Drought-Enhanced Xylem Sap Sulfate Closes Stomata by ...sulfate was independent of ABA biosynthesis,...

Drought-Enhanced Xylem Sap Sulfate Closes Stomata byAffecting ALMT12 and Guard Cell ABA Synthesis1

Frosina Malcheska, Altaf Ahmad, Sundas Batool, Heike M. Müller, Jutta Ludwig-Müller,Jürgen Kreuzwieser, Dörte Randewig, Robert Hänsch, Ralf R. Mendel, Rüdiger Hell, Markus Wirtz,Dietmar Geiger, Peter Ache, Rainer Hedrich, Cornelia Herschbach*, and Heinz Rennenberg

Professur für Baumphysiologie, Institut für Forstwissenschaften, Albert-Ludwigs-Universität Freiburg,79110 Freiburg, Germany (F.M., J.K., D.R., C.H., H.R.); Department of Botany, Faculty of Life Sciences, AligrahMuslim University, Aligrah 202002, India (A.A.); Department IV Molecular Biology of Plants, Centre forOrganismal Studies Heidelberg University, 69120 Heidelberg, Germany (S.B., Rü.H., M.W.); Julius-von-Sachs-Institut für Biowissenschaften, Julius-Maximulians-Universität Würzburg Lehrstuhl für MolekularePflanzenphysiologie und Biophysik, 97082 Würzburg, Germany (H.M.M., D.G., P.A., Ra.H.); Institut fürBotanik, Technische Universität Dresden, 01062 Dresden, Germany (J.L.-M.); Institut für Pflanzenbiologie,Technische Universität Braunschweig, 38106 Braunschweig, Germany (Ro.H., R.R.M.); and King SaudUniversity, Riyadh 11451, Saudi Arabia (H.R.)

ORCID IDs: 0000-0002-9403-5153 (J.L.-M.); 0000-0002-5251-9723 (J.K.); 0000-0002-6238-4818 (Rü.H.); 0000-0001-7790-4022 (M.W.);0000-0003-0715-5710 (D.G.); 0000-0002-2902-7552 (P.A.); 0000-0003-3224-1362 (Ra.H.); 0000-0003-1930-4576 (C.H.);0000-0001-6224-2927 (H.R.).

Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For this purpose, progressing soil waterdeficit is communicated from roots to shoots. Abscisic acid (ABA) is the key signal in stress-induced stomatal closure, but ABAas an early xylem-delivered signal is still a matter of debate. In this study, poplar plants (Populus 3 canescens) were exposed towater stress to investigate xylem sap sulfate and ABA, stomatal conductance, and sulfate transporter (SULTR) expression. Inaddition, stomatal behavior and expression of ABA receptors, drought-responsive genes, transcription factors, and NCED3 werestudied after feeding sulfate and ABA to detached poplar leaves and epidermal peels of Arabidopsis (Arabidopsis thaliana). Theresults show that increased xylem sap sulfate is achieved upon drought by reduced xylem unloading by PtaSULTR3;3a andPtaSULTR1;1, and by enhanced loading from parenchyma cells into the xylem via PtaALMT3b. Sulfate application causedstomatal closure in excised leaves and peeled epidermis. In the loss of sulfate-channel function mutant, Atalmt12, sulfate-triggered stomatal closure was impaired. The QUAC1/ALMT12 anion channel heterologous expressed in oocytes was gatedopen by extracellular sulfate. Sulfate up-regulated the expression of NCED3, a key step of ABA synthesis, in guard cells. Inconclusion, xylem-derived sulfate seems to be a chemical signal of drought that induces stomatal closure via QUAC1/ALMT12and/or guard cell ABA synthesis.

Leaves of vascular plants are equipped with stomatathat actively control the exchange of CO2, O2, andwatervapor between the leaf interior and the atmosphere.

Drought induces stomatal closure via the release of K+

and anions from the guard cells of the stomata (Hedrich,2012; Kollist et al., 2014). In this process the phytohor-mone abscisic acid (ABA), a drought-induced messenger,addresses the anion release via S-type/slow activatedanion channel (SLAC1), SLAC1Homolog 3 (SLAH3), andR-type/quick-activating anion channel1 (QUAC1/ALMT12) anion channels (Imes et al., 2013). Percep-tion of ABA by the ABA receptors PYR/PYL/RCARresults in PP2C phosphatase (ABI1) inactivation. In turn,Open Stomata1 (OST1), a Ser/Thr-protein kinase, andcalcium-dependent protein kinases released from ABI1inactivation, phosphorylate and activate the anion chan-nels SLAC1 and QUAC1/ALMT12. SLAC1 conducts therelease of Cl2 and NO3

2, and QUAC1/ALMT12 the re-lease of several anions such as Cl2, NO

3

2, and SO422

(Hedrich and Marten, 1993; Frachisse et al., 1999). Therelease of these anions depolarizes the plasma membraneof guard cells giving rise to voltage activation of the K+

release channel GORK (Ache et al., 2000) and deactivation

1 This work was supported by the Deutsche Forschungsgemein-schaft mainly under contract number RE 515/36-1 and also HE 1848/14-1, 15-1, WI 3560/1-1, and SFB1036.

* Address correspondence to [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Cornelia Herschbach ([email protected]).

H.R., C.H., Ra.H., and M.W. conceived the work; F.M., J.K., S.B.,H.M.M., J.L.-M., A.A., and D.G. performed the experiments; F.M.,C.H., S.B., H.M.M., D.G., and D.R. analyzed the data; Ro.H., R.R.M.,and Ra.H. contributed reagents/materials and analytical tools; C.H.,H.R., and Ra.H. wrote the manuscript; H.R., C.H., M.W., Ra.H., P.A.,D.G., and Rü.H. conceived the experiments; F.M., C.H., H.R., M.W.,P.A., and D.G. designed the experiments.

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of Kin channels (Hedrich, 2012). Inhibited influx of potas-sium along with enhanced efflux of potassium and asso-ciated osmotic water cause guard cell turgor loss andstomatal closure.Roots perceive restricted soil water availability and

communicate this environmental constraint as a stresssignal toward the shoot, most likely via the xylem.Chemical (Schachtman and Goodger, 2008), hydraulic(Christmann et al., 2007, 2013), and electrical (Gramset al., 2007; Gil et al., 2008) signaling have been con-sidered in drought stress signal transduction. Drought-related chemical signals, like ABA, are expected to besynthesized in the roots, then transported via the xylemto the leaves to induce stomatal closure (Hartung et al.,2002; Goodger and Schachtman, 2010; Wilkinson andDavies, 2010; Osakabe et al., 2014). However, graftingexperiments with ABA-deficient tomato plants indi-cated the independency of stomatal closure on root-derived ABA (Holbrook et al., 2002). Besides ABA,also plant S-metabolism responds to drought (García-Mata and Lamattina, 2010; Chan et al., 2013; Misraet al., 2015). Recent experiments with maize (Zea mays;Ernst et al., 2010; Goodger and Schachtman, 2010) andflowering hop (Humulus lupulus; Korovetska et al.,2014) provided the first evidence, to our knowledge,that xylem-transported sulfate could be a root-to-shoot-transported chemical signal of water deficiency. Ernstet al. (2010) additionally reported that the significantdecline in stomatal conductance was not accompaniedby leaf hydraulic changes. Therefore, they concludedthat at early stages of water deprivation, stomata re-spond to chemical rather than hydraulic signals fromthe roots. Enhanced sulfate contents in the xylem sapcan originate from increased sulfate uptake by the roots(Ernst et al., 2010), remobilization of sulfate from stor-age pools inside the roots, or from diminished sulfateunloading from the xylem sap along the transport path.Thus, irrespective of the mechanism involved, sulfatetransporters (SULTRs) provide for SO4

22 homeostasisand stimulus-dependent sulfate concentration changesin the xylem sap (Takahashi et al., 2011; Rennenbergand Herschbach, 2014). Important SULTRs in poplar(Populus3 canescens) are (1) PtaSULTR1,2, which is solelyexpressed in roots and, thus, thought to be responsiblefor sulfate uptake from the soil; (2) PtaSULTR1;1 andPtaSULTR3;3a, which are both abundant in parenchymacells around xylem vessels of the wood and leaf veins and,thus, responsible for the xylemunloading of sulfate; and (3)SULTRs from group 4, PtaSULTR4;1 and PtaSULTR4;2,which are putative tonoplast localized and, thus, respon-sible for sulfate release from vacuoles (Kataoka et al., 2004;Dürr et al., 2010). In addition, sulfate permeation by anionchannels has to be considered for xylem-loading of sulfate(Frachisse et al., 1999; Roberts, 2006).Import of sulfate into the chloroplasts coregulates

ABA biosynthesis. Sulfate reduction and Cys produc-tion in the chloroplasts feed-forward the sulfuration ofthe molybdenum cofactor (Moco) by Moco sulfurase(ABA3; Mendel, 2013; Cao et al., 2014). In Arabidopsis,oxidation of abscisic aldehyde to abscisic acid by the

aldehyde oxidase (AAO3), the last step in ABA synthesis,requires the molybdenum cofactor activated by a Mocosulfurase (ABA3; Nambara and Marion-Poll, 2005). Fur-thermore, sulfate-promoted Cys synthesis can also act instomatal movement via the gasotransmitter hydrogensulfide (H2S). H2S is produced in the cytosol in enzy-matic desulfuration of L-Cys, by L-Cys desulfurylase,and promotes stomatal closure in an ABA-dependentmanner (García-Mata and Lamattina, 2010; Scuffi et al.,2014). Thus, it has been hypothesized that increasedxylem sap delivered sulfate during drought can eithertrigger ABA synthesis in guard cells or/and inducestomatal closure in an ABA-dependent manner.

This study tested whether sulfate is an early xylem-delivered signal communicating drought stress to theshoot. Sulfate andABAconcentration changes in the xylemsap were monitored and related to stomatal conductance.Expression of SULTRs and putative SO4

22-permeableALMT12/QUAC1 anion channels were investigated toelucidate the origin of sulfate in the xylem sap duringdrought stress. Sulfate andABA feeding experimentswereperformed to test their effects on stomatal conductanceand to identify molecular targets of xylem-delivered sul-fate. The loss of sulfate-channel functionmutant, Atalmt12,was used to analyze sulfate impact on the ALMT12/QUAC1 anion channel. Effects on anion gating of theguard-cell-specific AtALMT12 channel were investigatedby heterologous expression in Xenopus laevis oocytes.Furthermore, sulfate responses on guard-cell-specific geneexpression of ABA receptors, drought-responsive genes,and transcription factors, as well asNCED3 (a key gene ofABA biosynthesis) were studied.

RESULTS

Sulfate, But Not ABA, Increases in the Xylem Sap duringEarly Drought

When poplar plants grown in sand culture were ex-posed to progressingwater deprivation, sulfate starts toaccumulate in the xylem sap after 48 h of drought. Theincrease in xylem sap sulfate preceded both ABA ac-cumulation in the xylem sap that increased after 64 h ofdrought and the decline in stem water potential thatincreased after 72 h of drought (Fig. 1). Stomatal conduc-tance, however, declined after 48 h of water deprivation,when xylem sap sulfate concentrations almost doubled(Fig. 1C), but ABA concentrations were not affected (Fig.1D). Important, at this time, changes in water potentialwere not observed (Fig. 1B). Drought-induced xylem sapsulfate was independent of ABA biosynthesis, becausepoplar RNAi lines down-regulated in the expression ofAAO and ABA genes also showed drought-enhancedxylem sap sulfate (Supplemental Fig. S1). The pH valueof the xylem sap slightly decreased to pH 5.756 0.33 andpH 5.65 6 0.20 after 48 h and 56 h of water deprivation,respectively, compared to pH 5.99 6 0.22 at the onset ofthe drought stimulus. In contrast to xylem sap sulfate thatconsistently increased over time, xylem sap phosphate

Plant Physiol. Vol. 174, 2017 799

Sulfate Affects Stomatal Aperture at Early Drought

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increased after 72 h of drought when twig water potentialincreased (Fig. 2A) and stomatal conductance was ;1/10th compared to the control. Malate that might also be asignal for stomatal closure (Hedrich and Marten, 1993;Hedrich et al., 1994) increased after 56 h of drought butdeclined thereafter (Fig. 2B). Potassium declined after 48,56, and 64 h of water deprivation (Fig. 2C).

Drought Affects Sulfate Transporter Expression

Changes in the xylem sap sulfate can be attributed tochanges in the activity of distinct SULTRs responsible

for sulfate uptake into the roots (PtaSULTR1;2; Dürret al., 2010), unloading of xylem sap sulfate into xylemparenchyma cells (PtaSULTR1;1 and PtaSULTR3;3a;Dürr et al., 2010), and/or remobilization of sulfatestored in the vacuoles of xylem parenchyma cells(PtaSULTR4;1 and PtaSULTR4;2; Dürr et al., 2010).Therefore, SULTR expression was investigated in dif-ferent root fractions (according to Herschbach et al.,2010) of drought-exposed poplar plants. Long whiteroots are roots emerging into the soil without devel-oping side roots. Fine roots, which are roots with adiameter of approx. 0.5–1 mm that developed highdensity of side roots, were the most important rootfraction within the root system of poplar (Herschbachet al., 2010). In addition, roots showing secondarygrowth were expected to be involved in nutrient stor-age. Expression of PtaSULTR1;2, the only root-specificSULTR expected to be responsible for sulfate uptake

Figure 1. Influence of water deprivation on stomatal conductance,stem water potential, and xylem sap composition of poplar. Stomatalconductance of the 10th leaf (A), stem water potential (B), sulfateconcentration (C), and ABA concentration (D) of the xylem sap at0 (control, n = 11), 24 (n = 4), 48 (n = 10–11), 56 (n = 5), 64 (n = 6), and72 (n = 9) h of water deprivation. The presented values6 SD are relativevalues compared to the control at 0 h of water deprivation. Data of thecontrols without water stress (0 h) of both experiments are presented inthe Supplemental Table S1. Asterisks indicate statistical significant dif-ferences of mean values 6 SD (Student’s t test or Mann-Whitney U test)at P , 0.05 compared to the well-watered plants (control, 0 h).

Figure 2. Influence of water deprivation on phosphate, malate, andpotassium in the xylem sap of poplar. Phosphate (A), malate (B),and potassium (C) in the xylem sap of poplar at 0 (control, n = 10–11), 24 (n = 4), 48 (n = 11), 56 (n = 5), 64 (n = 6), and 72 (n = 8) h ofwater deprivation. Relative values of ion concentrations comparedto their levels at 0 h of water deprivation are presented. Data of thecontrols without water stress (0 h) of both experiments are pre-sented in the Supplemental Table S1. Asterisks indicating statisticalsignificant differences (Student’s t test or Mann-Whitney U test) ofmean values 6 SD at P , 0.05 compared to the well-watered plants(control, 0 h).

800 Plant Physiol. Vol. 174, 2017

Malcheska et al.





(Dürr et al., 2010), showed a temporarily increased ex-pression after 24 h in fine and elongating roots that wasnot statistically significant, as well as after 48 h ofdrought in elongating roots (Fig. 3). Thus, the contin-uously increasing sulfate level in the xylem sap duringwater deprivation is unlikely to originate from en-hanced sulfate uptake especially after prolonged waterstress. mRNA levels of PtaSULTR1;1, responsible forxylem unloading (Dürr et al., 2010; Malcheska et al.,2013), significantly declined only after prolonged (72 h)water deprivation, but not in all three root fractions, i.e.white elongating roots, fine roots, and in roots showingsecondary growth (Fig. 3). However, transcript levels ofthe second SULTR responsible for xylem unloading ofsulfate, PtaSULTR3;3a (Dürr et al., 2010; Malcheskaet al., 2013), declined after 24 h of water deprivation inelongating roots and after 48 h in elongating roots aswell as in roots showing secondary growth (Fig. 3).After 56 h of water deprivation, PtaSULTR3;3a declinedin all root fractions and thereby indicated reducedxylem unloading of sulfate.Transcript levels of PtaSULTR3;1b, a homologous

SULTR gene of Arabidopsis group 3 SULTRs expressedin the chloroplast (Cao et al., 2013), start to increasedsignificantly after 48 h infine roots, and in addition, in bothelongating roots and roots showing secondary growthafter 56 h of water deprivation (Fig. 4). PtaSULTR3;1btranscript levels also increased in leaves after 64 h of waterdeprivation. Root expression of PtaSULTRs, which are

likely localized in the tonoplast (PtaSULTR4;1 andPtaSULTR4;2), was observed for PtaSULTR4;1 thatincreased transiently but not continuously over time inelongating roots and roots showing secondary growth(Fig. 4). In leaves, increased expression of SULTRs wasobserved for both PtaSULTR3;1b and PtaSULTR4;2,after 64 and 72 h of drought (Fig. 4).

Expression of QUAC1/ALMT12-Type Sulfate Channel IsDrought Sensitive

Sulfate uptakeby SULTRsdependson theproton-motiveforce established by the plasma membrane H+-ATPase(Lass and Ullrich-Eberius, 1984; Hawkesford et al., 1993).Thus, SULTRs can only transport sulfate from the apoplastinto xylemparenchymaand/or guard cells.Anion channels(Frachisse et al., 1999; Roberts, 2006) must thereforeperform efflux of sulfate from xylem parenchyma cells.In poplar, three putative ALMT channels that groupedwithAtALMT12 in clade 3 of theALMT1 anion channels/transporters family (and probably are able to gatesulfate) were identified (Barbier-Brygoo et al., 2011;Dreyer et al., 2012). In elongating roots, PtaALMT3aexpression increased after 56 and 64 h of drought but re-covered to the controlmRNA level thereafter. PtaALMT3b,however, increased in elongating roots even after 24 h ofwater deprivation and remained high throughout thedrought treatment (Fig. 5). Also, in fine roots, PtaALMT3bexpression was higher than in the control, i.e. at 0 h of

Figure 3. Transcript abundance ofSULTRs in roots and leaves of poplar inresponse to drought. Relative expressionlevels of PtaSULTR1;2, PtaSULTR1;1,and PtaSULTR3;3a compared to theirmRNA gene copies per mg RNA at 0 h ofwater deprivation in leaves, elongatingroots, fine roots, and roots with sec-ondary growth are presented. Data ofcontrols without water stress (0 h) ofboth experiments are presented in theSupplemental Table S1. Asterisks indi-cate statistically significant differencesof mean values6 SD (24 h, n = 3–4; 48 h,n = 9–11; 56 h, n = 5; 64 h, n = 6; and72 h, n = 8–9; Student’s t test or Mann-WhitneyU test) at P, 0.05 compared towell-watered poplar (control, 0 h ofwater deprivation; n = 10–11).






drought stress. As fine root biomass comprises morethan 50% of the root’s biomass (Herschbach et al.,2010), PtaALMT3b seems to be involved in the sulfaterelease from xylem parenchyma cells upon drought

and contributes to enhanced sulfate levels in xylemsap. In leaves, expression of PtaALMT3a continuouslydecreased after 48 h of drought, but PtaALMT3b wasenhanced after 72 h of water deprivation (Fig. 5).

Figure 4. Transcript abundance ofSULTRs in roots and leaves of poplar inresponse to drought. Relative expressionlevels of PtaSULTR3;1b, PtaSULTR4;1,and PtaSULTR4;2 compared to theirmRNA gene copies per mg RNA at 0 h ofwater deprivation in leaves, elongatingroots, fine roots, and roots with sec-ondary growth are presented. Data ofcontrols without water stress (0 h) ofboth experiments are presented in theSupplemental Table S1. Asterisks indi-cate statistically significant differencesof mean values6 SD (24 h, n = 3–4; 48 h,n = 9–11; 56 h, n = 5; 64 h, n = 6; and72 h, n = 9; Student’s t test or Mann-WhitneyU test) at P, 0.05 compared towell-watered poplar (control, 0 h ofwater deprivation; n = 10–11).

Figure 5. Expression of ALMT channels.Transcript abundance of PtaALMT3aand PtaALMT3b that grouped withAtALMT12 in the clade 3 (Dreyer et al.,2012) and of the housekeeping genePtaEF1B (Wildhagen et al., 2010) inleaves, elongating roots, fine roots, androots with secondary growth. Data pre-sented are relative mRNA abundance ofgene copies per mg RNA of the respec-tive gene compared to the control levelat 0 h of water deprivation. Data ofcontrols without water stress (0 h) ofboth experiments are presented in theSupplemental Table S1. Asterisks indi-cate statistically significant differencesof mean values6 SD (24 h, n = 3–4; 48 h,n = 9–11; 56 h, n = 5; 64 h, n = 5–6; and72 h, n = 9; Student’s t test or Mann-WhitneyU test) at P, 0.05 compared towell-watered poplar (control, 0 h ofwater deprivation; n = 10–11).


Malcheska et al.





Xylem-Delivered Apoplastic Sulfate InducesStomatal Closure

To test whether sulfate directly affects stomatal move-ment, sulfate, ABA, and combinations of both were fed todetached poplar leaves and stomatal conductance wasmeasured. Sulfate applied together with Mg at 2 mM

(Fig. 6; Supplemental Fig. S2) significantly reducedstomatal conductance compared to the control [1 mM

MgCl2 plus 60 mM Mg(NO3)2]. When 3 mM ABA wasfed, a similar decline in stomatal conductance wasobserved (Fig. 6). This effect was even stronger when3 mM ABA was applied in combination with 2 mM

sulfate (Fig. 6), suggesting that sulfate could serve as apositive regulator of ABA.To confirm the impact of sulfate on stomatal con-

ductance in poplar, similar feeding experiments wereperformed with Arabidopsis. When sulfate was fed todetached Arabidopsis leaves via the petiole, relativetranspiration dropped within 40 min by ;35% (Fig. 7).Thus, sulfate does not only trigger decreased stomatal

conductance in the perennial woody poplar, but also inthe annual herbaceous Arabidopsis.

In search for a molecular explanation of the sulfate-induced decrease of stomatal conductance, the effect ofapoplastic sulfate on the aperture of stomata in epi-dermis sections of Arabidopsis leaves was quantified.First, the interactive effect of sulfate on the action ofABA shown by Ernst el al. (2010) was confirmed usingthe identical experimental setup, but Arabidopsis ep-idermal peels were used instead of epidermal peels ofmaize (Fig. 8A). This experimental setup included ahigh potassium concentration (50 mM) in the incuba-tion solution to ensure opening of stomata after peel-ing. Next, we showed that sulfate in the absence ofABA closed the stomata when this high potassiumchloride concentration was decreased to zero (Fig. 8B)or to a potassium concentration found in the apoplasticspace of leaf cells (10 mM potassium; SupplementalFig. S3; Long and Widders, 1990). Without addition ofartificially high potassium chloride in the experimen-tal setup, sulfate induced stomatal closure in a dose-dependent manner (Fig. 8C). Remarkably, stomatalclosure was significantly induced by apoplastic ap-plication of 2 mM sulfate, a concentration found in thexylem sap of drought-stressed maize plants (Ernstet al., 2010). These data demonstrate that sulfate fedvia the petiole induce stomatal closure and suggestthat drought-induced increase of sulfate in the xylemcontributes to stomatal closure and may act as a long-distance transport signal from the root to the shoot(Fig. 1). These results do not exclude a role of othermetabolites (e.g. malate, ABA) as additional root-to-shoot signals.

Figure 6. Stomatal conductance of detached poplar leaves fed withsulfate and ABA via the petiole. The feeding solution containing 1 mM

MgCl2 plus 0.06 mM Mg(NO3)2 (pH 5.5) was taken as a control. Treat-ments were exposed to different concentrations of magnesium sulfate;2 mM MgSO4 plus 3 mM ABA; 3 mM ABA, 2 mM MgSO4 plus 0.3 mM ABA;and 0.3 mM ABA via the petiole (n = 4–6). Mean values 6 SD presentedshowed relative stomatal conductance after 60 min of incubation thatwere calculated in comparison to the stomatal conductance deter-mined during preincubation when stable values are reached. Stomatalconductance was determined by gas exchange measurements andmean 6 SD values of stomatal conductance (mmol m22 s21) are pre-sented in the Supplemental Table S1. Asterisks indicate significant dif-ferences compared to the control feeding (*P , 0.05; **P , 0.01;***P , 0.001; Student’s t test). The sulfate effect was independentlyobserved if sulfate was either added to the control solution (SupplementalFig. S2) or applied solely as MgSO4. Capital letter (A) indicates statisticalsignificant differences of mean values 6 SD at P , 0.05 (Student’s t test)between two treatments as indicated. Stomatal conductance of leaves fedwith the solution consisting of 1 mM MgCl2 plus 0.06 mM Mg(NO3)2ranged from 76 to 100 mmol m22 s21.

Figure 7. Transpirational water loss was determined by gas exchangemeasurements of detached leaves of Arabidopsis (Col-0). Partial sto-matal closure was provoked by feeding MgSO4 (final concentration10 mM) via the petiole. The control feeding solution contained 0.06 mM

MgNO3 plus 1 mM MgCl2. The gap in the x axes resulted from differenttime spans that individual plants needed to gain steady-state transpi-ration rates (n = 6; 6 SD). Values were related to leaf area and normal-ized to the time point of MgSO4 feeding (t = 0).











Sulfate-Induced Stomatal Closure InvolvesQUAC1/ALMT12

In the search for the molecular target of sulfate-induced stomatal closure, the responses of stomatafrom wild-type Arabidopsis and the loss-of-ALMT12function mutant (Atalmt12) to sulfate were analyzed.Whereas stomatal aperture of wild-type Arabidopsissignificantly decreased after sulfate application, sto-mata lacking ALMT12 failed to close in response tosulfate. Furthermore, Atalmt12 stomata were insensi-tive to ABA and a combination of ABA and sulfate(Fig. 8D). The insensitivity toward ABA is a charac-teristic feature of Atalmt12 (Meyer et al., 2010). To testif the sulfate insensitivity of the Atalmt12 mutant alsoaffects its response to drought stress, the mutant lineand wild-type Arabidopsis were subjected to waterdeprivation. When the gravimetric soil water content(water content per weight dried soil) dropped from;2.3 g g21 (well-watered) to ;0.7 g g21 (drought-stressed; Supplemental Table S2), a significant reduc-tion in stomatal conductance and transpiration wasobserved in wild-type Arabidopsis, but not in the

mutant line (Fig. 9; Supplemental Fig. S4). Interest-ingly, sulfate fed to detached leaves of the mutantline Atalmt12 did not affect stomatal conductance(Fig. 9C).

Sulfate Gates QUAC1/ALMT12 Open

The experiments presented above (Figs. 8D and 9)clearly show that the Atalmt12 mutant is less sensitiveto sulfate. Studies showed that QUAC1/ALMT12represents a sulfate-, nitrate-, and chloride-permeableanion channel with fast activation and deactivationkinetics (rapid-type anion channel, R-type; Hedrichand Marten, 1993; Meyer et al., 2010; Mumm et al., 2013)but onlymalatewas tested in activatingQUAC1/ALMT12by shifting its voltage-dependent open probability tohyperpolarized membrane potentials. Therefore, theresponse of extracellular sulfate on QUAC1/ALMT12steady-state conductance was investigated here.QUAC1/ALMT12was coexpressed in X. laevis oocyteswith its activating kinase OST1 (Imes et al., 2013).After 3 d of expression, two-electrode voltage-clamp

Figure 8. Sulfate-induced stomatal closure in peeled epidermis of Arabidopsis. Peeled epidermal layers with intact stomata wereprepared from 5-week-old Arabidopsis wild-type and Atalmt12 mutants as described in Ernst et al. (2010). A, Arabidopsis epi-dermal layers were floated on an artificial buffer (50 mM KCl and 10 mM MES, pH 5.5) with stomata facing the ambient air for 2 h.Subsequently, epidermal layers were exposed to increasing concentrations of ABA (0–0.3 mM) in the absence (white) or presenceof 15 mM sulfate (black, MgSO4) dissolved in potassium-containing solution (50 mM KCl and 10 mM MES, pH 5.3). B, Stomatalaperture of isolated epidermal cell layers in absence (white) or presence of 15 mM sulfate (black, MgSO4) dissolved in water orwater supplemented with 50 mM potassium and/or 10 mM MES at pH 5.3. C, Impact of different sulfate concentrations (0–15 mM

MgSO4) dissolved in potassium-buffered solution or water on stomatal aperture. D, Application of sulfate (15mMMgSO4) in wateror sulfate and ABA (0.3mM) in potassium-buffered solution towild-type (gray) or Atalmt12mutants (black-striped). Data representmean values6 SD of five to seven epidermal layers (number of counted stomata for each epidermal layer = 10, number of analyzedstomata = 50–60). Statistical differences between treatments were analyzed by ANOVA on ranks followed by Student-Newman-Keuls and are marked with different letters (n = 5–7).


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experiments were conducted in the presence of eitherchloride- or sulfate-based media. In the presence ofsulfate and a stimulating voltage pulse, QUAC1/ALMT12 activated with typical R-type channel fast-activation kinetics (Fig. 10A). Steady-state currents(ISS) plotted as a function of the applied membranepotential underline the sulfate-dependent induction ofanion efflux currents (Fig. 10B). Moreover, the peak cur-rents in sulfate-based buffers exhibited similar am-plitudes to those found in malate-based buffers. Toelucidatewhether the presence of sulfate shifts the voltage-dependent gating properties of QUAC1/ALMT12 tomorenegative membrane potentials, we inferred the relativeopen probability (rel. PO) and described the curves with aBoltzmann equation. Likemalate, sulfate shifted the rel. POof the anion channel to more negative membrane poten-tials following Boltzmann characteristics (Fig. 10C). Theassumed long-distance signal sulfate thus shifted the half-maximal activation potential (V1/2) by250 mV comparedto chloride controls.

Sulfate Triggers Gene Expression in Guard Cells in anABA-Like Manner

Besides sulfate directly affecting stomatal closure,ABA-sulfate interaction could result fromSO4

22 feeding-forward ABA production or sensitivity. To test this hy-pothesis, Arabidopsis leaves were treated either withABA, sulfate, or both stimuli together and changes intranscript abundance of typical ABA-regulated geneswere analyzed in epidermis fraction enriched withguard cells (Geiger et al., 2011; Fig. 11). Two ABA-responsive genes, the PP2C protein phosphatase(HAI1) and ABAR (Bauer et al., 2013), the ABA down-regulated transcription factor MYB60 (Bauer et al., 2013),and the ABA receptors PYL2 and PYL4 (Gonzalez-Guzman et al., 2012), were quantified by qRT-PCR. AfterABA or sulfate application, the ABA receptor genes (PYL2and PYL4) and the ABA down-regulated transcriptionfactor MYB60 were significantly down-regulated showingcomparable expression levels with both stimuli as well asin combination (Fig. 11, C to E). TheHAI1 andABAR geneswere significantly up-regulated only when feeding ABAand increased as well by sulfate feeding but not signifi-cantly. Combined treatment with ABA and sulfate didnot further affect gene expression compared to solelyABA application. These responses indicate that bothsulfate and ABA act via the same pathway. To test thisassumption, we analyzed the expression of NCED3, akey enzyme of the ABA biosynthesis (Iuchi et al., 2001;Wan and Li, 2006; Melhorn et al., 2008) that catalyzesthe cleavageof cis-xanthophylls (NambaraandMarion-Poll,2005) during ABA synthesis and is feed-forward-regulated

Figure 9. Drought and sulfate effects on stomatal conductance andphotosynthesis of wild-type Arabidopsis and the Atalmt12 mutantline. The effect of drought on wild-type Arabidopsis and the mutantAtalmt12 on stomatal conductance (A) and on rate of photosynthesis(B) of leaves still attached. Whole plants of wild-type Arabidopsis(n = 10) and Atalmt12 mutants (n = 11) growing on commercial soilwere continuously watered or exposed to drought (wild-type, n = 13;Atalmt12, n = 10) until the gravimetric soil water content reaches;0.7 g g21 for both lines (for biometrical data, see SupplementalTable S2). C, The effect of sulfate on stomatal conductance of de-tached leaves of wild-type Arabidopsis and the mutant Atalmt12.Detached leaves of wild-type Arabidopsis or the Atalmt12 mutantwere exposed to deionized water adjusted to pH 5.5 (by NaOH and/or HCl) for 2 h preincubation and were subsequently exposed eitherto deionized water (n = 10) or 10 mM MgSO4 (n = 10). Stomatalconductance was calculated from monitored weight loss after 60,90, and 120 min, when water loss reaches stable values. Stomatalconductance was calculated mathematically (see SupplementalMaterials and Methods). Statistics were prepared with one-wayANOVA Tukey after test for normal distribution with the Kolmogorov-Smirnov and Shapiro-Wilk tests. Different letters indicate significantdifferences of mean 6 SD values between groups at P , 0.05. Data onstomatal conductance of detached leaves of wild-type Arabidopsisand the Atalmt12 mutant exposed to 1 mM MgCl2 plus 0.06 mM

Mg(NO3)2 (control feeding solution; pH 5.5 adjusted by NaOH and/or

HCl) for 2 h preincubation and subsequently exposed either to thefeeding solution (n = 10) or to feeding solution supplemented with10 mM MgSO4 (n = 10) are presented as Supplemental Fig. S4.










by ABA (Barrero et al., 2006). NCED3 was almost 3-foldup-regulated upon sulfate exposure, but not affected byABA. Moreover, combined application of sulfate togetherwith ABA prevents the sulfate effect. These data suggest

that xylem-delivered sulfate enhances ABA biosynthesis inguard cells (Fig. 11F).

DISCUSSION

Is Sulfate a Xylem-Delivered Chemical Signal?

The presented experiments suggest that stomatalclosure during drought is promoted by sulfate deliv-ered via the xylem to the leaves. When stomatal con-ductance declined after 48 h of drought, only sulfatewas increased in the xylem sap whereas ABA increasedafter 64 h and malate after 56 h of water stress. Fur-thermore, this drought-related sulfate signal precededhydraulic signaling (Fig. 1). Velocity of xylem sap flowof 1.5-m-tall poplar plants (same clone as used in thisstudy) was ;5 to 6 m per hour (Windt et al., 2006),suggesting that sulfate can reach guard cells upondrought within 10minwith this approach, using poplarplants that are;80 cm in height. Although in the studyof Christmann et al. (2007) water stress was evokedsuddenly and hydraulic changes were observed, waterstress in this study was imposed gradually and did notaffect hydraulic conductivity. Holbrook et al. (2002)already showed that stomata close as the soil dries,independent of leaf-water deficit and root-producedABA; these authors hypothesized that a chemical sig-nal from the roots leading to a change in ABA levels inleaves may be responsible for stomatal closure. In thisstudy, feeding of sulfate triggered stomatal closure, andthus, established causal relationship between sulfate andstomatal closure (Figs. 6–9; Supplemental Comment). In-dependent evidence for sulfate as a signal for stomatalcontrol came from SO2 fumigation experiments with Pop-ulus 3 canescens (Randewig et al., 2014). SO2 reacts withwater by dissociation to sulfite. Inside plant cells, sulfite isdetoxified to sulfate by oxidation via sulfite oxidase (SO)in peroxisomes (Nowak et al., 2004; Brychkova et al., 2007;Lang et al., 2007) or directly in the apoplastic space byapoplastic peroxidases (Hamisch et al., 2012). Accumula-tion of SO2-derived sulfate in the apoplastic solutionaround guard cells may trigger guard-cell turgor lossby opening of QUAC1/ALMT12. Recalculation of datafrom SO2 exposure experiments (Randewig et al., 2014)revealed strong correlation between sulfate accumula-tion after SO2 exposure and the relative decrease of sto-matal conductance (Supplemental Fig. S5).

Irrespective of the species and methods, ABA-inducedreduction in stomatal conductance was only slightlystrengthened by sulfate (Figs. 6 and 8; Ernst et al., 2010;Korovetska et al., 2014) and vice versa, the sulfate-inducedstomatal closure was only slightly promoted by ABA (Fig.6). However, during drought when sulfate increased in thexylem sap of poplar, ABA remained low (Fig. 1). Aftercontinuing drought stress, sulfate in the xylem sap furtherincreased, whereas ABA also starts to increase. Malate andpotassium levels, both involved in stomatal movement,were affected in the xylem sap during early drought, i.e.malate increased whereas potassium decreased. Hence,this data cannot exclude that malate and/or potassium

Figure 10. Sulfate activates QUAC1/ALMT12 anion channels. A, After 3 dof expression, representative whole-oocyte currents of QUAC1/ALMT12-expressing oocytes were recorded with the two-electrode voltage-clamptechnique in thepresenceof either 10mM sodiumchloride or 10mM sodiumsulfate. In response to voltage jumps from the holding potentials of2200 to280 mV, anion efflux currents were elicited. B, Steady-state currents (ISS)recorded with QUAC1/ALMT12-expressing oocytes in either chloride-,sulfate-, or malate-based buffers (10 mM each) were plotted as a function ofthe applied membrane potential (n = 5, 6SD). C, The inferred rel. PO wasplotted as a function of the oocytes’membrane potential (n= 4,6SD). Fittingof the curveswith a Boltzmann equation revealed a half-maximal activationpotentials (V1/2) of 268 6 22 mV in sodium chloride-, 2118 6 34 mV insodium sulfate-, and21596 4 mV in sodium malate-based buffers.


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(Hedrich et al., 1994, Schachtman and Goodger 2008)could contribute to early effects on stomatal conductanceunder drought stress. However, this investigation indicatesthat sulfate might be one xylem-delivered chemical signalof stomatal closure in response to drought. It may be as-sumed that plants cultivated with GSH as their sole sulfursource would show impaired stomatal responsiveness todrought due to a lack of sulfate. However, such an effectwas not observedwhenArabidopsis plantswere cultivatedwith GSH as sole sulfur source (data not shown). Both,plants cultivated on sulfate and on GSH contained com-parable sulfate levels (data not shown) probably due to theoxidation of Cys to sulfate upon GSH degradation viasulfite oxidase (Rennenberg et al., 1982;Hänsch et al., 2007).Plant growth at low sulfate may be an alternative possi-bility to reduce sulfate levels in roots. However, cultureconditions that support normal growth and prevent sulfateaccumulation in the roots have so far not been achieved.

How Does Drought Trigger Enhanced Sulfate Levels in theXylem Sap?

Malcheska et al. (2013) suggested that sulfate in thexylem sap can originate from sulfate uptake by roots or

by sulfate release from xylem parenchyma and pith raycells of the wood. Lateral roots of Quercus robur seed-lings, however, showed sulfate uptake rates being in-dependent of predrawn shoot water potential, butxylem loading strongly declined with decreasing leafwater potential (Seegmüller and Rennenberg, 2012).This effect should not increase (as found in this study),but instead decrease sulfate in the xylem sap upondrought. Expression analyses of different SULTRs inpoplar roots further indicate that increasing sulfateuptake is not an important source of the sulfate accu-mulation in the xylem sap upon drought, but insteaddiminished unloading of sulfate from the xylem sap.mRNA abundance of PtaSULTR3;3a at early droughtand after prolonged drought stress PtaSULTR1;1 de-creased in all root fractions upon drought (Fig. 3). BothSULTRs are expressed in xylem parenchyma cells andthought to be involved in xylemunloading of sulfate (Dürret al., 2010). However, at early drought PtaSULTR3;3aseems to possess a predominant role because it decreasedfirst, whereas PtaSULTR1;1 may mediate reducedsulfate retrieval at later stages of drought. Expressionof PtaSULTR3;3a decreased first in elongating rootsexploiting new soil areas and then after 48 h in roots

Figure 11. Sulfate causes ABA-like geneexpression regulation. Arabidopsis leaveswere fed with 6 mM 6 ABA (equivalent to3 mM biologically active ABA), 10 mM

MgSO4, or both fed via the petiole for 4 h.Subsequently guard cell enrichment wasperformed as described by Bauer et al.(2013). A to E, Genes that are highly regu-lated by ABA showed the same features assulfate. F, Strong induction of NCED3 indi-cates induced ABA synthesis. qPCR datawere normalized to control plant values at100% (mean6 SD, n$ 10) and analyzed byANOVA on ranks followed by Tukey test.Only differences at P , 0.05 were takeninto consideration and marked with differ-ent letters.





showing secondary growth. Because of the low half-life of sulfate transporter protein (Rennenberg et al.,1989), such a regulation would allow for a rapid re-sponse to low water availability.

Higher contents of sulfate in the xylem sap attributedto slower sap flow rates seems unlikely because duringearly water deprivation, consistent changes in the xy-lem sap concentrations of several other ions were notrecorded (Fig. 2). PtaALMT3b, a homolog of AtALMT12(Barbier-Brygoo et al., 2011; Dreyer et al., 2012), in-creased during early drought in elongating roots andfine roots (Fig. 5) and may mediate progressive releaseof sulfate from xylem parenchyma cells. The increasedexpression of PtaALMT3b in elongating roots exploitingnew soil areas coincided with reduced expression ofPtaSULTR3;3a that removes sulfate from the xylem. InArabidopsis roots, expression of AtALMT12 also in-creased after 9 d of water stress (Rasheed et al., 2016).Because AtALMT12 is expressed in the root stele ofArabidopsis (Sasaki et al., 2010), sulfate gating from thecytosol of xylem parenchyma cells into the xylem sap

by this channel may contribute to increased xylem sapsulfate concentrations. Thus, decreased sulfate retrievalfrom the xylem sap together with enhanced mobiliza-tion of sulfate from xylem parenchyma cells may jointlycontribute to enhance sulfate levels in the xylem sapupon drought (Fig. 12). Increased sulfate in the xylemsap probably originated mostly from the cytosolic pool,because putative tonoplast-localized sulfate transporters(group 4) were not consistently affected by drought inany root fraction. mRNA of PtaSULTR4;2 increased inelongating roots only after 56 and 64 h of water with-drawal, whereas expression of PtaSULTR4;1 remainedunaffected. Therefore, enhanced sulfate efflux from thevacuoles seems unlikely.

Xylem-Delivered Sulfate Interacts with QUAC1/ALMT12and ABA Synthesis in the Leaves

The Arabidopsis mutant Atalmt12 is impaired in sto-matal closure to different signals, including ABA (Meyeret al., 2010; Sasaki et al., 2010). This study showed that this

Figure 12. Sulfate enrichment in the xylem sap during early drought in poplar results in enhanced apoplastic sulfate levels aroundguard cells. A, Under well-watered conditions, PtaSULTR3;3a (blue) is highly expressed in roots showing secondary growth,elongating, and fine roots. In combination with low expression of PtaALMT3b (gray), a putative sulfate-permeable channel(Barbier-Brygoo et al., 2011;Dreyer et al., 2012) in elongating and fine roots results in low sulfate concentrations in the xylem sap.The low expression of the putative chloroplast/plastid-localized PtaSULTR3;1b (gray, Cao et al., 2013, 2014) prevents enhancedsulfate transport into chloroplasts/plastids. B, Water stress results in enhanced sulfate concentration in the xylem sap and, as aconsequence, in higher sulfate levels in the leaf apoplast. Increasing sulfate concentrations in the xylem sap are realized byenhanced sulfate efflux from root parenchyma cells, indicated by increased PtaALMT3b (orange) expression in elongating rootsand fine roots, together with decreased sulfate uptake from the xylem sap, indicated by reduced expression of PtaSULTR3;3a(gray) in all three root fractions. In both leaves and roots, expression of the putative chloroplast/plastid-localized PtaSULTR3;1b(green; Cao et al., 2013, 2014) is enhanced during water stress as well. For a detailed view on the guard cells, see Figure 13.Enhanced expression is indicated in color, whereas low expression is indicated in gray.


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mutant is insensitive toward sulfate and as well towardjoint sulfate-ABA-induced stomatal closure (Figs. 8 and 9).However, in contrast to a recently published study onAtalmt12 mutants (Medeiros et al., 2016), the rate of pho-tosynthesis (Fig. 9; Supplemental Fig. S4) and growth(Supplemental Table S2) was not different between wild-type plants and the mutant in this study. Nevertheless,transpiration during drought remained higher in theAtalmt12 mutant in both studies (Fig. 9; Medeiros et al.,2016), supporting the view of impaired stomatal closure.Electrophysiological studies with QUAC1/ALMT12-expressing X. laevis oocytes revealed that extracellularand cytosolic malate and cytosolic sulfate activate theQUAC1/ALMT12 channel (Meyer et al., 2010; Sasakiet al., 2010). In addition, this study also showed that ex-tracellular sulfate activates the QUAC1/ALMT12 channel(Fig. 10; Mumm et al., 2013). Enhanced xylem-deliveredsulfate that enters the substomatal apoplastic space andgets into contact with guard cells either can activateQUAC1/ALMT12 (Figs. 12 and 13A) or can be taken upinto guard cells. In the presence of extracellular sulfate,QUAC1/ALMT12 was activated with typical R-typechannel fast-activation kinetics (Fig. 10A). Thus, anionsreleased from guard cells by xylem-derived sulfate due toQUAC1/ALMT12 activation can depolarize the guardcell plasma membrane which, subsequently, leads toopening of potassium outward channels, such asGORK, releasing K+. This results in stomatal turgorloss and, consequently, stomatal closure (Ache et al.,2000). However, sulfate is not the sole signal affectingguard cell turgor. Other signals such as an endogenouscontrol mechanism, the combination of several ionchannels, and a complex signal transduction networkalso contribute to stomatal movement (Kim et al., 2010;Hedrich, 2012;Munemasa et al., 2015;Minguet-Parramonaet al., 2016).

Another way of sulfate signaling becomes evidentafter sulfate enters the guard cells and may stimulateguard cell-autonomous ABA synthesis upon drought(Bauer et al., 2013; Fig. 13, B and C). A stimulating effectof sulfate on ABA synthesis has been shown withArabidopsis seedlings by applying different sulfateconcentrations to the growth medium (Cao et al., 2014).By working with a loss-of-function mutant deficient inthe chloroplast-localized sulfate transporter SULTR3;1,Cao et al. (2014) demonstrated diminished AAO ac-tivity and ABA levels in Arabidopsis seedlings. Thislast step in ABA synthesis (Nambara and Marion-Poll,2005) needs Cys in the cytosol that is synthesized viasulfate reduction in chloroplasts (Takahashi et al., 2011;Fig. 13D). Cys is also the precursor of H2S, synthesizedvia L-Cys desulfhydrase, which affects stomatal closure

Figure 13. Overview of four possible mechanisms by which xylem-delivered sulfate could trigger stomatal closure. Three sulfate-mediatedmechanisms can result in the release of anions such as chloride, nitrate,and sulfate that depolarize the plasma membrane, which further leadsto opening of potassium outward channels such as GORK (gray). ThenGORK can release K+, thereby mediating stomatal turgor loss and, con-sequently, stomatal closure (Ache et al., 2000). A, First, xylem-deliveredsulfate reaching the apoplast of guard cells can change voltage-dependentgating and thus triggers QUAC1/ALMT12 (pink) opening. B, Second,sulfate taken up into guard cells might trigger ABA synthesis via NCED3(marked in blue), which is a key step in ABA synthesis in the chloroplasts(Bauer et al., 2013), thereby causing higher ABA concentration in theguard cell cytosol. C, Third, sulfate delivered through the xylem sapcan be taken up into guard cells and further into the chloroplast byPtaSULTR3;1b (green). Here sulfate can be used for sulfate reductionand Cys synthesis. Increased expression of SULTR3;1, PtaSULTR3;1bin poplar, may enhance sulfate transport into chloroplasts. Cys isneeded in the cytosol for the Moco sulfurase reaction (ABA3) thatsulfurylates Moco, which is used by AAO3 in the last step of ABAsynthesis (Seo and Koshiba, 2002; Nambara and Marion-Poll, 2005).This assumption is supported by a sultr3;1 loss-of-function Arabidopsismutant that synthesizes less ABA (Cao et al., 2013) whereas highsulfate availability facilitates ABA synthesis. ABA can accumulate inthe cytosol due to higher ABA synthesis in guard cells via (B) and/or (C)and can bind to the ABA receptors PYR/PYL/RCAR (gray). The acti-vated ABA receptor inhibits PP2C (gray) phosphatases like ABI1 orABI2. Ser/Thr-protein kinases, like OST1 (gray), are then released frominhibition and are able to activate target proteins like QUAC1/ALMT12 (pink) by phosphorylation (Imes et al., 2013; Osakabe et al.,2014). D, Sulfate uptake into guard cells and further into chloroplasts,indicated by enhanced expression of PtaSULTR3;1b (Cao et al., 2013),can result in Cys accumulation (Calderwood and Kopriva, 2014). In anadditional mechanism, Cys can be used in the cytosol for H2S syn-thesis by L-Cys desulfhydrase activity (Calderwood and Kopriva,2014). H2S is able to inhibit inward-rectifying K+ channels, thereby

preventing stomatal opening (Papanatsiou et al., 2015). Labeling: Allfactors such as enzymes, proteins, channels, and metabolites andmetabolic pathways not investigated in this study, are labeled in gray.Factors analyzed and/or expected to be affected, are marked either bycolor (investigated) or in black (assumed to be affected).







as well (Calderwood and Kopriva, 2014; Scuffi et al.,2016). Although contradicting results, i.e. stomatalopening (Lisjak et al., 2010, 2011) and closure (García-Mata and Lamattina, 2010; Jin et al., 2013), have beenreported, H2S seems to affect stomatal closure in anABA-independent way by inhibiting inward-rectifyingK+ channels (Papanatsiou et al., 2015). Sulfate could betaken up into guard cells and further transported intothe chloroplasts for Cys synthesis. Enrichment in Cysmay also trigger H2S formation (Fig. 13D; Calderwoodand Kopriva, 2014) and, thus, stomatal closure fromxylem-delivered sulfate.

In this study, a stimulating effect of sulfate on guard-cell autonomous ABA synthesis by increased NCED3expression was strongly evident from feeding sulfate toArabidopsis leaf petioles (Fig. 11). Even in Arabidopsisroots and shoots, expression of NCED3 increased after3 and 5 d of water stress, respectively (Rasheed et al.,2016). ABA- or sulfate-treated Arabidopsis guard cellsshowed identical expression patterns of several ABA-controlled genes, but also revealed that the key tran-script of ABA biosynthesis (NCED3; Wan and Li, 2006;Melhorn et al., 2008) is ;3-fold induced by sulfate; thisinduction was counteracted by the addition of ABA(Fig. 11).

In summary, although other signals such as malate,potassium, and/or ABA might be involved in long-distance drought signaling, this data indicate thatxylem-delivered sulfate contributes to stomatal closureby different mechanisms, or processes. For process 1,extracellular sulfate can promote stomatal closure bygating the guard cells’ anion channel QUAC1/ALMT12open (Figs. 10 and 13A). For process 2, xylem-deliveredsulfate taken up into guard cells can induce NCED3expression and thereby up-regulates the key step inABA synthesis in the chloroplasts (Figs. 11 and 13B).For process 3, xylem-delivered sulfate can enter thechloroplast of guard cells and can enhance Cys syn-thesis that in turn may promote ABA synthesis (Fig.13C). For process 4, sulfate-induced Cys synthesis canresult in H2S production that effects inward-rectifyingK+ channels (Fig. 13D). Via processes 1, 2, and 3, severalion channels, e.g. SLAC1, SLAH3, andQUAC1/ALMT12,on the plasma membrane of guard cells, are targeted thatlead to ion efflux and plasma membrane depolarization(Imes et al., 2013). In turn, plasma membrane depolari-zation activates efflux of K+ from the guard cells byGORK(Ache et al., 2000). Efflux of ions and potassium fromguard cells leads to turgor loss and subsequent stomatalclosure upon drought. In contrast, process 4 will preventstomatal opening. Which of these signaling pathways/processes is of superior importance during early droughtneeds further investigation.

Increasing ABA concentrations in the xylem sapafter prolonged water stress (ABA in the xylem sapincreased after 64-h drought in this study) could be theresult of long-distance ABA cycling after ABA syn-thesis in leaves and phloem-to-xylem exchange alongthe transport to the roots (Goodger and Schachtman,2010). Another explanation refers to the suggestion of

Goodger and Schachtman (2010) that ABA accumu-lation at prolonged water stress may result fromenhanced ABA synthesis in roots. In this study, thepredicted chloroplast-localized sulfate transportPtSULTR3;1b showed low expression under well-watered conditions in all three root fractions whereasit continuously increased during ongoing water stress(Fig. 4). As the fine root fraction accounts for ;50% oftotal root biomass (Herschbach et al., 2010), PtSULTR3;1bmay contribute to sulfate transport into plastids of rootparenchyma cells to promote the roots’ own ABA syn-thesis at ongoing drought as shown for salt stress in theroots of Arabidopsis (Cao et al., 2014; Ruiz-Sola et al.,2014). Further studies are required to unravel the contri-bution of such amolecular mechanism on sulfate-inducedstomatal closure and to address the importance of xylem-delivered sulfate as an early drought stress signal fromroots to the shoot as well as the contribution of sulfate tothe roots’ own ABA synthesis.

MATERIALS AND METHODS

Plant Material

Poplar plants (Populus tremula3 Populus alba; synonym P.3 canescens) weremicropropagated as described by Strohm et al. (1995) from sterile cultures.Cuttings were planted into quartz sand (1.0–2.0 mm size of the particles) andwere cultured in a greenhouse under long-day conditions (Scheerer et al., 2010).The plants were fertilized once per week with 200 mL Hoagland solution(Honsel et al., 2012). If required, they were additionally watered with distilledwater. After 3 months, plants at a height of ;70 to 90 cm and an age of 15 to18 weeks were placed into a controlled environmental growth chamber (HPS1500; Heraeus Industrietechnik) for 2 weeks of acclimation (23/20°C d/n, aphotoperiod of 16 h light) at a photosynthetic photon flux density of 150 64 mmol m22 s21 at plant level and 60% relative air humidity. Arabidopsis(Arabidopsis thaliana) wild-type (Col-0) and Atalmt12 mutants (Meyer et al.,2010) were grown from seeds on commercial soils (Anzuchtsubstrat; FloragardVertriebs) under short-day conditions (8-/16-h light/dark) in a controlledenvironmental growth chamber (23/20°C, 60% relative air humidity, 70 65 mmol m22 s21 at plant level).

Leaf Gas Exchange Measurements

Transpiration, stomatal conductance, andphotosynthesis rates of poplar andArabidopsis (wild-type andAtalmt12mutant) plants were measured at the 10thattached poplar leaf of four to six plants or at a mature Arabidopsis leaf from11 to 14 plants using a portable gas exchange system (GFS 3000;Walz; Liu et al.,2015). Attached poplar leaves were placed into a leaf enclosure with 8-cm2 leafareas and attached mature Arabidopsis leaves into a leaf enclosure with 3-cm2

leaf area (Walz). Enclosures were flushedwith ambient air containing 400mL L21

CO2 and 10,000 mL L21 water vapor at a flow rate of 650 mL min21. Leaf tem-perature was maintained at 25°C and light intensity at 1,000 mmol m22 s21

photosynthetic photon flux density. After adaptation to enclosure conditions(;5–10 min), gas exchange was determined for 10 min for both poplar andArabidopsis.

The effect of sulfate on stomatal conductance of detached leaves of Arabi-dopsis wild-type (Col-1) and the Atalmt12 mutant was investigated by moni-toring water loss over time by weighting the tubes containing the feedingsolution plus two leaves. Stomatal conductance was then calculated as waterloss per unit leaf area and time (mmol m22 s21). For detailed information on thecalculation, see Supplemental Materials and Methods.

Furthermore, the effect of SO422 application on transpiration of detached

5- to 6-week-old Arabidopsis wild-type (Col-0) leaves was measured with adifferent custom-made setup. Water vapor was recorded using two parallelwater-cooled cuvettes with a gas stream of 1 L min21. The gas compositionwas controlled by mass flow meters, adjusted to 52.5% relative humidity at20°C and 380 mL L21 CO2, and detected by a HCM100 (Walz) as described by


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Bauer et al. (2013). This setup was designed to monitor stomatal movementonly by relative measurement of transpiration related to the leaf surface.Leaves were illuminated by two LEDs providing light at 655 nm (3-WWEPDR3-S1 Power LED Star tiefrot; Winger) and 455 nm (Luxeon Royal Blue;Philips) at a photon flux density of 100 and 8 mmol m22 s21, respectively. Thelight beams were collected by two dichroic mirrors (Q525 LPXR and DCLP425; Chroma Technology) and directed to the cuvettes via two fiber optics(Fiber Optic Illuminator FL-460). Detached Arabidopsis leaves from the wildtype (Col-0) were cut under water and placed into tubes containing thefeeding solution [1 mM MgCl2 and 0.06 mM Mg(NO3)2] in the gas exchangecuvettes. After 30 to 60 min in darkness when the water vapor concentrationreached equilibrium, leaves were illuminated and increased water loss wasmonitored. After stabilization, MgSO4 (10 mM final concentration) or feedingsolution [as control, 1 mMMgCl2 and 0.06 mMMg(NO3)2] was added via a tubeinto the feeding solution reservoir.

Drought Stress Experiments

Fifteen- to eighteen-week-old poplar plants were well watered up to thecontrol day (day 0) and thereafter exposed to drought by terminating watersupply. Plants were harvested at 0 h of water deprivation (day 0, control) and at24, 48, 56, 64, and 72 h of water deprivation. The water status of plants wasmonitored (1) by measuring stomatal conductance of the 10th attached leaf and(2) by determining the stem water potential according to the method ofScholander et al. (1965) using a pressure vessel (Soilmoisture). Xylem sapsamples were collected as described by Rennenberg et al. (1996). The 10th leafand three different root fractions, i.e. elongating roots without side roots (up to5 cm from the root tip), fine roots, and roots showing secondary growth, wereharvested, frozen in liquid N2, and stored at 280°C. The pH of the xylem sapwas measured with a pH-Meter (pH 526 Multical using a SenTix Mic-D elec-trode;WTW) andwas at 0 h 5.996 0.22, at 24 h 5.956 0.13, at 48 h 5.756 0.33, at56 h 5.65 6 0.2, at 64 h 5.85 6 0.18, and at 72 h 5.6 6 0.3. Data presented arecombined normalized data of two experiments with four to eleven replicates ateach harvest. Original data of all parameters for the controls at 0 h of waterdeprivation of both experiments are presented in the Supplemental Table S1.Therefore, respective mean values received at the control, i.e. day 0, were set to100% and used as reference to calculate relative values.

Drought stress of Arabidopsis plants was followed by determining potweight. First visible symptoms of drought stress were detected when thegravimetric water content of the soil reached 0.676 0.22 g g21 for the wild-typeand 0.73 6 0.14 g g21 for the mutant (Supplemental Table S2).

Feeding of Detached Leaves via the Petiole

Stomatal conductanceof the10thattached leafofpoplarwasmeasuredbeforethe feeding experiment of detached poplar leaves. After detaching, the leafpetiolewas directly placed into a solution consisting of 1mMMgCl2 and 0.06mM

Mg(NO3)2 adjusted to pH 5.5 with NaOH (pH 5.5 was chosen according to thepH of the xylem sap of poplar) for equilibrating stomatal conductance (pre-incubation). The solutionwaswithout potassium to avoid influence on stomatalbehavior by this ion. After detached leaves reached a comparable stomatalconductance to that of being attached, after;60 min the solution was replacedby solutions containing different concentrations of MgSO4 (0.2, 1, and 2 mM),ABA (0.3 and 3 mM), or a mixture of MgSO4 plus ABA (2 mM MgSO4 plus 3 mM

ABA; 2mMMg2SO4 plus 0.3mMABA) adjusted to pH 5.5. Stomatal conductancewas recorded for 1 h in four to six biological replicates. The stomatal conduc-tance measured after 60 min of incubation was calculated as percentage com-pared to the stomatal conductance determined during the preincubation.

In accordance to the experiment with epidermis peels of Arabidopsis (seebelow), comparable treatments were performed with excised leaves. Therefore,40 leaves of the wild-type Arabidopsis and of the Atalmt12 mutant were ran-domly collected and two of them were placed into a 0.5-mL tube (Eppendorf).Each tube contained 400 mL of deionized water at pH 5.5. Tubes were coveredwith Parafilm to avoid water loss by evaporation. After 2 h of preincubation,leaves were placed into new tubes with 400 mL of the solution containing eitherdeionized water or to 10 mM MgSO4 adjusted to pH 5.5 (n = 10 per line andtreatment). Water loss from leaves and, thus, transpiration was determined byweighing the leaves together with the tubes every 30min over 5 h. Control tubeswithout leaves did not show any evaporation if covered with Parafilm. Tem-perature and relative air humidity were recorded to calculate stomatal con-ductance (see below). Leaf area was determined from images taken afterexperiments by using the open source software ImageJ (National Institutes of

Health). Transpiration rates were calculated as mmol of water loss per leaf areain s (mmol m22 s21). Stomatal conductance for water vapor (gH2O) was cal-culated based on the transpiration rates, relative air humidity, and tempera-tures as described by De Kok et al. (1989) and Rennenberg et al. (1996)(equations are given in Supplemental Materials and Methods). The experimentwas performed twice with comparable results that were combined.

Measurements of Stomatal Aperture in Experiments withEpidermis Peels

The impact of sulfate andABAon stomatal aperture of 5-week-oldwild-typeArabidopsis and Atalmt12mutants grown on soil was determined according toErnst et al. (2010) with minor modifications. Epidermal peels were floated for2 h at room temperature and constant white light (200 mmol m22 s21) on wateror on incubation buffer (50 mM KCl and 10 mM MES, pH 5.3) with the stomataside of the epidermal peel facing the ambient air. The effect of MgSO4 (2–15mM)and ABA (0.1–0.3 mM) on stomatal aperture was tested by transfer of epidermalpeels to water or incubation buffer supplemented with respective compoundsfor 3 h. The stomatal aperture was determined with a conventional wide-anglemicroscope (DMIRB; Leica Microsystems).

Determination of the Anions

Sulfate and P contents in the xylem sap were determined by ion-exchangechromatography (DX-120 Ion Chromatograph; Dionex) as described byHerschbach et al. (2000). The xylem sap was diluted from 1:2 up to 1:20 withdeionized water.

Potassium and malate was determined in 10-fold ultrapure-water-dilutedxylem sap before isocratic separation of cations with 30 mM methanesulfonicacid at 43°C and a flow rate of 0.36 mL min21 for 27 min on an IonPac CS16Column (2 mm; Thermo Fisher Scientific) connected to an ICS-1000 System(Dionex). Quantification was performed by conductivity detection after anionsuppression (CERS-500 2 mm, suppressor current 43 mA) with the softwareChromeleon version 6.6 (Dionex). Ions were quantified from the same sampleaccording to Wirtz and Hell (2007).

Quantitative RT-PCR

Total RNA was isolated from 100 mg homogenized root and leaf materialaccording to Kolosova et al. (2004) and cDNA synthesis was performed asdescribed in Dürr et al. (2010), Honsel et al. (2012), and Malcheska et al. (2013).Gene expression analyses of PtaSULTRs, PtaALMT3a, and PtaALMT3b wereperformed by RT-qPCR, using a LightCycler 480 System (Roche Applied Sci-ence) as described byDürr et al. (2010), Honsel et al. (2012), andMalcheska et al.(2013). Lengths of the PCR fragments and primer sequences of all analyzedgenes, and for the selected housekeeping gene PtaEf1b, are given inSupplemental Table S3.

Transcript Analysis of Guard Cells Isolated fromArabidopsis Leaves Treated with ABA and/or Sulfate

Five to six leaves of 6- to 7-week-oldArabidopsis Col-0were cut underwaterto avoid embolism, then transferred to incubation (50 mM KCl and 10 mM MES,pH 5.3) for 1 h. Then 6 mM 6 ABA (equivalent to 3 mM biologically active ABA)or 10 mM MgSO4, or both, were added (final concentrations). After 4 h incu-bation, guard cells were extracted using the blendermethod (described in detailin Hedrich et al. [1989] and Raschke and Hedrich [1989]) and qPCR was per-formed as described by Geiger et al. (2011). Transcripts were normalized to10,000molecules of actin 2:8 using standard curves calculated for the individualPCR products.

Determination of ABA

Two technical replicates of 50 mL of xylem sap were used for ABA extraction.Before the extraction procedure, 100 ng of [2H6]-ABA (Plant Biotechnology In-stitute, National Research Council of Canada) were added to each sample, whichwas then incubated for 30 min under continuous shaking at 4°C. Subsequently,the aqueous sample was acidified to pH 3 with 1 M HCl and extracted two timeswith 600 mL ethyl acetate. For phase separation, the samples were centrifugedfor 10 min at 13,000g. The ethyl acetate phases were carefully removed and









combined. Ethyl acetatewas then evaporated under a streamofN2 and the sampleswere resuspended in 20 mLmethanol. Methylationwas performed by adding equalsample amounts of a 1:10 diluted solution (in diethylether) of trimethylsilyldiazo-methane (Sigma-Aldrich) for 30 min at room temperature. The mixture was evap-orated and resuspended in 50 mL ethyl acetate for GC-MS analysis.

GC-MSanalysiswas carried outon aSaturn2100 Ion-trapMass Spectrometerusing electron impact ionization at 70 eV, connected to a gas chromatograph(model no. CP-3900) equipped with an autosampler (model no. CP-8400; all byVarian). For analysis, 1 mL of the methylated samples was injected in thesplitlessmode (splitter opening 1:100 after 1min) onto amodel no. ZB-5 column(30 m3 0.25 mm3 0.25 mm; Phenomenex) using He carrier gas at 1 mL min21.Injector temperature was 250°C and the temperature program was 60°C for1 min, followed by an increase of 25°C min21 to 180°C, 5°C min21 to 250°C, and25°C min21 to 280°C, then 5 min isothermically at 280°C. For higher sensitivity,the mSIS mode (the Varian Manual; Wells and Huston, 1995) was used. Theendogenous hormone concentrations were calculated by the principle of iso-tope dilution (Cohen et al., 1986), using the ions at m/z 190:194 (endogenousand labeled standard; note that during fragmentation of ABA, two deuteriumare lost) for methylated ABA (Walker-Simmons et al., 2000).

Two-Electrode Voltage-Clamp Experiments with Xenopuslaevis Oocytes

For functional analyses, cRNA of QUAC1/ALMT12 and OST1 was preparedusing theAmpliCap-MaxT7HighYieldMessageMakerKits (EPICENTRE).OocytepreparationandcRNAinjectionhavebeendescribedbyBecker et al. (1996).Oocyteswere injected with 50 nL cRNA of QUAC1 (500 ng mL21) and OST1 (250 ng mL21)and incubated for 3 d at 16°C inND96 solution. Beforemeasurements, oocyteswereinjected with 50 nL of a 200 mM NaH-malate solution (pH 7.5) resulting in a finalcytosolic malate concentration of 18 mM. Whole oocyte currents were recordedusing the two-electrode voltage-clamp technique. The holding potential wasclamped to 2200 mV. Five-hundred-milliseconds-lasting test pulses ranged from60 to2200 mV. The rel. Po was inferred from instantaneous current responses at aconstant voltage pulse of 2200 mV after to the test pulses. The half-maximal acti-vation potential (V1/2) was calculated by fitting the experimental data pointswith asingle Boltzmann equation. Rel. PO curveswere normalized to the saturation valuesof the calculated Boltzmann distribution. Oocytes were perfused with solutioncontaining 10 mM MES/Tris, pH 5.6, 1 mM Ca-Gluconate2, 1 mM MgGluconate2,1 mM LaCl3 and either 10 mM of NaH-malate, NaCl, or NaHSO4. The osmolalitywas adjusted to 220 mOsmol kg21 with D-sorbitol.

Statistics

Statistics were performed according to the experimental design and data dis-tribution.Statisticalanalysesandgraphswerepreparedwith thesoftwareSigmaPlotversion 11 (Systat Software) or with the software OriginPro version 9.1 (http://www.originlab.de). Comparisons between the control (day 0) and different periodsof water deprivation in the drought-stress experiment with poplar were analyzedby Student’s t test for data with normal distribution (Figs. 1–5). The effects of dif-ferent solutions on stomatal conductance of detached leaves were analyzed usingStudent’s t test for datawith normal distribution (Fig. 6).Mann-WhitneyU test wasusedwhen datawere not normally distributed (Figs. 1–6).Normal distributionwastested with Shapiro-Wilk. One-way ANOVA using a Tukey test after testing fornormal distribution with the Kolmogorov-Smirnov and Shapiro-Wilk tests wasapplied for statistical analyses between wild-type Arabidopsis and mutants ex-posed to drought (Fig. 9). ANOVA on ranks followed by the Tukey test was ap-plied for the statistical analyses of relative expression levels of severalABA-inducedgenes in guard-cell-enriched extracts from peeled epidermis (Fig. 11). One-wayANOVA on ranks followed by Student-Newman-Keuls was used to identify sig-nificant differences between treatments of Arabidopsis epidermis peels. Significantdifferences atP, 0.05were considered (Fig. 8). The strength of association betweenthe relative difference in stomatal conductance for water vapor g(H2O) and sulfatecontent of SO2-exposed plants of poplar were tested with Pearson’s product-moment correlation analyses. Data were log-transformed to achieve normal dis-tribution and constant variance of the residuals. The correlation analyses and test ofassumptions were performedwith the software language R, version 3.0.1 (package“lme4”; R Core Team, 2013).

Accession Numbers

Sequences of PtaSULTR of P.3 canescens are available in the NCBI database(https://www.ncbi.nlm.nih.gov) under the following accession numbers:

DQ906929 (PtaSULTR1;1, Potri.005G169300), DQ174472 (PtaSULTR1;2,Potri.002G092500), DQ906928 (PtaSULTR3;1b, Potri.005G213500), DQ906924(PtaSULTR3;3a, Potri.008G130400), DQ906930 (PtaSULTR4;1, Potri.008G049500),DQ906935 (PtaSULTR4;2, Potri.010G211400), and FJ372570 (PtaEf1b,Potri.015G094200).ALMT familymembers of clade 3 for analyses inP.3 canescenswere taken from P. trichocarpa (Barbier-Brygoo et al., 2011) from Phytozomeversion 9 (http://www.phytozome.net/). Arabidopsis ALMT12 homologs are:POPTR_0001s02700, Potri.001G144300 (PtaALMT3a), POPTR_0005s23000, andPotri.005G208500 (PtaALMT3b). Genes of ABA synthesis down-regulated byRNAi constructs in P. 3 canescens (Supplemental Fig. S1) are according to theP. trichocarpa analogs POPTR_007s08330, Potri.007G066400 (PtaABA3),POPTR_0004s20280, and Potri.004G191300 (PtaAO2). Arabidopsis transcriptsthat have been quantified by qPCR are PYL2 (At2g26040), PYL4 (At2g38310),MYB60 (At1g08810), HAI1 (At5g59220), ABAR (At3g02480), and NCED3(At3g14440).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Stomatal conductance and xylem sap sulfate ofpoplar aba3 (Ptaba3-4, Ptaba3-7) and ao2 (Ptao2-22, Ptao2-53) RNAi lines(with halved ABA contents compared to wild-type poplar) during waterdeprivation.

Supplemental Figure S2. Stomatal conductance of detached poplar leavesfed with sulfate.

Supplemental Figure S3. Potassium dependency of sulfate-induced stoma-tal closure in epidermis layers of Arabidopsis.

Supplemental Figure S4. Transpiration of wild-type Arabidopsis and ofthe Atalmt12 mutant in response to drought as well as stomatal conduc-tance of detached leaves in response to sulfate.

Supplemental Figure S5. Correlation analyses between stomatal conduc-tance for water vapor g(H2O) at daytime and sulfate concentrations ofP. 3 canescens in response to SO2 exposure.

Supplemental Table S1. Experimental raw data (mean values 6 SD) for thecontrol plants of the drought (Figs. 1–5) and the feeding experimentswith poplar (Fig. 6).

Supplemental Table S2. Biometrics of wild-type Arabidopsis and of theAtalmt12mutant as well as basic parameters of soil water content duringdrought of the gas exchange experiment presented in Figure 9.

Supplemental Table S3. Primer sequences and length of amplified genefragments in the real-time PCR approach.

Supplemental Comment.

Supplemental Materials and Methods.

Supplemental References.

ACKNOWLEDGMENTS

We thank Silvia Heinze, TechnischeUniversität Dresden, Germany, for tech-nical assistance with ABA measurements and Prof. E. Martinoia for providingthe Atalmt12 mutant.

Received February 10, 2017; accepted April 18, 2017; published April 26, 2017.

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