Persulfidation-based Modification of Cysteine Desulfhydraseand the NADPH Oxidase RBOHDControls Guard Cell AbscisicAcid Signaling

Jie Shen,a,1 Jing Zhang,a,1 Mingjian Zhou,a,1 Heng Zhou,a,1 Beimi Cui,b Cecilia Gotor,c Luis C. Romero,c Ling Fu,d

Jing Yang,d Christine Helen Foyer,e,f Qiaona Pan,b,g Wenbiao Shen,a and Yanjie Xiea

a Laboratory Center of Life Sciences, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR Chinab Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdomc Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, 41092Sevilla, Spaind State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences/Beijing, BeijingInstitute of Lifeomics, Beijing 102206, ChinaeCentre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdomf School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, B15 2TT, United KingdomgThe Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School of Life Science, Jiangsu Normal University,Xuzhou 221116

ORCID IDs: 0000-0001-7267-1725 (J.S.); 0000-0003-4242-2677 (J.Z.); 0000-0002-0899-0120 (M.Z.); 0000-0001-7780-1023 (H.Z.);0000-0002-4901-1988 (B.C.); 0000-0003-4272-7446 (C.G.); 0000-0002-2414-4813 (L.C.R.); 0000-0002-4005-915X (L.F.); 0000-0001-8486-273X (J.Y.); 0000-0001-5989-6989 (C.H.F.); 0000-0002-9142-1905 (Q.P.); 0000-0003-1525-9472 (W.S.); 0000-0002-3503-1267(Y.X.).

Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation,which involves the post-translational modification of specific Cys residues to form persulfides. However, the mechanismsthat underlie this important redox-based modification remain poorly understood in higher plants. We have, therefore,analyzed how protein persulfidation acts as a specific and reversible signaling mechanism during the abscisic acid (ABA)response in Arabidopsis (Arabidopsis thaliana). Here we show that ABA stimulates the persulfidation of L-CYSTEINEDESULFHYDRASE1, an important endogenous H2S enzyme, at Cys44 and Cys205 in a redox-dependent manner. Moreover,sustainable H2S accumulation drives persulfidation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOGPROTEIN D (RBOHD) at Cys825 and Cys890, enhancing its ability to produce reactive oxygen species. Physiologically,S-persulfidation-induced RBOHD activity is relevant to ABA-induced stomatal closure. Together, these processes form anegative feedback loop that fine-tunes guard cell redox homeostasis and ABA signaling. These findings not only expand ourcurrent knowledge of H2S function in the context of guard cell ABA signaling, but also demonstrate the presence of a rapidsignal integration mechanism involving specific and reversible redox-based post-translational modifications that occur inresponse to changing environmental conditions.

INTRODUCTION

Through evolution and diversification, land plants have adoptedrobust mechanisms to perceive and relay signals that conveyinformation about water stress. The stomatal aperture in theepidermis of plant leaves is formed by a pair of specialized guardcells, which can sensemultiple stimuli and, consequently, controlgas exchange and transpirational water loss, which is critical forplant growth and sustenance in stressful and ever-changingenvironments (Blatt, 2000; Kim et al., 2010). Importantly, underdrought stress, the hyperosmotic signal causes the accumulation

of the phytohormone abscisic acid (ABA), which is regulatedtranscriptionally and post-translationally and integrates into acomplex signaling network that regulates stomatal aperture (Zhu,2016).Reactive oxygen species (ROS) act as second messengers in

guard cell responses to most of the stimuli that induce stomatalclosure (WangandSong,2008).NADPHoxidase (respiratoryburstoxidase homolog, RBOH) is a homolog of a mammalian 91-kDglycoprotein subunit of phagocyte oxidase (gp91phox), and a keyenzyme in the production of ROS from the outer leaflet of theplasma membrane (Anderson et al., 2011; Marino et al., 2012).Among the 10RBOHgenes found in theArabidopsis (Arabidopsisthaliana) genome, the RBOHD and F isoforms play pivotal roles inROS production, which is rate-limiting to guard cell ABA signaltransduction (Kwak et al., 2003). Similarly to mammalian RBOHs,these plant polytopic membrane proteins possess a core C-terminal region containing the functional NADPH-oxidizing de-hydrogenase domain responsible for superoxide generation

1 These authors contributed equally to this work.2Address correspondence to yjxie@njau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Yanjie Xie (yjxie@njau.edu.cn).www.plantcell.org/cgi/doi/10.1105/tpc.19.00826

The Plant Cell, Vol. 32: 1000–1017, 2020, www.plantcell.org ã 2020 ASPB.

(Suzuki et al., 2011). This C terminus also functions as a toggleswitch that affects the access of the NADPH substrate to theenzyme (Magnani et al., 2017). Additionally, plant RBOHs containan N-terminal intracellular region that carries two EF-hand motifsand a number of phosphorylation sites,which canbe activated viaCa21 binding and protein phosphorylation events that occur in-dividually or synergistically and involve protein kinases (Marinoet al., 2012). Nevertheless, mutagenesis experiments have in-dicated that Ser343 and Ser347 residues are necessary but notsufficient for elicitor-induced full activation of AtRBOHD (Nühseet al., 2007). Apart from these canonical post-translationalmodifications of the N-terminal, the Cys890 amino acid residuein the RBOHD C-terminal is susceptible to modification by nitricoxide, to form a Cys S-nitrosylation, which suppresses ROSproduction during the defense response to infection by Pseu-domonas syringae (Yun et al., 2011). Despite a long-establishedcorrelation between ABA-induced stomatal closure and a ROSburst, through the activation of RBOHD/F, the mechanisms un-derpinning this activation remain unclear.

Over recent years, tremendous progress has been made inunveiling the biological relevance of hydrogen sulfide (H2S), asmall gaseous molecule that functions as an important devel-opmental and stress-responsive signal in prokaryotes and eu-karyotes (Lai et al., 2014; Guo et al., 2016, 2017). H2S regulatesmany physiological processes throughout the plant life cycle,including seed dormancy and germination, root growth, cell se-nescence, autophagy, stomatal aperture/closure, and immunity(Xie et al., 2013, 2014; Aroca et al., 2018; Corpas et al., 2019). H2Ssignaling has been implicated in plant stress responses to highsalinity, drought, heavymetals, high temperature, osmotic stress,and oxidative stress (Gotor et al., 2019). A considerable number ofreports highlight the importance of H2S and the pathways to itsproduction in plants (Xie et al., 2013; Guo et al., 2016; Gotor et al.,

2019; Shen et al., 2019). Although H2S production occurs pre-dominantly via the photosynthetic sulfate-assimilation pathwayin chloroplasts, most chloroplastic sulfide dissociates to its ionicform, HS2, as the pH is basic and H2S is unable to cross the chlo-roplast membrane. Therefore, the largest proportion of endogenouscytosolic H2S is generated from L-cysteine by cysteine-degradingenzymes (Gotor et al., 2019), of which L-cysteine desulfhydrase1(DES1) is the first and most characterized (Álvarez et al., 2010).Recently, anumberof studieshave reported thatH2SproducedbyDES1 is an important player in guard cell ABA signaling and plantdrought tolerance (García-Mata and Lamattina, 2010; Jin et al.,2013; Du et al., 2019). In wheat (Triticum aestivum), ABA bio-synthesis and signaling are activated by H2S upon drought stress(Ma et al., 2016). DES1 activity is required for ABA-dependentstomatal closure, and the enzyme acts early in ABA-mediatedsignal transduction (Scuffiet al., 2014;Du et al., 2019; Zhanget al.,2019). The induction of stomatal closure by sulfurating moleculesis impaired in rbohD and rbohF mutants, indicating that NADPHoxidaseactsdownstreamofH2S inABA-inducedstomatal closure(Scuffi et al., 2018). However, the biochemical and molecularmechanisms by which H2S regulates downstream targets involvedin guard cell ABA signaling have been elusive.Signaling by H2S is proposed to occur via persulfidation—the

post-translationalmodification of proteinCys residues (R-SHs) bycovalentadditionof thiolgroupsto formpersulfides (R-SSHs;Arocaet al., 2018). Similar to but more widespread than S-nitrosylation(Hancock, 2019), protein persulfidation is a redox-based modi-fication that regulates diverse physiological and pathologicalprocesses. This action provides the framework on which to buildan understanding of the physiological effects of H2S (Paul andSnyder, 2012; Filipovic and Jovanovic, 2017). The covalent mod-ification that occurs through persulfidation can be reversed byreducing agents such as DTT. Persulfidation modulates protein

Persulfidation in Guard Cells 1001

activities by a range of mechanisms, including alterations tosubcellular localization, biochemical activity, protein–proteininteractions, conformation, and stability (Aroca et al., 2017b;Filipovic et al., 2018). As a typical example of the biological rel-evance of persulfide modification, increased expression of H2

S-producing enzymes and concomitant H2S production inducepersulfidation of Cys38 in the p65 subunit of NF-kB, which en-hances the binding of NF-kB subunits to the co-activator ribo-somal protein S3. The activator complex then migrates to thenucleus, where it upregulates the expression of several anti-apoptotic genes (Sen et al., 2012).

In Arabidopsis, a number of persulfidated proteins involved ina variety of biological pathways have been functionally charac-terized (Aroca et al., 2015, 2017a, 2018). For instance, H2S-triggered persulfidation disturbs actin polymerization, resultingin stunted root hair growth (Li et al., 2018). Persulfidation regulatesthe activities of key enzymes involved in the maintenance of ROShomeostasisand redoxbalance, includingascorbateperoxidase1and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) iso-formC1 (GAPC1). Thenuclear localization ofGAPC1was found tobemodulated byDES1-producedH2S (Aroca et al., 2015, 2017b).Therefore, it is reasonable to infer that the intracellular dynamicprocesses of persulfidation and persulfidation oxidation may bemodulated by the redox state in plant cells. The spatio-temporalcoordination of H2S andROSproduction is critical to the initiation,amplification, propagation, and containment of H2S/persulfida-tion signaling.

In this study, we report the fine-tuned regulation of guard cellredox homeostasis and ABA signaling through persulfidation. Inthe presence of ABA, DES1 itself was activated by H2S throughpersulfidation at Cys44 and Cys205, which led to the transientoverproduction of H2S in guard cells. This could facilitate theoveraccumulation of ROS by persulfidation of NADPH oxidaseRBOHD on Cys825 and Cys890 residues, thereby inducing sto-matal closure. The overaccumulated endogenous ROS may pre-vent continuous activation of ABA signaling in guard cells, whichwas achieved by a negative feedback mechanism throughpersulfide-oxidation of DES1 and RBOHD.

RESULTS

ABA Triggers Stimulation of Activity and Persulfidationof DES1

DES1 is a component of the ABA signaling pathway in guard cellsand responsible for intracellular H2S levels and proteome-widepersulfidation (Scuffi et al., 2014; Aroca et al., 2017b, 2018).Proteomic analysis of persulfidatedproteins inArabidopsis leavesshowed that DES1 is susceptible tomodification by persulfidation(Aroca et al., 2017a). We hypothesize that the activity of DES1might be regulated by H2S through persulfidation. To test thispossibility, we measured the activities of DES1 recombinantprotein treated with either NaHS or DTT followed by dialysis. Asexpected, NaHS stimulated the enzymatic activity of DES1, in-dicating that the effects of H2S on DES activity may rely on thethiol-based redox status (Figure 1A). Also, DES1 activity wasdecreased significantly by DTT treatment. To determine whether

DES1 is persulfidated in the presence of its product, H2S, weused a tag-switch assay in which persulfidated Cys was labeledwith cyan-biotin and specifically detected by anti-biotin immune-blot analysis (Supplemental Figure 1; Aroca et al., 2017a; Filipovicet al., 2018). NaHS induced persulfidation of DES1 recombinantprotein. This was then fully abolished by DTT, which is con-sistent with the occurrence of a reversible thiol modification(Figure 1A).To determine whether DES1 could be persulfidated in guard

cells, we generated a GFP-tagged DES1 transgenic line pMYB60:DES1-GFP des1, in which DES1 was expressed specifically inmature guard cells under the control of the guard cell-specific pro-moter of the MYB60 transcription factor (Supplemental Figure 2;Chater et al., 2015; Zhang et al., 2019). Transgenic Arabidopsis

Figure 1. Regulation of DES1 Protein by Persulfidation.

(A) Sulfurating molecule-induced persulfidation and activity of the DES1recombinant protein. Purified DES1 recombinant protein was treated withsulfurating molecule NaHS (1 mM) for 1 h, dialyzed and then divided intotwo aliquots; one was used to measure the DES enzyme activity and theother was then subjected to a tag-switch assay for the analysis of theprotein persulfidation (DES1-SSH). TreatmentwithDTT (1mM) is shown asnegative control. The DES activity data represent mean 6 SD (n 5 3) fromthree biological replicates. Different letters indicate significantly differentat P < 0.05 according to one-way ANOVA (post-hoc Tukey’s HSD test).(B) Analysis of persulfidation of DES1-GFP in guard cells. Total proteinswere extracted from pMYB60:DES1-GFP des1 transgenic plants afterexposure to different concentrations of NaHS or DTT (1 mM) for 1 h, andsubjected to the tag-switch assay.(C) ABA-induced persulfidation of DES1 in guard cells. Total proteinsextracted from pMYB60:DES1-GFP des1 transgenic plants were sub-jected to the tag-switch assay after exposure to ABA treatment (10mM) forthe indicated times.For in vitro assay, the persulfidated recombinant protein was detectedby an anti-biotin antibody after tag-switch labeling. Con, control; CBB,Coomassie Brilliant Blue protein stain; IB, immunoblotting. For in vivoassay, 4-week–old plants grown in the soil were treated and harvestedat the indicated times. The persulfidated DES1-GFP protein in guardcells was detected by an anti-GFP antibody after tag-switch labelingand streptavidin purification. The bottom representations show thatthe total DES1-GFP used for the tag-switch assay. The numbers abovethe bottom representations indicate the relative abundance of thecorresponding persulfidated protein compared with that of the con-trol sample (1.00). Signals from two independent experiments werequantified.

1002 The Plant Cell

plants were treated with a wide range of sulfurating molecules,slow-release H2S donors, or DTT. Subsequently, endogenouspersulfidatedproteinswere labeledwithcyano-biotin, andpurifiedwith streptavidin beads, which were then immunoblotted with ananti-GFP antibody. DES1-GFP expression was detected with ananti-GFPantibody.Moreover, the labeledenzymewas found tobepersulfidated invivo,and further strengthenedor fullyabolishedbya wide range of sulfurating molecules or slow-release H2S donorconcentrations or DTT, respectively (Figure 1B; SupplementalFigure 3). However, there was a low but detectable persulfidationsignal found in the GFP itself (Supplemental Figure 4). Next, weinvestigated whether DES1 was persulfidated during guard cellABA signaling. Time course results revealed that DES1-SSHformation was promoted in the early stage of the ABA response,i.e., 10 min after initiation of the response, in pMYB60:DES1 des1plants, indicating the biological relevance of DES1 persulfidationin guard cell ABA signaling (Figure 1C).

Redox-based Regulation of Persulfidation and EnzymaticActivity of DES1

Apart fromthealterationsofenzymeactivity, another featureof thenucleophilicity of persulfides is that they react strongly with two-electron oxidants such as H2O2 (Filipovic et al., 2018). Thus,persulfidated proteins undergo oxidation reactions to form per-thiosulfenic acids in the presence of excess oxidant (Gotor et al.,2019), thereby reducing the effective level of persulfidated pro-teins. To determine whether DES1 persulfidated residues can beoxidized in the presence of excess ROS, we analyzed the per-sulfidated levels of DES1 recombinant protein after treatmentwithH2O2 at awide range of concentrations. The results demonstratedthat H2O2 led to a dose-dependent decrease in persulfidationof DES1 recombinant protein in vitro (Figure 2A) and DES1-GFPprotein in guard cells (Figure 2B), concurrently with a dose-dependent decrease in DES1 recombinant protein activity.

Figure 2. Regulation of DES1 Protein by Persulfide-Oxidation.

(A) and (C) H2O2-triggered oxidation and downregulation of activity of the persulfidated DES1-His recombinant protein in vitro. Persulfidated DES1-Hisrecombinant protein was treated with H2O2 at a wide range of concentrations for 1 h (A) or increasing time period (10 mM of H2O2 for C), and then dividedinto two aliquots; one was used to measure the DES enzyme activity and the other was subjected to a tag-switch assay for the analysis of the levelof persulfidation of DES1 (DES1-SSH). The DES1 activity data represent mean 6 SD (n 5 3) from three biological replicates. Different letters indicatesignificantly different at P < 0.05 according to one-way ANOVA (post-hoc Tukey’s HSD test).(B)H2O2-triggeredoxidation of persulfidatedDES1 inguard cells. Total proteins extracted frompMYB60:DES1-GFPdes1 transgenic plantswere subjectedto the tag-switch assay after exposure to H2O2 at different concentrations for 1 h. Two images of the same blot with different times of exposure are shown.(D)EnhancementofABA-inducedDES1persulfidationbyDPI inguardcells. Total proteinsextracted frompMYB60:DES1-GFPdes1 transgenic plantsweresubjected to the tag-switch assay after exposure to ABA at 10 mM for 30 min, with or without pretreatment of DPI at 10 mM for 2 h.(E) ABA- and H2O2-regulated persulfidation of DES1 in guard cells. Total proteins extracted from pMYB60:DES1-GFP des1 and pMYB60:DES1-GFP des1rbohD transgenic plants were subjected to the tag-switch assay after exposure to ABA treatment at 10 mM for 30 min.(F)and (G)Reversiblepersulfidationandpersulfide-oxidationofDES1-His recombinantprotein.PurifiedDES1 recombinantproteinwasNaHS-pretreatedat10mMfor1h, followedbydialysisand then treatedwithH2O2at10mMfor the indicated times (F), orH2O2-pretreatedat10mMfor1h, followedbydialysis andtreated with NaHS at 10 mM for the indicated times (G), and then subjected to a tag-switch assay for the analysis of the persulfidation level (DES1-SSH).For in vitro assay, thepersulfidated recombinant proteinwasdetectedbyananti-biotin antibodyafter the tag-switch labeling.CBB,CoomassieBrilliantBlueprotein stain; IB, immunoblotting. For in vivo assay, 4-week–oldplants grown in the soil were treatedandharvested at the indicated times. ThepersulfidatedDES1-GFP protein in guard cells was detected by an anti-GFP antibody after tag-switch labeling and streptavidin purification. The bottom representationsshow that the total DES1-GFPused for the tag-switch assay as loading control. The numbers above thebottompanel indicate the relative abundance of thecorresponding persulfidated protein compared with that of the control sample (1.00). Signals from two independent experiments were quantified.

Persulfidation in Guard Cells 1003

Time-dependent oxidation of persulfidated DES1 recombinantprotein at both low and high concentrations of H2O2 was alsoobserved (Figure 2C; Supplemental Figure 5). The DES1-SSHprotein level in guard cells increased by 12.9-fold in the presenceof diphenyleneiodonium (DPI; Figure 2D), an inhibitor of plantflavoenzymes, including the ROS-producing enzyme NADPHoxidase, implying that persulfidated DES1 might be oxidized byROS produced from NADPH oxidase. To further verify this pos-sibility, the persulfidation levels of guard cell DES1 proteins weremeasured in the background of the RBOHD mutation, which hasbeen proved to be an important source of ROS in guard cellsignaling (Kwak et al., 2003). Under normal conditions, the level ofpersulfidated DES1 in guard cells was higher in the rbohDmutantbackground than in the parental line pMYB60:DES1 des1. Bothlines were further enhanced by ABA treatment, indicating that thepersulfidation of DES1 was negatively regulated by RBOHD-derived ROS in ABA signaling (Figure 2E). It is plausible that theDES1 on-off switch, controlled by persulfidation and persulfide-oxidation, is reversible. Our time-course experiments revealedthat the enhanced persulfidation of DES1 recombinant protein byNaHS pretreatment was diminished by the application of H2O2,and vice versa (Figures 2F and 2G; Supplemental Figure 6).

Persulfidation of DES1 at Cys44 and Cys205 Isa Prerequisite for ABA-stimulated Activity andStomatal Closure

Three Cys residues in DES1 are putative targets for persulfi-dation (Cys30, Cys44, andCys205; Supplemental Figure 7).Massspectrometric analysis further unequivocally confirmed that C44and C205 underwent persulfidation in purified recombinant DES1protein upon NaHS treatment (Figures 3A and 3B). To determinewhich Cys would affect H2S-mediated persulfidation and thestimulation of DES1 activity, we mutated each Cys to Ala sepa-ratelyorsimultaneously.Asshown inFigures3Cand3D, twoof themutant proteins, the Cys44Ala and Cys205Ala mutations, sig-nificantly lost the inducing effect of NaHS on persulfidation ofDES1 recombinant protein, with Cys205Ala presenting dominantfunction. Meanwhile, the Cys44Ala mutation decreased the levelof DES1 recombinant protein persulfidation, which could bepartially rescued by treatment with NaHS, but eliminated by theaddition of DTT (Supplemental Figure 8). In contrast, after thesubstitution ofCys205withAla, virtually nopersulfidation ofDES1recombinant protein was detected, even after NaHS treatment.Subsequently, we prepared protoplasts from plants transientlyexpressing the different DES1 protein versions fused to GFP andexamined the level of persulfidation of the DES1 mutant proteinsin vivo (Figure 3E).WhileDES1-GFPwas found tobepersulfidatedin protoplasts, the amount of persulfidated protein (DES1-GFP-SSH) was reduced partially in protoplasts expressing themutatedversion of Cys40Ala and was totally undetectable in Cys205Alaand Cys30/44/205Ala mutants, which was in agreement with theresults from the in vitro observations of mutant DES1 recombi-nant protein. Moreover, the formation of DES1-GFP-SSH couldbe enhanced by ABA or NaHS treatment of wild-type plants(Figure 3F).

In this study, we focused on the functional characterizationof Cys44 and Cys205, because a mutation at Cys30 did not

significantly affect the persulfidation of DES1 recombinant pro-tein. Treatment with NaHS led to marked induction of DES1recombinant protein activity, but did not affect DES activity in theCys44/205Ala double mutant and Cys30/44/205Ala triple mutant(Figure 3G). These mutants displayed a slight reduction in DES1activity when untreated compared with unmutated DES1 re-combinant protein. Similarly, treatment with NaHS or DTT did notalter the persulfidation of either the Cys44/205Ala- or Cys30/44/205Ala-mutated versions (Supplemental Figure 8). Taken to-gether, these results indicated that both Cys44 and Cys205 arekey to DES1 protein function and affect persulfidation-inducedDES1 activity.To explore the regulatory role of persulfide-oxidation at Cys

residueson theactivityofDES1,we furtherexamined theeffectsofH2O2 on its activity. H2O2 treatment of wild-type DES1 proteinpretreatedwith NaHS resulted in a significant decrease in activity,whereas DES1 activity in the Cys44/205Ala mutant was un-changed (Figure 3H). This suggests that the two cys residuescoordinate to regulate DES1 activity by persulfide-oxidation.To examine the biological consequences of DES1 persulfida-

tion, the impact of this modification on ABA-induced stomatalclosure was investigated. To do this, we generated the trans-genic lines pMYB60:DES1 des1, in which DES1 and its mutatedversions were specifically expressed in mature guard cells. Whentreated with ABA, guard cells of pMYB60:DES1 des1, pMYB60:DES1Cys30Ala des1, andpMYB60:DES1Cys44Ala des1plants showedincreased H2S production and stomatal closure, similar to that ofthe wild type (Figures 3G and 3H; Supplemental Figure 9). How-ever, these ABA-induced responses were abolished in pMYB60:DES1Cys205Ala des1 and pMYB60:DES1Cys44/205Ala des1 plants(Figures 3I and 3J). Consistent with this, the ABA treatment timecourse showed that the guard cell-impaired ABA responses inpMYB60:DES1Cys44/205Ala des1 were similar to those of des1,which can be restored by application of NaHS (Figures 3K and 3L;Supplemental Figure 10).

RBOHD-derived ROS in Guard Cells Are GeneticallyRequired for DES1-regulated Stomatal Closure

RBOHD is responsible for the autopropagation wave of ROSproduction in each cell along its systemic path, which is requiredfor required for system-acquired acclimation (Miller et al., 2009;Mittler et al., 2011; Gilroy et al., 2014). To elucidate the tissue-specific function of RBOHD, we expressed RBOHD protein in therbohD background under the control of ProML1, ProCAB3, andProMYB60 promoters, which have been used widely to expressproteins of interest in epidermal (includingguard cells),mesophyll,and guard cells, respectively (Supplemental Figure 11; Chateret al., 2015; Kirchenbauer et al., 2016; Bernula et al., 2017). Astomatal bioassay was conducted using various tissue-specificcomplementation lines togetherwithcorrespondingwild-typeandrbohD mutant plants, under ABA, NaHS, or H2O2 treatments.Interestingly, the impaired production of ROS triggered by ABAin the guard cells of rbohD was rescued when RBOHD wasexpressed in either epidermal or guard cells (Figure 4A, top;Supplemental Figure 12). Accordingly, the rbohD, stomatal re-sponses of tissue-specific complementation lines were inducedby ABA and NaHS (Figure 4A, bottom).

1004 The Plant Cell

Figure 3. Persulfidation of DES1 on Cys44 and Cys205 Regulates ABA-Induced Stomatal Closure.

(A) and (B) Fully annotated MS/MS spectra of IAM-derivatized DES1 peptides containing persulfided cysteines. Purified recombinant DES1 protein wastreated with NaHS (1 mM for 1 h), alkylated with IAM, and analyzed by LC-MS/MS. The localization of IAM-derivatized persulfides was unequivocallyannotated according to the modification-specific b- or y-ions .

Persulfidation in Guard Cells 1005

Although therewereanumberofmesophyll cells attached to thestrips after peeling, we observed that the mesophyll transgenicline behaved similarly to the guard cell complementation line.Importantly, a significant net ROS-associated influx was found ineither ABA- or NaHS-treated guard cell of pCAB3:RBOHD rbohDplants, while a small net ROS-associated influx was detected inrbohDmutants (Figure 4B). These results suggested that RBOHDactivity in both epidermal andmesophyll cells contributed toABA-induced ROS production in guard cells and, hence, stomatalclosure.

Previouspharmacological results showed that stomatal closurewas impaired in wild-type epidermal strips treated with NaHS inthe presence of DPI, an inhibitor of plant flavoenzymes, includingthe NADPH oxidase, indicating the interplay between H2S and H2

O2 (Scuffi et al., 2018). The NADPH oxidase isoforms RBOHD andRBOHF are responsible for the majority of guard cell H2O2 pro-duction (Kwak et al., 2003). To further evaluate the contribution ofRBOHD to H2S-induced stomatal closure, we crossed des1 withrbohD mutant plants and a homozygous des1 rbohD line wasidentified by genotyping-based PCR (Supplemental Figure 13).Our genetic data further demonstrated that, compared withthe des1mutant, NaHS-induced ROS production and stomatalclosure were almost fully abolished in des1 rbohD (Figure 4C),indicating that RBOHD is required genetically for DES1/H2

S-induced stomatal closure. The stomatal response and ROSproduction within des1 rbohD guard cells were less ABA-sensitive, but the corresponding responses to H2O2 were notaltered.

Our results indicated that the regulation between intercellularROS production and ABA signaling may occur during stomatalclosure (Figures 4A to 4C). To further investigate whether in-tercellular ROS signaling was regulated by DES1, we expressedRBOHDprotein in thedes1 rbohDbackgroundunder thecontrol oftissue-specific promoters. ABA treatment failed to induce sto-matal closure when RBOHD was expressed in either epidermal,mesophyll, or guard cells of the line carrying the des1 rbohDbackground (Supplemental Figure 14). Together with the resultsshown in Figure 4A, our observations suggested that functionalDES1 activity is involved in ABA-triggered guard cell ROS pro-duction and intercellular ROS relay.

H2S-triggered Stimulation of NADPH Oxidase Activityof RBOHD

Next, to examine whether NADPH oxidase activity could beinfluenced directly by H2S in vitro, we performed enzyme activityassays of NADPH oxidase. Total crude membrane proteins fromwild-type and des1mutant plants were extracted and treatedwithdifferent concentration of sulfurating molecules, followed by di-alysis, before determination of NADPH oxidase activity. In bothwild-type and des1 plants, we clearly observed direct and sig-nificant activation of NADPH oxidases by two sulfurating mole-cules, inadose-dependentmanner (Figure4D).NADPHoxidase inthe des1 mutant was present in much lower amounts than in thewild type (Columbia-0 [Col-0]) at basal level, suggesting the

Figure 3. (continued).

(C)and (D) In vitropersulfidationanalysis ofDES1andmutant recombinantproteinsby the tag-switchmethod. Thepersulfidatedproteinwasdetectedbyananti-biotin antibody.(E) In vivo persulfidation analysis of themutated versions of DES1 protein. Total proteins from Arabidopsis protoplasts transiently expressed DES1 and itsmutant derivatives fused to GFP were subjected to the tag-switch assay for the analysis of the level of persulfidation of different versions of DES1(DES1-SSH). The bottom representation shows the total DES1-GFP used for the tag-switch assay as loading control.(F) Effect of ABA and NaHS on the persulfidation level of DES1 in vivo. Total proteins from Arabidopsis protoplasts transiently expressed unmutated DES1fused toGFPwere treatedwithorwithoutABA (10mMfor 30min) orNaHS (1mMfor1h) beforebeing subjected to the tag-switch assay for theanalysisof thelevel of persulfidation (DES1-SSH). The bottom representation shows the total DES1-GFP used for the tag-switch assay as loading control.(G)DESenzymeactivityof different versionsof theDES1-His recombinantproteins in thepresenceorabsenceofNaHSorH2O2.RecombinantproteinswerepretreatedwitheitherNaHS (10mM)orH2O2 (10mM) for 1h.Con, untreatedconditions. Thedata representmean6 SD (n53) from threebiological replicates.Bars denoted by the different letters were different significantly at P < 0.05 according to one-way ANOVA (post-hoc Tukey’s HSD test).(H) Reversibility of the enzyme activity of DES1-His recombinant protein (wild type) and its mutant derivative Cys44/205Ala. Recombinant proteins werepretreated with either NaHS (10 mM) or DTT (1 mM) for 1 h, followed by dialysis and then treated with the H2O2 (10 mM) at the indicated times. The datarepresentmean6 SD (n5 3) from three biological replicates. Different letters indicate significantly different values at P < 0.05 according to one-way ANOVA(post-hoc Tukey’s HSD test).(I)and (J)EffectsofABAandNaHSon theguardcellH2Sproductionandstomatal closureofwild type, thedes1, and thedes1withdifferentmutatedvarieties.H2S production was monitored using the fluorescent dye 7-azido-4-methylcoumarin. Epidermal strips of each line were preincubated for 3 h in openingbuffer (10 mM of MES at pH 6.15, and 10 mM of KCl) under light (120 mE m22 s21) and loaded with 7-azido-4-methylcoumarin (15 mM) for 40 min beforewashing in MES buffer three times for 15 min each. Subsequently, samples were treated for 30 min with ABA (10 mM), NaHS (100 mM), or H2O2 (10 mM),respectively. For (G), the value 100% corresponds to the fluorescence of wild type in control conditions. R.U., relative units. The corresponding stomatalaperture sizes were also measured. The data represent mean6 SD from three biological replicates (n$ 90 in each replicate). Bars denoted by the differentletters indicate significantly different values at P < 0.05 according to two-way ANOVA (post-hoc Tukey’s HSD test).(K) Stomatal closure and H2S production rate of different plant lines under ABA treatment for the indicated times. Treatment andmeasurement details aredescribed in (I) and (J). The data represent mean6 SD from three biological replicates (n$ 90 in each replicate). Asterisks represent significant differencesbetween pMYB60:DES1 des1 and pMYB60:DES1Cys44/205Ala des1 according to Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).Four-week–oldplantsgrown in the soilwere treatedandharvested at the indicated times. For the immunoblot, thenumbersupon thebottompanels indicatethe relative abundance of the corresponding persulfidated protein compared with that of the control sample (1.00). Signals from two independent ex-periments were quantified. Con, control; IB, immunoblotting; wild type, unmutated version of DES1 protein.

1006 The Plant Cell

important role of endogenous H2S produced by DES1 in regulatingNADPH oxidase activity in vivo.

To verify the contribution of RBOHD function to H2S-inducedNADPH oxidase, changes to NADPH oxidase activity in thepresence or absence of NaHSwere determined in a rbohDmutantand rescued line pRBOHD:RBOHD rbohD. In this case, while lowbasal levels of NADPH oxidase activity in the rbohDmutant were

still detectable compared with the wild type (Col-0), the NaHS-induced activity of NADPH oxidase observed in the wild type wasalmost fully abolished in this mutant (Figure 4E). This low level ofNADPHoxidase activity in the rbohDmutantwaspartially rescuedin pRBOHD:RBOHD rbohD, indicating the predominant contri-bution of RBOHD to NaHS-induced NADPH oxidase activityin vivo.

Figure 4. H2S Requires RBOHD Activity for the Induction of Stomatal Closure.

(A)ROSproduction in guard cells and stomatal responsesofwild-type, rbohD, and rbohD rescued lines. RBOHDwas rescuedby expressingRBOHDunderthe control of tissue-specific promoters, in which RBOHDwas expressed in guard cells (pMYB60:RBOHD), epidermal (pML1:RBOHD), or mesophyll cells(pCAB3: RBOHD) individually. ROS production was monitored using florescent dye H2DCFDA (upper representation). Epidermal strips of each line werepreincubated for3h inopeningbuffer (10mMofMESatpH6.15,and10mMofKCl) under light (120mEm22 s21) and loadedwithH2DCFDA (15mM) for15minbefore washing inMES buffer three times for 15min each. Subsequently, samples were treated for 1 h with ABA (10mM), NaHS (100mM), or H2O2 (10 mM),respectively. Thevalue100%corresponds to the fluorescenceofwild type (Col-0) incontrol conditions.Con, control; R.U., relative units. Thecorrespondingstomatal aperture sizeswere alsomeasured (lower representation). The data representmean6 SD from three biological replicates (n$ 90 in each replicate).Bars denoted by the different letters were significantly different at P < 0.05 according to two-way ANOVA (post-hoc Tukey’s HSD test).(B)ABA-andNaHS-inducednetH2O2 influxes in guardcells of rbohD andpCAB3:RBOHD rbohDplants. Leaves from4-week–oldplantswerepreincubatedfor 3h inopeningbuffer (10mMofMESatpH6.15, and10mMofKCl) under light (120mEm22 s21) andwashed inMESbuffer three times for 15min each. Thenet H2O2 influxes were observed after ABA (10 mM) or NaHS (100 mM) treatment during the indicated times (n 5 6).(C) ROS production in guard cells and stomatal responses of wild-type, des1, des1/rbohD, and des1/rbohD rescued lines. Treatment and measurementdetails are shown in (A). Con, control.(D) to (F)Effects of sulfuratingmolecules onNADPHoxidase activity ofwild-type,des1, rbohD, and rbohD rescued lines. ForNADPHoxidase activity assay,the crudemembrane extracts fromeach line (4weeks old) were extracted and treatedwithNaHSorNa2S at the indicated concentrations (0mM to1mM; forF, NaHSat 100mM) for 30min. NADPHoxidase activitywas determined after dialysis. The data representmean6 SD (n5 3) from three biological replicates.Different letters indicate significantly different values at P < 0.05 according to two-way ANOVA (post-hoc Tukey’s HSD test).

Persulfidation in Guard Cells 1007

ABA Induces Persulfidation of RBOHD on Cys825and Cys890

RBOHD protein contains 10 cysteines that might serve as po-tential sites for persulfidation-based modification by H2S in theN-terminal portion (N-terminal; Cys208), carboxy-terminal portion(C-terminal; Cys825 and Cys890), extracellular portion (Cys410,412, 432, 651, and 695), and transmembrane portion (Cys387 andCys480). To determine whether the cytosolic sides of RBOHDmight be persulfidated, the C- and N-terminal portions of thisprotein were expressed and exposed to sulfurating moleculestypically used to score for persulfidation in vitro. A tag-switchanalysis revealed that persulfidation of the C-terminal portion ofRBOHD recombinant protein was induced by two sulfuratingmolecules individually (Figure 5A), which was in agreement withthe results from the analysis of the persulfidation level of RBOHD-C-GFP in protoplast (Figure 5B; Supplemental Figure 15), andtreated with two H2S donors as well (Supplemental Figure 16).However, no persulfidation of the N-terminal portion was de-tected, regardless of the addition of sulfurating molecules (Figure5C).

We next determined whether the RBOHD-C-terminal waspersulfidated during the plant response to ABA. After ABAtreatment, the level of persulfidated RBOHD-C-SSH protein wasincreased, but this was largely impaired in des1 plants, furthersuggesting a regulatory role for DES1 in modulating the persul-fidation the RBOHD C-terminal (Figure 5D). Subsequently, weanalyzed RBOHD-C-SSH formation after treatment with H2O2 ata wide range of concentrations. The results suggested that H2O2

led to a time and dose- dependent decrease in the persulfidationof RBOHD-C-SSH protein both in vitro and in vivo (Figures 5Eand5F;Supplemental Figure17). The level ofRBOHD-C-SSHwasconsistently higher in the presence DPI, regardless of the pres-ence of ABA (Figure 5G). Moreover, similarly to the persulfidationof DES1 recombinant protein, persulfidation and persulfide-oxidationprocesses were reversible on the thiol groups of RBOHD-C-terminalprotein (Supplemental Figure 18).

TheCys residueswithin theRBOHD-C-terminalwere, therefore,mutated either individually or in combination and the resultingproteins were expressed, treated with sulfurating molecules, andanalyzedby tag-switchassay.Both themutationsofCys825AlaorCys890Ala significantly impaired persulfidation of RBOHD-C-terminal recombinant protein, particularly Cys825Ala (Figure 5H),but this could be reversed by treatment with two sulfuratingmolecules, separately (Supplemental Figure 19). Similarly, in vivoresults demonstrated that persulfidation was additive on Cys825andCys890,andwasalmostcompletelyeliminated in theCys825/890Ala doublemutant, which was not affected by treatments witheither ABA or NaHS (Figures 5I and 5J). Unfortunately, we failed toidentify any persulfided sites on RBHOD-C recombinant protein,probably due to the low abundance and/or poor purity of thepurified protein. Collectively, these findings imply that RBOHD isspecifically persulfidated at bothCys825 andCys890,withCys825being the preferred target.

TheArabidopsis genomecontain 10genes that encodeNADPHoxidase, of which RBOHDA, B, and C contain Cys residues that arehomologous to both Cys825 and Cys890 of RBOHD, indicatingthat different RBOH proteins might also be persulfidated in this

species. We therefore expressed recombinant RBOHC-C-terminalmutated versions and exposed them to either NaHS or DTT. Aswas thecasewith theRBOHD-C-terminal, the unmutatedRBOHC-C-terminal was specifically persulfidated, but mutations of theconserved cysteines blocked this process of protein persulfida-tion (Supplemental Figure 20). The conservation of Cys residuesand the persulfidation-dependent regulation of RBOH familyproteins suggest that persulfidation-mediated activation is ageneral regulatory mechanism adopted by plants.

RBOHD Persulfidation Controls ABA-stimulated ROSProduction and Stomatal Closure

We investigated whether the persulfidation-mediated activationof RBOHD plays a role in ABA-stimulated ROS production andstomatal responses. To explore the possible biological con-sequences of Cys825 and Cys890 persulfidation on RBOHDactivity, we monitored NADPH oxidase activities in mutant rbohDplants expressing wild-type RBOHD, Cys825Ala, Cys890Ala, orCys825/890Ala driven by the native RBOHD promoter (Supple-mental Figure 21). NaHS-induced leaf NADPH oxidase activitywas significantly impaired in the pRBOHD:RBOHDCys825Ala rbohDand pRBOHD:RBOHDCys890Ala rbohD lines, compared withpRBOHD:RBOHD rbohD (Figure 6A). Inducible NADPH oxidaseactivity and ROS accumulation in guard cells were fully abolishedin the pRBOHD:RBOHDCys825/890Ala rbohD line, implying an ad-ditive effect of RBOHD persulfidation at Cys825 and Cys890during ABA/H2S-triggered NADPH oxidase activity, which resultsin increased ROS generation in guard cells (Figure 6B, top).Next, we analyzed the responsiveness of the transgenic rbohD

lines (expressing either wild-type RBOHD or mutant derivatives)to ABA or NaHS during stomatal responses. When treated withNaHS or ABA, pRBOHD:RBOHD rbohD exhibited stomatal clo-sure similar to that of the wild type (Figure 6B, bottom). However,this inducible stomatal closure was markedly reduced in thepRBOHD:RBOHDCys825Ala rbohD, pRBOHD:RBOHDCys890Ala

rbohD, and pRBOHD:RBOHDCys825/890Ala rbohD lines.To confirm that ABA-activated RBOHD function in stomatal

closure and ROS production in guard cells are consequences ofDES1-triggered persulfidation, we introduced RBOHD and itssubstitutional mutant derivatives under the control of the nativeRBOHD promoter into the des1 rbohD mutant, and tested theROS production and guard cell responses to either ABA or NaHSin the resulting transgenic plants. All of the transgenic plants withthe des1 rbohD background exhibited reduced ROS productionand stomatal responses to ABA, which further reinforces theconclusion that DES1 is genetically required for ABA-inducedRBOHD activation and stomatal closure (Figure 6C). Whentreated with NaHS, pRBOHD:RBOHD des1 rbohD guard cellsexhibited an increase in ROS and stomatal closure, similar to thatof the wild type. However, NaHS-induced ROS activation andstomatal closure were abolished in pRBOHD:RBOHDCys825Ala

des1 rbohD,pRBOHD:RBOHDCys890Ala des1 rbohD, andpRBOHD:RBOHDCys825/890Ala des1 rbohD plants. Collectively, these resultssuggested thatDES1/H2S-triggeredRBOHDpersulfidation atCys825andCys890 plays a critical role inmodulating ABA-induced guardcell ROS production and stomatal closure.

1008 The Plant Cell

Figure 5. ABA-Induced Persulfidation of RBOHD at Cys825 and Cys890.

(A)TheCterminusofRBOHD ispersulfidated in thepresenceor absenceof twosulfuratingmolecules. RBOHD–C-terminal recombinantproteinwas treatedwith NaHS or Na2S at 1 mM for 1 h, followed by the tag-switch assay.(B) Analysis of persulfidation of RBOHD–C-terminal in vivo. Total proteins from Arabidopsis wild-type protoplast transformed with 35S:RBOHD-C-GFPwere extracted and exposed to NaHS for 1 h at the indicated concentrations, then subjected to the tag-switch assay.(C) The N terminus of RBOHD is not persulfidated. RBOHD-N-terminal recombinant protein was treated with NaHS 1 mM for 1 h, followed by the tag-switch assay.(D) ABA-induced persulfidation of RBOHD–C-terminal in vivo. Total proteins from Arabidopsis wild-type (Col-0) and des1mutant protoplasts transformedwith 35S:RBOHD-C-GFP were extracted and treated with or without ABA at 10 mM for 30 min, then subjected to the tag-switch assay.(E) and (F) H2O2-triggered persulfide-oxidation of the RBOHD–C-terminal recombinant protein in vitro (E) or RBOHD-C-GFP protein in vivo (F). Sampleswere treated with H2O2 at indicated concentrations for 1 h, then subjected to the tag-switch assay.(G)Enhancement of ABA-inducedRBOHD-C-GFPpersulfidation byDPI. Total proteins fromArabidopsis protoplast transformedwith 35S:RBOHD-C-GFPwere subjected to the tag-switch assay after exposure to ABA at 10 mM for 30 min, with or without pretreatment of DPI at 10 mM for 2 h.(H) to (J) Persulfidation analysis of the C terminus of wild-type and mutant RBOHD derivatives in vitro (H) and in vivo (I) and (J).For in vitro assay, the persulfidated recombinant proteins were detected by an anti-biotin antibody after the tag-switch labeling. Proteins were detected byimmunoblotting using an anti-his antibody served as a loading control. Con, control.

Persulfidation in Guard Cells 1009

DISCUSSION

In dry conditions, plants rapidly synthesize the drought stresshormone ABA at high levels to trigger signal transduction eventsthat lead to stomatal closure and reduced water loss. AlthoughABA is known to activate stress responses, the mechanisms bywhich ABAsignals are relayed in guard cells remain unresolved. Inthis study, we unraveled the molecular framework for DES1/H2

S-triggered protein persulfidation, which links ABA signaling path-ways with stomatal closure.

Persulfidation of DES1 and RBOHD during ABA-inducedStomatal Closure

Maintaining the redox status is a conspicuous feature of signalingcascades and precisely controlled in plant cells (Hancock, 2019;Smirnoff and Arnaud, 2019). Interestingly, the reducing and oxi-dizingmoleculesH2SandH2O2,whicharegeneratedbyDES1andNADPH oxidase, are both critically involved in the ABA response(Kwak et al., 2003; Scuffi et al., 2018). However, the molecularmechanisms that mediate the synthesis and function of thesesignaling molecules are poorly understood. Perhaps the bestexample of the convergent action of DES1 and NADPH oxidase isthe control of stomatal aperture, where H2S is suggested to serveas a gasotransmitter in stomatal closure (Honda et al., 2015;Papanatsiou et al., 2015). Our data are entirely consistent withthese observations (Scuffi et al., 2018). While closure was notevident in the des1 rbohDmutant line after treatment with NaHS,the guard cell response to H2O2was the same as that of wild type.The impairment inABA/NaHSsensitivity ofdes1 rbohD, in termsofstomatal closure, was not altered by the introduction of DES1driven by guard cell promoterMYB60, which illustrated a positiveregulatory role for RBOHDdownstream of DES1/H2S in guard cellABA signaling. RBOHD was shown to mediate long-distancesignaling via a dynamic autopropagating wave of ROS that cantravel at a rate of up to 8.4 cm min21 through the apoplast (Milleret al., 2009). In support of this finding, both ABA and NaHStriggered a ROS-associated influx into guard cells and stomatalclosure when RBOHD was tissue-specifically expressed in therbohDmutant background. Our results infer that plants retain theability toproduceROSsignalsbyRBOHDand facilitate cell-to-cellcommunications to trigger downstream events in guard cells inresponse to environmental changes.

H2S physiologically persulfidates a diverse range of proteins.As persulfidation occurs frequently, this post-translational mod-ification has been proposed to affect a variety of biologicalpathways (Aroca et al., 2017a, 2017b, 2018). Here, we demon-strated that ABA-activated DES1-endogenous H2S physiologically

persulfidatedDES1 to regulate its activity,withCys44andCys205as themain sites of thismodification. Our results revealed that theactivity of persulfidatedDES1can return to thebasal state quickly,depending on the dynamic redox status, e.g., endogenous H2O2

homeostasis, which can lead to reversible persulfide-oxidation ofpersulfidated DES1, thus fine-tuning the strength and duration ofthe ABA signal. Therefore, ROS accumulation may function asa negative feedback mechanism to prevent the continuous trig-gering of ABA signaling in guard cells, which is achieved bypersulfide-oxidationofpersulfidatedDES1.Moreover, persulfidesmay serve a protective function of protein thiols in highly oxidizedenvironments (Gotor et al., 2019). It is therefore a matter of im-portance as DES1-produced H2S contributes far more to the ABAsignaling than previously imagined. The regulatory mechanismis physiologically related to the guard cell ABA response. InpMYB60:DES1Cys44/205Ala des1, ABA was less effective in in-ducing guard cell H2S production and stomatal closure than inwild-type plants within the treatment period. This observationsupports a positive regulatory role for ABA in the persulfidationand activation of DES1, whichmay facilitate the downstreamH2Ssensing, which is essential in relaying the guard cell ABA signals.The rapid production of ROS in guard cells acts as systemic

secondary messenger to trigger stomatal closure (Song et al.,2014). Plant NADPH oxidases belong to the RBOH family, andcatalyze the production of superoxide that can then be convertedinto H2O2 spontaneously or by superoxide dismutase or perox-idases (Smirnoff and Arnaud, 2019). In many cases, RBOHsregulation occurs on the N-terminal extension that carries EF-hand motifs and potential phospho-sites through Ca21 bindingand phosphorylation, which are thought to be important for ac-tivation of NADPH oxidase (Ogasawara et al., 2008; Chen et al.,2017). Notwithstanding the long-established correlation betweenABA signaling and ROS burst activation in guard cells, themechanisms that underpin the activation of a ROS burst, and areassociated with the RBOHD C-terminal, are largely unknown. Inthis study, we discovered that H2S regulates guard cell ABAsignaling through persulfidation of two cysteines in theC-terminalof RBOHD. Persulfidation triggers RBOHD to facilitate a burst ofROS, which, in turn, causes stomatal closure. This might repre-sent amechanism for raised ABA-triggeredROS signaling in guardcells.des1plantsshowed reducedNADPHoxidaseactivityand lowpersulfidation at the C-terminal of RBOHD, supporting a positiveregulatory role for H2S in the ABA-activated RBOHD.Our findings are reminiscent of those in plant immunity, where

S-nitrosylation at a conserved Cys (890, not 825) in RBOHDpromotes cell death during plant immune function (Yun et al.,2011). While S-nitrosylation on Cys890 disrupts the side-chainposition of Phe921, impeding FAD binding affinity, persulfidation

Figure 5. (continued).

For in vivo assay, total proteins from Arabidopsis protoplasts transiently expressed the RBOHD C terminus and its mutant derivatives fused to GFP weretreated with or without ABA (10 mM for 30 min) or NaHS (1 mM for 1 h) before being subjected to the tag-switch assay and streptavidin purification. Thepersulfidatedproteinsweredetectedbyananti-GFPantibody. Thebottom representations show the total GFP fusedproteins used for the tag-switch assayas loading control. Wild type, the unmutated version of RBOHD-C protein; Con, untreated conditions. The numbers above the bottom representationsindicate the relative abundance of the corresponding persulfidated protein compared with that of the control sample (1.00). Signals from two independentexperiments were quantified.

1010 The Plant Cell

enhances the reactivity of the Cys890 thiol and may render itmore nucleophilic, and thus may facilitate its binding with FAD.Importantly, under our conditions, Cys825 was predominantlypersulfidated and additional effects of Cys825 and Cys890 onguard cell ROS production and responses were seen. Thus, wecould not fully reject the hypothesis that nitrosylation of Cys890affects the guard cell response. Further investigations using theRBOHD native protein and 3D structure will help to unveil themechanisms of persulfidation-regulated RBOHD activity. Nota-bly, we observed that the RBOHDCys825/890Ala mutation abolishedthe H2S and ABA responses, suggesting that the persulfidation ofRBOHDplays a regulatory role in guard cell ABA responses. It hasbeen reported that persulfidation precedes nitrosylation on the

same Cys residue in several instances (Sen et al., 2012; Vandiveret al., 2013). This opens the possibility of crosstalk betweenpersulfidation and nitrosylation in the regulation of ROS pro-duction and ABA signaling in guard cells. Further clarification ofthe temporarysequencesofpersulfidationandnitrosylationwouldoffer new insights into the sophisticated mechanisms of ABA-controlled guard cell signaling.

Redox-based Reversible Persulfidation Controls Guard CellABA Signaling

The on–off switch for RBOHD function controlled by persulfidationand persulfide-oxidationmay fine-tune the strength or duration of

Figure 6. DES1-Triggered Persulfidation of RBOHD at C825 and C890 Is Involved in ABA-Induced ROS Activation and Stomatal Closure.

(A) Effects of NaHS or DTT on the activity of NADPH oxidase of mutant rbohD plants expressing either a wild-type RBOHD or Cys825Ala, Cys890Ala, andCys825/890Ala mutant derivatives driven by its native promoter. Treatment and measurement details are represented in Figures 4D to 4F. Con, control.(B) and (C)Effects of ABAorNaHSonROSproduction and stomatal closure of wild type (Col-0), rbohD, des1 rbohD, and lines expressing either awild-typeRBOHDor Cys825Ala, Cys890Ala, andCys825/890Alamutant derivatives driven by its native promoter. Treatment andmeasurement details are shown inFigure 4A. Con, control; R.U., relative units.

Persulfidation in Guard Cells 1011

RBOHD activation and ROS signals in response to ABA. Our re-sults indicate that H2S and ROS produced by DES1 and RBOHD,respectively, can fulfill this role. Although the kinetic and structuraldetails of DES1/RBOHD persulfidation and persulfide-oxidationremain limited, our findings suggest the following: ABA causesrapid and strong activation of DES1 in guard cells, which canpersulfidate downstream functional proteins, including itself onCys44 and Cys205, thus amplifying guard cell ABA signals. Theinducible DES1/H2S also persulfidates NADPH oxidase RBOHDon Cys825 and Cys890 to facilitate the rapid induction of aROS burst and stomatal closure (Figure 7 and the graphic ab-stract).WhenROSaccumulates, it triggers persulfide-oxidation ofRBOHD and DES1 to form perthiosulfenic acid, leading to ABAdesensitivity. The reducible S–S bond present in the formedperthiosulfenated Cys can be reduced by disulfide reductasessuch as thioredoxin, which has been demonstrated in the formedS-sulfocysteine bond in the active site of 39-phosphoadenosine-59-phosphosulfate reductase (Palde and Carroll, 2015) Thestimulated RBOHD activity may be returned to the basal statequickly through this redox-based posttranslational modification,based on dynamic redox homeostasis. The finding that ROS-triggered persulfide-oxidation of persulfidated RBOHD and DES1suggests that plants possess yin-and-yang mechanisms to or-chestrate redox signaling coupledwith hormonal response,whichmay feedback toprevent thecontinual activationofABAsignals inguard cells. As H2S and ROS are ubiquitous, this yin-and-yanginterplay is most likely to be a rapid regulatory mechanism thatprimes theparadigmaticABA-triggered regulationofplantRBOHsand ROS production in guard cells. Correspondingly, tippingthe DES1/H2S-RBOHD/ROS balance toward increased endog-enous ROS production can trigger protein persulfidation oxida-tion, further leading theseactivatedproteins to return to their basalstates. Plants endowed with this negative feed-back loop couldsense the redox status and control ROS homeostasis under ev-erchanging environments.

METHODS

Plant Materials and Growth Conditions

TheArabidopsis (Arabidopsis thaliana)des1 (SALK_103855; Col-0)mutantwas obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/abrc); rbohD was a gift from Prof. Wenhua Zhang,Department of Plant Science, Nanjing Agricultural University.

The des1 rbohD double mutant line was obtained by crossing rbohDwithdes1 (SupplementalDataSet 1).Homozygousmutantswere identifiedbysequencing, combinedwithPCR-basedgenotyping andcorrespondingphenotypes (including reduced plant size and vigor).

Seedswere surface-sterilized andwashed three timeswith sterilewaterfor 20 min, then cultured in Petri dishes on solid half strength Murashige andSkoog (MS)medium (pH 5.8). Plants were grown in a growth chamber witha 16-h/8-h (23°C/18°C) day/night regime using bulb-type fluorescent lightat a light intensity of 100 mmol photons m22 s21 irradiation.

Molecular Cloning

For Expression in Escherichia coli

PCR-amplified fragments of AtRBOHD-N (1 bp to 1,068 bp), AtRBOHD-C(2,269bp to2,763bp), and full-lengthAtDES1were introduced intoPET28avector (for His fusion) using homologous recombination technique (Va-zyme), with the enzyme digestion site of NdeI and XhoI.

Site-directed mutagenesis was performed with Mut Express II FastMutagenesis Kit (Vazyme). All procedures followed the manufacturer’smanual. The specific primers used for AtRBOHD-N, RBOHD-C, and full-length AtDES1 are listed in Supplemental Data Set 1.

For Transient Expression in Arabidopsis Protoplasts

PCR-amplified fragments were cloned into PAN580 vector (from AyingZhang’s lab,Collegeof LifeSciences,NanjingAgriculturalUniversity) at thesides of XbaI and BamHI using a homologous recombination technique(Vazyme).

Arabidopsis protoplasts were isolated according to the method de-scribed previously by Yoo et al. (2007). The transient expression vector

Figure 7. Working Model Shown in This Study.

Whendrought stress signalwas sensed by theplant, ABAwas synthesized and triggered rapid and strong expression ofDES1 in guard cells by anunknownmechanism. Inducible DES1 expression can persulfide many downstream signaling proteins, including itself, on Cys44 and Cys205—leading to theamplification of guard cell H2S signals. The inducible DES1/H2S also persulfidates NADPH oxidase RBOHD on Cys825 and Cys890 to facilitate the rapidinduction of ROSburst, which in turn causes stomatal closure.WhenROS accumulates to high levels, it further causes persulfide-oxidation of RBOHDandDES1 (2SSOnH), leading toABAdesensitivity. Theoxidizedpersulfide (2SSOnH)maysubsequentlybe reduced to a thiol group (2SH)by thioredoxin,whichformed a feedback prevention of ABA signaling continuously activated in guard cells.

1012 The Plant Cell

PAN580 was delivered into Arabidopsis protoplasts using a PEG-calcium–mediated approach and protoplasts were cultured for 12 h.Protoplasts expressing the targeted GFP driven by the constitutive 35Senhancer under fluorescence microscopy were used for transient ex-pression analysis.

For Expression In Planta

Promoter cloning of CAB3, ML1, MYB60, and RBOHD was done usingArabidopsis wild-type DNA as template. The promoter regions of CAB3(2,000 bp),ML1 (1,500 bp),MYB60 (1,500 bp), andRBOHD (1,500 bp)wereamplified by PCR (Supplemental Data Sets 1 and 2). CAB3, pML1, andMYB60 fragments were cloned into pCAMBIA 1305 vector (from AyingZhang’s lab, College of Life Sciences, Nanjing Agricultural University). TheRBOHD products were cloned into 130221-flag vector (from Yiqun Bao’slab, College of Life Sciences, Nanjing Agricultural University).

The constructed plasmids were transferred into an Agrobacteriumtumefaciens strain, and then the inflorescence of Arabidopsiswas affectedby dipping. Transgenic plants were selected on half strength MS mediumcontaining Hygromycin B (50 mg/mL). Identification of transgenic plantswas conducted by genotyping combined with PCR, fluorescence obser-vation, and immunoblot analysis.

Expression and Purification of Recombinant Protein

Expression and purification of the recombinant protein was performed inBL21 cells (Vazyme). A quantity (0.1 mM) of IPTG was added and thebacteria grown to OD600 5 0.4 to 0.6, for 12 h at 16°C. After enrichingthe bacterial solution, the precipitation was suspended in PBS buffer,the protein broken by ultrasonic crusher, and was centrifuged at 12,000gfor 30 min, with the extract then taken for purification. The HIS-tagged pro-teins were purified using a NI-NTA prepacked gravity column (SangonBiotech); the procedure for protein purification was performed in accor-dance with this column specification.

Direct Mass Spectrometry Analysis of Persulfidated Cys Siteson DES1

Directmass spectrometry analysis of persulfidatedCys sites onDES1wasperformedwithmodificationsasdescribedbyBogdándietal. (2019)andFuet al. (2019). In brief, purified recombinant proteins (;0.1mg/mL, 120mL) in50 mM of HEPES at pH 7.6 were incubated with 1 mM of NaHS at roomtemperature for 1 h. Protein samples were then alkylated with 10 mM ofiodoacetamide (IAM) in the dark at room temperature for 30 min. Next,alkylated proteins were resolved by SDS-PAGE gel and stained withCoomassie Brilliant Blue. Gel bands corresponding to DES1 (;40 kd) weresliced, destainedwith50mMofNH4HCO3 in40%methanol, andsubjectedto in-gel digestion as described by Shevchenko et al. (2006). The resultingpeptides were extracted, desalted, and evaporated to dryness until liquidchromatography tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS was performed with the same settings as previously described byAkter et al. (2018) and Huang et al. (2019) on a Q Exactive Plus (ThermoFisher Scientific) instrument operated with an Easy-nLC1000 System(Thermo Fisher Scientific). The resulting mass spectrometry data weresearched against the protein sequence of DES1 (UniProt accessionnumber: F4K5T2) using the software pFind 3 (Chi et al., 2018). Theprecursor-ion mass and fragmentation tolerance were 10 microliters perliter and 20 microliters per liter, respectively. A specific-tryptic search wasused with a maximum of three missed cleavages allowed. Mass shifts of115.9949 D (Met oxidation, M), 157.0214 D (C2H3N1O1, carbamidome-thylation, C), and188.9935D (C2H3N1O1S1, IAM-derivatized persulfide, C)were searched for dynamic modifications.

Enzyme Activity Assay of DES1 Recombinant Protein

TheDESactivity forDES1weredeterminedusing themethodsdescribed inÁlvarez et al. (2010). The DES activity wasmeasured by themonitoring therelease of sulfide from L-cysteine in a total volume of 3 mL containing0.8mMof L-cysteine, 100mMof Tris-HCl at pH 9.0, and 300mL of purifiedDES1 protein. The reaction was initiated by adding L-cysteine. After in-cubation at 37°C for 15min, the reaction was terminated by the addition of300 mL of 30 mM of FeCl3 dissolved in 1.2 M of HCl and 300 mL of 20-mMN,N-dimethyl-P-phenylenediamine dihydrochloride dissolved in 7.2 M ofHCl. The formation of methylene blue was determined at 670 nm ina spectrophotometer. The DES enzymatic activity was calculated bycomparison to a standard curve of Na2S. The amount of total protein wasdetermined by the method of Bradford (1976).

SDS-PAGE and Immunoblotting

Proteinextractswereseparatedby12%SDS-PAGE.Afterelectrophoresis,the gel was transferred to a polyvinylidene difluoridemembrane (Roche) at100 V for 45 min at 4°C. The membrane was blocked with 5% skimmedblocking solution (5% [w/v] BSA in TBS with 0.5% [v/v] Tween-20) andincubated for 2 h with shaking at room temperature or overnight at 4°C.Immunoblot analysis was performed with antibodies (Supplemental DataSet 2) diluted in blocking solution at the following dilutions: anti-GFP-HRP-DirecT, 1:4,000 (MBL); and anti-biotin-HRP, 1:10,000 (Abcam). Forquantification of protein abundance, the software ImageJ (https://imagej.nih.gov/ij/) was used and signals from two independent experiments werequantified. Full-sized membrane scans are presented in SupplementalFigure 22.

Immunochemical Detection of S-Persulfidated Proteins

S-persulfidated proteins were detected using a modified Tag-switchmethod as described by Aroca et al. (2017a).

For in vitro assays, the purified recombinant proteins were treated withNaHS, DTT, or H2O2 at the indicated concentrations, respectively. Theproteins were blocked with methylsulfonylbenzothiazole, and the S-persulfidated cysteines were labeled by CN-biotin. The S-persulfidatedproteins were detected by immunoblot using an anti-biotin antibody (anti-biotin-HRP, 1:10,000; Abcam).

For in vivo assay, Arabidopsis leaves (;1 g) were ground in a mortarunder liquid nitrogen. Extract and Arabidopsis protoplasts were ho-mogenized in buffer (25 mM of TRIS, 100 mM of NaCl, and 0.2% [v/v]Triton X-100, at a pH of 8.0) containing a complete protease inhibitorcocktail (Sigma-Aldrich). The extract was centrifuged at 4°C for 30 min.Blocking buffer consisting of 50mMofmethylsulfonylbenzothiazole thatwas dissolved in tetrahydrofuran was added to the extract and the so-lution was incubated at 37°C for 1 h to block free sulfhydryl groups.Proteins was precipitated with acetone and resuspended pellet in buffer(50 mM of TRIS, 2.5% [w/v] SDS, and 20 mM of CN-biotin, at a pH of 8.0)and incubated 3 h to 4 h 37°C. Protein was precipitated with acetone,washed with acetone 70% (w/v) twice, and resuspended pellet in buffer(50 mM of TRIS and 0.5% [w/v] SDS, at a pH of 8.0). The biotinylatedproteins were purified by immunoprecipitation for 1 h at 25°C with 80 mLof streptavidin magnetic particles (Roche). Particles were washed threetimes and bound proteins were eluted with a suitable buffer, and sep-arated by 12% (w/v) SDS-PAGE, transferred to a polyvinylidene fluoridemembrane. Proteins was detected with an antibody against GFP (anti-GFP-HRP-DirecT, 1:4,000; MBL).

Measurement of H2S and ROS in Guard Cells and Stomatal Aperture

Epidermal strips of each ecotype were preincubated for 3 h in openingbuffer (10 mMMES at pH 6.15, and 50mM of KCl) under light (100 mEm22

Persulfidation in Guard Cells 1013

s21) and loaded with a fluorogenic probe useful for the detection of H2S(7-Azido-4-methylcoumarin; Sigma-Aldrich) for 20 min, or loaded with29,79-Dichlorofluorescein diacetate (an oxidation-sensitive fluorescentprobe tomeasureROS;Sigma-Aldrich) for 15minbeforewashing inMESbuffer three times for 15 min each, subsequently treated for 1 h with10 mMof ABA, 100 mMof NaHS (the sulfurating molecule), or 10 mMof H2

O2 and then analyzed using a TCS-SP2 Laser Scanning Confocal Mi-croscope (Leica; excitation at 405 nm, emission at 454 nm to 500 nm forthe detection of H2S; excitation at 488 nm, emission at 500 nm to 530 nmfor the detection of ROS). Stomatal aperture measurements were per-formed as previously described byXie et al. (2016); epidermal stripsweresoaked in to the opening buffer, which was 10 mM of ABA, 100 mM of HT(the scavenger of H2S), 100 mM of NaHS (the sulfurating molecule), or10 mM of ABA1100 mM HT (the scavenger of H2S) for 1 h. All manipu-lations were performed at 25 6 1°C. Data were analyzed using thesoftware ImageJ. Each value represents the mean of at least 90 stomatataken from different leaves, bars (when present) represent the SE of eachtreatment.

Noninvasive Microtest Technique Measurement

The basic principles of noninvasive microtest technique (NMT) mea-surement are described by Newman (2001). The net guard cells ROS-associated fluxes were measured using the Scanning Ion-SelectiveElectrode Technique System (BIO-001A; Younger USA). A ROS-sensitivemicrosensor was purchased from the Xuyue Beijing NMT Service Center.Theexperimental voltage is1600mV.Theprimaryposition (M1)ofanROS-sensitivemicrosensorwasplaced10mmfrom the guard cells’ surface, andthe further-away position (M2) was 45mm. Net guard cell ROS-associatedfluxes were calculated by Fick’s law of diffusion: J (pmol/cm2/s)52Do3

(dc/dx), where Do indicates the diffusion constant, dc the concentrationgradient (calibration with H2O2 standard curve), and dx the distance (30mm). Experiments were conducted at room temperature (246 1°C) underambient light. Data (means6 SD, n5 6). Leaves from a 1-month–old plantwereplaced inaPetri dishand immersed inMES(atpH6.15)assaysolution,after preconditioning, steady-state ion fluxes were recorded over a periodof 5 min. Then, an ABA/NaHS-containing stock solution was applied andmixed to reach the required final concentration of 10 mM and 100 mM,respectively.

Determination of NADPH Oxidase Activity

Crude membrane extracts were isolated. Two grams of leaf tissues wereground in liquid nitrogen and dissolved in four volumes of the extractionbuffer (25mMof HEPES at pH 7.5, 5mMof EDTA, 2mMof EGTA, 1mMofDTT, 150ofmMKCl, 250mMofmannitol, and0.5mMof PMSF). The crudeextract was filteredwith four layers of gauze and centrifuged at 10,000g for20 min and the resulting supernatant was ultracentrifuged at 50,000g for30min. The resulting pellet was resuspended in suspension buffer (2.5mMof HEPES at pH 7.5, 5 mMof EDTA, 1mMof DTT, 150mMof KCl, 250mMof mannitol, and 1 mM of PMSF), and used as the membrane fraction tomeasure NADPH oxidase activity.

The NADPH-dependent O22 generating activity in isolated crude

membrane was determined by following the reduction of sodium 39-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benze-nesulfonic acid hydrate (XTT) by O2

2. The assaymixture of 1mL contained50mMof Tris–HCl buffer at pH 7.5, 0.5mMof XTT, 100 mMofNADPH, and15 mg to 20 mg of membrane proteins. The reaction was initiated with theaddition of NADPH, and XTT reduction was determined at 470 nm. Cor-rections were done for background production in the presence of 50 unitsSOD. Rates of O2

– generation were calculated using an extinction co-efficient of 2.16 3 104 M21 cm21.

Statistical Analysis

Statistical analysis and plotting of graphs were performed using thesoftware SPSS 16.0 (https://www.ibm.com/products/spss-statistics). Todetermine statistical significance, we used one-way or two-way ANOVA(corrected with post-hoc Tukey’s HSD test). Differences were consideredsignificant at P < 0.05. Statistical data are provided in Supplemental DataSet 3.

Accession Numbers

DNA and derived protein sequence data from this article are in the Ara-bidopsis Genome Initiative database under the following accessionnumbers: DES1 (At5g28030), RBOHD (At5g47910), RBOHF (At1g64060),MYB60 (At1g08810), ML1 (At5g61960), CAB3 (At1g29910).

Supplemental Data

Supplemental Figure 1. Analysis of persulfidation of DES1-GFPin vivo (supports Figure 1).

Supplemental Figure 2. Relative expression of DES1 in wild type(Col-0), des1, and lines expressing either a wild-type DES1 orCys30Ala, Cys44Ala, Cys205Ala, and Cys30/44/205Ala mutant deriv-atives driven by MYB60 promoter (supports Figures 1 and 3).

Supplemental Figure 3. Detection of persulfidation of the DES1protein in vivo (supports Figure 1).

Supplemental Figure 4. Analysis of persulfidation of DES1-GFPin vivo (supports Figure 1).

Supplemental Figure 5. Time-dependent persulfidation oxidation ofDES1-His recombinant protein (supports Figure 2).

Supplemental Figure 6. Persulfidation of DES1-His recombinantprotein by H2O2 pretreatment (supports Figure 2).

Supplemental Figure 7. Amino acid sequence of DES1 protein. DES1protein has 323 amino acids in total (supports Figure 3).

Supplemental Figure 8. The persulfidation formation of DES1-Hisrecombinant protein and its derivatives Cys44Ala, Cys205Ala, Cys44/205Ala, and Cys30/44/205Ala (supports Figure 3).

Supplemental Figure 9. Representative images of ABA on the guardcell H2S production of des1 rescued lines shown in Figure 3G(supports Figure 3).

Supplemental Figure 10. The effects of NaHS on the guard cell H2Sproduction (A) and stomatal closure (B) of wild type, des1, and des1rescued lines (supports Figure 3).

Supplemental Figure 11. Relative expression of RBOHD in wild type(Col-0), rbohD and lines expressing RBOHD under the control oftissue-specific promoters, in which RBOHD was expressed in meso-phyll cells (pCAB3:RBOHD), epidermal (pML1:RBOHD), or guard cells(pMYB60:RBOHD) individually (supports Figure 4).

Supplemental Figure 12. Representative images of guard cell ROSproduction for rbohD and its rescued lines shown in Figure 4A(supports Figure 4).

Supplemental Figure 13. Genotyping for the des1 rbohD mutant(supports Figure 4).

Supplemental Figure 14. Stomatal responses of wild-type, des1rbohD, and des1 rbohD rescued lines (supports Figure 4).

Supplemental Figure 15. Subcellular localization of RBOHD-C-GFP(supports Figure 5).

1014 The Plant Cell

Supplemental Figure 16. Detection for the persulfidation of theRBOHD-C-terminal in vivo (supports Figure 5).

Supplemental Figure 17. A time-dependent decrease of persulfida-tion of RBOHD-C recombinant protein in vitro (supports Figure 5).

Supplemental Figure 18. Reversible persulfidation of RBOHD-Crecombinant protein in vitro (supports Figure 5).

Supplemental Figure 19. Persulfidation of RBOHD-C recombinantprotein and relative derivatives (supports Figure 5).

Supplemental Figure 20. Persulfidation of the RBOHC-C recombi-nant proteins and related derivatives (supports Figure 5).

Supplemental Figure 21. Relative expression of RBOHD in wild type(Col-0), rbohD, and lines expressing either a wild-type RBOHD orCys825Ala, Cys890Ala, and Cys825/890Ala mutant derivatives drivenby its native promoter individually (supports Figure 6).

Supplemental Figure 22. Immunoblot membrane scans for this study(supports Figures 1–3, and 5).

Supplemental Data Set 1. Oligonucleotide primers used in this study.

Supplemental Data Set 2. Materials used in this study.

Supplemental Data Set 3. Detailed statistical analyses of the resultsin this study.

ACKNOWLEDGMENTS

Theauthors thankHonghongWufromHuazhongAgriculturalUniversity forhis helpful advice on the NMT experiment. Y.X. thanks his wife Dan Chen,daughterMengmeng, and family for their unwavering support and encour-agement, without which this article could not have been published. Thework was supported by the National Natural Science Foundation of China(grants31670255and21922702), theFundamentalResearchFunds for theCentral Universities (grant KYZ201859), the National Natural ScienceFoundation of Jiangsu Province (grant BK20161447), and theEuropeanRegional Development Fund through the Agencia Estatal de Investigaciónof Spain (grant BIO2016-76633-P).

AUTHOR CONTRIBUTIONS

J.S., J.Z., H.Z., B.C., J.Y., and Y.X., designed the study; J.S., J.Z., M.Z.,H.Z., and L.F., performed the experiments; B.C., Q.P., C.G., L.C.R., L.F.,J.Y., W.S., and Y.X. analyzed the data; C.G., L.C.R., C.H.F., and Y.X. wrotethe article.

Received October 22, 2019; revised January 17, 2020; accepted February5, 2020; published February 5, 2020.

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Persulfidation in Guard Cells 1017

DOI 10.1105/tpc.19.00826; originally published online February 5, 2020; 2020;32;1000-1017Plant Cell

Jing Yang, Christine Helen Foyer, Qiaona Pan, Wenbiao Shen and Yanjie XieJie Shen, Jing Zhang, Mingjian Zhou, Heng Zhou, Beimi Cui, Cecilia Gotor, Luis C. Romero, Ling Fu,

Controls Guard Cell Abscisic Acid SignalingPersulfidation-based Modification of Cysteine Desulfhydrase and the NADPH Oxidase RBOHD

 This information is current as of August 15, 2020

 

Supplemental Data /content/suppl/2020/02/05/tpc.19.00826.DC1.html

References /content/32/4/1000.full.html#ref-list-1

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