Guard Cells Integrate Light and Temperature Signals toControl Stomatal Aperture1[OPEN]

Kalliopi-Ioanna Kostaki ,a Aude Coupel-Ledru,a Verity C. Bonnell,a Mathilda Gustavsson,a Peng Sun,a

Fiona J. McLaughlin,a Donald P. Fraser,a Deirdre H. McLachlan,a Alistair M. Hetherington,a

Antony N. Dodd,b and Keara A. Franklina,2,3

aSchool of Biological Sciences, University of Bristol, Bristol BS8 1TQ, United KingdombJohn Innes Centre, Norwich NR4 7RU, United Kingdom

ORCID IDs: 0000-0003-2097-5924 (A.C.-L.); 0000-0002-5951-6365 (D.P.F.); 0000-0001-6060-9203 (A.M.H.); 0000-0001-6859-0105 (A.N.D.);0000-0002-1859-640X (K.A.F.).

High temperature promotes guard cell expansion, which opens stomatal pores to facilitate leaf cooling. How the high-temperature signal is perceived and transmitted to regulate stomatal aperture is, however, unknown. Here, we used areverse-genetics approach to understand high temperature-mediated stomatal opening in Arabidopsis (Arabidopsis thaliana).Our findings reveal that high temperature-induced guard cell movement requires components involved in blue light-mediated stomatal opening, suggesting cross talk between light and temperature signaling pathways. The molecular playersinvolved include phototropin photoreceptors, plasma membrane H1-ATPases, and multiple members of the 14-3-3 proteinfamily. We further show that phototropin-deficient mutants display impaired rosette evapotranspiration and leaf cooling athigh temperatures. Blocking the interaction of 14-3-3 proteins with their client proteins severely impairs high temperature-induced stomatal opening but has no effect on the induction of heat-sensitive guard cell transcripts, supporting the existence ofan additional intracellular high-temperature response pathway in plants.

Plants experience constant fluctuations in tempera-ture and are increasingly exposed to global heatwaves(Perkins et al., 2012). Despite the importance of tem-perature for plant growth and development, the mo-lecular mechanisms controlling temperature perceptionin angiosperms have only recently started to emerge.High temperature-induced hypocotyl elongation hasbeen attributed, in part, to accelerated reversion of theplant photoreceptor phytochrome B (phyB) to its inac-tive Pr form during long nights and at low light levels(Jung et al., 2016; Legris et al., 2016). PhyB inactivationelevates the abundance of the bHLH transcription fac-tor PHYTOCHROME INTERACTING FACTOR4

(PIF4), driving auxin biosynthesis and stem elonga-tion (Koini et al., 2009; Franklin et al., 2011; Jung et al.,2016). PIF4 additionally promotes the accumulation ofFLOWERING LOCUS T (FT) transcript to accelerateflowering in short photoperiods (Kumar et al., 2012).High temperature-mediated up-regulation of FT andHEAT SHOCK PROTEIN70 (HSP70) transcripts in-volves eviction of the alternative histone, H2A.Z, fromnucleosomes. Mutants deficient in the ACTIN-RE-LATED PROTEIN6 (ARP6) subunit of the SWR1 chro-matin-remodeling complex are unable to insert H2A.Zinto nucleosomes and therefore display a constitutivewarm-temperature transcriptome (Kumar and Wigge,2010). Induction of HSPs by heat shock in Arabidopsis(Arabidopsis thaliana) protoplasts has further beenshown to involve the heat-activated calcium channelCYCLIC NUCLEOTIDE-GATED ION CHANNEL6(CNGC6; Gao et al., 2012).

Elucidation of plant temperature signaling networksis confounded by the fact that commonly measuredphysiological outputs of temperature change (stemelongation and flowering time) can be temporally andspatially distant from the temperature perceptionevent, requiring intercellular, intertissue, and interor-gan signaling networks. To address this constraint, wehave used the Arabidopsis guard cell as a single-cellmodel to study high temperature-regulated physio-logical and gene expression responses in parallel.Guard cells surround epidermal pores, termed stomata.They can adjust their turgor in response to environ-mental stimuli to regulate stomatal aperture, thereby

1This work was supported by the Leverhulme Trust (grant no.RPG-2014-178), the Biotechnology and Biological Sciences ResearchCouncil (grant nos. BB/L01369X/1 and BB/N001168/1), and a MarieCurie Fellowship from the European Research Council (MSCA-IFH2020) to A.C.-L.

2Author for contact: kerry.franklin@bristol.ac.uk.3Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Keara A. Franklin (kerry.franklin@bristol.ac.uk).

A.M.H., A.N.D., and K.A.F. conceived the project and supervisedexperiments; K.-I.K., A.C.-L., V.C.B., M.G., P.S., F.J.M., and D.H.M.performed experiments; K.-I.K., A.C.-L., V.C.B., M.G., P.S., F.J.M.,D.P.F., A.M.H., A.N.D., and K.A.F. analyzed data; K.-I.K., A.C.-L.,and K.A.F. wrote the article with contributions from all authors.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.01528

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controlling gas and water exchange between leavesat the atmosphere (Hetherington and Woodward,2003). Stomatal opening is promoted by blue lightand involves the phototropin photoreceptors andtheir downstream target BLUE LIGHT SIGNALING1(BLUS1; Kinoshita et al., 2001; Takemiya et al., 2013).When grown in well-watered conditions at elevatedtemperature (28°C), Arabidopsis plants display in-creased transpiration and architectural adaptations toenhance evaporative cooling (Crawford et al., 2012;Bridge et al., 2013). Greater stomatal conductance hasbeen observed in multiple species exposed to temper-atures up to 40°C, although interpreting the effects oftemperature on stomatal function in planta is con-founded by the effects of temperature on vapor pres-sure deficit and photosynthesis (Urban et al., 2017).Stomatal aperture can be quantified in isolated epi-dermises via stomatal bioassays. This approach pro-vides a single-cell system that offers unique advantagesfor studying thermosensory signaling; cells heat upuniformly, and complications arising from the interplaybetween increased temperature and humidity are re-moved. Epidermal strip bioassays have shown thattemperatures between 30°C and 45°C drive stomatalopening in faba bean (Vicia faba; Rogers et al., 1979,1980). Further analyses in protoplasts have shown thatelevated temperature (36°C) increases the currentsthrough hyperpolarization-activated K1 channels inguard cell membranes (Ilan et al., 1995). Molecularunderstanding of this process is limited, but stomatalopening in response to heat stress (38°C) has beenshown to involve the production of reactive oxygenspecies by RESPIRATORY BURST OXIDASE PRO-TEIN D (RBOHD) in Arabidopsis (Devireddy et al.,2020). Furthermore, FT has been reported to promotestomatal opening (Kinoshita et al., 2011), providing alink between high temperature and stomatal aperture.In this study, we report the existence of a high-

temperature signaling pathway controlling Arabi-dopsis guard cell movement that requires phototropinsand plasma membrane (PM) H1-ATPase activity forfull stomatal opening, yet operates independently fromknown high-temperature signaling components. Per-turbation of this pathway disrupts stomatal openingbut not heat-induced transcript accumulation, sug-gesting either the existence of multiple thermosensingmechanisms or bifurcation of a thermosensory path-way upstream of PM H1-ATPase activation.

RESULTS

Isolated Guard Cells Respond to High Temperature in theLight and the Dark

Stomatal responses to high temperature were inves-tigated using epidermal peel bioassays to ensure thattemperature effects could be analyzed in isolation,without confounding alterations in humidity. Hightemperature-induced stomatal opening was clearly

visible in isolated epidermal tissue containing viableguard cells (Fig. 1A). As the epidermis can no longerreceive signals from the mesophyll layer and adjacentpavement cells are ruptured, guard cells must pos-sess the necessary molecular machinery to perceivetemperature changes. High temperature-mediatedstomatal opening occurred in the light and the dark,with 35°C in the light resulting in the greatest re-sponse (Fig. 1B). High temperature-induced stomatalopening was observed in isolated barley (Hordeumvulgare) and Commelina communis epidermises at 40°C(Supplemental Fig. S1), suggesting conservation ofthis pathway in angiosperms. In Arabidopsis, 35°Ctreatment had no effect on guard cell viability, asidentified by staining with fluorescein diacetate(Supplemental Fig. S2, A–G). Shifting peels from 35°Cto 22°C led to statistically significant stomatal closurein the light and the dark, confirming that the responseis reversible (Supplemental Fig. S2H). The rapidity ofthe response was demonstrated in time-course anal-yses, whereby significant stomatal opening was ob-served within 45 min (Fig. 1C).

High Temperature-Mediated Stomatal Opening in IsolatedGuard Cells Requires Phototropins and PMH1-ATPase Activity

The involvement of known high-temperature sig-naling components in high temperature-mediated sto-matal opening was investigated via stomatal bioassaysusing the cngc, arp6, pif4, and ft null mutants (Fig. 2,A–C). We considered phyB to be an unlikely candidatefor multiple reasons. PhyB is a weak positive regulatorof stomatal opening (Wang et al., 2010), so inactivationof phyB at warm temperatures would promote stoma-tal closure rather than opening (Jung et al., 2016).Thermal reversion of phyB is additionally mostly ef-fective in the dark and at low-light levels (Jung et al.,2016; Legris et al., 2016), whereas the opening of Ara-bidopsis stomata by 35°C treatment is maximally ef-fective at high-light levels. All of the mutants tested hadwild-type apertures at 35°C, suggesting that hightemperature-mediated stomatal opening does not in-volve these known thermosensory mechanisms. Mu-tants deficient in FTdisplayed reduced stomatal aperturesfollowing transfer from the dark to (red 1 blue) light,consistent with previous studies (Supplemental Fig. S3;Kinoshita et al., 2011), but showed wild-type stomatalapertures whenmaintained in white light. The reportedthermosensory activity of phototropin photoreceptors(Fujii et al., 2017) led us to additionally investigate therole of phototropins and their downstream target,BLUS1, in high temperature-mediated stomatal open-ing. Loss of phototropins and BLUS1 resulted in sig-nificantly reduced stomatal apertures at 22°C (Fig. 2D).This is consistent with the established roles of theseproteins in blue light signaling (Kinoshita et al., 2001;Takemiya et al., 2013). Impaired stomatal opening wasmost severe in the phot1/2 mutant (Fig. 2D), confirming

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the redundant action of these photoreceptors (Kinoshitaet al., 2001) and suggesting the existence of a phototropin-mediated, BLUS1-independent pathway controllingstomatal opening in plants maintained in white light.All blue light signaling mutants displayed a stomatalopening response to high temperature, but this wasstrongly impaired in phot1/phot2 mutants, where a sig-nificant interaction between genotype and temperaturewas recorded (Fig. 2D). These data suggest that a smallamount of guard cell movement can occur in responseto 35°C independently of phototropin, but completestomatal opening requires phototropin activation.blus1-3 mutants displayed significantly smaller stoma-tal apertures than wild-type plants at 35°C, suggestinga partial involvement in this response.

Influx of K1 and water into guard cells increasesturgor, increasing stomatal aperture (Outlaw andLowry, 1977; Kwak et al., 2001). A role for inward K1

channels in guard cell responses to high temperature

has been reported (Ilan et al., 1995), so we decided toinvestigate potential upstream regulators. A require-ment for the activation of inward K1 channels is thehyperpolarization of the PM through the activation ofthe PM H1-ATPase (Assmann et al., 1985; Kinoshitaand Shimazaki, 1999; Roelfsema et al., 2001). Treat-ment of epidermal peels with the ATPase and phos-phatase inhibitor sodium orthovanadate (Ueno et al.,2005; Kinoshita et al., 2011) resulted in reduced sto-matal apertures at 35°C, suggesting a possible role forthe PM H1-ATPase downstream of high-temperatureperception (Fig. 3A), although the broad-spectrum ac-tivity of this compound required supporting data fromreverse-genetics studies to substantiate this conclusion.There are 11 functional members of the PMH1-ATPasefamily in Arabidopsis (AHA1–AHA11), all of which areexpressed in guard cells (Ueno et al., 2005). AHA1,AHA2, and AHA5 show the highest transcript accu-mulation in guard cell protoplasts (Ueno et al., 2005)

Figure 1. Isolated Arabidopsis guard cells sense elevations in temperature. A, High temperature induces stomatal opening inisolated epidermal tissue. Representative images of guard cells treated at 22°C and 35°C are shown. Bars5 5 mm. B, Guard cellsrespond to a range of temperatures in white light and dark conditions. Stomatal bioassayswere performed on isolated epidermisesfrom fully expanded rosette leaves. Peelswere incubated at 22°C for 2 h followed by incubation at 22°C, 30°C, 35°C, or 40°C for afurther 2 h. WL, White light. Error bars indicate SE. Asterisks indicate significant differences by Tukey’s posthoc test at P , 0.05(n5 86–90, measured from three separate leaves, all from different plants). P values from a two-way ANOVA comparing stomatalaperture, with temperature and light as factors, are shown below graphs to highlight whether a significant interaction betweenlight and response to temperature exists. n.s., Not significant. C, Changes in stomatal aperture in response to high temperature arestatistically significant within 45min (P, 0.01). Stomatal bioassays were performed on isolated epidermises maintained in whitelight. Peels were incubated at 22°C for 2 h before transfer to 35°C. Error bars indicate SE (n5 60–90, measured from three separateleaves, all from different plants).

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and thus were selected for further analysis. Impairmentin stomatal opening was detected in aha1-8 under bothtemperature regimes, suggesting that AHA1 mediateslight-induced guard cell movement and that AHA1function is required for complete stomatal openingunder high temperatures (Fig. 3B). The aha1-6 alleledisplayed a similar phenotype to aha1-8 (SupplementalFig. S4). By contrast, the two mutant alleles of aha2(aha2-5 and aha2-4) displayed reduced stomatal aper-tures at 35°C but not at 22°C (Fig. 3B; Supplemental Fig.S4). Together, these data suggest a specific role forAHA2 in high temperature-mediated stomatal open-ing. Wild-type stomatal opening phenotypes were ob-served in the aha5 mutant at both temperatures(Fig. 3B). The aha1aha2 double mutant is embryonic le-thal and thus could not be investigated (Haruta et al.,2010). PROTON ATPASE TRANSLOCATION CON-TROL1 (PATROL1) encodes a translocator protein thatmoves AHAs in and out of the PM and was found to beessential for stomatal opening in all conditions (Fig. 3C;Hashimoto-Sugimoto et al., 2013).PROTON PUMP INTERACTOR1 (PPI1) activates

AHA1 (Morandini et al., 2002). We investigated hightemperature-mediated stomatal opening in ppi1 andppi2 mutants and detected only a mild stomatal open-ing defect for ppi1 at 22°C, consistent with a role forPPI1 in activating AHA1 in response to light (Fig. 3C;Morandini et al., 2002; Anzi et al., 2008).

14-3-3 Proteins Mediate Stomatal Opening in IsolatedGuard Cells in Response to High Temperature

One of the best-characterized mechanisms for theactivation of the PM H1-ATPase involves interactionwith a 14-3-3 protein. There are 13 members of the 14-3-3 protein family in Arabidopsis (Sehnke et al., 2002),all of which are expressed in guard cells according to theArabidopsis eFP browser (Winter et al., 2007). We used14-3-3 single mutants to systematically investigatewhether these regulators participated in a hightemperature-specific stomatal opening pathway. Fivemutants (pi, phi, chi, omega, and psi) displayed signifi-cantly reduced stomatal apertures at 35°C, but not at22°C, when compared with wild-type controls (Fig. 4, Aand B). These observations suggest that multiple mem-bers, particularly of the nonepsilon clade, share a role inhigh temperature-mediated guard cell movement. 14-3-3epsilon was not included in this analysis because wewere unable to generate a homozygous line. To furtherestablish the importance of 14-3-3 proteins in hightemperature-induced stomatal opening, we incubatedwild-type epidermal peels with 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR), which is a59-AMP mimetic successfully used in plants to inhibitthe interaction of 14-3-3s with their client proteins(Toroser et al., 1998; Camoni et al., 2000; Paul et al., 2005;Lozano-Durán et al., 2014). This treatment blockedhigh temperature-mediated stomatal opening by ;70%

Figure 2. High temperature-induced sto-matal opening in isolated guard cells re-quires phototropin but not components ofhigh-temperature signaling pathways. Ato C, Loss-of-function mutants of genesinvolved in high-temperature signalinghave no impairment in stomatal openingat 35°C. D, Phototropins are required forstomatal opening in response to hightemperature. Stomatal bioassays wereperformed on isolated epidermises fromfully expanded rosette leaves exposed towhite light. Peels were incubated at 22°Cfor 2 h followed by incubation at 22°C or35°C for a further 2 h. Columbia-0 (Col-0)controls were carried out in parallel. Errorbars indicate SE. Asterisks indicate signif-icant differences by Tukey’s posthoc testat P, 0.05 (n5 90, measured from threeseparate leaves, all from different plants).P values from a two-way ANOVA com-paring stomatal apertures, with genotypeand temperature as factors, are shownbelow graphs to highlight whether a sig-nificant interaction between genotypeand response to temperature exists. n.s.,Not significant.

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(Fig. 4C). Higher order mutants showed reduced sto-matal apertures at 22°C in some combinations (klpc andunpc) but did not reveal any additive phenotypes athigh temperature, suggesting the existence of complexinteractions between 14-3-3 proteins (Fig. 4D).

Calcium Influx Does Not Drive HighTemperature-Mediated Stomatal Opening in IsolatedGuard Cells

Calcium channels have an established role in heatsensing and thermotolerance in land plants (Saidi et al.,2009; Finka et al., 2012). Movement of calcium from theapoplast, however, promotes stomatal closure (De Silvaet al., 1985). To establish whether high-temperatureresponses of guard cells involve Ca21 signaling, epi-dermal peels were incubated with increasing calciumconcentrations. Addition of 50 mM CaCl2 to the incu-bation buffer was sufficient to impair high temperature-induced stomatal opening, whereas treatment with100 mM CaCl2 at 35°C resulted in the same absolute

aperture values as untreated stomata at 22°C (Fig. 5A).Incubation of peels with chelating agents 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid (BAPTA)and EGTAhindered the inhibition of stomatal opening byadded extracellular calciumand allowed formuch greaterapertures to be achieved at 35°C than in the absence ofBAPTA or EGTA (Fig. 5, B and C). Stomata subjected toadded CaCl2 did not, however, open as expected in re-sponse to high temperature in the solvent control condi-tion (Fig. 5B).We attribute this to the presence of dimethylsulfoxide (DMSO; 0.4% [v/v]), which can induce theformation of water pores on the PM (Notman et al., 2006),thus increasing dramatically the uptake of calcium. Wefurther observed that calcium import leading to stomatalclosure occurred through verapamil-insensitive channelsat both temperatures (Fig. 5D). Collectively, the applica-tion of chelating agents and calcium channel blockersshowed that influx of external calcium reduces stomatalaperture and therefore can be excluded as a high-temperature signal driving opening. In line with thisconclusion, analyses of the inward-rectifying calciumchannelmutants cngc6 and reduced hyperosmolality-induced

Figure 3. High temperature-inducedstomatal opening in isolated guardcells requires PM H1-ATPase activ-ity. A, Treatment with the PM H1-ATPase inhibitor vanadate reduces hightemperature-induced stomatal open-ing in Arabidopsis (Col-0). B, Mutantsdeficient in the PM H1-ATPases AHA1and AHA2 display reduced stomatalopening at high temperature. C, PA-TROL1 is essential for stomatal open-ing. The PM H1-ATPase-interactingPPI1 and PPI2 proteins are not re-quired for high temperature-mediatedstomatal opening. Stomatal bioassayswere performed on isolated epider-mises from fully expanded rosetteleaves, exposed to white light. Peelswere incubated at 22°C for 2 h, fol-lowed by incubation at 22°C or 35°Cfor a further 2 h. For mutant analysis,Col-0 controls were carried out inparallel. Error bars indicate SE. Aster-isks indicate significant differencesby Tukey’s posthoc test at P , 0.05(n5 90, measured from three separateleaves, all from different plants). Pvalues from a two-way ANOVA com-paring stomatal apertures, with tem-perature and vanadate treatment (A) orgenotype (B) as factors, are shownbelow graphs to highlight whether asignificant interaction between eachfactor and response to temperatureexists. n.s., Not significant.

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[Ca21]i increase1 (osca1-4) revealed no impairment of hightemperature-mediated guard cell movement (Figs. 2Aand 5E). Conversely, treatment with the membrane-permeable form of the calcium chelator BAPTA(BAPTA-AM; Young et al., 2006) inhibited hightemperature-induced stomatal opening, suggestinga possible signaling role for intracellular calcium (Fig. 5F).The vacuolarmembrane two-pore calcium channelmutanttwo-pore calcium channel1 (tpc1-2) retained wild-type sto-matal apertures in response to high temperature, showingthat TPC1 is not required for the response (Fig. 5E).

Phototropins Promote Evapotranspiration and LeafCooling at High Temperature

The involvement of phototropins and AHA2 in hightemperature-mediated stomatal opening was further

explored in planta, through gravimetric measurementsof transpiration and thermal imaging. Wild-type, aha2-5, and phot1/phot2 rosettes were grown in a similarmanner to plants used for epidermal strip bioassays for4.5 weeks. Consistent with previous reports (Sakamotoand Briggs, 2002), phot1/phot2mutants displayed severeleaf curling (Supplemental Fig. S5), which results inaltered boundary layer conductance. We measuredwhole-plant transpiration, whole-plant temperature,and calculated rosette conductance as E/VPDleaf-air. Onday 1 of the experiment, measurements were recordedat a constant temperature of 22°C. In contrast to aha2-5mutants that resembled wild-type controls, phot1/phot2plants displayed elevated rosette temperature (Fig. 6, Aand B) and lower rosette conductance (Fig. 6, C and D).On day 2, the same protocol was repeated for 3 hpostdawn to enable transpiration to stabilize. Halfthe plants were then transferred to identical light

Figure 4. Multiple members of the 14-3-3 family contribute to high temperature-induced stomatal opening. A, Hightemperature-induced stomatal openingin epsilon group mutants. B, Hightemperature-induced stomatal openingin nonepsilon group mutants. C, Dis-rupting the interaction of 14-3-3 pro-teins with their client proteins inhibitshigh temperature-induced stomatalopening in Arabidopsis (Col-0). D, 14-3-3 proteins are not functionally re-dundant, as quadruple 14-3-3 mutantsdo not display additive impairment inhigh temperature-induced stomatalopening (c, chi; k, kappa; l, lambda; n,nu; p, phi; u, upsilon). Stomatal bio-assays were performed on isolatedepidermises from fully expanded ro-sette leaves exposed to white light.Peels were incubated at 22°C for 2 hfollowed by incubation at 22°C or 35°Cfor a further 2 h. For mutant analysis,Col-0 controls were carried out in par-allel. Error bars indicate SE. Asterisksindicate significant differences byTukey’s posthoc test at P , 0.05(n 5 90, measured from three separateleaves, all from different plants). Pvalues from a two-way ANOVA com-paring stomatal apertures, with tem-perature and genotype (A, B, and D) orAICAR treatment (C) as factors, areshown below graphs to highlightwhether a significant interaction be-tween each factor and response totemperature exists. n.s., Not significant.

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Figure 5. High temperature-mediated stomatal opening in isolated guard cells is not mediated by import of extracellular calcium.A, Larger stomatal apertures are observed at 35°C in the presence and absence of calcium chloride. Extracellular calcium dose-response curves at 22°C and 35°C are shown. The experiment was performed in Col-0. The concentration of calcium with noadded CaCl2 was estimated to be approximately 35 mM. B, Incubation with the calcium chelator BAPTA leads to increasedstomatal apertures in response to high temperature. The experiment was performed in Col-0. C, Incubation with the divalent

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conditions at 33°C. This was themaximum temperaturethat could be used without generating a large change inair vapor pressure deficit (VPDair) between 22°C andhigh-temperature treatment. Transfer to 33°C resultedin a rapid increase in rosette temperature, which wasmaintained at approximately 29°C in wild-type andaha2-5 plants (Fig. 7, A and B). This elevation was ac-companied by a large increase in rosette conductance(Fig. 7, C and D). In phot1/phot2 plants, rosette tem-perature reached higher values and the increase in ro-sette conductance was reduced when compared withwild-type controls (Fig. 7). No differences in stomataldensity or index were recorded in aha5-2 or phot1/phot2mutants (Supplemental Fig. S6), suggesting that pho-totropins contribute to high temperature-mediatedstomatal opening in planta.

Stomatal Opening and Transcriptional Responses to HighTemperature Are Not Mediated by a LinearThermosensing Pathway in Guard Cells

To explore signaling cross talk between differenthigh-temperature responses, we analyzed the tran-script abundance of high temperature-induced markergenes in isolated guard cells treated with AICAR.We hypothesized that if high temperature-mediatedstomatal opening and transcription were controlledby a linear thermosensory pathway, then perturba-tion of this signaling cascade would impair bothresponses. The abundances of two high temperature-induced transcripts, namely HSP70 and HSP18.2,were measured in epidermal peels at 22°C and 35°C inthe presence or absence of AICAR, a pharmacologicalinhibitor of the 14-3-3-PM H1-ATPase interactionand of high temperature-induced stomatal opening(Fig. 8A). Consistent with previous reports, the guardcell-specific transcripts GC1 and MYB60 (Yanget al., 2008) displayed higher relative transcript abun-dances in epidermal peels than in correspondingleaf tissue, demonstrating that the method used forRNA extraction resulted in guard cell-enrichedsamples (Supplemental Fig. S7A). Quantification ofrelative transcript abundance with two separate con-trol genes showed that HSP70 and HSP18.2 transcriptsaccumulated in response to high temperature, evenin the presence of AICAR (Fig. 8A; SupplementalFig. S7B). Leaf disc transcript abundances displayed

the same trends as the guard cell-enriched samples(Supplemental Fig. S7, C and D). HSP70 promoter ac-tivity was additionally recorded using transgenic linesexpressing the HSP70::LUCIFERASE reporter (Kumarand Wigge, 2010). The HSP70 promoter had similarhigh-temperature responses in the presence or absenceof AICAR, consistent with the transcript data (Fig. 8B).

DISCUSSION

When atmospheric relative humidity is not a re-strictive factor, plants respond to high temperature byopening their stomata. This physiological response oc-curs under laboratory and field conditions in a numberof species (Willmer and Mansfield, 1970; Rogers et al.,1979, 1980; Sadras et al., 2012; Mendes and Marenco,2017; Urban et al., 2017). Despite the conservationof this response, little is known about how guardcells perceive and transduce temperature signals. Here,we show that high temperatures induce stomatalopening inArabidopsis, barley, andC. communis (Fig. 1;Supplemental Fig. S1). We have further characterizedthe temperature range and kinetics of the Arabidopsisresponse (Fig. 1, B and C). Incubation of epidermalpeels at 35°C led to a statistically significant increase instomatal aperture without affecting tissue viability andwas therefore chosen as the most appropriate treatmentto study high-temperature signaling in isolated guardcells (Fig. 2B; Supplemental Fig. S2).Mutants deficient in known thermosensory signaling

components (cngc6, arp6, and ft) displayed wild-typeapertures at 35°C, suggesting that these componentsare not required for high temperature-mediated sto-matal opening (Fig. 2A). Intriguingly, and in contrast tostudies using dark-to-(red 1 blue) light transfer, ftmutants maintained in white light displayed wild-typestomatal apertures at 22°C (Fig. 2C; SupplementalFig. S3). It is therefore possible that FT functions pre-dominantly to promote stomatal opening followinga dark-to-light transfer. In this study, plants weregrown in 10-h photoperiods. A striking reduction in FTtranscript abundance has been recorded in guard cellprotoplasts isolated from plants grown in short pho-toperiods compared with protoplasts extracted fromplants grown in long photoperiods (Aoki et al., 2019).The similar stomatal apertures observed in the wildtype and ft mutants under white light in this study

Figure 5. (Continued.)calcium chelator EGTA leads to increased stomatal apertures in response to high temperature. D, The effect of calcium on hightemperature-induced stomatal opening is not mediated by verapamil-sensitive calcium channels. E, The calcium channels TPC1and OSCA1 are not required for high temperature-induced stomatal opening. F, Treatment with the intracellular calcium chelatorBAPTA-AM impairs stomatal opening in response to high temperature. Carrier, 0.1% (v/v) DMSO1 0.025% (v/v) Pluronic F-127.Stomatal bioassays were performed on isolated epidermises from fully expanded rosette leaves exposed to white light. Peels wereincubated at 22°C for 2 h followed by incubation at 22°C or 35°C for a further 2 h. Formutant analysis, Col-0 controls were carriedout in parallel. Error bars indicate SE. Asterisks indicate significant differences by Tukey’s posthoc test at P , 0.05 (n 5 90,measured from three separate leaves, all from different plants). P values from a two-way ANOVA comparing stomatal apertures,with temperature and pharmacological treatment (B–D and F) or genotype (E) treatment as factors, are shown below graphs tohighlight whether a significant interaction between each factor and response to temperature exists. n.s., Not significant.

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might, therefore, be due to low FT levels in wild-typeguard cells. Increased ambient temperature elevatesauxin biosynthesis in Arabidopsis seedlings via a pro-cess mediated by PIF4 (Franklin et al., 2011). Auxinshave been shown to promote stomatal opening bylimiting hydrogen peroxide production in guard cells(Dodd, 2003; Song et al., 2006). Mutants deficient inPIF4 displayed wild-type stomatal opening responsesto elevated temperature, suggesting that auxin eleva-tion is not a key regulatory mechanism underlying thisresponse (Fig. 2A).

A thermosensory role for the phototropin blue lightphotoreceptors has been suggested following observa-tions that the lifetime of photoactivated phototropin istemperature dependent in multiple species (Kodamaet al., 2008; Nakasone et al., 2008; Fujii et al., 2017).Phototropins contain two light, oxygen, or voltage(LOV) domains at their photosensory N-terminal do-main that bind FMN chromophores. In Marchantia pol-ymorpha, the single MpPHOT photoreceptor has beenshown to regulate cold-induced chloroplast move-ment, with low temperature prolonging the lifespanof the active LOV2 domain. Warmer temperaturescause the reversion of LOV2 to an inactive state,

preventing the response (Fujii et al., 2017). As photo-tropins promote blue light-induced stomatal open-ing (Kinoshita et al., 2001), we hypothesized thatthey might function as thermosensors driving hightemperature-mediated guard cell movement. Althougha small, phototropin-independent response to hightemperature was observed, complete stomatal open-ing required both phot1 and phot2 (Fig. 2D). This resultwas further supported by in planta data showing re-duced evapotranspiration and leaf cooling in hightemperature-treated phot1/phot2mutants (Figs. 6 and 7).As warm temperatures have been shown to inactivatephototropin signaling in Marchantia spp. (Fujii et al.,2017), it is unclear how exposure to high temperaturewould lead to enhanced phototropin activity. The sit-uation in Arabidopsis is, however, more complex, withthe lifespan of LOV2 altered through interactions withother phototropin domains (Kasahara et al., 2002). Al-ternatively, high temperature may regulate signalingpathways downstream of phototropin rather than theactivity of the photoreceptor itself (Fig. 9). The existenceof an additional phototropin-independent pathwaymay explain why some high temperature-mediatedstomatal opening can also be observed in the dark

Figure 6. Phototropins promote leaf cooling androsette conductance at 22°C. A, Dynamics of ro-sette temperature in Col-0, aha2-5, and phot1/phot2 plants maintained at a photon irradiance of150 mmol m22 s21 and a constant temperature of22°Cwith a VPDair of 0.93 kPa. Error bars indicateSE (n5 7). B, Thermal images of plants shown in Aat 5 h following light onset. Bar 5 2 cm for allimages. C, Dynamics of rosette conductance (E/VPDleaf-air) in plants grown as in A. This was cal-culated from gravimetric whole-plant transpira-tion (E) measurements and VPDleaf-air. Error barsindicate SE (n5 7). D, Average E/VPDleaf-air at 8.5 hafter light onset. Error bars indicate SE (n 5 7).Statistical analyses were performed by one-wayANOVA with Tukey’s posthoc test, and differentletters indicate significant differences at P, 0.01.

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(Fig. 1B). It is possible that this response is mediated byRBOHD-mediated reactive oxygen species production(Devireddy et al., 2020).Blue light activates the PM H1-ATPase, causing

PM hyperpolarization that in turn activates inward-rectifying K1 channels and stomatal opening (Shimazakiet al., 1986; Schroeder et al., 1987; Kinoshita andShimazaki, 1999; Ueno et al., 2005). In fava bean, hightemperature regulates the activity of inward- andoutward-rectifying K1 channels, consistent with theinduction of stomatal opening (Ilan et al., 1995). Here,we provide genetic evidence suggesting that AHA1and AHA2 are required for a full stomatal opening re-sponse to high temperature in epidermal peels. Anadditional role for AHA1 in light-induced stomatalopening was also observed (Fig. 3B; SupplementalFig. S4). Consistent with our findings, AHA1 was re-cently reported to play a major role in blue light-induced stomatal opening, whereas aha2 and aha5mutants showed no impairment of the response(Yamauchi et al., 2016). In addition to the C-terminalautoinhibitory domain, PM H1-ATPase pump activ-ity is controlled by N-terminal domain variations,which may provide a mechanism for posttranslational

regulation by different factors (Ekberg et al., 2010).Recruitment of AHA1, and possibly other AHA iso-forms, to the PM in guard cells is mediated by theMunc13-like protein PATROL1 (Hashimoto-Sugimotoet al., 2013). We provide evidence that PATROL1 isessential for stomatal opening in response to hightemperature, as is the case for fusicoccin, low CO2, andblue light (Fig. 3C; Hashimoto-Sugimoto et al., 2013).To address how AHAs become activated in the

high-temperature pathway, we investigated potentialroles for AHA-regulating 14-3-3 proteins and theAHA-interacting proteins PPI. In response to bluelight, 14-3-3 proteins, acting as dimers, bind to theautoinhibitory C-terminal region of the PMH1-ATPaseand promote pumping (Jahn et al., 1997; Oeckinget al., 1997; Wu et al., 1997; Emi et al., 2001). Geneticanalyses of single and higher order 14-3-3 mutantsrevealed that they collectively contribute to hightemperature-mediated stomatal opening with poten-tially antagonistic interactions between isoforms (Fig. 4).Isoform-specific redundancy and antagonism between14-3-3 proteins has previously been reported in Arabi-dopsis root growth (van Kleeff et al., 2014). Of the 14members present in Arabidopsis, 14-3-3 PHI and

Figure 7. Phototropins promote leafcooling and rosette conductance athigh temperature. A, Dynamics of ro-sette temperature in Col-0, aha2-5, andphot1/phot2 plants maintained at aphoton irradiance of 150mmolm22 s21

and a constant temperature of 22°Cwith a VPDair of 0.93 kPa (left) andtransferred at 3 h to 33°C with a VPDair

5 1.2 kPa (right). Error bars indicate SE

(n 5 7). B, Thermal images of plantsshown in A at 5 h following light onsetand 2 h after transfer to high tempera-ture. Bar 5 2 cm for all images. C,Dynamics of rosette conductance (E/VPDleaf-air) in plants grown as in A. Thiswas calculated from gravimetric whole-plant transpiration (E) measurementsand VPDleaf-air. Error bars indicate SE

(n 5 7). D, Average E/VPDleaf-air 8.5 hafter light onset. Error bars indicate SE

(n 5 7). Statistical analyses were per-formed by one-way ANOVA withTukey’s posthoc test, and different let-ters indicate significant differences atP , 0.01.

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LAMBDA have been shown to be important for bluelight-induced stomatal opening (Kinoshita and Shimazaki,1999; Ueno et al., 2005; Tseng et al., 2012). 14-3-3 iso-forms of the nonepsilon group are more effective atactivating the proton pump when compared with theepsilon group, a result that mirrors our data (Fig. 4;Pallucca et al., 2014). The C andN termini of each 14-3-3isoform are highly specific, and members of the familydemonstrate variation in binding affinity to the protonpump AHA2 (Rosenquist et al., 2000; Alsterfjord et al.,2004). Different family members may therefore activatethe proton pump in response to environmental stimuli.Furthermore, 14-3-3 proteins can homodimerize andheterodimerize. Their subcellular localization dependson cell type and may be driven by client interactions,presenting multiple input points for environmental

factors to exert their influence (Oecking et al., 1997; Wuet al., 1997; Paul et al., 2005; Chang et al., 2009).

Treatment with AICAR severely impaired hightemperature-induced stomatal opening, highlightingthe importance of 14-3-3 interactions in this response(Fig. 3A). It is interesting that AICAR treatment has alsobeen shown to suppress pathogen-associatedmolecularpattern-triggered stomatal closure (Lozano-Duránet al., 2014), suggesting that the function of 14-3-3s iscontext dependent. 14-3-3 proteins have several clientsin addition to the PM H1-ATPase, and it is possiblethat AICAR effects on high temperature-mediated sto-matal opening may be related to other channels (Ichidaet al., 1997; Sottocornola et al., 2006; Gobert et al.,2007; Latz et al., 2007). Further analysis is required todetermine whether interaction of AHA2 with 14-3-3proteins is enhanced at high temperature (Fig. 9). De-spite clear evidence suggesting a role for AHA2 in hightemperature-mediated stomatal opening in epidermalpeels, aha2-5 mutants displayed wild-type levels of ro-sette conductance and leaf cooling at high temperature(Fig. 7). This discrepancy may result from the reducedsensitivity of in planta assays and redundancy betweenfamily members (e.g. AHA1) and/or the masking ofAHA2 signaling in leaves resulting from signals fromneighboring cells.

Figure 8. Transcriptional induction of high temperature-regulatedgenes in guard cells is not inhibited by pharmacologically impairing14-3-3-PM H1-ATPase interaction. A, Transcript abundance of HSP70and HSP18.2 following AICAR and temperature treatments. Epidermalpeels from fully expanded rosette leaves were incubated at 22°C for 2 hbefore incubation at 22°C or 35°C for 2 h. TIP41 was used as a controlgene. Error bars indicate SD (n5 3). B,HSP70::LUC bioluminescence indetached leaf epidermises following AICAR and temperature treat-ments. The arrow denotes the temperature shift. An equimolar amountof mannitol was used as an additional osmotic control. Error bars in-dicate SD (n 5 12).

Figure 9. Possible sites of high-temperature signal integration duringstomatal opening inwhite light. Stomatal opening in isolated guard cellsfollowing dark-to-blue light transfer involves phototropin-mediatedphosphorylation of BLUS1, which then activates PM H1-ATPases. PA-TROL1 is required for AHA1 insertion into the PM. In prolonged whitelight, we observed a prominent role for phototropins and redundantinteractions between H1-ATPases and 14-3-3 proteins in promotingstomatal opening. A partial role for BLUS1was observed, suggesting theexistence of a phototropin-mediated, BLUS1-independent pathway inthese conditions. Complete stomatal opening in isolated guard cells athigh temperature requires phototropins, PM H1-ATPase activity, andredundant activities of 14-3-3 proteins. A partial role for BLUS1 wasidentified. A requirement for phototropins in elevating stomatal con-ductance at high temperature was additionally observed in wholeleaves. Some phototropin-independent increases in stomatal openingand rosette conductance at high temperature were also observed. Hightemperature may stimulate phototropin activity directly, phototropinsignaling, or H1-ATPase activity. Increased H1-ATPase activity mayresult from enhanced 14-3-3 binding.

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External calcium antagonizes high temperature-induced stomatal opening (Fig. 5A), which may resultfrom the reversible inhibition of PM H1-ATPase activ-ity (Kinoshita et al., 1995). Pharmacological evidencehas suggested a role for intracellular calcium stores inblue light-induced stomatal opening (Shimazaki et al.,1992). In addition, low CO2 produces more [Ca21]cyttransients compared with elevated CO2 and also in-creases stomatal apertures (Young et al., 2006). Bothstomatal movements in response to CO2 and [Ca21]cyttransients were inhibited by BAPTA-AM treatment(Young et al., 2006). In our study, BAPTA-AM treat-ment reduced high temperature-mediated stomatalopening, supporting a role for intracellular calcium(Fig. 5D), although the existence and source of [Ca21]cyttransients at high temperature remain to be identified.Increased calcium concentration in the micromolar re-gion promotes the association of 14-3-3 proteins withPM H1-ATPases (Manak and Ferl, 2007). Conversely,millimolar concentrations of calcium inhibit the di-merization of the 14-3-3 protein family member PSIand its interaction with client proteins (Abarca et al.,1999). It is therefore possible that local changes in cal-cium concentration may regulate the function of 14-3-3proteins during high temperature-induced stomatalopening.Although AICAR inhibited high temperature-

induced guard cell movement, it did not affect the ac-cumulation of heat-responsive transcripts (Fig. 8;Supplemental Fig. S7). This is in agreement with astudy demonstrating that PECTIN METHYLESTER-ASE34 is required for acquired thermotolerance andstomatal movements but is not required for the induc-tion of heat shock genes or the accumulation of heatshock proteins (Huang et al., 2017). CNGC6 facilitatesthe expression of heat shock proteins during acquiredthermotolerance (Gao et al., 2012), but loss of thischannel protein does not impair high temperature-induced stomatal opening (Fig. 2A). Furthermore,chromatin remodeling mediated by ARP6 is involvedin high temperature-mediated transcription, yet arp6mutant stomata opened fully in response to hightemperature (Fig. 2A; Kumar andWigge, 2010). Thesedata suggest either the existence of discrete thermo-sensing mechanisms in different cellular compart-ments of guard cells or the divergence of a singlethermosensory pathway upstream of 14-3-3-PM H1-ATPase interaction.In conclusion, we demonstrate that Arabidopsis

guard cells integrate light and temperature informationto control stomatal aperture. The point at which thesesignals converge has yet to be elucidated but may in-volve direct stimulation of phototropin, amplificationof phototropin signaling, and/or enhancement of AHAactivity, possibly through enhanced 14-3-3 binding(Fig. 9). This study also establishes the Arabidopsisguard cell as a tractable system for studying intracel-lular thermosensing in angiosperms. Future work inthis area is essential to facilitate crop production andbiodiversity management in a warming climate.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plant material is described inSupplemental Table S1. SALK and SAIL lines were obtained from the Not-tingham Arabidopsis Stock Centre and were tested for homozygosity withgenomic DNAPCR. For previously uncharacterized lines, exact insertion pointswere determined by sequencing and transcript levels were quantified by re-verse transcription quantitative PCR (RT-qPCR). Seeds were sterilized withchlorine gas according to the protocol of Lindsey et al. (2017) and germinated onone-half-strength Murashige and Skoog medium plates (0.22% [w/v] Mura-shige and Skoog salts, 1% [w/v] Suc, and 0.6% [w/v] agar, pH 5.8). Seedlingswere transplanted to a 3:1 mixture of compost (all-purpose growing medium;Sinclair) and sand (horticultural silver sand; Melcourt) andwere grown under a10-h photoperiod in controlled-environment chambers (microclima; Snijders)irradiated by fluorescent tubes at 150mmolm22 s21. Chambers were set to 22°Cduring the day, 20°C during the night, and 70% relative humidity.

Genotyping

To verify the genotype of T-DNA mutants, a 10-mm-diameter leaf disc wasflash frozen in liquid nitrogen and homogenized with 5-mm stainless-steelbeads using a TissueLyser II (Qiagen). Ground samples were resuspendedwith 400 mL of extraction buffer (200 mM Tris Cl, pH 7.5, 250 mM NaCl, 25 mM

EDTA, and 0.5% [w/v] SDS), centrifuged at 15,000g for 1 min, and the super-natant was transferred to a new tube. An equal volume of ice-cold isopropanolwas added, and DNA was left to precipitate on ice for 30 min. Samples werecentrifuged at 15,000g for 7 min, and the pellet was resuspended in 100 mL ofdeionized water. One microliter of isolate was used as the template in a 25-mLPCR with DreamTaq polymerase (Thermo Fisher Scientific). PCR productswere analyzed by electrophoresis on a 1% (w/v) agarose gel. In the case ofpreviously uncharacterized insertions, PCR products using the T-DNA primerwere isolated using the Wizard SV Gel and PCR Clean-Up System (Promega)and sequenced. Primer sequences used for genotyping are provided inSupplemental Tables 2A and 2B.

Epidermal Peel Bioassays

Plants at 4.5 to 6 weeks old were used for epidermal bioassays. Abaxialepidermal peels were isolated from fully expanded rosette leaves. An incisionwas performed near the leaf base, between the leaf edge and themidvein. Tissuewas pulled from the incision site toward the leaf tip. Peels were trimmed withVannas scissors to produce segments from the middle of the lamina blade. Toprevent stomatal opening during sample preparation, peels were kept in 10/0buffer (10 mM MES, pH 6.15). Unless specified, peels from three leaves pergenotype, per condition, derived from different plants, were removed and in-cubated in 10/50 buffer (10 mM MES and 50 mM KCl, pH 6.15) at 22°C for 2 h,illuminated with white light (100 mmolm22 s21) or kept in the dark. These werethen transferred to fresh 10/50 buffer prewarmed to the desired temperatureand incubated for a further 2 h, unless different time points are specified, underthe same light regime. For analyses of ft mutant responses to blue light, peelsfrom dark-adapted plants were either maintained in the dark (610 mM fusi-coccin) or transferred at dawn to a mixture of blue light (lmax 470 nm, 10 mmolm22 s21)1 red light (lmax 660 nm, 50mmolm22 s21) provided by light-emittingdiodes. Following treatments, peels were transferred to microscope slides andimmediately covered with a cover slip before imaging. Unless otherwisespecified, apertures for 10 stomata per peel were measured with an OlympusBX50 microscope attached to a Moticam 580 camera (Motic). No more thanthree neighboring stomata and no stomata near the edge of peels were mea-sured. Each experiment was performed independently three times, and datapresented are the pooled values. Chemical stock solutions used for pharma-cological treatments during bioassays were made up with either 10/50 buffer(100 mM AICAR [Santa Cruz Biotechnology], 1 M CaCl2, and 20 mM verapamilhydrochloride [Santa Cruz Biotechnology]), water (250 mM EGTA [pH adjustedto 8 with KOH] and 10 mM sodium orthovanadate [pH adjusted to 10 withHCl]), ethanol (10 mM fusicoccin [Santa Cruz Biotechnology]), or DMSO(62.5 mM BAPTA [Santa Cruz Biotechnology] and 25 mM BAPTA-AM [SantaCruz Biotechnology]). Chemicals made up with water, ethanol, or DMSO werediluted with 10/50 buffer to the final working concentrations. Chemical treat-ments were added to the fresh 10/50 buffer used during the second incubation

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with the exception of BAPTA-AM,which included an additional loading step asdescribed previously (Young et al., 2006).

Viability Staining

To evaluate guard cell viability, epidermal peels were stained with fluo-rescein diacetate immediately after temperature incubations. Peels weretransferred to fresh 10/50 buffer containing 0.001 (w/v) fluorescein diacetate(SantaCruzBiotechnology; 1% [w/v] stock solution in acetone) and incubated atroom temperature for 10 min. Peels were briefly washed with 10/50 buffer toremove excess dye, and fluorescence was imaged using a Zeiss Axiovert 200Mmicroscope equippedwith anXBO75fluorescent lampandaGFPfilter. Volocitysoftware (Perkin-Elmer) was used to analyze the images.

Gravimetric Analysis of Transpiration andThermal Imaging

In all experiments, plants were maintained at 150 mmol m22 s21. Fromgermination to the end of the experiment, each pot was weighed daily andwatered at a target weight to maintain soil water content at 0.65 g water g21 drysoil, previously determined as a well-watered level nonlimiting for growth andcorresponding to 80% of field capacity (Rymaszewski et al., 2018). Gravimetrictranspiration measurements were undertaken 4.5 weeks after sowing, during 2consecutive days on seven plants per genotype. Pots were sealed with a doublelayer of Parafilm to prevent water evaporation from the soil. On day 1, all plantswere maintained at 22°C and relative humidity at 65%, resulting in a constantVPDair of approximately 0.93 kPa. Each pot was weighed with 1023 g accuracy(Precisa balances XB; Precisa) every hour, starting 1.5 h before light onset.Weight losses between measurements were used to calculate transpiration rateon a leaf area basis (E), using the total rosette area on the same day for eachplant. Each weight measurement was coupled to an individual thermal-infrared image of each plant, taken from above (FLIR E60 Thermal ImagingCamera; FLIR Systems). Reflected temperature was measured with a multidi-rectional mirror and processed using Thermimage R (Tattersall, 2019). Plantpixels were separated from the image background using temperature thresh-olds, and the plant temperature was calculated as the average temperature ofplant pixels. On day 2, the same plants were measured following the sameprotocol (hourly weighing and thermal imaging) in the same conditions,starting 1.5 h before light onset. Three hours after light onset, when transpira-tion was stabilized, plants were transferred to a second cabinet adjusted to 33°Cand 75% relative humidity, yielding a VPDair of approximately 1.2 kPa (as closeas possible to the VPDair in the control cabinet). Hourly weighing and thermalimaging were then performed for a further 6 h. Measurements of air tempera-ture, air relative humidity, and leaf temperature were used to calculate the leaf-to-air vapor pressure deficit (VPDleaf-air). E/VPDleaf-air was then calculated foreach plant at each time point as an estimate of total rosette conductanceaccording to the diffusion equation of water vapor in the air.

Determination of Rosette Area

Daily estimates of projected rosette area were calculated for each plant usingimages taken from above every 2 d and analyzed with Ilastik software, version1.21.7 (Sommer et al., 2011). Total pixels corresponding to plants were extractedfrom background using a random forest classification method based on colorand texture and later converted to millimeters. Total rosette area was measured4.5 weeks after sowing by dissecting the plants and was compared with pro-jected rosette area on the same day to determine genotype-dependent coeffi-cients for the ratio of total versus projected rosette area.

Stomatal Density

Plantsweregrown in the control conditionsdescribedabove for transpirationanalyses. Five weeks after sowing, impressions of the abaxial surface of twomature rosette leaves from seven plants per genotype were made with dentalresin (President Jet Light Body; Coltène/Whaledent). Clear nail varnish wasapplied to the set impression after removal from the leaf. The varnish impres-sions were viewed on an Olympus BX51 inverted microscope, fitted with anOlympus DP70 camera, and analyzed with ImageJ software version 1.43U(National Institutes of Health). Stomata were counted within a 0.3-mm2 areanear the middle of the leaf. Where possible, two measurements were recordedper leaf. Stomatal density was calculated as the number of stomata per mm2.

Stomatal index (S.I.) was calculated as follows: S.I. 5 [(number of stomata)/(number of other epidermal cells1 number of stomata)]3 100. A minimum offive leaves were analyzed per genotype.

Bioluminescence Imaging

Epidermal peels were incubated in 10/50 buffer containing 5 mM luciferin(D-luciferin potassium salt; Melford) at 22°C for 2 h in the dark. A black 96-wellcell culture plate was loaded with 350 mL of 10/50 buffer and 5mM luciferin perwell, and the chemical treatments were randomly distributed along the plate.Peels were transferred so that each peel occupied one well, and biolumines-cence was measured with a Photek HRPCS intensified CCD camera system(Photek) equipped with Peltier temperature control. Peels were imaged for 1 hat 22°C and then for another 4 h and 45 min either at 22°C or 35°C. A clearplastic cover was used to minimize evaporation. Images were captured every15 min, with 14-min integration time. Acquisition and image analysis wereperformed with the IMAGE32 software (Photek).

RNA Extraction

For characterization of mutant lines, 30 10-d-old seedlings (;100 mg oftissue) were flash frozen in liquid nitrogen and homogenized with 3-mmtungsten carbide beads using a TissueLyser II (Qiagen). RNA from groundsamples was then extracted using the Spectrum Plant Total RNA kit (Sigma-Aldrich). Samples were treated with DNase I (Sigma-Aldrich) to remove re-sidual DNA. For analyses ofHSP transcripts, RNAwas extracted from isolatedepidermises (to enrich samples in guard cell transcripts) and leaf discs, bothderived from fully expanded rosette leaves. Each epidermis sample consisted of20 abaxial epidermal peels, with each peel approximately corresponding to halfthe surface area of the leaf blade. Peels were kept in 10/0 buffer during samplepreparation and were then processed similarly to a stomatal bioassay. Fol-lowing treatment, peels were flash frozen in liquid nitrogen. Leaf controlsconsisted of four 5-mm-diameter leaf discs andwere treated as described above.Frozen sampleswere homogenized at 4°Cwith 400-mL glass beads (glass beads,acid washed, 425–600 mm; Sigma-Aldrich) and 350 mL of buffer RULT (Qiagen)containing 1% (v/v) b-mercaptoethanol using a TissueLyser II (Qiagen). Ho-mogenization treatment comprised five cycles of 1 min of homogenization at30 Hz followed by 1 min of rest on ice. RNA was extracted using the RNeasyUCP Micro Kit (Qiagen).

Transcript Abundance

Isolated RNA was quantified using a NanoDrop spectrophotometer andreverse transcribed using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems) according to the manufacturer’s instructions. qPCR wasperformedwith 23 Brilliant III SYBRGreen QPCR (Agilent Technologies). Datawere collected and analyzed using the MxPro software (Agilent Technologies).Three biological and three technical repeats were performed for each sample,and relative transcript abundance was calculated using the 22DDCT method(Livak and Schmittgen, 2001). Biological repeats refer to an independent ex-periment, using different tissue samples. Primer sequences for RT-qPCR ex-periments are provided in Supplemental Table S3.

Statistical Analyses

SigmaPlot v13 (Systat Software), IBM-SPSS-Statistic23 (IBM), and R (RCore Team, 2013) were used to analyze quantitative data.

Accession Numbers

Accession numbers are as follows: 14-3-3 CHI (At4g09000), 14-3-3 KAPPA(At5g65430), 14-3-3 LAMBDA (At5g10450), 14-3-3 MU (At2g42590), 14-3-3 NU(At3g02520), 14-3-3 OMEGA (At1g78300), 14-3-3 OMICRON (At1g34760), 14-3-3 PHI (At1g35160), 14-3-3 PI (At1g78220), 14-3-3 PSI (At5g38480), 14-3-3 RHO/IOTA (At1g26480), 14-3-3 UPSILON (At5g16050), AHA1 (At2g18960), AHA2(At4g30190), AHA5 (At2g24520), ARP6 (AT3G33520), BLUS1 (At4g14480),CNGC6 (At2g23980), FT (At1g65480), GC1 (At1g22690),HSP18.2 (AT5G59720),HSP70 (At3g12580), MYB60 (At1g08810), OSCA1 (At4g04340), PATROL1(At5g06970), PHOT1 (AT3G45780), PHOT2 (At5g58140), PIF4 (At2g43010),

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PP2AA3 (At1g13320), PPI1 (At4g27500), PPI2 (At3g15340), and TPC1(At4g03560).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Barley and C. communis guard cells sense tem-perature in the light and the dark.

Supplemental Figure S2. Stomata remain viable after treatment at 35°C.

Supplemental Figure S3. ft mutants display impaired stomatal openingresponses when transferred from dark to light.

Supplemental Figure S4. High temperature-induced stomatal opening inisolated guard cells requires PM H1-ATPase activity (additional mutantalleles for AHA1 and AHA2).

Supplemental Figure S5. Morphology of Col-0, aha2-5, and phot1/phot2mutants under experimental treatments.

Supplemental Figure S6. aha-5 and phot1/phot2 mutants do not displayaltered stomatal density or index.

Supplemental Figure S7. Transcriptional induction of high temperature-regulated genes is not inhibited by pharmacologically impairing 14-3-3-PM H1-ATPase interaction.

Supplemental Table S1. Mutant lines.

Supplemental Table S2A. Primers used for genotyping: T-DNA spanning.

Supplemental Table S2B. Primers used for genotyping: T-DNA specific.

Supplemental Table S3. Primers used for RT-qPCR.

ACKNOWLEDGMENTS

We thank Phil Wigge (University of Potsdam), Christian Fankhauser (Uni-versity of Lausanne), and Dr. Bert de Boer (Vrije Universiteit Amsterdam) forthe donation of seed. We also thank Dr. Llorenç Cabrera-Bosquet (Institut Na-tional de la Recherche Agronomique Montpellier) for help with thermal imageanalysis.

Received December 12, 2019; accepted January 6, 2020; published January 16,2020.

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