Ion Transport at the Vacuole during Stomatal Movements1[OPEN] · Institut de Biologie Intégrative de la Cellule, Centre National de la Recherche Scientiﬁque, 91190 Gif-sur-Yvette,

Update on Vacuolar Transport

Ion Transport at the Vacuole duringStomatal Movements1[OPEN]

Cornelia Eisenach* and Alexis De Angeli

Department of Plant and Microbial Biology, University of Zurich, Zurich CH-8008, Switzerland (C.E.); andInstitut de Biologie Intégrative de la Cellule, Centre National de la Recherche Scientifique,91190 Gif-sur-Yvette, France (A.D.A.)

ORCID ID: 0000-0003-4237-2278 (C.E.).

Plant gas exchange with the environment is facili-tated by stomata, small pores found on most aerialsurfaces of land plants. Stomatal pores are formed be-tween a pair of specialized guard cells. In C3 plants,open stomata allow the uptake of CO2 for photosyn-thesis during the day and at the same time the loss ofwater vapor, maintaining the transpiration stream. Atnight and during drought, plants close their stomata toconserve water, while they open them in response tolow CO2 and at high temperatures to allow for evapo-rative cooling. The opening and closure of stomatadepends on guard cell turgor, which, in turn, relies onfluxes of osmotically active solutes in and out of theguard cell. During stomatal opening, osmotically activesolutes enter guard cells via the plasma membrane orare produced inside the cell and ultimately are stored inthe vacuole (Roelfsema and Hedrich, 2005; Kollist et al.,2014; Santelia and Lawson, 2016). This causes waterinflux, a concomitant increase in turgor and swelling ofthe guard cell pair, resulting in the opening of the sto-matal pore. K+ and its charge-balancing anions Cl2,NO3

2, and malate (Mal22), as well as sugars, are theosmotica accumulating in guard cell vacuoles for sto-matal opening. The ions are transported across thevacuolar membrane (also, the tonoplast) by ion chan-nels and secondary active transporters, while vacuolarproton pumps generate the necessary proton motiveforce and acidify the vacuolar lumen.

With the dawn of the patch-clamp technique, manyion channels were characterized in the 1980s and1990s. A lot of these early studies were conductedwith the broad bean Vicia faba and the monocotyledon

dayflower plant Commelina communis because of thelarge size of their guard cells. It was only with the era ofmolecular genetics that the proteins responsible for thecharacterized transport were identified in Arabidopsis(Arabidopsis thaliana), and this review focuses almostexclusively on vacuolar transport proteins of this spe-cies with a role in stomatal physiology (Table I; Fig. 1).

Nevertheless, many discoveries about guard cellfunction were made using V. faba, C. communis, andother species but were carried over into Arabidopsisguard cell research and now shape our reasoning. Forexample, Mal22 is often listed as an important osmoti-cum. This is true for some species such as V. faba(Outlaw and Lowry, 1977; Outlaw and Kennedy, 1978;Van Kirk and Raschke, 1978a, 1978b) and in certaingrowth conditions (Raschke and Schnabl, 1978; VanKirk and Raschke, 1978a). But in Arabidopsis guardcells, on average, K+ is charge balanced to 50% by Cl2

and only to 5% by Mal22, with Mal22 concentrations inthe range of 1 to 2 mM (Negi et al., 2008; Monda et al.,2011, 2016; Takahashi et al., 2015). Nevertheless,

1 This work was supported by the European Union’s SeventhFramework Programme for research, technological development,and demonstration (grant no. GA-2010-267243, PLANT FELLOWS,to C.E.), the Forschungskredit of the University of Zurich (to C.E.), theSwiss National Foundation (grant no. 31003A_141090/1 to A.D.A.),and LabEx Saclay Plant Sciences (grant no. ANR-10-LABX-0040-SPSto A.D.A.).

* Address correspondence to [email protected]. and A.D.A. both reviewed the literature and synergistically

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Tab

leI.

Ove

rview

oftheionch

annels,

tran

sporters,

andpumpsthat

functionat

thetonoplast

duringstomatal

move

men

ts,theirtran

sport

mode,

tran

sported

substrates,

biophysical

and

bioch

emical

properties,an

dassociated

mutantphen

otypes

G,Single-ch

annel

conductan

ce;n/a,notavailable;nH,Hillco

efficien

t.

Nam

eTypeofTran

sporter

Substrates

Biophysical

andBioch

emical

Properties

ArabidopsisMutantPhen

otype(s)

Referen

ces

AtALM

T9

Ionch

annel

Cl2

(1),Mal

22(1,2)

G(Cl2)=32pS;

nH(Cl2)=2.5;

KmMal2-=27m

M(2)

Red

uce

dtran

spiration,wiltingin

drough

tstress

andstomatal

aperture

dep

endingonCl2

(2,3)

(1)Koverm

annet

al.(2007);(2)DeAnge

liet

al.(2013);(3)Bae

tzet

al.(2016)

AtALM

T6

Ionch

annel

Mal

22,Fu

m22

n/a

Nophen

otypewas

found

Meyer

etal.(2011)

AtCLC

aAntiporter

anion/H

+NO

32.

.Cl2

2NO

32:1

H+stoichiometry

(1)

Red

uce

dstomatal

open

ingan

dim

pairedABA-induce

dclosure

(2)

(1)DeAnge

liet

al.(2006);(2)Weg

eet

al.

(2014)

AtCLC

cProbab

lyan

tiporter

anion/H

+MaybeCl2

n/a

Red

uce

dstomatal

open

ingan

dim

pairedABA-induce

dclosure

Jossieret

al.(2010)

AtTPC1/SV

chan

nel

Ionch

annel

K+,Na+,Ca2

+G

(K+)=56pS(1)or43pS(2)

Red

uce

dstomatal

closure

by10m

M

CaC

l 2(1,3)

(1)Peiteret

al.(2005);(2)Ran

fet

al.

(2008);(3)Islam

etal.(2010)

AtTPK1/VK

chan

nel

Ionch

annel

K+

G(in)=45pS;

G(out)=20pS

Delayed

butco

mplete

reductionof

tran

spirationin

response

toABA

Gobertet

al.(2007)

AtN

HX1an

dAtN

HX2

Antiporter

cation/H

+K+(N

HX1,NHX2),

Na+

(NHX1)

KmK+=12–4

0m

M(2)

Red

uce

dstomatal

open

ingin

ligh

tan

dclosure

inABA,an

dreduce

dwater

loss

duringdrough

t(1)

(1)Andreset

al.(2014);(2)Barraganet

al.

(2012)

AtCAX1an

dAtCAX3

Antiporter

cation/H

+Ca2

+KmCa2

+=13.1

mM;Vmax

=12.4

nmolmg2

1protein

min

21(3)

Red

uce

dstomatal

open

ingin

ligh

t(1,

2)an

dim

pairedau

xininhibitionof

ABA-inducedclosure

(1)

(1)Choet

al.(2012);(2)Connet

al.

(2011);(3)Hirschiet

al.(1996)

AtVHA

Vac

uolarproton

ATPase

H+

H+-ATPco

uplingratio=1.75–3

.28

(1);KmATP=0.6

mM(2)

Red

uce

dABA-induce

dstomatal

closure

(3)

(1)Davieset

al.(1994);(2)Hed

rich

etal.

(1989);(3)Bak

etal.(2013)

AtAVP1

Vac

uolarproton

PPi-ase

H+

H+-PPico

uplingratio=1(1);KmPPi

=15–2

0mM(2)

Red

uce

dABA-induce

dstomatal

closure

(3)

(1)Schmidtan

dBriskin

(1993);(2)

Hed

rich

etal.(1989);(3)Bak

etal.

(2013)

AtM

RP5

ABCCtran

sporter

IP6(1)

KmIP6=263–3

10nM;Vmax

=1.6–

2.5

mmolmin

21mg2

1protein

(1)

Impairedstomatal

open

ingan

dclosure;reduce

dwhole-plant

tran

spirationan

ddrough

tsensitivity

(2)

(1)Nag

yet

al.(2009);(2)Klein

etal.

(2003)

Plant Physiol. Vol. 174, 2017 521

Vacuolar Transport in Stomatal Movement



Arabidopsis guard cell Mal22 concentration increasesduring stomatal opening 2- to 3-fold and generallycorrelates with stomatal aperture (Monda et al., 2011;Ding et al., 2014; Takahashi et al., 2015; Medeiros et al.,2016).

We have gained good understanding of the impor-tance of individual transport processes at the vacuole.But, by contrast with events at the plasma membrane,the succession of events at the vacuole leading to sto-matal opening and closure in response to environ-mental signals is hardly established. Stomata close inresponse to drought, which is signaled by the phyto-hormone abscisic acid (ABA). ABA triggers signal-ing cascades that regulate plasma membrane ionchannels by phosphorylation, increases in cytosolicfree Ca2+ concentration, and other second messengers(Roelfsema et al., 2012; Munemasa et al., 2015; Jezekand Blatt, 2017). The changed activities of these ionchannels result in large changes in the electric poten-tial gradient across the plasma membrane (i.e. theplasma membrane potential), which depolarizes fromabout 2110 to 250 mV during stomatal closure(Roelfsema and Hedrich, 2005; Roelfsema et al., 2012).By contrast, there are no in vivo measurements of thevacuolar membrane potential in guard cells and no

information regarding how it changes during stomatalopening and closure.

Because our knowledge of the events at the plasmamembrane is so much more advanced, these eventsare commonly thought of as initiating and changes invacuolar membrane potential and vacuolar transportprocesses are thought of as reactionary, as it were.But there is no actual evidence to assume this isthe case. In fact, it is more likely that fluxes at thevacuolar and plasma membranes are coordinated.For example, we know that kinases such as Ca2+-dependent kinases (CDPKs) and the SNF1-related ki-nase2.6, AtOST1, regulate ion channels at the plasmamembrane (Kollist et al., 2014; Munemasa et al., 2015;Jezek and Blatt, 2017), but it recently became clearthat such kinases also have targets at the tonoplast(Latz et al., 2013; Wege et al., 2014). Furthermore,changes in ATP-dependent transport, at both thetonoplast and the plasma membrane, together seemto be required to generate intracellular Ca2+ signalsthat initiate stomatal closure (Minguet-Parramonaet al., 2016).

During stomatal closure, the guard cell volumedecreases and, accordingly, so does the vacuolarvolume, in Arabidopsis by about 20% (Gao et al.,

Figure 1. Identified transport systems at the vacuole during stomatal opening (left) and closure (right) and their regulation. Majortransporters at the plasma membrane that might link with vacuolar transport processes are indicated in light gray, and theirregulation is indicatedwhere it links with that of vacuolar transporters. Positive regulation is green, and negative regulation is red.CLCc and proton pumps in dark gray indicate that there is no direct evidence for the transported substrate or that there is no directevidence for involvement in stomatal opening, respectively. Dotted lines indicate that the reduction of this transport is probablynecessary for the maintenance of vacuolar pH and/or tonoplast potential and that this maintenance constitutes the transporter’srole in stomatal closure. Dashed lines indicate that the role of the channel is in dispute or only minor. Changes in luminal pH areindicated by a color code. The vacuolar membrane potential is indicated by algebraic signs.

522 Plant Physiol. Vol. 174, 2017

Eisenach and De Angeli



2009). In V. faba and Arabidopsis, the decrease invacuolar membrane surface concomitant with thevolume decrease is accomplished by indentation ofthe tonoplast and the formation of intravacuolarstructures (Gao et al., 2009). While, in Arabidopsis,the singular vacuole of open guard cells does notseem to fragment during stomatal closure, in V. faba,the formation of separate small vacuoles has beenobserved in closed stomata (Gao et al., 2009; Andréset al., 2014).

VACUOLAR K+ TRANSPORT DURINGSTOMATAL MOVEMENT

Only recently, it was established that guard cell-expressed K+ and Na+ transporters of the NHX (Na+/H+

exchangers) family are essential for K+ accumulationin Arabidopsis guard cell vacuoles during stomatalopening (Barragán et al., 2012; Andrés et al., 2014). Overa decade ago, NHX-type transporters were shown tobe responsible for vacuolar pH homeostasis in morn-ing glory (Ipomoea nil or Pharbitis nil) petals, wherethe vacuolar pH determines flower color (Yamaguchiet al., 2001). In fact, a double mutant of AtNHX1 andAtNHX2 shows impaired vacuolar pH homeostasisand reduced K+ accumulation in Arabidopsis roots(Bassil et al., 2011; Barragán et al., 2012). This doublemutant also displays impaired stomatal opening andreduced whole-plant transpiration in the light, and itsvacuolar pH in guard cells wasmore acidic (by;0.3 pHunits) compared with a wild-type pH of about 5.8/5.9(Andrés et al., 2014). Thus, K+ is accumulated in thevacuole during stomatal movement via NHX-type H+

antiporters and relies on the proton motive forceestablished by vacuolar proton pumps.Interestingly, the nhx1 3 nhx2 double mutant also

shows impaired stomatal closure in response to dark-ness, ABA, and during drought stress (Andrés et al.,2014). While a role for AtNHX in vacuolar K+ uptakeduring opening is expected, the reason for the effectobserved during closure is unclear. It could be that thealtered vacuolar pH in nhx1 3 nhx2 double mutantsinterferes with other vacuolar transport during closureor that the altered K+ concentrations in vacuole andcytosol affect tonoplast voltage.The transport process involved in K+ loss from the

vacuole during closure probably involves ion channelsof the TPK (Two-Pore K+) family. AtTPK1 was discov-ered in 2007 (Gobert et al., 2007) and mediates an ioncurrent formerly known as VK (Vacuolar Potassium;Ward and Schroeder, 1994). This channel presents amarked selectivity for K+ and is voltage independent.Instead, AtTPK1 is activated by cytosolic Ca2+ and is atarget of CDPKs and 14-3-3 proteins (Latz et al., 2007,2013). Interestingly, TPK1 channels from Arabidopsis,rice (Oryza sativa), and barley (Hordeum vulgare) areactivated by changes in cross-tonoplast osmoticgradients (Maathuis, 2011). During stomatal closure,as the cytosol loses solutes, hypoosmotic situations

might occur, activating AtTPK1 to induce K+ releasefrom the vacuole. Even though AtTPK1 is expressedin guard cells and mediates K+ efflux, analysis ofleaf water conductance indicates that the knock-out mutant tpk1 shows only a moderately delayedABA-induced stomatal closure, and no whole-plantdrought stress phenotype was reported (Gobertet al., 2007). Other AtTPKs might make up for theloss of AtTPK1 function in the tpk1 mutant, but therole of K+ loss through AtTPKs in stomatal closureand whole-plant water balance needs more detailedinvestigation.

An important K+ conductance in the vacuole is due tothe vacuolar cation channel TPC1 (Peiter et al., 2005).AtTPC1 is the only tandem-pore Ca2+ channel genepresent in Arabidopsis. The gene product mediates theso-called Slow Vacuolar (SV) current. This ion channelpresents a complex ion selectivity mediating monova-lent as well as divalent cation currents, such as K+ andCa2+ currents (Hedrich and Marten, 2011). This dualpermeability fueled a controversy on the intracellularfunction of AtTPC1 in controlling K+ and Ca2+ fluxes(Ranf et al., 2008; Islam et al., 2010; Rienmüller et al.,2010; Hedrich and Marten, 2011; see below). Despitethe importance of AtTPC1 currents, from an electro-physiological point of view, their role in guard cells isstill uncertain. The voltage dependence of AtTPC1-mediated currents is puzzling; it activates at positivetonoplast potentials that are commonly thought not toexist in plants (see below). The point mutation D454/fou2 in a vacuolar loop of AtTPC1 disrupts an intra-vacuolar Ca2+-binding site. This point mutation shiftsthe activation threshold toward negative, physiologi-cal, transtonoplastic potentials, making the channelsconstitutively conductive. This induces an unbalance inthe K+ and Ca2+ cytosolic/vacuolar distribution (Beyhlet al., 2009), indicating that the control of AtTPC1opening is tightly regulated in plant cells. However, nostomatal opening analysis has been reported for fou2.

VACUOLAR ANION TRANSPORT DURINGSTOMATAL MOVEMENT

One of the major advances of the last decade lies inthe molecular identification of anion transport proteinsat the vacuolar and plasma membranes and theirrole in stomatal movement (Roelfsema et al., 2012).In the vacuolar membrane, two different families ofanion transporters have been found to function duringopening and closure: the AtCLC and the AtALMTfamilies. CLC stands for chloride channel and ALMTstands for aluminum-activated malate transporter. Butthe ALMT channels investigated in Arabidopsis guardcells are not aluminum activated. The plasma mem-brane ALMTs also are referred to as AtQUAC, forquick-activating channel, which references their func-tion in mediating the so-called rapid-type current.AtALMTs form a family of membrane proteins that ispresent only in land plants. In Arabidopsis, there are





13members of the ALMT family, and five of them form aclade that seems to be specific to the vacuolar membrane(Kovermann et al., 2007; clade 2 in Dreyer et al., 2012).So far, two of the vacuolar AtALMTs have been shownto be expressed in stomata. AtALMT6 is expressed spe-cifically in guard cells, and its properties were inves-tigated by patch clamp (Meyer et al., 2011). It was foundthat AtALMT6mediates Mal22 and Fum22 (fumarate)currents but not NO3

2 and Cl2 currents. Interestingly,AtALMT6 is activated by cytosolic Ca2+ in themicromolarrange, and an acidic vacuolar pH shifted the threshold ofactivation of this channel. Because of this shift in activa-tion, at a vacuolar pH around 5, AtALMT6 can mediateMal22 efflux from the vacuole to the cytosol. However, sofar, it has not been possible to detect a stomatal aperturephenotype in almt6 knockout plants. Therefore, the exactrole of AtALMT6 in guard cells is still unknown.

More recently, AtALMT9 was found to be importantfor light-induced stomatal opening (De Angeli et al.,2013). AtALMT9 is localized at the vacuolar membraneand is expressed in both shoots and roots. In the shoots,AtALMT9 is expressed in several cell types, includingguard cells. Electrophysiological analysis of AtALMT9shows that this channel is a voltage-gated inward rectifier,opening at negative membrane potentials. AtALMT9 canmediate fluxes of both Mal22 and Cl2 from the cytosol tothe vacuole. Cytosolic Mal22 concentrations are estimatedto range from 0.5 to 2 mM (De Angeli et al., 2013), and atsuch concentrations, no AtALMT9-mediated Mal22 in-ward current can be detected in patch-clamp experiments(Km

malate = 27 mM; Table I). However, Mal22 in the 0.5 to2 mM range acts on the opening probability of AtALMT9and, consequently, stimulates AtALMT9-mediated Cl2

currents. The fact that Mal22 regulates the activity of ionchannels suggests that, in vivo, cytosolicMal22might serveas a signaling molecule. In guard cells, AtALMT9 partici-pates in stomatal opening in response to light, as almt9knockout mutants display reduced stomatal opening.Moreover, the functionofAtALMT9 in stomatal opening isdependent on the presence of Cl2 in the medium. Thissuggests that, in guard cells, AtALMT9 mediates the ac-cumulation of vacuolar Cl2 rather than Mal22 during sto-matal opening. Recent data show that, also in other tissues,such as root vasculature, AtALMT9 is involved in Cl2

rather than Mal22 transport (Baetz et al., 2016).Another family of anion transport systems identified

in guard cells is the CLC family. CLCs are a family ofmembrane proteins that is present in all eukaryotic andprokaryotic organisms. In eukaryotes, CLC proteinscan be localized in intracellular membranes and at theplasma membrane (Jentsch, 2015). So far, all intracel-lular CLCs were found to be anion/H+ exchangers,while the plasma membrane CLCs are anion chan-nels. In Arabidopsis, there are seven CLCs (AtCLCa–AtCLCg), all localized in intracellular membranes, andAtCLCa and AtCLCc have identified roles in guard cellphysiology (Jossier et al., 2010; Wege et al., 2014).AtCLCc was the first putative vacuolar anion trans-porter shown to be involved in regulating stomatalmovements (Jossier et al., 2010). The results obtained

from stripped epidermis show that AtCLCc has a dualfunction in light-induced stomatal opening and ABA-induced stomatal closure. However, no direct data on thetransport properties of this channel are available to date,but based on the amino acid sequence, it is possible thatAtCLCc is an anion/H+ exchanger with a preference forCl2. The only Arabidopsis CLC expressed in guard cellsfor which functional data are available is AtCLCa.AtCLCa was the first NO3

2 transport system identified inthe tonoplast (De Angeli et al., 2006). It was shown thatAtCLCa is an anion/H+ exchanger with a marked selec-tivity for NO

3

2 over Cl2 and an exchange stoichiometry of2NO3

2:1H+. The exchange reaction catalyzed byAtCLCaallows the accumulation of NO3

2 into the vacuole up toconcentrations 30 to 50 times the ones found in the cytosol.In guard cells, the capacity of AtCLCa to mediate anionaccumulation in the vacuole made it a likely actor duringstomatal opening, and indeed, clca knockout mutantplants show reduced stomatal opening (Wege et al., 2014).Unexpectedly, AtCLCa also is involved in mediatingstomatal closure. Stomata of clca plants show impairedABA-induced closure, and rosettes dehydrate fasterthan wild-type rosettes during drought stress. Notably,AtCLCa is phosphorylated by the AtOST1 kinase. Thephosphorylation of AtCLCa was found to stimulate itsactivity at transtonoplastic membrane potentials below0 mV. These are thought to be physiological, and at thesepotentials, AtCLCa is able to mediate the efflux of anionsfrom the vacuolar lumen into the cytosol. AtOST1 is acentral player of ABA signaling in guard cells (Munemasaet al., 2015); thus, AtCLCa is part of this signaling pathwaycontrolling the stomatal aperture.

The regulation of AtCLCa by AtOST1 and AtTPK1regulation by CDPK are probably only the beginning ofour understanding of how vacuolar anion transportersare regulated by posttranslational modification. A furtherregulator of vacuolar anion transport was found in cyto-solic nucleotides such asATP.ATPs are voltage-dependentinhibitors of AtALMT9 (Zhang et al., 2014), and also,AtCLCa is inhibited by ATP (De Angeli et al., 2009).

The finding that AtCLCa andAtCLCc are involved instomatal closure is intriguing. It has been suggestedthat AtCLCa also mediates the efflux of anions fromthe vacuole during stomatal movements (Wege et al.,2014), and so couldAtCLCc. However, vacuolar solutesmove passively downhill their chemical gradient to theapoplast, making it questionable why secondary activetransporters might be required during closure. A morelikely explanation could be that, as in the nhx1 3 nhx2mutant, clca and clcc mutants are disturbed in vacuolarpH homeostasis or the maintenance of tonoplast po-tential, and this, in turn, affects other transport systemsrequired for stomatal closure.

VACUOLAR CA2+ TRANSPORTERS DURINGSTOMATAL MOVEMENT

Ca2+ is an important second messenger in guard cellsignaling. The cytosolic concentration of free Ca2+ in a





resting state is below 1 mM, but it increases and oscil-lates in response to environmental cues leading tostomatal closure. Details of these intracellular Ca2+ sig-nals are reviewed elsewhere (Pottosin and Schönknecht,2007; Dodd et al., 2010; Roelfsema and Hedrich, 2010;Jezek and Blatt). Suffice it to say that cytosolic freeCa2+ oscillations rely on Ca2+ release from and rapidreuptake into the vacuole and/or other endomem-brane stores, where the concentration of free Ca2+

is about 1,000 times higher than in the cytosol(see refs. above). Quantitative modeling suggests thatmore than 95% of the Ca2+ required for cytosolic sig-nals is of vacuolar/endomembrane origin (Minguet-Parramona et al., 2016). As is the case for the plasmamembrane, our knowledge of themolecular identity ofguard cell Ca2+ transporters in the tonoplast is poor.There are two Ca2+ uptake systems in plant vacuoles:P-type Ca2+ pumps of the ACA (Autoinhibited Ca2+

ATPases) family, with a high affinity, and H+/Ca2+

antiporters of the CAX (Cation Exchanger) family,with a lower Ca2+ affinity (Pottosin and Schönknecht,2007). However, to date, a clear role for these trans-porters in guard cell Ca2+ signaling has not beenestablished. AtACA4 and AtACA11 are known to lo-calize to the vacuole, but no data regarding stomatalbehavior are available for the aca4 3 aca11 doubleknockout mutant (Boursiac et al., 2010). AtCAX1 andAtCAX3 are highly expressed in guard cells and arelocalized to the Arabidopsis vacuole (Cheng et al.,2003, 2005). The double knockout mutant cax1 3 cax3was analyzed by two research groups that found im-paired light-induced stomatal opening (Conn et al.,2011; Cho et al., 2012). The double mutant sequesteredless Ca2+ into mesophyll vacuoles, and its apoplasticCa2+ activity was higher compared with the wild type(Conn et al., 2011). Conn et al. (2011) found that theincreased apoplastic Ca2+ is responsible for the re-duced stomatal aperture. Thus, the stomatal pheno-type in the cax1 3 cax3 mutant is likely to be indirect,as a consequence of reduced Ca2+ accumulation intomesophyll vacuoles, rather than a direct result of im-paired guard cell Ca2+ signaling. Cho et al. (2012) alsoposited an indirect effect, as they found AtCAX1 andAtCAX3 tomodulate apoplastic pH, excluding a directfunction of these proteins in the generation of guardcell Ca2+ signals.To date, we do not know if and/or how Ca2+ is

released from the vacuole during stomatal closure.Because the AtTPC1/SV channel is permeable to Ca2+

and activated by the cation, it was suggested that thechannel might be involved in intracellular Ca2+ sig-naling (Hedrich andMarten, 2011). However, the tpc1mutant is not affected in cytosolic Ca2+ oscillationsand signaling (Ranf et al., 2008; Islam et al., 2010). It isnot affected in the inhibition of opening induced byABA, methyl jasmonate, and CO2 (Islam et al., 2010),does not show impaired stomatal closure in ABA(Ranf et al., 2008), and even the impairment of sto-matal closure in 10 mM external Ca2+ (Peiter et al.,2005) is not associated with any severe whole-plant

mutant phenotype with regard to plant water bal-ance. In brief, the analysis of stomatal responses intpc1 knockout mutants failed to clarify the functionsof AtTPC1 in guard cells, but it is unlikely that guardcell Ca2+ signaling relies on AtTPC1-mediated Ca2+

release from the vacuole. Ligand-gated Ca2+ channelsthat are sensitive to inositol triphosphate, inositolhexakisphosphate (IP6), and cADP ribose are thoughtto exist at the guard cell vacuolar membrane. How-ever, while Ca2+ currents sensitive to these ligandshave been described for vacuoles, no channel proteinshave been identified to date (Roelfsema and Hedrich,2005).

OTHER VACUOLAR TRANSPORT DURINGSTOMATAL MOVEMENT

It was shown recently that starch breakdown inguard cells is necessary for stomatal opening in Arabi-dopsis (Horrer et al., 2016). It is assumed that starchconversion to sugars, but also the import of sugars viathe plasma membrane, is required to achieve or main-tain open stomata, replacing or adding to K salts asosmotica (Daloso et al., 2016; Santelia and Lawson,2016; Santelia, 2017). How might sugars be importedinto and released from the guard cell vacuole? No dataexist addressing exactly these questions; however,AtTMT1 and AtTMT2 are two transporters that havebeen shown to accumulate Glc and Suc into the vacuolein other cell types (Wormit et al., 2006; Schulz et al.,2011), but their role in stomatal guard cells is notknown.

The accumulation of solutes in the vacuole causesmassive fluxes of water into the lumen. Althoughwater can permeate membranes by diffusion, aqua-porins are probably necessary to mediate the largeand fast water fluxes that we have to assume acrossthe vacuolar membrane during stomatal movement.Vacuolar aquaporins are referred to as tonoplast in-trinsic proteins (TIPs), and few data on their role inguard cell movement exist. Pou et al. (2013) studiedTIP expression in grapevine (Vitis vinifera) and founda strong positive correlation between VvTIP2;1 ex-pression and stomatal conductance, suggesting thatTIPs are necessary to achieve maximal stomatal aper-ture. This view is strengthened by the observationthat constitutive overexpression of SlTIP2;2 in tomato(Solanum lycopersicum) increases whole-plant transpi-ration (Sade et al., 2009). Interestingly, under droughtstress, overexpressing plants transpired more and hada lower relative water content compared with the wildtype. This indicates that SlTIP2;2 is important for sto-matal opening but not for stomatal closure, suggestingthat water flux through SlTIP2;2 might be gated orregulated to allow flux in only one direction. Regula-tion by posttranslational modification was describedrecently for the plasma membrane-localized aqua-porin AtPIP2;1, the activity of which is required forstomatal closure in response to ABA but not for





stomatal opening (Grondin et al., 2015). This raisesinteresting new aspects of how we commonly think ofguard cells: ion flux is regulated and water followspassively. Could there be situations in which waterfluxes are regulated and are the driver of guard cellmovements and the ions follow? It was suggested re-cently that this happens in response to changes in airhumidity (Peak and Mott, 2011; Mott and Peak, 2013).Water-driven changes in cytosolic water potentialcould alter the tension of the vacuolar membrane, in-ducing ion release or uptake through osmosensitive ormechanosensitive channels (Peyronnet et al., 2014).MacRobbie (2006) observed osmotic effects on vacu-olar ion release of guard cells and hypothesized theosmosensor to be an aquaporin. In addition, as men-tioned above, TPK channels respond to osmotic effects(Maathuis, 2011), and a recent advance identifiedOSCA1, a Ca2+-permeable channel at the plasmamembrane that is gated by osmotic pressure (Yuanet al., 2014).

A vacuolar transport protein with an intriguing roleduring stomatal movement is AtMRP5/AtABCC5. It isa guard cell-expressed ABC-type transporter thatmediates high-affinity uptake of IP6 into vacuoles(Nagy et al., 2009). Although AtMRP5/AtABCC5also is expressed in other cell types, mrp5 knockoutmutant plants show strong guard cell mutant phe-notypes (Klein et al., 2003). The mrp5 mutant is im-paired in light- and auxin-induced stomatal openingas well as in ABA- and Ca2+-induced stomatal clo-sure. Whole plants of mrp5 knockout mutants showreduced transpiration and, hence, increased droughttolerance (Klein et al., 2003). In patch-clamp experi-ments, it was found that mrp5 mutant guard cellsdisplayed reduced ABA activation of plasma mem-brane Ca2+ and S-type anion currents as well as re-duced activation of anion currents by cytoplasmicCa2+ (Suh et al., 2007). While it was initially thoughtthat the transporter localizes to the plasma mem-brane, it was later reasoned that it is vacuole localized(Nagy et al., 2009). To explain the observed mutantphenotypes, the authors hypothesized that excesscytosolic IP6 in the mrp5 mutant might complex cy-tosolic Ca2+ or increase its release from the vacuolethrough the stimulation of putative IP6-sensitivevacuolar Ca2+ channels.

VACUOLAR pH DURING STOMATAL MOVEMENT

The transport of protons across the vacuole is ener-gized by two proton pumps, a vacuolar H+-ATPase(AtVHA) and an Arabidopsis V-PPi-ase (AtAVP). Afunctional knockout in two transmembrane domainisoforms of the V-typeATPase, vha-a23 vha-a3, showedincreased root vacuolar pH by 0.5 units with respect tothe wild-type pH of 5.9 (Krebs et al., 2010). In fugu5-1,a knockout of the PPi-ase AtAVP1, the vacuolar pHwas increased by 0.25 units compared with the wild-type pH of 5.8 (Ferjani et al., 2011). The triple mutant

vha23 vha33 avp1 shows that these two kinds of protonpumps present an additive function only in some celltypes (Kriegel et al., 2015). In petunia (Petunia hybrida)petals, the vacuolar membrane also contains ATPases ofthe P-type, the type found at the plasma membrane(Verweij et al., 2008; Faraco et al., 2014), but there is noreport for such a function in Arabidopsis guard cells.

The nhx1 3 nhx2 double knockout mutants displaymore acidic guard cell vacuoles and reduced opening(Andrés et al., 2014). The same study showed that light-induced stomatal opening was accompanied by an al-kalinization of guard cell vacuolar pH, from pH 5.8 to5.9, when epidermal strips were incubated in 10 mM

KCl. The fact that mutants of the H+-coupled antipor-ters AtNHX1, AtNHX2, AtCLCa, and AtCLCc all showimpaired stomatal opening phenotypes demonstratesthat the existence of a proton gradient is necessary foropening. Therefore, it is surprising that AtVHA andAtAVP1 knockout mutants do not show impaired sto-matal opening (Bak et al., 2013), and triple mutantsdefective in both proton pumps show vacuolar pHvalues that are still slightly acidic (Schumacher, 2014;Kriegel et al., 2015). Hence, part of the proton gradientnecessary for stomatal opening must be generatedelsewhere. It has been suggested that vacuolar acidityoriginates during vacuolar biogenesis and, thus, relieson other acidic compartments, such as the trans-Golgi network/early endosome (Schumacher, 2014;Kriegel et al., 2015). The det3 mutant, a knockout of thetrans-Golgi network/early endosome-localized V-typeATPase, however, shows impaired stomatal closure(Allen et al., 2000; Brüx et al., 2008), but stomatalopening was not reported to be affected in this mutant.Therefore, it is still unclear how exactly the cross-tonoplast proton gradient necessary for stomatal open-ing is established.

With regard to stomatal closure, it now seems clearthat, in response to ABA, guard cell vacuoles acidify inArabidopsis and in V. faba (Bak et al., 2013; Andréset al., 2014). The respective AtVHA and AtAVP1 mu-tants are slightly impaired inABA-induced closure, andAtAVP1 mutants show a reduced acidification duringclosure (Bak et al., 2013), suggesting that this acidifi-cation results, at least in part, from active pumping.This notion is supported by the observation that over-expression of ThVHAc, a tamarisk (Tamarix hispida)VHA subunit, in Arabidopsis led to reduced water lossunder cadmium stress (Yang et al., 2016). The abovestudies indicate that active proton pumping aids sto-matal closure, but pumping might not be required onlyfor vacuolar acidification but also to maintain a steadytonoplast potential (Hirata et al., 2000). The fact that theantiporters mentioned above show stomatal closingdefects supports the notion that guard cell vacuolar pHalso may be regulated through these proton antiportsystems (Pittman, 2012).

In summary, recent evidence suggests that, in Ara-bidopsis, guard cell vacuolar pH becomes less acidicduring stomatal opening and more acidic during clo-sure. It is not entirely clear how the proton gradient





necessary for opening is established, but it seems evi-dent that, in contrast to the plasmamembrane, vacuolarproton pumping is required for stomatal closure.

THE VACUOLAR MEMBRANE POTENTIAL DURINGSTOMATAL MOVEMENT

To date, we do not know the resting value of thevacuolar membrane potential in guard cells. Measure-ments on isolated vacuoles are problematic, as thetranstonoplast potential would be artificially deter-mined by the measuring solutions. In intact guard cells,it is technically difficult to access the tonoplast for po-tential measurements, because of the small size and thedynamic nature of the guard cell vacuole. Thus, mea-surements were performed on cell types amenable tomicroelectrodes, like root cells, mesophyll cells, orunicellular green algae, and using fluorescent dyes onisolated vacuoles. The reported transtonoplast po-tential values varied between 0 and 266 mV (Thomand Komor, 1985; Bethmann et al., 1995; Walker et al.,1996; Miller et al., 2001; Wang et al., 2015). In Arabi-dopsis, measurements of tonoplast potentials inmesophyll and root hair cells yielded a value of about230 mV (Miller et al., 2001; Wang et al., 2015). Basedon these data, similar resting state values are com-monly assumed in guard cell vacuoles. However, it isunknown if and how much the vacuolar membranepotential undergoes variations in response to extra-cellular and intracellular stimuli such as ABA or Ca2+.Like any other membrane potential, the vacuolarpotential is theoretically defined by the combinationof the permeabilities and the gradients of the differentionic species able to permeate the membrane. TheGoldman-Hodgkin-Katz equation provides a theo-retical framework to understand the behavior ofmembrane potentials (Hille, 2001). It highlights thatthe membrane potential is governed by the mem-brane permeability for one or more ions. Since theactivity of vacuolar ion channels and transporters (i.e.membrane permeability) is strictly regulated, thiswill translate into changes in membrane potential. Intheory, the vacuolar membrane potential could becalculated, and based on the theoretical frameworkmentioned above, the guard cell modeling platformOnGuard calculates vacuolar transport parametersand retrieves tonoplast potential values between215and 235 mV (Chen et al., 2012; Hills et al., 2012). TheOnGuard model predicts that the vacuolar mem-brane potential depolarizes during stomatal open-ing and hyperpolarizes during closure (Chen et al.,2012). By contrast, in the literature, the vacuolarmembrane potential often is hypothesized to depo-larize during closure: an activation of the voltage-independent TPK1 by cytosolic Ca2+ would lead to K+

efflux, which, it being the dominant conductance atthis moment, would lead to a depolarization of themembrane potential toward less negative potentialsbelow 0 (Roelfsema and Hedrich, 2005; Hedrich and

Marten, 2011; Kollist et al., 2014). Because our knowl-edge of the activity gradients and permeabilities ofall ions, as well as the regulation of tonoplast transport,is limited, it is difficult to make any valid statementon the behavior of the vacuolar membrane potentialduring stomatal movement. In the future, the use oftonoplast-targeted, voltage-sensitive genetic reportersmight help to shed light on this important aspect ofguard cell biology.

CONCLUSION

In the last decade, our knowledge of vacuolar iontransport in guard cells has increased significantly.Several lines of evidence now indicate that the transportof ions across the vacuolar membrane is crucial for theregulation of the stomatal aperture. In guard cells, thetonoplast does not appear to be a simple follower ofthe plasmamembrane but is likely to be a primary actorof stomatal movements. The transport of ions acrossthe tonoplast emerges to be tightly controlled byposttranslational modifications and intracellular mole-cules such as phosphorylation, dicarboxylates, andnucleotides. These controls may be required for thecoordination of ion fluxes across both the plasma andvacuolar membranes and for efficient stomatal open-ing and closure. Several open questions exist, and ourknowledge of the molecular actors operating at thetonoplast is still incomplete. It will be a challenge for thenext years to address issues such as the behavior ofthe guard cell vacuolar membrane potential, the sourceof luminal acidification, the release of anions duringstomatal closure, and the role of vacuolar transport inguard cell Ca2+ signals.





ACKNOWLEDGMENTS

We thank Enrico Martinoia for discussion and critical reading of the article.

Received February 2, 2017; accepted April 3, 2017; published April 5, 2017.

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Ion Transport at the Vacuole during Stomatal Movements1[OPEN] · Institut de Biologie Intégrative de la Cellule, Centre National de la Recherche Scientiﬁque, 91190 Gif-sur-Yvette,