Debate about this theoryhas seen apparently contradictory evidence presented

* Address correspondence to timothyb@utas.edu.au.T.J.B. and S.A.M.M. contributed equally to this work.[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.17.00078

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from researchers using diverse approaches. Genetic tech-niques are seen as the ideal means of resolving such de-bates, but so far this evidence has also provided confusingresults about the presence and localization of necessarycomponents required for ABA signaling in stomata (seebelow). In the face of equivocal physiological evidence, it iscritical to consider stomatal evolution fromfirst principles,in terms of how differences in stomatal regulation impactsplant performance. Here we focus on the evolution of sto-matal regulation of plant water content, from the perspec-tive of selection and adaptation, considering the functionalrole of stomata, and how this relates to variation in form,positioning, and macroscopic function observable acrossthe phylogeny of land plants. Thismacroscopic perspectiveis essential when considering core plant processes, such asstomatal action, because adaptive changes in function areexpected to have profound andmeasurable effects on plantperformance (McElwain and Steinthorsdottir, 2017).

The evolution of ABA responsiveness in land plantsrepresents a fascinating example of how different per-spectives can lead to profoundly different conclusions.For example, we know that ferns and lycophytes arecapable of very fast (,5 min) stomatal closure to,10%maximum aperture during dehydration, a necessaryresponse to prevent damage to the plant (see below).The size, speed, and shape of this closure response inferns and lycophytes appears to be well explained bypassive stomatal closure through guard cell dehydration,without any need for active processes of ion trafficking(Brodribb andMcAdam, 2011).When combinedwith thefact that unlike angiosperms, fern and lycophyte stomatado not respond to endogenous levels of ABA (McAdamand Brodribb, 2012), the conclusion that ABA-mediatedclosure is not important in basal vascular plants seemsrobust. However, reports regularly emerge of smallstomatal responses in fern and lycophyte guard cellsartificially exposed to ABA levels hundreds of thousandsto millions of times higher than endogenous levels(Ruszala et al., 2011; Cai et al., 2017; Hõrak et al. 2017).The reasonable conclusion from these data is that fernand lycophyte guard cells react to exceedingly highlevels of ABA, but the challenge remains to understandthe adaptive relevance of such observations.

In this review we document recent discoveries madeusing an evolutionary approach to reconstructing stomatalfunction and consider how this adds to the classical geneticmanipulation approach in a model angiosperm that hasdominated stomatal biology for the past three decades.

DEVELOPMENTAL HOMOLOGY BUTFUNCTIONAL DIVERGENCE?

The opening and closing of stomata is a conspicuousfeature of vascular land plant physiology (Darwin,1898), and the presence of stomata on moss and horn-wort sporophytes (Ziegler, 1987) aswell as the epidermesof species from the oldest vascular land plant fossilassemblage, the 410 million-year-old Rhynie Chert(Edwards andAxe, 1992) suggest that these features are

a critical tool for terrestrial plant survival. Despite nu-merous losses of stomata in the bryophytes, includingin the liverworts and a number of basal moss clades(Paton and Pearce, 1957; Haig, 2013), the structure anddevelopmental genes that guide epidermal cell fate andultimately the differentiation of guard cells appear to beancient and highly conserved (Vatén and Bergmann,2012; Renzaglia et al., 2017). This archaic developmen-tal origin of stomata raises the intriguing question ofwhat selective pressure drove the evolution of thesefirst adjustable pores in the earliest land plants. If thefunction of the earliest stomatawas the same as in vascularplants, facilitating the dynamic optimization of water useagainst carbon gain (Cowan, 1977), then common ele-ments of the stomatal control process likely evolved withthese first stomata. This angiosperm-centric hypothesishas been long held as the best explanation for stomatalevolution (Haberlandt, 1886; Paton and Pearce, 1957;Ziegler, 1987; Chater et al., 2011), but has been recentlychallenged by key differences in the general behavior andapparent role of early stomata aswell as recentmolecularand physiological evidence indicating a highly divergentfunctional role for stomata in these most basal stomata-bearing land plants (Haig, 2013; Pressel et al., 2014; Fieldet al., 2015; Chater et al., 2016; Renzaglia et al., 2017).

While the regulation of gas exchange by stomata tofacilitate photosynthesis is a canon of plant physiology,based on the well-described behavior of millions ofstomata concentrated on the primary photosynthetic or-gans of derived vascular plants (Ziegler, 1987), the place-ment and number of stomata in nonvascular plants andthe earliest, leafless vascular plants is vastly different (Fig.1). In the most basal land plant species, there are no sto-mata on the primary photosynthetic organ, the gameto-phyte, with guard cells located solely on the reproductive

Figure 1. Vast differences exist across land plant lineages in terms of sto-matal density and size. Images of stomata-bearing epidermis from twounexceptional and highly representative species of respective lineages arepresented to illustrate this phylogenetic difference. Left, The epidermis ofthe sporophyte of a temperate, globally distributed, stomata-bearinghornwort species (Phaeoceros carolinianus), the sporophyte is the onlyorgan of this nonvascular plant to bear stomata. All nonvascular plantspecies are characterized by a similar and extremely limited number ofstomata (Paton and Pearce, 1957; Field et al., 2015). Right, The epidermisof the angiosperm, canopy tree species (Elaeocarpus kirtonii) is the pri-mary stomata-bearing organ of this and most other angiosperm species.Stomatal density in this leaf is approximately 230 stomata mm22, which isquite modest for the leaves of an angiosperm tree (Franks and Beerling,2009; Brodribb et al., 2013). Images were taken at the same magni-fication, scale bar = 100 mm.

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sporophyte and, inmosses, on the apophysis at the base ofthe capsule (Haberlandt, 1886). This is similar to a numberof the earliest vascular land plant fossils, particularly thecooksonioids, which are likely the common ancestor ofextant vascular plants (Gonez and Gerrienne, 2010), andfrom which the evidence of the oldest stomata is recorded(Edwards and Axe, 1992). These species had stomata(rarely more than three) similarly clustered around thebase of the reproductive sporangia (Edwards et al., 1998).There is compelling evidence that the main sporophyteaxes of these plants were not capable of autonomousphotosynthesis given their anatomy and instead relied onprimary photosynthesis occurring in a basal gametophyte(Boyce, 2008), much like extant bryophytes, whichhave very low photosynthetic activity in the sporophyte(Paolillo and Bazzaz, 1968; Thomas et al., 1978).Suggestions, based on the frequency and positioning

of the earliest fossil stomata, that ancestral stomata didnot perform a role in photosynthetic gas exchange, aresupported by observations of living examples of horn-wort and moss stomata. In these plants, the stomatalpore forms and opens only once then never closes(Pressel et al., 2014; Field et al., 2015; Renzaglia et al.,2017). This single opening event corresponds to a sub-sequent evaporation of the fluid-filled intercellularspaces in the sporangial tissue, facilitating the desiccationof the sporophyte prior to spore release. Additionally, ithas been proposed that enhanced transpiration causedby stomatal opening may also drive a strong transpira-tionalflux of nutrients and photosynthates from the basalgametophyte into the developing sporophyte (Haig,2013). Recently, these observations have received consid-erable molecular support by a study in the moss speciesPhyscomitrella patens in which a major delay in the dehis-cence of capsules was observed in astomatal, guard celldevelopmental knockout mutants (Chater et al., 2016).Indeed, this lack of capsule dehiscence was the onlyreported functional difference between astomatal mu-tants and wild-type plants.

ANCIENT STOMATAL OPENING DRIVEN BYPHOTOSYNTHESIS IN THE GUARD CELLS

While a divergent role for stomata seems likely be-tween basal land plants and more derived vascularplants, the observation that all stomata are apparentlycapable of opening suggests that a conserved openingmechanism may operate across stomatal-bearing landplants. The one possible exception seems to be the basalmoss genus Sphagnumwhere stomatal opening is likelya derived mechanism, being triggered by the loss ofguard cell turgor (Duckett et al., 2009). This conspicu-ous inversion of the normal positive relationship be-tween aperture and guard cell turgor pressure in thestomata of Sphagnum, however, is not apparent in morederived mosses or hornworts, suggesting that, like vas-cular plants, stomatal opening in basal species other thanSphagnum requires an increase inguard cell turgor (Wiggans,1921; Heath, 1938). In vascular plants, particularly

angiosperms, active metabolic processes essential forincreasing guard cell turgor and opening the pore arewell described, particularly in the light (Schroederet al., 2001; Shimazaki et al., 2007; Inoue and Kinoshita,2017). This opening process is driven by the hyperpo-larization of the guard cell membrane potential throughthe activation of the plasmamembrane proton pump (H+-ATPase). Photosynthesis inside the guard cells (Zeigerand Field, 1982) provides a source of ATP (Tominagaet al., 2001; Lawson, 2009; Suetsugu et al., 2014); however,in angiosperms this guard cell response alone is notsufficient to fully open stomata (Willmer andPallas, 1974;Mumm et al., 2011; Chen et al., 2012). Photosynthesis inthe mesophyll also provides a strong additional signal foropening (Roelfsema et al., 2002; Sibbernsen and Mott,2010; Lawson et al., 2014), potentially via an unknownaqueous signal (Fujita et al., 2013), possibly combinedwithstarchmetabolism (Horrer et al., 2016). Like vascular plants,bryophyte stomata contain chloroplasts (Butterfass, 1979;Lucas and Renzaglia, 2002). Indeed, the presence of chlo-roplasts in guard cells appears to be an ancestral trait, withdistinctive coloring of the guard cells in comparison to ep-idermal pavement cells noted in rhyniophytoids (Edwardset al., 1998), suggesting that these earliest fossilized sto-mata similarly contained chloroplasts. While in angio-sperms photosynthesis in the mesophyll appears toprovide a major signal for driving stomatal opening in thelight (Roelfsema et al., 2002; Lawson et al., 2014), in basalvascular plants, stomatal opening in the light can bedrivenby guard cell-autonomous photosynthesis alone, withrapid stomatal opening occurring in the isolated epidermisof leptosporangiate ferns (McAdam and Brodribb, 2012),which lack stomatal responses to blue light and henceopen only by photosynthetic processes (Doi et al., 2006,2015; Doi and Shimazaki, 2008). These data indicate that amesophyll signal for stomatal opening in the light likelyevolved after the divergence of seed plants (McAdam andBrodribb, 2012). The ancestral ability of nonseed plantstomata to respond to light in the absence of themesophyllmay be due to the comparatively high number of chloro-plasts in the guard cells of these species (Butterfass, 1979).

An association between guard cell photosynthesisand stomatal opening in the earliest stomatal-bearingland plants provides the ideal mechanism for activelytriggering stomatal opening to desiccate the sporo-phyte. 390 million years ago, however, the rise of extantclades of basal vascular land plants including thelycophytes, and followed by the ferns, marked a majoranatomical transition in land plants, namely the evo-lution of an independent, dominant sporophyte gen-eration with dedicated photosynthetic organs coveredin stomata. This evolutionary transition associatingstomata with photosynthesis required a major changein the way land plants used stomata, from facilitatingthe desiccation of the sporophyte to enhancing photo-synthetic gas exchange in the light, and the regulationof plant water status. Maximizing leaf gas exchange forphotosynthesis in the light likely required no evolution-ary transformation in guard cell control, as this processcould easily be achieved by allowing stomata to rapidly

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increase in turgor on exposure to light, presumably inthe same way basal vascular land plants open stomata.The apparent involvement of the H+-ATPase in the sto-matal opening of mosses appears to confirm a commonmechanism with vascular plants (Chater et al., 2011),although more work in bryophytes is required to con-firm this potentially ancient mechanism. Extant lyco-phyte and fern stomata have the capacity to openrapidly in the light (Mansfield and Willmer, 1969; Doiand Shimazaki, 2008; McAdam and Brodribb, 2012),with lycophyte stomata possessing both photosyn-thetically driven stomatal opening in red light- as wellas blue light-triggered opening, while leptosporangiateferns (which comprise more than 96% of fern specificdiversity; Palmer et al., 2004) have lost blue light sto-matal signaling (Doi et al., 2015). The reason behind thisloss of blue light stomatal signaling in derived ferns is,as yet, unknown, but may be due to a chimeric red-bluephotoreceptor, an adaptation associated with photo-synthesis in low light environments (Kawai et al., 2003).

STOMATA ON THE PRIMARY PHOTOSYNTHETICORGAN AND THE MAINTENANCEOF HOMEOHYDRY

Although a role for stomatal opening seems apparentacross all land plants, the role and process of stomatalclosure seems much less uniform, particularly if basalplants are considered. In vascular plants, however, aprimary stomatal function is the action to close the poreduring water stress to reduce transpiration and main-tain plant hydration thus avoiding damage to the plantvascular system (Fig. 2). The primary selective pres-sures driving the evolution of stomatal closure in thelight are typically cited as being a mechanism for in-creasing water use efficiency (during stomatal closure

at low humidity), or as a means of protecting tissuesfrom desiccation. It is hard to understand how selectionfor efficient water use can operate at the level of theindividual plant, becausewater conservation inevitablyprovides more water for competitors (Cowan, 2002).The alternative, protective role provides a much moreconvincing selective advantage to plants and seemslikely to underpin the evolution of stomatal responsesin the light (Wolf et al., 2016). However, as mentionedabove, several lines of evidence suggest that this actionmay not be an ancestral character in stomatal evolution,including the widespread capacity for desiccation tol-erance (Peñuelas and Munné-Bosch, 2010) in bryo-phytes and the fact that ancestral stomata probablyaided rather than inhibited desiccation (of sporangia) inbasal land plants (Caine et al., 2016). Desiccation tol-erance obviates the need to control evaporation fromleaves, but was likely to be incompatible with the ev-olution ofmassive plants that began to dominate forestsduring the radiation of land plants (Kenrick and Crane,1997). The reason for this is that the internal watertransport system in all plants becomes cavitated duringacute water stress, causing leaves to be cut off fromwater in the soil (Tyree and Sperry, 1989). This type ofdamage can be easily repaired in small plants wherecapillary action can redissolve air embolisms in thevascular system after rain (Rolland et al., 2015), butlarger woody plants are unable to repair cavitated xy-lem tissues by capillarity or root pressure (Charrieret al., 2016), and therefore xylem becomes irreversiblydamaged during water stress (Brodribb and Cochard,2009; Cochard and Delzon, 2013). Stomatal closure toprotect the xylem from cavitation must have thus beenan evolutionary prerequisite for the increase in plantsize that occurred during the radiation of vascularplants. Indeed, it has been demonstrated that the sto-mata of basal vascular plants such as ferns close before

Figure 2. Stomata in the earliest vascularplants probably first closed to prevent cavita-tion of the vascular system, a function thatappears to remain conserved until the present.Data here show the trajectory of stomatal clo-sure (blue line) with respect to leaf water po-tential as three tomato plantswere subjected togradual soil drying (green, red, and blackcrosses). Close to the water potential of com-plete stomatal closure, the process of xylemcavitation begins (three traces for leaves fromthree individuals are shown; black, red, andgreen circles). Cavitation curves show the ac-cumulation of cavitation events in the leafveins of three tomato plants as they desiccate(data from Skelton et al. (2017)).

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the decline in hydraulic function associated with xylemcavitation (Brodribb andHolbrook, 2003).New techniquesthat visualize the process of xylem cavitation clearlydemonstrate that a similar role of stomatal closure inprotecting the xylem from cavitation during water stress(Fig. 2) is also common to gymnosperms and angiosperms(Brodribb et al., 2016; Hochberg et al., 2017). There aregoodexamples of interactionbetweenopening and closingsignals to stomata during the early onset of plant desic-cation, for example the opening of stomata at high vaporpressure deficit (VPD) by exposure to low CO2 (Bunce,2006). However, the closure mechanism during dryingappears to override all other signals as plants approachturgor loss (Aasamaa and Sõber, 2011; Bartlett et al., 2016),and subsequent xylem cavitation (Hochberg et al., 2017).The simplest and most effective means of closing a

turgor-operated valve, such as the stomatal pore, whenleaf water status declines is to establish a strong hydraulicconnection between guard cells and surrounding leaftissue, such that the hydration of the guard cells is phys-ically linked to the hydration of the leaf. Such a linkagemeans that guard cells lose turgor as leaf water potentialdeclines, passively closing the pore and dramatically re-ducing evaporation (Brodribb and McAdam, 2011). Thisextremely simplemeans of stomatal closure in response todeclining leaf water status requires no metabolic input orcomplex signaling intermediates and is well described inlycophytes and ferns (Lange et al., 1971; Lösch, 1977, 1979;Brodribb and McAdam, 2011; Martins et al., 2016). Sto-matal responses to changes in vapor pressure deficit (orthe humidity of the air) are thus highly predictable inthese early vascular plants based on a passive model thatlinks leaf turgor with guard cell turgor (Brodribb andMcAdam, 2011;Martins et al., 2016). However, there is animportant prerequisite for this passive mechanism tofunction correctly in these basal species: a minimal influ-ence of epidermal cell mechanics on stomatal aperture,meaning that only guard cell turgor influences the aper-ture of the pore (Franks and Farquhar, 2007). This pre-requisite is very much absent in angiosperms.In angiosperms, the passive response of stomata to

rapid changes in leaf hydration is completely inverted,causing stomata to passivelymove in thewrongdirection,openingwhen evaporation increases and closing as leaveshydrate. This peculiar characteristic arises because of acomplicated mechanical relationship between guard cellsand epidermal cells that results in these “wrong-way”passive responses to leaf water content (Darwin, 1898;Iwanoff, 1928). An absence of this mechanical advantagein ferns and lycophytes appears consistent with passivecontrol in these early clades. Furthermore, cuticle analysisof the rhyniophytoids suggests that this lack of epidermalmechanical advantage is ancestral in stomatophytes. Thestomata in these ancient plants apparently opened towardthe periclinal wall of the guard cell (Edwards and Axe,1992), much the same way as extant lycophytes and ferns(Franks and Farquhar, 2007; Apostolakos et al., 2010) forwhich there is nomovement of the dorsal guard cell wallsinto the surrounding epidermal cells, a major contrastwith modern angiosperms (Fig. 3).

METABOLICALLY CONTROLLED STOMATALCLOSURE IN RESPONSE TO LEAF WATER DEFICIT

A passive linkage between leaf water status andguard cell turgor observed in extant basal vascularplants appears to be sufficient to prevent xylem cavi-tation during diurnal changes in evaporative demand(Martins et al., 2015). However, without more sophis-ticated mechanisms to reduce guard cell turgor andproduce complete stomatal closure, it has been hypoth-esized that passive closure does not provide a sufficientlytight stomatal seal capable of preventing ferns and lyco-phytes from rapidly reaching critical leafwater potentialswhen soil water is depleted during drought (McAdamand Brodribb, 2013). As a result, ferns and lycophytes indry environments rely on either a high plant capacitanceor low stomatal density (McAdam and Brodribb, 2013),desiccation tolerance (Hietz, 2010), and in some cases,rather cavitation-resistant xylem (Baer et al., 2016) tosurvive drought. An active stomatal closing signal hasthe potential to restrict transpiration to rates approachingcuticular transpiration (Tardieu and Simonneau, 1998;Brodribb andHolbrook, 2003, 2004), providing the abilityfor species to preserve plant water even if leaves havelow capacitance and large numbers of stomata. In seedplants, the presence of an active regulator of stomatalresponses to leaf water status is evident from diverseobservations from the field (Schulze et al., 1974), undercontrolled conditions (Tardieu and Davies, 1993), and inelectrophysiological experiments on isolated guard cells(Grabov and Blatt, 1998; Pei et al., 2000; Raschke et al.,2003; Negi et al., 2008; Jezek and Blatt, 2017) all of whichindicate the presence of a metabolic signal driving stoma-tal closure. This signal is the phytohormone ABA,which issynthesized as leaves lose turgor and actively closesseed plant stomata (Mittelheuser and Van Steveninck,1969; Pierce and Raschke, 1980; Davies et al., 1981).

CO-OPTION OF AN ANCIENT AND HIGHLYCONSERVED ABA SIGNALING PATHWAY INTO THEGUARD CELLS OF SEED PLANTS

The molecular signaling pathway eliciting an ABAresponse is well described, initiated by RCAR/PYR/PYL receptors, which in the absence of ABA bind toPP2Cs (Ma et al., 2009; Park et al., 2009). When ABA ispresent RCAR/PYR/PYLbinding to PP2Cs is eliminated,

Figure 3. A schematic representation illustrating the difference in themain mode of guard and epidermal cell deformation during stomatalopening in nonangiosperms compared to angiosperms. The arrows in-dicate the direction of cell movement.

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allowing PP2Cs to activate SnRK2 phosphorylators(Yoshida et al., 2006). In seed plants, once activated byPP2Cs in the presence of ABA these SnRK2s phosphory-late guard cell-specific membrane-bound anion channels(the best described of these are the S-type anion channels,SLACs), initiating the efflux of ions and consequentlydecreasing cell turgor and closing the stomatal pore(Geiger et al., 2009). There is an array ofmembrane-boundchannels and transporters that facilitate the efflux of mostosmolytes once SnRK2s signal the presence of ABA andactivate anion channels (Hills et al., 2012). The dominantrole for SnRK2s in this ABA signaling cascade is stronglysupported by experimental data. The phenotype of singlegene mutants in the key SnRK2 for stomatal responsesto ABA, OST1, are profound, displaying no sensitivityto ABA (Mustilli et al., 2002) or VPD (Merilo et al., 2013;Merilo et al., 2015). While other SnRK2-independent sig-naling pathways for ABA perception and SLAC activa-tion have been suggested (Geiger et al., 2010; Brandt et al.,2012; Pornsiriwong et al., 2017), these are redundant andmany either converge or require cross-talkwith functionalOST1 toactivate anionchannels (Brandt et al., 2015).Whetherthese alternative SnRK2-independent ABA signalingpathways play an adaptively relevant role in stomatalfunction is yet to be determined. The core SnRK2-centric ABA signaling pathway is highly conservedacross land plants with all lineages, including thosespecies without stomata, such as liverworts, displayingfunctional physiological responses to ABA (Ghoshet al., 2016), RCAR/PYR/PYL ABA receptors presentin genomes (Hauser et al., 2011), and functional PP2Cs(Tougane et al., 2010). This highly conserved ABA sig-naling pathway is known to regulate desiccation toler-ancemechanisms (Tougane et al., 2010), spore dormancy,and sex determination in nonseed plants (McAdam et al.,2016), among other processes.

Three essential evolutionary steps (Fig. 4) were re-quired for the co-option of the ancient ABA signal into afunctionally relevant metabolic regulator of gas ex-change in the earliest seed plants: (1) an operationalSnRK2-SLAC pairing, (2) guard cell-specific expressionof this pairing, and (3) the ability to synthesize ABAover a time-frame relevant to stomatal responses. Oneor more of these requirements for ABA-driven stomatalresponses does not occur in nonseed plants. In lyco-phytes and ferns, native SnRK2s are unable to activatenative SLACs (McAdam et al., 2016), while a functionalSnRK2-SLAC pairing, albeit weak, observed in P. patens(Lind et al., 2015) is not specific to the guard cells(Chater et al., 2011; Vesty et al., 2016) and likely plays arole in nitrate homeostasis. In well-studied angio-sperms there is a potent pairing of native SnRK2s andSLACs that are specifically expressed in guard cells(Li and Assmann, 1996; Geiger et al., 2009; Fujii et al.,2011). Whether this also occurs in the first group of landplants to possess functional stomatal responses to endog-enous ABA, the gymnosperms, remains to be tested. Thethird requirement relates to the speed of ABA synthesisover a timeframe that matches apparent ABA-driven sto-matal responses. In terms of changing soil water deficit

(over days), this condition ismet in all vascular landplants(Kraus and Ziegler, 1993; Hoffman et al., 1999; Kong et al.,2009; McAdam and Brodribb, 2013), but only in angio-sperm does ABA synthesis appear to occur over a time-frame that corresponds to the dynamics of stomatalresponse to VPD (McAdam and Brodribb, 2015).

DIVERSITY IN THE REGULATION OF WATER USEAMONG SEED PLANTS IS DRIVEN BY DIFFERENCESIN ABA METABOLISM

The first group of land plants to unequivocally re-spond to endogenous ABA, the gymnosperms, is ableto synthesize ABA in leaves after at least 6 h of sus-tained water stress (McAdam and Brodribb, 2014) andutilize these high levels to close stomata during drought(Brodribb et al., 2014). Evidence suggests that the ear-liest gymnosperms used ABA to prevent cavitation ofthe xylem when growing in seasonally dry environ-ments (Brodribb et al., 2014), unlike fern and lycophytespecies, which appear incapable of dominating dryforest communities. While survival in dry environmentslikely provided the selective pressure to co-opt ABAsignaling into the guard cells, the evolution of this traitappears to have become an important axis of variation inwater use strategies and responses to leaf water status.Diversity in water use strategies stemming from differ-ences in ABA metabolism is apparent in the gymno-sperms. Species that have vulnerable xylem to cavitationsynthesize high levels of ABA in order to close stomataand persist in seasonally dry environments (Brodribband McAdam, 2013). By contrast, other conifer speciesproduce cavitation-resistant xylem (Pittermann et al.,2010; Larter et al., 2015) that allows plants to survivewhen leaf water potentials drop below24.5 MPa duringdrought. Once leaves reach this low leaf water potential,stomata will close passively due to declining guard cellwater contenteven in the absence of ABA (Brodribbet al., 2014; Deans et al., 2017). In these cavitation-resistant species, drought stress beyond this criticalpoint leads to a decline in ABA levels to prestress valuesdespite the plant experiencing extreme but recoverablewater stress. This alternative strategy is also associatedwith anisohydric stomatal responses to drought and isthought to prolong gas exchange and photosynthesisduring drought (Brodribb and McAdam, 2013).

While ABA metabolism provides an important ex-planation for variation in stomatal behavior within thegymnosperms, it also appears to explain differencesbetween gymnosperms and angiosperms in their sto-matal responses towater deficit. In gymnosperms, ABAsynthetic rates are too slow to effectively regulate sto-matal responses to changes in VPD. This is either due toa lack of specific enzymes late in the ABA biosyntheticpathway (Hanada et al., 2011; McAdam et al., 2015), ora general delay in the up-regulation of critical rate-limiting enzymes for ABA biosynthesis (McAdam andBrodribb, 2015), both of which require further testing.The result of this slow ABA synthesis in gymnosperms

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is that conifer stomata have predictable and passive,non-ABA-mediated responses to short-term changesin VPD (McAdam and Brodribb, 2014), just like thetwo most basal lineages of vascular plants. In angio-sperms, however, ABA biosynthesis is extremely rapid(Christmann et al., 2005; Waadt et al., 2014; McAdamet al., 2016) and can be up-regulated by a drop in leafturgor over the time frameofminutes (Pierce andRaschke,1981; McAdam and Brodribb, 2016). Thus, angiospermsrespond to VPD using ABA, as is evidenced by thechange inABA levels in angiosperm leaves in response tostep changes in VPD (Bauerle et al., 2004; McAdam andBrodribb, 2015), while mutants in ABA biosynthetic andsignaling genes have compromised stomatal responses to

VPD (Xie et al., 2006; Merilo et al., 2013; McAdam et al.,2016). The result of this regulation of stomatal responsesto VPD by ABA is that, unlike all other vascular landplant clades, the stomatal response to VPD in angio-sperms is often hysteretic and unable to be predictedby passive hydraulic processes (O’Grady et al., 1999;McAdam and Brodribb, 2015). Contributing to this hys-teresis in stomatal response to VPD is an internal balancebetween the rates of ABA biosynthesis and catabolism,both of which are regulated in different tissues. WhileABA biosynthesis in response to a drop in leaf turgor ishypothesized to occur very near to the vascular tissue(Kuromori et al., 2014), ABA catabolism in an Arabi-dopsis leaf is primarily controlled by two CYP707 genes,

Figure 4. Top, Three key evolutionary transitions are required for ABA to regulate diurnal gas exchange in land plants. 1, Theability of native SnRK2 kinases (as in AtOST1 from Arabidopsis) to interact with, and activate, native anion channels (like theS-type anion channel AtSLAC1 from Arabidopsis). Representative plots of a positive activation of Arabidopsis proteins are takenfrom McAdam et al. (2016). 2, If native SnRK2s can activate native SLACs then the genes encoding these proteins must beuniquely expressed in guard cells, and this is the case in angiosperms (Li and Assmann, 1996), including Arabidopsis, whereAtOST1 is expressed in the guard cells as illustrated by the profound GUS staining of the promoter of AtOST1 in these cells (Fujiiet al., 2007). 3, To regulate diurnal leaf gas exchange, foliar ABA levels must change over a timeframe that is relevant to thestomatal response to changes in VPD. Data for the gymnosperm Metasequoia glyptostroboides, taken from McAdam andBrodribb (2014), show strong increases in ABA level in branches that are dehydrated and maintained at specific leaf waterpotentials for a minimum of 6 h. This contrasts with data from the angiosperm species Pisum sativum taken from McAdam et al.(2016), which like most angiosperms displays a strong increase in ABA level after only 20 min following a doubling in vaporpressure deficit from 0.7 to 1.5 kPa. Bottom, Mapping these key transitions to a phylogeny of land plants, which assumes thatmosses are sister to liverworts (Wickett et al., 2014) and divergent from hornworts, shows that only in angiosperms do all three ofthese essential physiological and molecular requirements for diurnal gas exchange control by ABA occur. As gymnosperms havefunctional stomatal responses to ABA (McAdam and Brodribb, 2014) and ferns do not have guard cell-specific expression ofnative SnRK2 or SLAC genes (McAdam et al., 2016) it is likely that seed plants were the first land plants to evolve a guard cellspecific expression of these genes. Only in angiosperms is ABA synthesis the same speed as the stomatal response to an increasein VPD (McAdam and Brodribb, 2015).

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one expressed in vascular tissue, the other predominantlyin guard cells (Okamoto et al., 2009). These two geneshave different rates of expression in leaves that are ex-posed to high humidity, suggesting temporal varia-tion in ABA catabolism across the leaf (Okamoto et al.,2009). Whether alternative expression profiles forthese two key leaf ABA catabolism genes, or indeedABA transport genes (Kuromori et al., 2011; Kannoet al., 2012) occurs across angiosperm species to ex-plain reported differences in the sensitivity of species toVPD remains to be tested.

ONGOING QUESTIONS IN THE FIELD OFSTOMATAL EVOLUTION

Given the ever-growing multitude of genomic datafrom representative species spanning the land plantphylogeny, in silico analyses are becoming an increas-ingly popular means of discussing physiological evo-lution (Pabón-Mora et al., 2014; Yue et al., 2014; Chenet al., 2016). Stomatal evolution, however, provides someexcellent examples of why gene phylogenies shouldalways be used in combination with experimentalstudies of stomatal behavior in situ. Evidence of highlyconserved stomatal developmental genetics amongland plants (Vatén and Bergmann, 2012; Caine et al.,2016) appears to support the long-held notion of

conserved stomatal function (Haberlandt, 1886; Patonand Pearce, 1957; Ziegler, 1987; Chater et al., 2011).However, a recent combination of molecular and wholeplant experiments by Chater et al. (2016) suggests a di-vergent functional role for stomata in bryophytes. Allthat is required to transform the function of guard cellsfrom passive to functionally ABA sensitive is the reloca-tion or concentration of an ancestral and highly con-served ABA signaling pathway into the guard cells(McAdam et al., 2016). However, this evolutionary tran-sition could never be realized by in silico analysis of ge-nomes alone, which instead finds all stomata-bearingplants have the genetic capacity to perceive and respond toABA, therefore providing the foundation for the as-sumption that all stomata respond to ABA. Whether re-cent in silico suggestions of major differences in the ABAbiosynthetic pathwayacross landplant lineages (McAdamet al., 2015) explain differences in ABA synthetic rates(McAdam and Brodribb, 2014) remains to be tested.

There are a number of environmental signals regu-lating stomatal aperture that have not been discussed inthis review for which either we have little mechanisticunderstanding of, like inverted stomatal rhythms inCAM plants (Ting, 1987), or little functional under-standing of, like stomatal responses to phytohormonesother than ABA (Dodd, 2003). Future work to resolvethese questions and place these responses in the contextof evolution is essential. There remains much debate inthe literature on the evolution of the stomatal responseto CO2 (Brodribb et al., 2009; Franks and Britton-Harper, 2016). A common feature of all studies in thisarea is a conserved tendency to respond to CO2, par-ticularly low CO2, which likely reflects a commonphotosynthetic signaling in all stomata (Brodribb et al.,2009; Franks and Britton-Harper, 2016). However, ma-jor differences across lineages in the way stomata in-stantaneously respond to CO2 in the dark (Doi andShimazaki, 2008; Brodribb and McAdam, 2013) as wellas the influence of ABA on the sensitivity of stomata toCO2 (McAdam et al., 2011) reflect as yet undescribedand potentially important mechanistic differences inthe way stomata respond to more natural endogenousand environmental signals. Further work in this field isrequired to reveal these key mechanistic differences.Received January 20, 2017; accepted April 11, 2017; published April 12, 2017.

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