Hydrogen Sulfide-induced Barley Resilience to Drought and Salinity through Protein Persulfidation

Plan Physiology and Biochemis y 221 (2025) 109644 A ailable online 11 Feb ua y 2025 0981-9428/© 2025 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Hyd ogen sul ide-induced ba ley esilience o d ough and salini y h ough p o ein pe sul ida ion Reyes Ca illo , Inmaculada Mo eno , Luis C. Rome o , Angeles A oca ** , Cecilia Go o * Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones Cien í icas and Uni e sidad de Se illa, Se illa, Spain ARTICLE INFO Keywo ds: Amino acids ABA Abio ic s ess Hyd ogen sul ide Na+/K+ a io Pe sul ida ion ROS ABSTRACT Ba ley (Ho deum ulga e) is a widely cul i a ed ce eal c ops, and i s p oduc ion is inc easingly h ea ened by en i onmen al s esses such as d ough and salini y. Hyd ogen sul ide is es ablished as a signaling molecule ha p omo es ole ance o plan s ess h ough pe sul ida ion, a pos - ansla ional modi ica ion o cys eine esidues in p o eins. The pu pose o his s udy is o explo e he impac o NaHS (sul ide dono ) p e ea men on ba ley plan s in enhancing ole ance o d ough and salini y s esses, and de e mine i pe sul ida ion is in ol ed. In p e ea ed-plan s, pheno ypical ai s and pigmen con en s showed an imp o emen in he su i al o he plan s unde s ess condi ions. Quan i ica ion o s ess-ma ke s such as an hocyanin, p oline, and eac i e oxygen species also showed signi ican dec eased con en s in p e ea ed compa ed o un ea ed plan s. In addi ion, he accumula ion o amino acids unde d ough s ess was signi ican ly educed when plan s we e p e ea ed wi h NaHS. Simila ly, he inc ease o ABA con en as a ypical d ough esponse was educed in he p e ea ed plan s. When plan s a e exposed o sal s ess, he Na + /K + a io was main ained low in NaHS-p e ea ed plan s, by inc easing K + le els. The sul ide amelio a i e e ec o sal was also obse ed du ing ge mina ion in p e iously NaHS-soaked seeds. Ou indings sugges ha sul ide p e ea men p epa es ba ely plan s o be e deal wi h d ough and salini y. Mo eo e , pe sul ida ion was analyzed unde all condi ions, exhibi ing enhanced le els unde s ess when plan s we e p e ea ed wi h NaHS. Ou indings indica e ha sul ide p e ea men induces a p e ious s a e in ba ley o espond mo e e icien ly o s ess and p opose pe sul ida ion is he unde lying mechanism. 1. In oduc ion D ough s ands as he mos des uc i e en i onmen al limi a ion, in lic ing signi ican ly highe yield losses han any o he abio ic s esso , co e ing mo e han 20% o ag icul u al a eas wo ldwide (Rojas, 2020). Mo eo e , p ojec ions indica e an escala ion in d ough equency and in ensi y wi hin cu en ly a id egions owing o clima ic change. Addi- ionally, soil salini y is ano he c i ical abio ic s ess ha has become a majo conce n, hinde ing ag icul u al p oduc ion and posing isks o en i onmen al heal h and economic s abili y (Hassani e al., 2021). Bo h abio ic s esses, salini y and d ough , subs an ially in luence he ood supply’s sa e y wo ldwide (O as e al., 2021). Ba ley (Ho deum ulga e L.), one o he mos widely cul i a ed ce eal c ops, plays a c ucial ole in global ood secu i y and ag icul u al economies. Howe e , ba ley p oduc ion is inc easingly h ea ened by en i onmen al s esses such as d ough and salini y (Binde ei e al., 2021). These abio ic s esses can lead o signi ican educ ions in c op yield and quali y, which pose a challenge o sus ainable ag icul u e. D ough and salini y s esses induce oxida i e s ess in plan s, cha ac e ized by he o e p oduc ion o eac i e oxygen species (ROS) ha can damage cellula s uc u es, p o eins, and nucleic acids (C uz de Ca alho, 2008). To su i e and h i e unde hese ad e se condi ions, ba ley plan s ha e e ol ed a a ie y o physiological and biochemical s a egies. These include he syn hesis o osmop o ec an s, he ac i a- ion o an ioxidan de ense sys ems, and he modula ion o s ess- esponsi e genes (Abdel ady e al., 2024). Despi e hese na u al de ense mechanisms, he se e i y o d ough and salini y s esses o en exceeds he plan ’s in insic ole ance le els, necessi a ing he Abb e ia ions: ABA, abscisic acid; ICP-OES, induc i ely coupled plasma op ical emission spec oscopy; PBS, phospha e-bu e ed saline; PEG, polye hylene glycol; PTM, pos - ansla ional modi ica ion; ROS, eac i e oxygen species; UPLC-MS/MS, ul a pe o mance liquid ch oma og aphy andem mass spec oscopy. * Co esponding au ho . ** Co esponding au ho . E-mail add esses: [email p o ec ed] (A. A oca), [email p o ec ed] (C. Go o ). Con en s lis s a ailable a ScienceDi ec Plan Physiology and Biochemis y jou nal homepage: www.else ie .com/loca e/plaphy h ps://doi.o g/10.1016/j.plaphy.2025.109644 Recei ed 21 Oc obe 2024; Recei ed in e ised o m 15 Janua y 2025; Accep ed 10 Feb ua y 2025 Plan Physiology and Biochemis y 221 (2025) 109644 2 de elopmen o addi ional p o ec i e s a egies. Recen s udies ha e iden i ied hyd ogen sul ide (H₂S) as a p omising molecule o enhancing plan s ess ole ance (A oca e al. 2018,2021). H 2 S has been demons a ed o play a c ucial ole in nume ous biological unc ions wi hin plan s, including di e se de elopmen al and physio- logical p ocesses, as well as s ess esponses aimed a managing chal- lenges such as hea y me al oxici y, salini y, and hea s ess, among o he s (Zhang e al. 2015,2021;Zhao e al., 2018). Despi e i s adi- ionally known oxici y a high concen a ions, H₂S has gained ecog- ni ion as an impo an signaling molecule in ol ed in a ious plan physiological p ocesses. Recen esea ch highligh s he po en ial o H₂S as a signaling molecule in plan s ha can mi iga e he ad e se e ec s o d ough and salini y. The key p o ec i e mechanism o H₂S is h ough pe sul ida ion (A oca e al., 2015), a pos - ansla ional modi ica ion (PTM) o cys eine esidues in p o eins. This PTM can enhance p o ein unc ion, s abili y, and in e ac ion, he eby imp o ing he plan ’s s ess esponse. Pe sul- ida ion helps o egula e he ac i i y o an ioxidan enzymes (A oca e al., 2015), ion channels, and ansc ip ion ac o s (A oca e al. 2017, 2021;Zhang e al., 2024), which a e c i ical o main aining cellula homeos asis unde s ess condi ions. By modula ing hese p o eins, H₂S can imp o e he plan ’s abili y o cope wi h d ough and salini y s esses, educing oxida i e damage and enhancing o e all s ess ole - ance. In ac , esea ch in di e en o ganism sys ems has p o ed ha a e sul ide ea men , pe sul ida ion le els expe ienced a no able in- c ease, consequen ly sa egua ding cys eines om o e oxida ion (Ga cia-Calde on e al., 2023;Ju ado-Flo es e al. 2023a,2023b;Zi a- no ic e al., 2019). The e o e, H₂S has been p oposed as an e ec i e cellula mechanism o p o ein p o ec ion agains i e e sible oxida ion imposed by abio ic s esses in plan s. Ne e heless, no p e ious s udies a e a ailable abou he in luence o H 2 S-induced pe sul ida ion and he ou come o H 2 S p e ea men in he compa ison o d ough and salini y s ess ole ance in ba ley. In his con ex , his s udy explo es he ole o hyd ogen sul ide in enhancing ba ley ole ance o d ough and salini y s esses, and he mechanism o pe sul ida ion in ol ed. By elucida ing he molecula and physiological pa hways in luenced by H₂S, we aim o p o ide insigh s in o how his gaseous molecule can be ha nessed o imp o e c op esilience and p oduc i i y unde challenging en i onmen al condi ions caused by clima e change. Unde s anding hese mechanisms no only con ibu es o he undamen al knowledge o plan s ess biology, bu also o e s p ac ical applica ions o ag icul u al managemen and c op imp o emen s a egies. 2. Ma e ial and me hods 2.1. Plan ma e ial, g ow h condi ions and ea men s Ba ley (Ho deum ulga e, Sca le cul i a , Spain) kindly p o ided by D . Gonz´ alez-And ´ es, Uni e si y o Le´ on, was used in his s udy. The seeds we e s e ilized and ge mina ed in dis illed wa e o 2–3 days a 25 ◦C, and we e sown in soil. Ba ley plan s g own in soil and i iga ed wi h wa e o 15 days we e di ided in o wo ba ches, one ba ch i i- ga ed wi h wa e and he o he wi h 50 μ M NaHS o 7 addi ional days. Each ba ch was hen di ided in o wo new ba ches, one was wa e i iga ed and he o he was subjec ed o 20% PEG 6000 (w/ ) o 6 days ( o he d ough s ess s udy), o al e na i ely one i iga ed wi h wa e and he o he i iga ed wi h 300 mM NaCl o 15 days ( o he salini y s ess s udy). Fo ge mina ion expe imen s, ba ley seeds we e s e ilized and di ided in o wo ba ches, one was kep in dis illed wa e and he o he in 50 μ M NaHS, o 18 h a 4 ◦C in he da k. Subsequen ly, each ba ch was di ided in o wo new ba ches and he seeds we e placed in Pe i dishes con aining wa e o 300 mM NaCl and kep o 7 days in he da k a 20–22 ◦C. 2.2. De e mina ion o chlo ophyll and ca o enoid con en s Chlo ophyll and ca o enoids we e de e mined spec opho ome i- cally (Jana B aniˇ sa e al., 2014). Plan ma e ial p e iously collec ed and ozen a −80 ◦C, was g ound in liquid ni ogen and 50 mg o powde was ans e ed o a 1.5 mL mic o ube. Subsequen ly, we added 1 mL o 100% ( / ) me hanol, and kep i o 24 h on a o a y shake , a 4 ◦C unde da k condi ions. Then, 1/10 dilu ions o he samples we e made and he abso bance was measu ed a 666, 653 and 470 nm. Concen a ion was calcula ed using he ollowing equa ions: Chlo ophyll a( μ g mg −1 FW) =[(15.65 ×A 666 )–(7.34 ×A 653 )] / mg FW. Chlo ophyll b( μ g mg −1 FW) =[(27.05 ×A 653 )–(11.21 ×A 666 )] / mg FW. Ca o enoids (uni s mg −1 FW) =[(1000 ×A 470 –2.86 ×chlo ophyll a– 129.2 ×chlo ophyll b) / 221)] / mg FW. 2.3. De e mina ion o an hocyanin and p oline con en s To al an hocyanin con en o lea es was ex ac ed using p opanol/ HCl/wa e (18/1/81, / / ), kep in a boiling-wa e ba h o 3 min, and o 24 h on a o a y shake a 4 ◦C unde da k condi ions. The mix u e was hen cen i uged a 10,000 pm o 10 min. The abso bance o he supe na an was measu ed a 535 nm and 650 nm. An hocyanin con en was de e mined using he ollowing o mula: A 535 =A 535 –2.2 ×A 650 and e e ed o he esh weigh , as desc ibed (Ju ado-Flo es e al., 2023a). To al p oline con en o lea es was de e mined ollowing p e ious me hod (Ca illo e al., 2011). The plan issue was ex ac ed using a cold mix u e o e hanol/wa e (40/60, / ) and kep i o 24 h on a o a y shake , a 4 ◦C unde da k condi ions. Then, he mix u e was cen i uged a 15,000 pm o 5 min and 50 μ L o supe na an s was mixed wi h 100 μ L o eac ion mix: [1% ninhyd in (w/ ) in 60% ace ic acid ( / ) and 20% e hanol ( / )]. The mix u e was kep in a boiling-wa e ba h o 20 min, p o ec ed om ligh . A e cooling a oom empe a u e, 100 μ L o he mix u e we e ans e ed o a mic opla e well. The abso bance was measu ed a 520 nm. To de e mine he p oline con en , a calib a ion line o p oline solu ion anging om 0.4 o 1 mM was used, and e e ed o esh weigh . 2.4. Amino acid and abscisic acid de e mina ion by UPLC-MS/MS Fo ee amino acid de e mina ion, app oxima ely 100 mg o ozen plan issue was homogenized in liquid ni ogen. The me aboli es we e ex ac ed om each aliquo in 400 μ L o 0.1 N HCL and 0.1% o mic acid ( / ) wi h shaking o 30 min a 4 ◦C. Samples we e cen i uged 15 min, 15,000 pm a 4 ◦C. Samples we e immedia ely subjec ed o UPLC‒MS/ MS as desc ibed p e iously (Ju ado-Flo es e al., 2023a). Fo abscisic acid de e mina ion, app oxima ely 50 mg o ozen plan issue was homogenized in 1.5-mL Eppendo ubes o 2 min a maximum speed wi h a Re sch ball mill 153 (MM400; Re sch). The me aboli es we e ex ac ed om each aliquo in 0.8 mL o sol en mix u e con aining me hyl- e -bu yl-e he /me hanol (MTBE/MeOH, 3/ 1, / ) cooled o −20 ◦C, wi h shaking o 30 min a 4 ◦C. The samples we e sonica ed in an ice-cooled ba h o 15 min. An aliquo o 0.4 mL o acidi ied wa e (0.1% HCl) was added and ho oughly o exed o 1 min. A e ha , he samples we e kep on an o bi al shake o an addi ional 30 min a 4 ◦C. The samples we e cen i uged a 15,000 pm o 10 min a 4 ◦C. A ixed olume o he uppe supe na an was ans- e ed o a esh ube and d ied down using a Speed-Vac concen a o a oom empe a u e. The d ied pelle s we e esuspended in 100 μ L o wa e /me hanol (50/50, / ) solu ion, and he esuspended samples R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 3 we e immedia ely subjec ed o UPLC‒MS/MS ho monal analysis as desc ibed (Zhang e al., 2024). Ex e nal abscisic acid s anda d solu ions we e used o quan i a ion. 2.5. De e mina ion o cys eine con en To al Cys con en was quan i ied by e e se-phase HPLC a e de i a iza ion wi h monob omobimane as desc ibed p e iously (Ju ado-Flo es e al., 2023a). App oxima ely 100 mg o ozen plan issue we e homogenized in liquid ni ogen and Cys ex ac ed in 400 μ L o 0.1 N HCl and 1 mM EDTA. Samples we e cen i uged 15 min, 15,000 pm a 4 ◦C and an aliquo o 100 μ L was mixed wi h 200 μ L 200 mM CHES (pH 9.3) and incuba ed 30 min a 4 ◦C. Then, he de i a iza ion eac ion was pe o med by adding 20 μ L o 15 mM monob omobimane, incuba ing o 60 min, and s opped by adding 5 μ L 20% o mic acid ( / ). The mix u e was cen i uged a 12,000 pm o 10 min and immedia ely subjec ed o e e se-phase HPLC. 2.6. De e mina ion o hyd ogen pe oxide con en Hyd ogen pe oxide (H 2 O 2 ) was quan i ied using he Amplex ed- based me hod (The mo Fishe Scien i ic), as desc ibed p e iously (Ga cia-Calde on e al., 2023). B ie ly, 50 mg o ba ley lea es was g ound o a ine powde in liquid ni ogen, and H 2 O 2 was ex ac ed wi h 50 mM sodium phospha e (pH 7.4), cen i uged and he supe na an used o H 2 O 2 de ec ion. A 100 μ L o eac ion bu e con aining 25 μ M Amplex Red, 10 U mL −1 ho se adish pe oxidase (HRP) and 5 μ L o su- pe na an in 50 mM PBS (pH 7.4) was incuba ed o 30 min and changes in luo escence caused by he o ma ion o oxidized Amplex- ed (Ex/Em: 560 nm/590 nm) we e eco ded. S anda d calib a ion cu es we e ob- ained wi h known H 2 O 2 concen a ions. 2.7. De e mina ion o Na + and K + by ICP-OES The de e mina ion o hese elemen s was pe o med as p e iously desc ibed (Mos o a e al., 2015). Be o e analyzing he plan samples, he calib a ion cu e was ep esen ed using di e en concen a ions ( om 1 ppm o 100 ppm) o each elemen . App oxima ely, 1g o esh plan was d ied o 6 d a 65 ◦C. Subsequen ly, 15 mL o a solu ion o 1 M HNO 3 and 10 mM MgCl 2 was added, and kep o 7 d a oom empe - a u e o diges ion. The diges ed samples we e hen measu ed in ipli- ca e by induc i ely coupled plasma-op ical emission spec ome y (ICP-OES) using an iCAP 7200 ICP-OES Duo (The moFishe Scien i ic) a he A omic Emission Spec ome y Se ice o he ICMS (Ins i u e o Ma e ials Science o Se illa). 2.8. In-gel pe sul ida ion de ec ion P o ein pe sul ida ion pa e ns in plan issue we e analyzed wi h he dimedone swi ch me hod (A oca e al., 2022). In de ail, 150 mg o plan lea ma e ial was g ound in liquid ni ogen wi h 200 μ L o cold PBS lysis bu e [1 ×PBS pH 7.4, 1 mM EDTA, 2% SDS (w/ )] supplemen ed wi h 1×p o ease inhibi o (Pie ce™, The mo Scien i ic). Then, samples we e incuba ed wi h 5 mM 4-chlo o-7-ni obenzo u azan (Cl-NBF) a 37 ◦C o 30 min, p o ec ed om ligh . Me hanol/chlo o o m p ecipi a ion was pe o med o elimina e excess Cl-NBF, and he p o ein pelle s ob- ained we e washed wi h cold me hanol, d ied, and edissol ed in 1 × PBS wi h 2% (w/ ) SDS supplemen ed wi h 1 ×p o ease inhibi o . P o eins we e incuba ed wi h 25 μ M DAz-2/Cy-5 p eclick mix a 37 ◦C o 30 min. Following incuba ion, me hanol/chlo o o m p ecipi a ion was pe o med, and he pelle s we e washed wi h me hanol as desc ibed abo e. P o ein labeling was analyzed using SDS-PAGE. A e SDS PAGE, gels we e ixed o 30 min in 12.5% me hanol ( / ) and 4% ace ic acid ( / ) p o ec ed om ligh . The gel was imaged a 640 nm o he Cy5 signal and 488 nm o he NBF-Cl signal. The pe sul ida ion le els we e quan i ied by measu ing he Cy5/Cl-NBF luo escence signal a io. 2.9. S a is ical analysis All da a a e he means o a leas h ee independen expe imen s. The da a we e subjec ed o one-way analysis o a iance (ANOVA), and di e en le e s indica e signi ican di e ences be ween ea men s a p >0.05, acco ding o Dunne ’s mul iple compa isons es using he so wa e package o s a is ical analysis G aphPad P ism 9.5.0. 3. Resul s 3.1. Impac o hyd ogen sul ide p e ea men on ba ley plan s subjec ed o di e en s ess condi ions Signi ican e idence o he p o ec i e ole o H 2 S in plan s has al eady been gene a ed unde s ess condi ions (A oca e al., 2021; Go o e al., 2019), bu a compa a i e s udy in he same plan s subjec ed o di e en s ess condi ions is s ill lacking. To in es iga e his, ba ley was selec ed as a c op species and he pe o mance o NaHS-p e ea ed plan s was de e mined when hey we e exposed o d ough by PEG ea men o al e na i ely o salini y s ess by NaCl ea men . Ou s ablished expe imen al sys em consis ed o 15-day-old ba ley plan s g own in soil ha we e di ided in o wo ba ches, one was wa e ed, and he o he was ea ed wi h 50 μ M NaHS o 7 addi ional days. Then, each ba ch was di ided in o wo new ba ches, one subjec ed o wa e i i- ga ion, and he second one o s ess condi ions, which we e d ough by i iga ing wi h 20% PEG (w/ ) o 6 days, o salini y by i iga ing wi h 300 mM NaCl o 15 days. The e o e, a he end, we had wa e -i iga ed plan s (con ol), NaHS- ea ed plan s (NaHS), non ea ed-plan s i i- ga ed wi h PEG (d ough ), non ea ed plan s unde salini y (NaCl), NaHS- ea ed plan s subjec ed o PEG ea men (NaHS +d ough ) and NaHS- ea ed plan s subjec ed o sal (NaHS +NaCl). The ypical wil ing and b owning pheno ypes o plan s unde d ough we e obse ed ha ma ched a signi ican dec ease in esh weigh and a lowe educ ion in shoo leng h compa ed o con ol plan s. When plan s we e p e ea ed wi h NaHS, hey pa ially eco e ed om bo h pheno ypic pa ame e s unde d ough (Fig. 1A). When plan s we e subjec ed o NaCl s ess, a dec ease in esh weigh was also obse ed, al hough i was lowe han he e ec caused by d ough . Howe e , p e ea men wi h NaHS also had a signi ican posi i e e ec eco e ing pa ially he non-s essed pheno ype (Fig. 1B). 3.2. Biochemical cha ac e iza ion o he e ec o NaHS p e ea men on ba ley plan s unde s ess Fu he analysis o he impac o NaHS p e ea men was pe o med by quan i ying di e en biochemical pa ame e s in plan samples. We obse ed a s ong educ ion in bo h chlo ophylls (a and b) oge he wi h ca o enoids con en in plan s a e d ough compa ed o con ol condi- ions, which was imp o ed in NaHS-p e ea ed plan s unde d ough , wi hou eaching he alues obse ed unde con ol and NaHS condi- ions. Simila esul s we e ob ained when sal s ess condi ions we e s udied (Fig. 2). In addi ion, di e en biochemical pa ame e s ha a e well-known ma ke s in plan s ha med by s ess condi ions, such as an hocyanin and p oline, we e quan i ied. Unde d ough , he con en o bo h compounds was highe han in he con ol plan s, showing a e y s ong inc ease in p oline con en , which was indica i e o he plan s being a ec ed by s ess. Whe eas he le el o bo h ma ke s signi ican ly dec eased in NaHS-p e ea ed plan s unde d ough , eaching he same le el as plan s g own unde con ol condi ions. When sal s ess was examined, we also obse ed an inc ease in bo h s ess ma ke s, al hough in he case o p oline, he inc ease was signi ican ly lowe han ha obse ed unde d ough condi ions. Simila ly, NaHS-p e ea ed plan s showed a educ ion in p oline con en when we e subjec ed o sal s ess compa ed o he un ea ed plan s, howe e , no change was obse ed in he an hocyanin con en (Fig. 3). These indings demons a e ha p e- ea men wi h sul ide causes an imp o emen in he su i al o ba ley R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 4 Fig. 1. E ec o NaHS p e ea men on he pheno ype o ba ley plan s unde s ess condi ions. Pheno ypes and quan i ica ion o esh weigh and shoo heigh o ba ley plan s ea ed as indica ed. A) D ough and B) saline s ess. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e ences. P <0.05. R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 5 plan s unde s ess condi ions, being mo e e icien unde d ough . The high con en o p oline ound unde d ough migh no be due exclusi ely o he accumula ion o his amino acid, because he me hod used o p oline quan i ica ion was a non-speci ic chemical me hod, ho oughly used in se e al publica ions (Ca illo e al., 2011). In o de o de e mine he s eady-s a e le el o indi idual amino acids, we quan i ied hei accumula ion by UPLC-MS/MS in plan ex ac s unde ou expe - imen al condi ions. Cys eine con en was also de e mined by e e se-phase HPLC a e de i a iza ion wi h monob omobimane. We ound indeed a s ong accumula ion o p oline unde d ough s ess, ha was signi ican ly educed when plan s we e NaHS-p e ea ed, bu in addi ion, we clea ly obse ed he accumula ion o o he amino acids. The mos signi ican accumula ion unde d ough was obse ed o yp ophan, which was subs an ially educed when NaHS p e ea men was imposed (Fig. 4). Howe e , o he amino acids, such as glu amine, me hionine, cys eine, h eonine, phenylalanine, aline, isoleucine, leucine, and y osine, also accumula ed unde d ough s ess and dec eased in NaHS-p e ea ed plan s. These esul s indica e ha he accumula ion o p oline as a majo compa ible osmoly e unde d ough condi ions go oge he wi h he eadjus men o amino acid me abolism, as well as, ha NaHS p e ea men egula es hese al e a ions. Bo h wa e de ici and salini y a e s ess condi ions ha al e cell homeos asis, p oducing eac i e oxygen species (ROS) accumula ion and subsequen oxida i e damage (Mille e al., 2010). In acco dance, Fig. 2. E ec o NaHS p e ea men s on pigmen con en s unde di e en s esses. Chlo ophyll aand b, and ca o enoids con en o ba ley plan s ea ed as indica ed. A) D ough and B) saline s ess. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e ences. P <0.05. R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 6 H 2 O 2 le els we e quan i ied in ba ley plan s unde he di e en ea - men s. A signi ican p oduc ion o H 2 O 2 was de e mined when plan s we e subjec ed o any o hese s ess condi ions, ha was educed when plan s we e ea ed wi h NaHS p io o he s ess (Fig. 5). Thus, he esul s a e indica i e o he ole o sul ide alle ia ing he oxida i e s ess o he plan s. 3.3. E ec o NaHS p e ea men on s ess-speci ic plan esponses Al hough plan s sha e common esponses o cope wi h di e en s ess condi ions, some a e mo e speci ic o one condi ion han o he o he . Va ious en i onmen al s esses, p ima ily dehyd a ion, igge he p oduc ion o he phy oho mone abscisic acid (ABA) o ac i a e he ABA-signaling pa hway and induce ole ance o s ess (Cu le e al., 2010). The in e ela ion be ween sul ide and ABA has been widely e idenced and in pa icula , he ac ion o sul ide in ABA-dependen s oma al closu e (A oca e al., 2021). The e o e, he le el o ABA was quan i ied in he plan samples unde d ough condi ions and a s ong induc ion o ABA was de e mined in plan s subjec ed o PEG ea men compa ed o plan s unde con ol o sul ide-i iga ed condi ions. Signi ican ly, when plan s we e p e ea ed wi h NaHS, a educ ion in he le el o ABA was obse ed unde d ough condi ions (Fig. 6). These Fig. 3. E ec o NaHS p e ea men on biochemical s ess ma ke con en s o ba ley plan s unde di e en s esses. An hocyanin and p oline con en s o ba ley plan s ea ed as indica ed. A) D ough and B) saline s ess. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e ences. P <0.05. R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 7 Fig. 4. E ec o NaHS p e ea men on con en o ee amino acids unde d ough s ess in ba ley plan s. Plan s we e ea ed as indica ed and he le els o indi idual amino acid we e de e mined by UPLC-MS/MS. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e ences. P <0.05. R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 8 indings u he sugges ha sul ide p e ea men p epa es plan s o be e deal wi h d ough . A key adap i e esponse o cope wi h sal s ess is he main enance o he balance o cellula ions, which is dis up ed by he accumula ion o oxic Na + ha displaces essen ial ions such as K + (Pa k e al., 2016). Acco dingly, a low Na + /K + a io is indica i e o imp o ed plan ole - ance o sal s ess. In ou expe imen al sys em, we measu ed he Na + and K + con en s and de e mined e y low le els o Na + and signi ican high le els o K + in plan s ha we e g own unde con ol condi ions o a e i iga ion wi h NaHS. Upon salini y, he le els o bo h Na + and K + ions we e equally in un ea ed plan s, howe e , when plan s we e NaHS-p e ea ed, he K + con en signi ican ly inc eased o a le el simila o ha o wa e -i iga ed plan s, while Na + con en emained simila o ha o he un ea ed plan s. Consequen ly, he Na + /K + a io was educed by NaHS p e ea men (Fig. 7), sugges ing an imp o emen o plan ole ance o salini y by sul ide ac ion. 3.4. E ec o NaHS p e ea men on he pe sul ida ion le el The bes mechanism s udied by which sul ide plays i s ole as a signaling molecule in plan s is he pos ansla ional modi ica ion o p o ein cys eines named pe sul ida ion, which consis s o he o ma ion o cys eine pe sul ides (A oca e al., 2021;Go o e al., 2019). To analyze whe he pe sul ida ion is esponsible o he be e plan pe o mance obse ed in NaHS-p e ea ed plan s unde s ess, in gel-pe sul ida ion de ec ion was pe o med. We de e mined a sligh inc ease in he pe - sul ida ion le el when plan s we e i iga ed wi h NaHS compa ed o he plan s wa e ed. I hese NaHS-i iga ed plan s we e subjec ed o d ough s ess, he le el o pe sul ida ion inc eased signi ican ly (Fig. 8A). Likewise, we obse ed a signi ican induced le el o pe sul ida ion in plan s unde sal s ess when hey we e p e iously ea ed wi h NaHS (Fig. 8B). This enhanced pe sul ida ion le el was accompanied by an imp o ed ole ance o d ough and salini y as p e iously obse ed, sugges ing ha p o ec i e ole o sul ide p e ea men unde s ess could be ela ed o an induced pe sul ida ion le el. 3.5. NaHS p e ea men imp o es ba ley seed ge mina ion unde sal s ess Di e en s udies ha e shown a p o ec i e ole o sul ide in seed ge mina ion o di e en plan species unde ce ain abio ic s esses, mainly he ay me al s ess (Zhang e al. 2008,2010). To ex end he s udy o o he abio ic condi ion, we in es iga ed he e ec o NaHS p e- ea emen on he abili y o ba ley seeds o ge mina e unde sal s ess condi ions. Ba ley seed ge mina ion was s ongly a ec ed by sal s ess, as was obse ed a i s sigh ; howe e , seeds p e ea ed wi h NaHS showed a clea imp o emen in ge mina ion a e (Fig. 9A). To quan i y he e ec o sal s ess and he ole o sul ide in ge mina ion, di e en a iables we e s udied, such as he pe cen age o ge mina ed seeds, he leng h o sp ou s and oo s and seed igou . Seeds ge mina ed unde sal s ess showed a s ong educ ion in all o hese a iables, demons a ing he nega i e e ec o sal on ge mina ion. Howe e , he e ec o NaHS in p e ea ed seeds unde salini y was amelio a ed, mainly obse ed in he pe cen age o ge mina ion and seed igou (Fig. 9B). Fig. 5. E ec o NaHS p e ea men on hyd ogen pe oxide con en o ba ley plan s unde di e en s esses. H₂O₂, con en o ba ley plan s ea ed as indi- ca ed. A) D ough and B) saline s ess. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e ences. P <0.05. Fig. 6. E ec o NaHS p e ea men on ABA con en o ba ley plan s unde d ough s ess. Plan s we e ea ed as indica ed and ABA con en was measu ed. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e - ences. P <0.05. R. Ca illo e al. Plan Physiology and Biochemis y 221 (2025) 109644 9 4. Discussion Subs an ial s udies ha e al eady demons a ed he ole o hyd ogen sul ide as induce o plan esis ance, mainly by sul ide exogenous applica ion combined o he s ess condi ion (A oca e al., 2021;Co pas, 2019;Li e al., 2016;Zhang e al., 2021). P o ein pe sul ida ion has been demons a ed as he molecula mechanism in ol ed in sul ide signaling (Go o e al., 2019;Huang and Xie, 2023), and se e al p o eomic ana- lyses ha e shown an in en o y o pe sul ida ed p o eins. This lis has been mainly ob ained om A abidopsis ei he unde non-s ess o s ess condi ions in he absence o sul ide ea men (A oca e al. 2015,2017; Ga cia-Calde on e al., 2023;Ju ado-Flo es e al. 2021,2023a,2023b; Lau eano-Ma in e al., 2020). As well, a ca alog o pe sul ida ed p o eins has been desc ibed in some c ops such as ice (Zhang e al., 2024), bean (Ma amo os e al., 2024) and peppe (Mu˜ noz-Va gas e al., 2024), also in he absence o sul ide ea men . The e o e, he endogenous sul ide is esponsible o he pe sul ida ion le el in hese condi ions and plan s, howe e , he e ec o he exogenous sul ide p e ea men on he pe - sul ida ion le el ei he in he absence o s ess o be o e imposing he s ess is unknown. This s udy explo es he e ec o p e ea men wi h NaHS as a sul ide dono on he plan pe o mance o a c op species as ba ley unde physiological g ow h condi ions, as well as on plan ole ance o d ough -associa ed osmo ic s ess and salini y. In gene al, no signi ican Fig. 7. E ec o NaHS p e ea men on he a io Na + /K + o ba ley plan s unde sal s ess. Plan s we e ea ed as indica ed, and A) Na + and K + con en s we e de e mined by ICP and B) Ra io Na + /K + was calcula ed. Da a a e means ±SD. Same le e s indica e no s a is ically signi ican di e ences. P <0.05. R. Ca illo e al.