Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity

Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity Denise Scuffi , Rosario Pantaleno , Paula Schiel , Jan-Ole Niemeier , View ORCID Profile Alex Costa , Markus Schwarzländer , Ana M. Laxalt , Carlos García-Mata doi: https://doi.org/10.1101/2025.02.14.638267 Denise Scuffi a Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata , Consejo Nacional de Investigaciones Científicas y Técnicas (IIB-UNMdP-CONICET), 7600, Mar del Plata, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rosario Pantaleno a Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata , Consejo Nacional de Investigaciones Científicas y Técnicas (IIB-UNMdP-CONICET), 7600, Mar del Plata, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paula Schiel a Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata , Consejo Nacional de Investigaciones Científicas y Técnicas (IIB-UNMdP-CONICET), 7600, Mar del Plata, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan-Ole Niemeier b Institute of Plant Biology and Biotechnology (IBBP), University of Münster , Schlossplatz 8, 48143 Münster, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alex Costa c University of Milan, Department of Biosciences , Milan, 20133, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alex Costa Markus Schwarzländer b Institute of Plant Biology and Biotechnology (IBBP), University of Münster , Schlossplatz 8, 48143 Münster, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ana M. Laxalt a Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata , Consejo Nacional de Investigaciones Científicas y Técnicas (IIB-UNMdP-CONICET), 7600, Mar del Plata, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carlos García-Mata a Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata , Consejo Nacional de Investigaciones Científicas y Técnicas (IIB-UNMdP-CONICET), 7600, Mar del Plata, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: camata{at}mdp.edu.ar Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Stomata are natural pores through which plants exchange gases with the environment, mainly carbon dioxide and oxygen required for photosynthesis and respiration, as well as water vapor through evapotranspiration. However, they also serve as entry points for microbial pathogens such as Pseudomonas syringae pv . tomato ( Pst ) bacteria. To prevent microbe invasion, guard cells detect pathogens-associated molecular patterns (PAMPs), including the bacterial peptide flagellin (flg22), triggering stomatal closure. This study identifies hydrogen sulfide (H 2 S) and its cytosolic source L-CYSTEINE DESULFHIDRASE 1 (DES1), as key players in stomatal immunity. We demonstrate that H 2 S and DES1 are involved in flg22- and bacterial-induced responses, including stomatal closure and modulation of reactive oxygen species (ROS) production. We have found that knock out mutants in DES1 gene exhibits reduced susceptibility to Pst spray-inoculation and lower apoplastic and cytosolic H 2 O 2 levels in response to flg22. Additionally, H 2 S independently induces cytosolic H 2 O 2 levels in guard cells without requiring RBOHD activity. All together, these findings establish H 2 S and its source, DES1, as critical components of the stomatal immune response. One Sentence Summary H 2 S and DES1 actively participate in flg22-induced stomatal closure modulating apoplastic and cytosolic ROS production. INTRODUCTION Plants are exposed to a wide variety of environmental challenges, including a diversity of pathogens. Stomata act as a hub of the exchange between the plant and its environment in the aerial part of land plants, because they enable and regulate the large fluxes of inorganic matter, i.e. water, carbon dioxide and oxygen that ultimately build and maintain the plant homeostasis. The size of the stomatal pore is regulated through variations of guard cell volume. Multiple stimuli, both environmental and endogenous, are sensed by plants and translated through a complex signalling network into redistribution of osmotically active solutes, resulting in the influx/efflux of water with the consequent change in cell volume. Since stomatal opening has direct impact on carbon fixation and water homeostasis, stomatal pore regulation is a central physiological process for the plant, and scales up to our global climate ( Blatt, 2000 ; Schroeder et al., 2001 ; Kim et al., 2010 ; Qi et al., 2018 ). A potential downside of “natural openings” in the epidermal layer of plants, such as stomata, hydathodes or wounds, is that they provide an entry point for many pathogens, including bacteria and some fungi. Upon contact with pathogens-associated molecular patterns (PAMPs), stomatal closure is triggered as an early defence response to avoid the infection of the endogenous tissues ( Underwood et al., 2007 ; Agurla et al., 2014 ; Melotto et al., 2017 ). In Arabidopsis, flagellin and flg22 (a conserved peptide from flagellin that is recognized as a PAMP) are recognized by the membrane immune receptor, FLAGELLING SENSING 2 (FLS2), to induce PAMP-triggered immunity (PTI). This signalling event, among others, involves the production of superoxide (O 2 . - ) via the activation of RESPIRATORY BURST OXIDATIVE HOMOLOGUE D (RBOHD) which then is converted by the superoxide dismutase to hydrogen peroxide (H 2 O 2 ) in the apoplast; calcium (Ca 2+ ) influx from the apoplast; the activation of mitogen activated-protein kinases (MAPKs) and stomatal closure ( Bethke et al., 2012 ; Daudi et al., 2012 ; Kadota et al., 2014a ; Li et al., 2014 ; Toum et al., 2016 ; Arnaud et al., 2017 ; Tian et al., 2019 ; Thor et al., 2020 ; Bjornson et al., 2021 ). The signalling triggered by PAMPs that leads to stomatal closure is referred to as stomatal immunity, one of the plant’s initial responses to pathogen attack to limit their entry into the leaf ( Melotto et al., 2024 ). Some pathogens, such as Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000), have developed mechanisms to suppress stomatal immunity by “rewiring” guard cell signalling network through the production of phytotoxins like coronatine (COR), to reopen the stomata after 3h of infection ( Melotto et al., 2006 ). Despite major advances in the understanding of the stomatal immunity signalling network, new actors and mechanisms of action are still emerging. Hydrogen sulfide (H 2 S) acts as a gasotransmitter in many biological systems, including plants, where it plays a key role in guard cell signalling ( Pantaleno et al., 2021 ). There is evidence that H 2 S modulates proteins by persulfidation, i.e. a posttranslational modification (PTM) of cysteine (Cys) residues in target proteins ( Wang et al., 2021 ; Pantaleno and Scuffi., 2024 ). Formation of persulfides (RSSH) on Cys thiol moieties is reversible, giving rise to a molecular switch in cell signalling that may be linked to the modulation of molecular structure and/or activity of the modified protein. This can be observed in proteomes of persulfidated proteins from Arabidopsis leaf extracts, where some of the hits are modified in a reversible manner ( Aroca et al., 2015 ; Aroca et al., 2017 ). Persulfidation is currently regarded as a mechanism that protects proteins from irreversible oxidation in persistent oxidative environments ( Filipovic et al., 2018 ; Aroca et al., 2021 ). Plants synthesize H 2 S enzymatically from sulfur-containing amino acids in different subcellular compartments (Gotor et al., 2019). Among them, the first characterized source, L-CYSTEINE DESULFHYDRASE 1 (DES1), which degrades L-cysteine to produce pyruvate, ammonia and H 2 S, is considered a major cytosolic source ( Álvarez et al., 2010 ). DES1-dependent H 2 S production participates in several physiological processes, including stomatal closure in response to different stimuli ( Scuffi and García-Mata, 2021 ). For example, in abscisic acid (ABA)-dependent stomatal closure, guard cell specific DES1 expression increases cytosolic H 2 S ( Scuffi et al., 2014 ; Du et al., 2019 ; Zhang et al., 2019 ) which, in turn, persulfidates and modulates the activity of key enzymes of the guard cell signalling network such as OPEN STOMATA 1 (OST1) and RBOHD, resulting in stomatal closure ( Chen et al., 2020 ; Shen et al., 2020 ). In addition, H 2 S was reported to inhibit inward-rectifying K + channels and increase the cytosolic levels of other second messengers such as nitric oxide (NO), phosphatidic acid (PA) and cytosolic H 2 O 2 specifically in guard cells ( Scuffi et al., 2014 ; Papanatsiou et al., 2015 ; Scuffi et al., 2018 ). However, the above-mentioned responses are not necessarily linked to ABA signalling. The involvement of H 2 S, and its enzymatic sources, in abiotic stress responses, has attracted the research attention of plant scientists. In contrast, the putative role of H 2 S in plant-pathogen interactions may have lagged behind. Fungal infection can boost H 2 S emission in different crop species through a process known as Sulfur Induced Resistance (SiR). SiR is intimately related to sulfur metabolism and to the nutrient status of the plant ( Bloem, 2004 ; Bloem et al., 2007 ; Bloem et al., 2012 ; Vojtovič et al., 2021 ). In addition, bacterial pathogens, such as Pst DC3000, were reported to modulate plant H 2 S dynamics, leading to resistance ( Shi et al., 2015 ). Furthermore, we have recently established that the mitochondrial source of H 2 S, β-cyanoalanine synthase (CAS-C1) participates in flg22-induced stomatal closure ( Pantaleno et al., 2024b ). Although, the involvement of H 2 S in stomatal immunity has been poorly investigated. In this work we set out to understand the involvement of cytosolic DES1/H 2 S production in stomatal immunity and its relationship with other components of the PTI response, such as the second messengers H 2 O 2 and Ca 2+ . To specifically dissect stomatal responses, we combined the genetic impairment of cytosolic H 2 S production with cell compartment-specific biosensing of responses in guard cell physiology. Our findings integrate cytosolic H 2 S into the stomatal signalling network. RESULTS Absence of DES1 affects Pseudomonas-induced stomatal closure and immunity To investigate the role of H 2 S signalling in stomatal immunity we used des1 mutant lines of Arabidopsis to abolish the main cytosolic H 2 S source. We first exploited this model to address the question of whether there is any involvement of H 2 S in stomatal movement in response to Pst DC3000 versus Pst DC3000 hrcC - , being the latter a strain with a defective type III secretion system as required for effector secretion. Pst DC3000 hrcC - represents a useful tool to study PTI responses without the interference of bacterial effectors ( Hauck et al., 2003 ). Epidermal peels from the abaxial side of Arabidopsis leaves from wild type (Col-0) and des1 mutant plants were isolated and incubated in opening buffer and subsequently treated with a bacterial suspension of Pst DC3000 ( Figure 1 A) or Pst DC3000 hrcC - ( Figure 1 B) for 1 h or 3 h. Stomata exposed to either of the bacterial suspensions close after 1 h and reopen after 3 h of treatment in Col-0 epidermal peels, as compared to the mock control. Although des1 stomata exhibited lower stomatal aperture values with the mock treatment, we have previously shown they have the capacity to close under H 2 S-donor and H 2 O 2 treatment ( Scuffi et al., 2014 ; Scuffi et al., 2018 ). However, in contrast to Col-0, no difference in aperture was observed in stomata from des1 plants among treatments, suggesting that DES1 is required for the full scale stomatal PTI response to Pst ( Figure 1 ). Download figure Open in new tab Figure 1: Absence of DES1 affects Pseudomonas-induced stomatal closure. Epidermal peels from 4- to 5-week-old wild type (Col-0) and des1 ( des1 ) mutant Arabidopsis plants were pre-incubated in opening buffer (5 mM MES pH 6.1, 50 mM KCl) for 3 h under light and subsequently treated with 24 h-grown bacteria (OD=0.1) Pst DC3000 (A) or Pst DC3000 hrcC - mutant (B) in the same buffer under light for 1 h or 3 h. The values of stomatal aperture are expressed in microns (µm) and represented in box-plots where the box is bound by the 25 th and 75 th percentile, whiskers span 10 th and 90 th percentile, and the line in the middle is the median. The individual points represent each measurement. Data is from four independent experiments (Table S5). Different letters indicate statistical differences among treatments (Tukey’s Method, p-value < 0.05). Given that there is no information on the involvement of DES1 and H 2 S in the response of leaf tissue to flg22, we asked how DES1/H 2 S may affect flg22-triggered stomatal signalling. To address this question, we isolated epidermal peels from Col-0 and des1 leaves and treated them with 0, 10, 100 and 1000 nM of flg22. Flg22 induced stomatal closure in Col-0 epidermal peels in a concentration-dependent manner while this response was disrupted in des1 stomata to the same flg22 concentrations ( Figure 2 A). To determine if the impaired stomatal immunity of des1 mutant is due to the reduction of H 2 S production, we challenged des1 epidermal peels with flg22 together with the H 2 S donor, GYY4137. Figure 2 B shows that exogenous addition of H 2 S restores stomatal response to flg22 indicating that H 2 S is required for flg22-triggered stomatal immunity response ( Figure 2 B). Finally, in order to see whether the response was specific to flg22, we tested other PAMP such as the bacterial elongation factor Tu (EF-Tu) peptide 18 (elf18), which consistently caused stomatal closure in Col-0 but not in des1 mutant (Figure S1). Taken together, these results strongly suggest that DES1 participates in stomatal closure elicited by PAMPs. Download figure Open in new tab Figure 2: DES1 is required for flg22-induced stomatal closure response. Epidermal peels from 4- to 5-week-old wild type (Col-0) and des1 ( des1 ) mutant Arabidopsis plants were pre-incubated in opening buffer (5 mM MES pH 6.1, 50 mM KCl) for 3 h under light and subsequently treated for 90 min with 0, 10, 100 or 1000 nM flg22 (A), or with opening buffer (Control), 1 µM flg22, 100 µM of the H 2 S donor, GYY4137 (GYY) or flg22 + GYY4137 (B) in the same buffer under light. The values of stomatal aperture are expressed in microns (µm) and represented in box-plots where the box is bound by the 25 th and 75 th percentile, whiskers span 10 th and 90 th percentile, and the line in the middle is the median. The individual points represent each measurement. Data is from at least three independent experiments (Table S5). Different letters denote statistical differences among treatments (Tukey’s Method, p-value < 0.05). Since DES1 participates in bacterial and elicitor-dependent stomatal closure, we inoculated des1 mutant plants with Pst DC3000 hrcC - bacteria by spray, in order to study the susceptibility of these plants taking into account the participation of the stomata in this response. Although an increase in bacterial growth is observed at 72 hours after inoculation compared to 24 hours in both genotypes, des1 mutant plants show less growth compared to the Col-0 plants indicating a lower susceptibility phenotype ( Figure 3 ) Download figure Open in new tab Figure 3: des1 mutant plants are less susceptible to Pst. DC3000 hrcC - spray inoculation. 5-6 week-old Arabidopsis wild type (Col-0) and des1 ( des1 ) plants were surface-inoculated by spray with a Pseudomonas syringae pv. tomato DC3000 hrcC - ( Pst. DC3000 hrcC - ) suspension (OD = 0.2 (λ = 600 nm)). Bacterial growth was assessed in leaf discs at 24- or 72-hours post-inoculation (hpi), and the number of colony-forming units (CFU) per cm 2 of leaf extracts was determined. Log CFU/cm 2 were calculated and represented in box-plots where the box is bound by the 25 th to 75 th percentile, whiskers span 10 th to 90 th percentile, and the line in the middle is the median. The individual points represent each measurement (Table S5). Asterisks denote statistical differences between treatment (Paired samples t -test, P-value <0.05 = (*), P-value <0.01 = (**)). DES1 is involved in flg22-induced apoplastic H 2 O 2 production Flg22 perception triggers an apoplastic, RBOHD-dependent, ROS burst within the first minutes of the defense response ( Felix et al., 1999 ; Kadota et al., 2014a ). To assess the involvement of DES1 in the RBOHD-branch of the stomatal PTI signalling network, we assayed apoplastic ROS production by flg22-treated leaf discs as a readout using luminol-based detection method. Strikingly, the apoplastic ROS burst was attenuated in des1 leaf discs as compared to Col-0 leaf discs ( Figure 4 ), pinpointing a role for DES1 in the RBOHD-mediated redox signaling elicited by flg22. Download figure Open in new tab Figure 4: Flg22-induced apoplastic ROS burst is diminished in des1 leaf discs. Leaf discs of 4- to 5-week-old wild type (Col-0) or des1 ( des1 ) mutant Arabidopsis plants were incubated with 100 nM flg22 and the ROS burst was measured with a luminol-based assay. The luminescence was recorded every 2 min for 30 min and expressed as relative light units (RLU). The curves show the mean + SE of 12 discs from three independent experiments (n=36) during time (Table S4). Inset: Total ROS production was calculated integrating areas under curve (AUC) from each leaf disc and represented in box-plots where the box is bound by the 25 th to 75 th percentile, whiskers span 10 th to 90 th percentile, and the line in the middle is the median. The individual points represent each measurement (Table S5). Asterisk denotes statistical differences between treatments ( t -test, p-value 0.01) Flg22-induced cytosolic H 2 O 2 signature is DES1 and H 2 S-dependent After the rapid apoplastic ROS burst, flg22 induces an increase in cytosolic H 2 O 2 which is required for stomatal closure induction ( Toum et al., 2016 ; Rodrigues et al., 2017 ; Nietzel et al., 2019 ; Arnaud et al., 2023a ). To study the role of DES1 in cytosolic H 2 O 2 dynamics in response to flg22, we isolated epidermal peels from Col-0 and des1 mutant Arabidopsis plants expressing the specific cytosolic H 2 O 2 biosensor roGFP2-Orp1 ( Nietzel et al., 2019 ) and floated them in opening buffer for at least 7 h to ensure full recovery from tissue injury-induced oxidation as we have previously described ( Scuffi et al., 2018 ; Pantaleno et al., 2024a ). Then, epidermal peels were treated with flg22 and the redox state of the cytosolic localised sensor in guard cells was determined. The first observation is that des1 exhibits a higher oxidation state of the sensor under control conditions ( Figure 5 ). Upon flg22 treatment, oxidation of roGFP2-Orp1 was induced in both Col-0 and des1 background, indicating an increase in cytosolic H 2 O 2 concentration. However, the magnitude of the response in des1 was significantly lower compared with Col-0, (p-value < 2.2e-16, Wilcoxon test in Col-0 vs p-value = 3.668e-10, Wilcoxon test in des1 ) ( Figure 5 ). Notably, flg22-dependent oxidation was abolished by pre-treatment with the H 2 S scavenger hypotaurine (HT) (Figure S2). Taken together, these results indicate that DES1 and, more broadly, endogenous H 2 S are required to induce cytosolic H 2 O 2 production in guard cells in response to flg22. Download figure Open in new tab Figure 5: Flg22 requires DES1 to induce cytosolic H 2 O 2 flux in guard cells. Epidermal peels from 4- to 5-week-old Col-0 or des1 mutant plants expressing H 2 O 2 specific biosensor roGFP2-Orp1 in the cytosol were incubated for 7-12 h in opening buffer (5 mM MES pH 6.1, 50 mM KCl). Then, were treated with opening buffer (Mock) or 1 µM flg22 (flg22) for 60-90 min. Moreover, epidermal peels were treated with 20 mM DTT or 10 mM H 2 O 2 for 10 min to estimate the dynamic range of the sensor in situ . Values are expressed as the ratio of 405/488 nm and are represented in the box plots where the box is bound by the 25 th to 75 th percentile, whiskers span 10 th to 90 th percentile, and the line in the middle is the median. The individual points represent each measurement. Red and blue bands indicate the 25 th to 75 th percentile of maximum and minimum ratio values obtained for treatments with external H 2 O 2 and DTT, to drive the sensor towards full oxidation and reduction. Data are from at least three independent experiments (Table S5). Different letters denote statistical differences among treatments (Tukey’s Method, p-value < 0.05) H 2 S-dependent cytosolic H 2 O 2 rise in guard cells is not via RBOHD We next asked the question of whether the H 2 S-dependent cytosolic H 2 O 2 response is dependent on RBOHD or rather on a different branch of the PTI pathway. We generated lines expressing the cytosolic roGFP2-Orp1 sensor in the rbohD background ( Torres et al., 2002 ) by crossing. Epidermal peels from Col-0 and rbohD expressing roGFP2-Orp1 were incubated in opening buffer and then treated with 100 µM of the H 2 S donor, GYY4137 for 15 minutes as previously established ( Scuffi et al., 2018 ). GYY4137-derived H 2 S induced an increase in roGFP2-Orp1 oxidation in Col-0 guard cells. However, roGFP2-Orp1 oxidation of rbohD guard cells was even greater than that of wild type plants ( Figure 6 ) demonstrating that RBOHD is not required for the cytosolic H 2 O 2 response to H 2 S. Download figure Open in new tab Figure 6: H 2 S-induced cytosolic roGFP2-Orp1 oxidation in guard cells does not require RBOHD. Epidermal peels from from 4- to 5-week-old Col-0 and rbohD Arabidopsis plants expressing the H 2 O 2 biosensor, roGFP2-Orp1 in the cytosol were incubated in opening buffer (5mM MES pH 6.1, 50 mM KCl) for 7-12 h and then treated with Dimethyl sulfoxide (DMSO) 0.01% (v/v) (Mock) or with 100 µM of the H 2 S donor GYY4137 (GYY4137) for 15 min. Moreover, epidermal peels were treated with 20 mM DTT or 10 mM H 2 O 2 for 10 min to estimate the dynamic range of the sensor in situ . Values are expressed as the ratio of 405/488 nm and are represented in the box plots where the box is bound by the 25 th to 75 th percentile, whiskers span 10 th to 90 th percentile, and the line in the middle is the median. The individual points represent each measurement. Red and blue bands indicate the 25 th to 75 th percentile of maximum and minimum ratio values obtained for treatments with external H 2 O 2 and DTT, to drive the sensor towards full oxidation and reduction. Data are from at least three independent experiments (Table S5). Letters denote statistical differences among treatments (Tukey’s Method, p-value < 0.05). H 2 S requires Ca 2+ to induce stomatal closure An additional hallmark of PTI signalling is a signature in cytosolic free calcium ([Ca 2+ ] cyt ) which triggers RBOH-activation directly by EF-hand binding and via Ca 2+ -dependent kinase signalling ( Köster et al., 2022 ). Ca 2+ signalling also contributes to other branches of the signalling network and shapes transcriptional re-programming. In guard cells, elicitors, like flg22, induce [Ca 2+ ] cyt oscillations, which are required for stomatal closure ( Dodd et al., 2010 ; Thor and Peiter, 2014 ; Arnaud and Hwang, 2015 ; Thor et al., 2020 ). To address the question of whether H 2 S affects the Ca 2+ -branch of the stomatal immunity signalling network, we treated Col-0 epidermal peels with the H 2 S donor, GYY4137, in presence or absence of the membrane-permeable Ca 2+ chelator BAPTA-AM used to buffer cytosolic Ca 2+ to suppress Ca 2+ signaling, or the extracellular Ca 2+ chelator, EGTA, to prevent any Ca 2+ influx from the apoplast. Chelation of either intra- or extracellular Ca 2+ prevented GYY4137 from inducing stomatal closure, suggesting that Ca 2+ signalling is required for H 2 S induced stomatal closure ( Figure 7 ). Download figure Open in new tab Figure 7: Apoplastic and cytosolic Ca 2+ availability is required for H 2 S-induced stomatal closure. Epidermal peels from 4- to 5-week-old wild type (Col-0) Arabidopsis plants were pre-incubated in opening buffer (5 mM MES pH 6.1, 50 mM KCl) for 3 h under light and subsequently treated for 90 minutes with opening buffer, 25 µM of membrane permeable Ca 2+ chelator, BAPTA-AM (BAPTA-AM), 200 µM of extracellular Ca 2+ chelator EGTA (EGTA) in absence (-GYY4137) or presence (+ GYY4137) of 100 µM H 2 S donor, GYY4137 under light. The values of stomatal aperture are expressed in microns (µm) and represented in box-plots where the box is bound by the 25 th and 75 th percentile, whiskers span 10 th and 90 th percentile, and the line in the middle is the median. The individual points represent each measurement. Data are from at least three independent experiments (Table S5). Different letters denote statistical differences among treatments in each genotype (Tukey’s Method, p-value < 0.05). DISCUSSION Stomatal closure is a key process of the plant immune response, since it generates a physical barrier that restricts pathogen entry to plant tissues. One of the first steps of this early response is the recognition of different PAMPs at the guard cell plasma membrane, an event that triggers PTI signalling involving several of the components that act as hubs within the guard cell signalling network ( Hetherington and Woodward, 2003 ) and induces several bona fide defense processes like callose deposition ( Zhang et al., 2020 ). In this study we demonstrated the involvement of H 2 S, and its main cytosolic source through DES1 activity, in the stomatal immunity response. We also present evidence that pinpoints where in the guard cell signalling network H 2 S may act. DES1 is required to induce stomatal closure under pathogen attack H 2 S and different enzymatic sources, have been found to be involved in guard cell response to ABA, and other hormones related to abiotic stress ( Liu and Xue, 2021 ; Pantaleno et al., 2021 ; Scuffi and García-Mata, 2021 ), while the role of H 2 S in the biotic stress response has been largely overlooked. Here, we use bacterial suspensions and PAMPs to show the involvement of DES1 in stomatal immunity. Mutant plants in DES1 gene were impaired to close stomata in response to both bacteria and elicitors (flg22 and elf18). Moreover, stomata of des1 closed when treated with flg22 together with H 2 S, supporting that DES1 participates in flg22-triggered response through the production of H 2 S ( Figure 1 and 2 ). These results are in line with recent findings involving the mitochondrial source of H 2 S β-cyanoalanine synthase CAS-C1, where mutants lacking CAS-C1 gene are unable to close stomata upon Pst and flg22 treatment ( Pantaleno et al., 2024b ). The involvement of DES1 in stomatal immunity suggests that Pst or flg22 might be modulating either the expression or the activity of DES1, as for ABA-dependent stomatal closure, where ABA induced DES1 expression specifically in guard cells, and both DES1 and cytosolic H 2 S are required for stomatal closure ( Scuffi et al., 2014 ; Zhang et al., 2019 ). In this case, however, there are not evident differences in DES1 expression levels in control or flg22-treated guard cells (Figure S4), suggesting that flg22 might be regulating DES1 activity through a mechanism unknown until now. Recent reports on the mode of action of H 2 S in signal transduction processes indicate that H 2 S modulates target proteins through persulfidation. In this context, guard cells are not an exception, since the activity of several of the components that act as hubs in the guard cell signalling network were reported to be modulated by persulfidation (Reviewed in Pantaleno et al., 2021 , Pantaleno and Scuffi., 2024 ). Such is also the case of the ABA-dependent response where DES1 is persulfidated and activated at the early stage of ABA treatment and can be reversibly and negatively regulated by H 2 O 2 ( Shen et al., 2020 ). Given the relevance of stomatal movement dynamics for plant immunity, we investigated the des1 response to Pst at the whole plant level by spray infection with Pst DC3000 hrcC - and we observed des1 are less susceptible than Col-0 plants ( Figure 3 ). This is in line with previous reports showing that plants lacking DES1 presented a resistance phenotype to Pst DC3000 when leaf are infiltrated. The authors associate this response with the higher total glutathione and cysteine content in des1 mutant, which can be linked with downstream immunity responses ( Álvarez et al., 2012 ). In addition, a new line of evidences show that water availability in the apoplast can be crucial for pathogenesis ( Aung et al., 2018 ). In consequence, it has been reported that when the stomata remain open, due to a lack of response to the closing stimulus, the apoplast water content which is necessary for sustain bacterial growth, is limited, generating phenotypes that are more resistant to pathogen’s attack ( Freeman and Beattie, 2009 ; Xin et al., 2016 ; Liu et al., 2022 ). H 2 S and H 2 O 2 involvement in stomatal immunity The immune response triggered by flg22 is well characterized, not only in terms of the actors that compose it, but also in the temporal sequence of the processes involved. From this characterization, it is known that flg22 rapidly triggers an apoplastic ROS burst, highly dependent on the activation of RBOHD ( Felix et al., 1999 ; Mersmann et al., 2010 ; Macho et al., 2012 ), which also has an active role in H 2 S and ABA-induced stomatal closure ( Kwak et al., 2003 ; Scuffi et al., 2018 ; Shen et al., 2020 ). In fact, RBOHD was found to be persulfidated at Cys825 and Cys890 and activated, upon ABA treatment and these residues are required for ABA and H 2 S-dependent stomatal closure ( Shen et al., 2020 ). Here we show that DES1 is partially required to induce apoplastic H 2 O 2 in response to flg22. It would be interesting to measure apoplastic H 2 O 2 production in response to exogenous application of H 2 S. Such an analysis is complicated given the inhibitory effect of H 2 S over the horseradish peroxidase (HRP) that is required for the luminol assay. After the fast and transient apoplastic ROS burst, H 2 O 2 is proposed to permeate into the cells through aquaporins, to amplify the defense response. Whether or not the RBOHD-produced apoplastic ROS are indeed the exactly same molecules that cause cytosolic oxidation, is still a matter of debate and deserves future dissection. However, there is agreement in that both are required to induce stomatal closure ( Kadota et al., 2014b ; Toum et al., 2016 ; Arnaud et al., 2017 ; Rodrigues et al., 2017 ; Nietzel et al., 2019 ; Yang et al., 2021 ; Arnaud et al., 2023b ). Here, we used the H 2 O 2 biosensor, roGFP2-Orp1, and observed that flg22 induces cytosolic H 2 O 2 increase with a time offset of about 50 min as compared to the apoplastic ROS burst. The cytosolic oxidation requires endogenous H 2 S and is partially dependent on DES1. Treatment with a general H 2 S donor also induces cytosolic H 2 O 2 in the first 15 min in an RBOHD-independent manner ( Figure 6 ) suggesting the involvement of an alternative source of cytosolic H 2 O 2 . These findings align with two recent studies; one demonstrating PAMP-mediated oxidation of roGFP2-Orp1 in the cytosol, independent of NADPH oxidases and apoplastic peroxidases (PRX), in Arabidopsis leaves ( Arnaud et al., 2023a ); and another one showing that roGFP2-Orp1 is oxidized by flg22 in Arabidopsis guard cells, even in rbohD and rbohF mutants ( Arnaud et al., 2023b ). In the latter study, RBOHF-dependent ROS release in guard cells was detected using the fluorescent dye H 2 DCFDA. This may suggest that ROS detected by H 2 DCFDA in undefined subcellular locations, but not by the cytosolic H 2 O 2 biosensor, may contribute to flg22-dependent cytosolic ROS signalling ( Arnaud et al., 2023a ; Arnaud et al., 2023b ). Similarly, PRX, rather than RBOHD or RBOHF, have been implicated in stomatal closure induced by cytokinin, and certain PRXs are strongly expressed in guard cells after flg22 treatment ( Arnaud et al., 2017 ). Furthermore, PAMP-INDUCED PEPTIDE 1 (PIP1)-mediated stomatal closure is PRX-dependent ( Hou et al., 2019 ). Weather PRXs, RBOHs, or other enzymes located in distinct subcellular compartments are involved in H 2 S signalling, and how their activity relates to stomatal immunity, remains to be elucidated. However, when rbohD plants are treated with the mitochondria-tagged-H 2 S donor AP39, a lower amount of cytosolic H 2 O 2 is observed, suggesting that RBOHD is required to induce cytosolic H 2 O 2 levels in response to mitochondrial H 2 S ( Pantaleno et al., 2024b ). The observed differences in H 2 O 2 dynamics to a general or mitochondrial donor, may be attributed to the fact that H 2 S is a very reactive molecule and therefore, it is particularly likely to act close to its site of release ( Filipovic et al., 2018 ; Benchoam et al., 2019 ). Furthermore, our findings suggest that different endogenous sources of H 2 S may operate at different levels to affect the stomatal response, highlighting the importance of the subcellular location in H 2 S-signalling. H 2 S participation in other PTI responses Ca 2+ is regarded as one of the few hubs in stomatal signaling network. Cytoplasmic Ca 2+ increase is regulated by Ca 2+ permeable channels, among them, CYCLIC NUCLEOTIDE GATED CHANNEL (CNGC) 2 and CNGC4, which assemble a channel that is phosphorylated upon flg22 perception, and the REDUCED HYPEROSMOLALITY INDUCED [Ca 2+ ] i INCREASE (OSCA) 1.3 which is also phosphorylated and required for pathogen-induced stomatal closure ( Tian et al., 2019 ; Thor et al., 2020 ). The regulation of [Ca 2+ ] cyt modulates the activity of downstream targets containing Ca 2+ -binding domains, including calcium dependent protein kinases (CDPKs), NADPH oxidases and PHOSPHOLIPASES D (PLD). Moreover, once activated, CDPKs phosphorylate key targets as RBOHD and slow-type anion channel SLAC1 to induce stomatal closure ( Qin and Wang, 2002 ; Ogasawara et al., 2008 ; Boudsocq et al., 2010 ; Geiger et al., 2010 ; Dubiella et al., 2013 ; Guzel Deger et al., 2015 ). It is known that the second messengers H 2 O 2 and Ca 2+ have a complex interplay involving crossed regulation, where RBOHD-mediated apoplastic H 2 O 2 production depends on flg22-triggered [Ca 2+ ] cyt increase, while Ca 2+ entrance depends on apoplastic ROS burst ( Marcec et al., 2019 ). Here we show that H 2 S is not able to close stomata when epidermal peels are pretreated with Ca 2+ chelating agents ( Figure 7 ) indicating that Ca 2+ is required for H 2 S-induced stomatal closure. In ABA-dependent signalling it has been demonstrated that H 2 S activates S-type anion currents in a [Ca 2+ ] cyt and OST1-dependent manner, while OST1 persulfidation is necessary for its activation and for the Ca 2+ influx from apoplast to the cytosol ( Wang et al., 2016 ; Chen et al., 2020 ; Chen et al., 2021 ). OST1, together with anion channels SLAC1 and SLAC1 homolog 3 (SLAH3), are also needed to induce flg22-induced stomatal closure ( Guzel Deger et al., 2015 ). Moreover, H 2 S inhibits inward-rectifying K + channels in a Ca 2+ -independent way in tobacco guard cells ( Papanatsiou et al., 2015 ). Interestingly, we show that DES1/H 2 S participate in these two processes that are core components of stomatal immunity response. Further studies will be needed to understand the mechanism by which H 2 S is acting upstream of Ca 2+ . Another well characterized process acting downstream of flg22 signalling is the activation of MAPK and transcriptional reprogramming as a part of the PTI response ( He et al., 2006 ; Boudsocq et al., 2010 ; Macho et al., 2012 ). Through guard cell specific-gene expression analysis and MAPK activity assay in seedlings, we show that DES1 is not involved in this pathway (Figure S3). Through this work, we incorporate H 2 S into the mechanistic framework underlying plant defense response. We provide evidence for the involvement of DES1/H 2 S in stomatal immunity triggered by Pst and flg22. Our findings further elucidate the pathways within the stomatal signalling network that may be modulated by H 2 S. In summary, we propose a model where the bacterial elicitor flg22 binds to its specific receptor FLS2, promoting the formation of FLS2-BAK1 complex, which triggers downstream signalling pathways, including the activation MAPK cascade. In parallel, DES1 is activated in the cytosol, catalyzing the production of ammonia, pyruvate, and, H 2 S from L-cysteine. DES1 is partially required to activate RBOHD via an unknown mechanism. RBOHD generates superoxide which is subsequently dismutated into H 2 O 2 in the apoplast and transported into the cytosol via aquaporins. Additionally, H 2 S contributes to cytosolic H 2 O 2 production through an undetermined source. Simultaneously, Ca 2+ channels in the plasma membrane are activated, enabling Ca 2+ influx into the cytosol to facilitate stomatal closure ( Figure 8 ). Download figure Open in new tab Figure 8: Working model of H 2 S and DES1-involvement in flg22-signalling in guard cells. Flagellin elicitor (flg22) from Pseudomonas syringae pv tomato DC3000 ( Pst DC3000) binds to the specific receptor FLAGELLIN SENSING 2 (FLS2) and induce the formation of complex FLS2 and co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) to triggers the signalling pathway which includes the activation of mitogen activated protein kinase (MAPK) cascade. On the other hand, upon flg22 perception L -CYSTEINE DESULFHIDRASE 1 (DES1) is activated generating ammonia, pyruvate and H 2 S from L-cysteine. DES1 is partially required to activate NADPH OXIDASE RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) via an unknown mechanism. RBOHD produces superoxide (O 2 - ) which is dismutated into H 2 O 2 in the apoplast and then entry into the cytosol through aquaporins (AQP). Furthermore, H 2 S induces cytosolic H 2 O 2 via an undetermined source. Ca 2+ channels in the plasma membrane allowing Ca 2+ to enter the cytosol to facilitate stomatal closure. As such this study opens up future research avenues to identify the specific molecular targets for H 2 S and how they are modulated to integrate the stomatal immunity response. MATERIALS AND METHODS Biologic Material and Chemicals Arabidopsis thaliana Columbia-0 (Col-0) wild type, des1-1 (SALK_103855), rbohD (D3) ( Torres et al., 2002 ) mutant and Col-0 expressing the roGFP2-Orp1 biosensor in the cytosol were available in our lab ( Scuffi et al., 2018 ). rbohD and des1 plants were crossed with the roGFP2-Orp1 biosensor lines. The F2 generation plants used was selected by fluorescence to ensure expression of the roGFP2-Orp1 sensor and by PCR-based genotyping to ensure homozygosity for the rbohD and des1 locus (Primers listed in Table S5). Seeds were germinated in soil (soil:vermiculite:perlite, 3:1:1) and kept at 4°C for 2 d. For stomatal aperture, apoplastic H 2 O 2 detection, Pst spray-inoculation and gene expression experiments, plants were grown at 22°C in an 8-h-light/16-h-dark photoperiod at 200 µmol photons m -2 s -1 (IIB-CONICET-UNMdP. Mar del Plata, Argentina). For biosensor analysis by epifluorescence microscopy, plants were grown at 22°C using an 8-h-light/16-h-dark photoperiod at 100 µmol photons m -2 s -1 (UNIMI, Milan, Italy). For biosensor analysis by confocal microscopy, plants were grown at 25°C using a 16-h-light/8-h-dark photoperiod at 100 µmol photons m -2 s -1 (IBBP, Münster, Germany). Pseudomonas syringae pv tomato DC3000 ( Pst DC3000) and Pseudomonas syringae pv tomato DC3000 hrcC - ( Pst DC3000 hrcC - ) were kindly provided by Dr. Georgina Fabbro from CIQUIBIC-CONICET-UNC, Argentina. Bacteria were grown in King’s B media (2% (w/v) proteose peptone, 1% glycerol, 0.15% (w/v) K 2 HPO4, pH 7.2) supplemented with 100 mg L -1 rifampicin (for Pst DC3000 hrcC - ) or 50 mg L -1 rifampicin and 50 mg L -1 kanamycin (for Pst DC3000) at 28°C. The elicitors peptides flg22 (QRLSTGSRINSAKDDAAGLQIA) and elf18 (SKEKFERTKPHVNVGTIG) were purchased from ProteoGenix (Schiltigheim, France). The H 2 S donor, GYY4137 (morpholin-4-ium4 methoxyphenyl(morpholino) phosphinodithioate), the H 2 S scavenger, hypotaurine (HT), the horseradish peroxidase type VI-A (HRP), 3-aminophthalhydrazide, 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol), MES buffer, EGTA and BAPTA-AM were purchased from Sigma-Aldrich. For gene expression analysis, primers were synthesized by Macrogen (Seoul, Republic of Korea) and the fast Universal SYBR Green Master mix used was from Roche (Merck, Darmstadt, Alemania). Stomatal aperture assay Stomatal aperture assays were performed according to Pantaleno et al. (2024a) . Briefly, epidermal peels from abaxial side of fully expanded leaves from 5 to 6-week-old Arabidopsis plants were excised with tweezers and immediately floating in opening buffer (5 mM MES pH 6.1, 50 mM KCl) for 3 h under light (200 µmol photons m -2 s -1 ) and subsequently maintained in the same buffer omitting the active component (‘mock’) or exposed to different treatments as indicated in the legends. Pst DC3000 and Pst DC3000 hrcC - strains were grown at saturation at 28°C for 24 h in King’s B-agar plate and resuspended in sterile 10 mM MgCl 2 until OD=0.1. Stomata were photographed using an AmScope MU1000 camera coupled to an Olympus CKX53 microscope with a 40x lens (LUCPlanFLN, 0.6 numerical aperture). The stomatal aperture width was quantified using the ImageJ analysis software (NIH, Bethesda, MD, USA) Bacterial Spray Inoculation Five to 6-week-old Col-0 and des1 plants were sprayed with a Pst DC3000 hrcC - suspension (OD = 0.2 [λ = 600 nm]) in 10 mM MgCl 2 , 0.02% (v/v) Silwet. Following spray inoculations, plants were kept covered with a transparent lid at 20°C under short-day conditions (8-h-light/16-h-dark photoperiod, 200 μmol photons m −2 s −1 ). Leaf discs samples (4 per plant) were taken at 24 and 72 hours (hpi) and the number of colony-forming units (CFU) was determined after serial dilution and plating as described (Johansson et al., 2014). Apoplastic H 2 O 2 detection Leaf discs from 4- to 5-week-old plants were cut and floated on deionized water overnight and then passed to 96-wells white plates. H 2 O 2 production was triggered with 100 nM flg22 applied together with 20 mM luminol and 0.02 mg/L -1 HRP. Luminescence was measured with a luminometer (Thermo Scientific Luminoskan Ascent Microplate). Each plate was measured over a period of 30 min with a 2 min interval. Epifluorescence Microscopy Biosensor analyses by epifluorescence microscopy was performed according to Pantaleno et al. 2024a . Briefly, epidermal peels from fully expanded leaf of Col-0 Arabidopsis 4- to 5-week-old plants expressing H 2 O 2 biosensor roGFP2-Orp1 in the cytosol were floated in opening buffer for at list 7 h under light for recovery as we previously described ( Scuffi et al., 2018 ). Peels were then treated with 1 µM flg22 preincubated or not for 10 min with 200 µM Hypotaurine (HT, H 2 S scavenger) for 1 h and to determine the dynamic range of the response of the roGFP2-based biosensors in situ , we used treatments with 10 mM H 2 O 2 and 20 mM DTT for 10 min to induce full sensor oxidation and reduction, respectively. The peels were analyzed in vivo using an inverted Nikon Ti-E fluorescence microscope coupled to a Hamamatsu ORCA-D2 Dual CCD camera. Excitation light was produced by a fluorescent lamp Prior Lumen 200 PRO (Prior Scientific) and samples were imaged using a 60x oil immersion objective (CFI Plan APO Lambda 60x 1.4 numerical aperture). roGFP2-Orp1 was excited sequentially with 470/40 nm and 405/40 nm and the emission was collected using a 505/530 nm bandpass filter (GFP-specific filter) for both excitation wavelengths with a 2 x 2-pixel binning. The ratio 405/470 nm was calculated for each guard cell using the Ratio Plus plugin ( https://imagej.net/ij/plugins/ratio-plus.html ) for ImageJ analysis software (NIH, Bethesda, MD, USA). The background was subtracted for each channel and then the ROI was delimited to guard cells. Fluorescence was measured as the mean pixel intensity. Confocal Laser Scanning Microscopy Biosensor analyses by CLSM was performed according to Pantaleno et al. 2024a . Briefly, epidermal peels from fully expanded leaf of Col-0, des1 or rbohD from 4- to 5-week-old Arabidopsis plants expressing H 2 O 2 biosensor roGFP2-Orp1 in the cytosol were floated in opening buffer for at least 7 h under light for recovery and then treated with 1 µM of flg22 or 100 µM of the H 2 S-donor GYY4137 according to the legend of the figure. To determine the dynamic range of the response of the roGFP2-based biosensors in situ , we used treatments with 10 mM H 2 O 2 and 20 mM DTT to induce full sensor oxidation and reduction, respectively. The epidermal peels were mounted under a LSM980 inverted microscope (Carl Zeiss Microscopy). Images were collected with a 40x water immersion (C-Apochromat, 1.2 numerical aperture) and the biosensors were excited sequentially at 405 and 488 nm (line-switching mode) and emission was detected at 508 to 526 nm. Ratiometric images were analyzed using the custom MatLab program package, Redox Ratio Analysis ( Fricker, 2016 ) (for rbohD ) or ImageJ (NIH, Bethesda, MD, USA) (for des1 ) and ROI was set to specifically cover the guard cell. Gene expression in guard cell-enriched samples (GC-e) Gene expression in GC-e was performed according to Pantaleno et al. 2024a . Briefly, Col-0 and des1 Arabidopsis epidermal peels (GC-e, guard cell enriched) were floated in opening buffer (5 mM MES pH 6.1, 50 mM KCl) for 3 h under light and then treated with 1 µM flg22 for 60 min in the same conditions as we previously described ( Scuffi et al., 2014 ). Total RNA was extracted using homemade reagent: Phenol saturated in buffer Tris pH 8 38 % v/v, Guanidine thiocyanate 11.8 % w/v, Ammonium thicyanate 7.91 % w/v, Sodium acetate 0.1 M pH 5, Glycerol 5% v/v. Subsequently, 1 µg of total RNA was used for the RT-qPCR reaction using an oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (M-MLV RT, SIGMA). For qPCR reaction, Fast Universal SYBR Green Master mix was employed, using a Step-One Real Time PCR machine from Applied Biosystems. The standard amplification program was used. The expression levels of the gene of interest were normalized to those of the constitutive ACT2 ( At3g18780 ) gene by subtracting the cycle threshold value of ACT2 from the cycle threshold value of the gene (ΔCT). The nucleotide sequences of the specific primers for qPCR analysis are listed in Supplemental Table S5. The annealing temperature for each primer was 60°C. LinRegPCR was the program employed for the analysis of RT-qPCR data ( Ruijter et al., 2009 ). MAPK Activation MAPK assay was performed on nine 2-week-old Arabidopsis Col-0 and des1 seedlings grown in liquid MS plus 1% (w/v) sucrose at 25°C using a 16-h-light/8-h-dark photoperiod at 100 µmol photons m -2 s -1 . Seedlings were treated with 1 µM flg22 for 0, 5, 15, or 30 min and flash-frozen in liquid nitrogen. MAPK activation was monitored by western blotting with antibodies that recognize the dual phosphorylation of the activation loop of MAPK (pTEpY). Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr-204) rabbit monoclonal antibodies (Cell Signalling) were used according to the manufacturer’s protocol (1:5,000). Blots were stained with Ponceau S to verify equal loading. Statistical analysis Data analyses were performed using RStudio (R Foundation for Statistical Computing, Vienna, Austria; URL: https://www.Rproject.org/ ). The statistically significant differences were analyzed using Student’s t-test, Generalized Linear Model or Generalized Linear Mix Model procedure, with the gls function from the nlme library. Multiple comparisons among individual means were performed by Tukey’s Method. The error distribution was Gaussian or Gamma and all effect were considered significative at p < 0.05 or p < 0.001 (Table S5). Author Contributions DS performed and designed most of the experiment, analyzed the data and wrote the article, RP performed the experiments from figure 3 and S2, PS performed the experiments from figure 3 and 7, J-ON performed the experiments from figure 5 , AC, MS and AL participate in the experimental design, discussion and writing and CGM conceived the project, analyzed data and wrote the article. All authors contribute to the writing of the article Acknowledgements We thank the Deutsche Forschungsgemeinschaft (DFG) for funding through the infrastructure grant INST 211/903-1 FUGG for the confocal microscope as operated by the Imaging Network of the University of Münster (RI_00497), the University of Mar del Plata (UNMdP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; PICT 2016 N° 2553, PICT 2017 N° 601, 2018 N° 1449, PICT 2019 N° 1040 and PICT 2021 N° 92), the DAAD for Research Stays for University Academics and Scientists, 2020 (57507437) fellowship to D.S. and Travelling Fellowship from The Company of Biologists to R.P. Part of the work was carried out with the support of the NOLIMITS Center of Excellence for Plant Biology and Other Life Sciences established by the University of Milan. 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( 2019 ) ABA-triggered guard cell L -cysteine desulfhydrase function and in situ H 2 S production contributes to heme oxygenase-modulated stomatal closure . Plant, Cell & Environment 1 – 13 View the discussion thread. Back to top Previous Next Posted February 19, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity Denise Scuffi , Rosario Pantaleno , Paula Schiel , Jan-Ole Niemeier , Alex Costa , Markus Schwarzländer , Ana M. Laxalt , Carlos García-Mata bioRxiv 2025.02.14.638267; doi: https://doi.org/10.1101/2025.02.14.638267 Share This Article: Copy Citation Tools Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity Denise Scuffi , Rosario Pantaleno , Paula Schiel , Jan-Ole Niemeier , Alex Costa , Markus Schwarzländer , Ana M. 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