Accepted Article

article as doi: 10. differencesbetween version lead to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not insunflower stress seedlings. salinity under activities enzyme various regulating thereby in NOsignaling and denitrosylation nitrosylation exhibit enhanced enzymeunder activity salinity stre stress leadstoa reversible inhibitionbothof theseenzymes cotyledons. in However,seedling roots showed that salt reductase and monodehydro-ascorbate dehydrogenase of glyceraldehyde-3-phosphate systems, including pectinesterase, phospholipase D 40 arecommon to both. Eighteen S-nitrosylated prot observed beto regulated by S-nitrosylation, 17 areunique to cotyledons, 4 areunique to roots whereas categoryof carbohydratemetabolism followedby other metabolic proteins. Ofthetotal 61 proteins denitrosylationof proteins.Highest numberof proteins havingundergone S-nitrosylation belonged to the androots. cotyledons revealed analysis LC-MS/MS assay. switch biotin using cotyledons and in roots analyzed was proteins ofcytosolic S-nitrosylation mediated stress Salt m 120 of conditions under inhibited is reversibly enzyme the that shows (DTT) dithioerythritol Germany Germany D-80939 München, Germany D-80939 München, Germany reductase (GSNOR) activity is eviden activity (GSNOR) reductase to salt stress sensitivity termsin of nitrite accumulation. Present aberrations. fi biochemical of avariety for responsible are conditions, stress under production enhanced their and conditions growth normal in cells in produced (RNS) species nitrogen reactive various and (NO) oxide Nitric * Correspondence c b Helmholtz Zentrum Muenchen, German Research Corresponding author, e- author, Corresponding S-nitrosylation/denitrosylation as mechan as a regulatory S-nitrosylation/denitrosylation a Research Unit Protein Science, Helmholtz Zentrum Muenchen, Heidemannstr. 1 1 Heidemannstr. Muenchen, Zentrum Helmholtz Science, Protein Unit Research Laboratory of Plant Physiology and Biochemistry Laboratory of PlantPhysiology Prachi Jain 1111/ppl.12641

Salt stress enhances S-nitrosylation of proteins in cotyledons whereas roots exhibit exhibit roots whereas incotyledons proteins of S-nitrosylation enhances stress Salt a , Christine von von , Christine Toerne mail [email protected] This articleisprotected bycopyright.Allrightsreserved. ndings demonstrate that sunflower seedling roots exhibit high t in response to salt stress. Rest inresponse saltstress. t to Delhi-110007, India Delhi-110007, b , Christian Lindermayr

and the Versionof Record. Please cite this alpha andcalmodulin. Furthe

opposite patterns of S-nitrosylation in seedling seedling in ofS-nitrosylation patterns opposite eins are beingreported fo ss. These observations implicatess. Theseobservations of role S- the Center Environmental for Health,Neuherberg, , Department of University Botany, of Delhi, ism ofsaltstress sensin

A significantA reductionin S-nitrosoglutathione c and CBhatla Satish oration of GSNOR activity oration ofwith GSNOR activity g in sunflower seedlings seedlings g in sunflower r the first time inplant r physiological analysis analysis physiological r a *

M NaCl. NaCl. This articleisprotected bycopyright.Allrightsreserved. Accepted Article N termed as reactive nitrogen species (RNS). These include peroxynitrite (ONOO peroxynitrite include These (RNS). species nitrogen as reactive termed mediated through signaling is Nitric oxide-induced a 2010) al. (Yadavet root formation adventitious 2017), Bhatla and (Jain tolerance stress biotic and abiotic 2005), al. et (Creus roots lateral of induction shown to play critical role inseed germination (Beligni and Lamattina 2000), flowering(He et al. 2004), been has NO 2013). et al. (Salgado tissues and organs plant various in levels nitrite of quantification ofnitric oxide. Nitrite is considered as a biomarkerof NOmetabolism, thereby necessitating the mechanisms(del Rioal. et 2004). Thus,nitrite content inplant tissues indirectly correlateswith the level enzymatic nitrite pathway, reduction by nitrate reductase or in the apoplast through non-enzymatic endogenously produced through established biochemical routes actionnamely, ofarginine- dependent (N plants processes in andphysiological biochemical various in involved molecule signaling ubiquitous cells. animal and plant in molecule signaling Nitric oxide is a bioactive, gaseous free radical which plays critical roles as a diffusible intracellular Introduction switch technique Biotin BST: oxide; Monodehydroascorbate Dithioerythritol;Nitric MDHAR, reductase; DTT, NO, GSNO, S-nitrosoglutathione;GAPDH, Glyceraldehyde-3-phosphate nitrosothiol; dehydrogenase; Abbreviations- enzyme known to control nitrosylation in both plant and animal systems (Benhar et al. 2009, Xu et al. Xuetal. etal.2009, systems (Benhar animal and plant in both nitrosylation control to known enzyme processes. Nitrosoglutath denitrosylation 2015).non-enzymatic(Benhar and Denitrosylation bothenzymatic can occur by and nitrosylation therateof ofby aproteinextent ofS-nitrosylation isgoverned SNOs, the of nature labile the of Because 2006). etal. (Wang oxide nitric of and transport in storage involved They are capable ofmediating S-nitrosylation (GSH). of specific target proteins and are glutathione like low-molecular weightthiols, by unstable diminished bequickly and canthus highly (SNOs) are nitrosothiols (SNOs) which further play roles in signaling, transport and storage of NO. S-nitrosothiols of S- production to the leading residues cysteine of groups NOto of thiol attachment covalent involves It plants. in signaling ofNO-mediated incontext PTM important and anestablished is S-nitrosylation which are caused by post-translational post-translational by which arecaused responsible is conditions understress production NOandvarious RNS produced cellsinandenhanced in damage/death. normalconditions their together with reactiveoxygen species (ROS), theiroverproduction may be involved in cell 2 O 3 (Corpas and Barroso 2013). All these RNS serve as nitrosative stress biomarkers in plants, and and plants, in biomarkers stress as nitrosative RNS serve Allthese Barroso2013). and (Corpas RNS, Reactive nitrogen species; GSNOR, S- RNS, Reactive ione reductase (GSNOR) is so far the the issofar (GSNOR) reductase ione modifications (PTMs) (PTMs) ofproteins. modifications nd various hormonal responses hormonal nd various for a variety most aberrations of for aofbiochemical variety generation of a variety of molecules collectively collectively ofmolecules a variety of generation eill et al. 2003, Mur et al. 2013). Nitric oxide oxide is al.2013).Nitric et et al. 2003, eill Mur nitrosoglutathione reductase; SNO, S- most worked out and an established most worked outandanestablished (Zhou et al. 2005). al. et (Zhou - ), NO ), 2 , GSNO , GSNO and This articleisprotected bycopyright.Allrightsreserved. Accepted Article by NaCl stress has been shown in Arabidopsis shown inArabidopsis NaCl stresshasbeen by A arelimited. NaCl of presence the in proteome of S-nitroso modulation species,on the reports plant various in proteome on total stressof salt impact the have reported studies several Although 2005). al. et (Lindermayr plantspecies various studied in under salineconditions. high light (Lin etal.2012). has Amongmost intensively examined been these, S-nitrosylation all and al. 2011) (Bai et dessication al. 2013), et Camejo 2009, et al. (Tanou saltstress 2009), Deswal and 2017).(Jain Bhatla of abioticstress conditionsbring A variety aboutno (Astierand stability et al.2012). by activity or regulates enzyme negatively positively et (Corpas temperatures high lowand darkness and has been activity etal. (Lamotte 2005). GSNOR resistance (Feechan 2005),et al. cell death (Chen et etal.2007). Plant responses (Rusterucci stress (Feechan etal.2005).Asignaling role for in GSNOR beenproposed plants has also and animals plant cells.NO andRS ofendogenous Homeostasis in Impaired2013). al. et (Kubienova activity of GSNOR le ofGSNO thereduction catalyzing by of GSNO levels intracellular the modulating by nitrosylation protein of extent the affects indirectly GSNOR 2013). Gupta and Bhatla 2005), phospholipase (Gupta and Bhatla 2007), lipoxygenase Bhatla (Yadav2007), and Bhatla and (Gupta phospholipase 2005), Gupta andBhatla 2003, al. (Gupta et detection oflipase biochemical and forlocalization methodologies andnovel tools developing 2003), 2002, and Bhatla (Sadegipour sunflower duringin seedling growth mechanisms signaling associated and its mobilization of oil body biochemistry and physiology understanding hasbeen engagedinextensiv laboratory Author’s S-nitrosylation. undergoing MS/MS analysis coupled with biotin allowsswitch method identification of target proteins LC- proteins. ofS-nitrosylated method identification for used widely most the is (2001), Snyder bioticstress. Biotin abioticand conditions of nitrosylation and denitrosylation t S- etal.1999). (Mannick activity enzyme shown toactivate been has denitrosylation However, leads generally to nitrosylation en of reduction pattern of S-nitrosylation (Fareset al. 2011, Came inthe exhibit changes been haveobservedto antioxidant enzymes andphotorespiration, respiration in nitrosylation response to sa sativum, Arabidopsis however, a detailed proteomic study of mitochondrial proteins revealed a reduction ofS- a reduction proteins revealed mitochondrial of study proteomic detailed a however, thereby indicating the roleof thisenzyme maintainingin the levelsof nitrosothiols in In vitro These include hypoxia (Perazzolli et (Perazzolli hypoxia These include S-nitrosylation of proteins using NO donors such as, GSNO, has been been has as, GSNO, of proteinssuch NOdonors S-nitrosylation using lt treatment (Camejo et al. 2013). Proteins of the pathways of of thepathways Proteins 2013). et al. (Camejo lt treatment ogether regulate the activities of various enzymes, especially under especially enzymes, various of activities regulate the ogether observed to be modulated by GSNOR include disease disease GSNORinclude by observedmodulated to be small proportion of S-nitrosylated proteomemodified proportionsmall ofS-nitrosylated switch technique (BST), developed by Jaffrey and by Jaffrey developed switch (BST), technique zyme activity activity in plants (Abatandzyme 2009). Deswal (Wawer et al. 2010, Fares et al. 2011). al. InFares et 2010, (Wawer etal. observed to be induced by mechanical wounding, wounding, mechanical by induced be to observed teworthy modulationteworthy ofS-nitrosylationproteins of NO appears to be strongly modulated by GSNOR GSNOR NO appears tobestrongly modulated by al. 2009) and protection against nitrosative stress nitrosative against andprotection 2009) al. e research over the past more than decadea in jo et al. 2013). Earlier reports suggest that S- suggest reports Earlier 2013). al. et jo affecting protein functi affecting protein toGSSGammonia and presence inthe of GSH al. 2008, Chaki et al. 2011). S-nitrosylation al. et Chaki 2008, al. ads to increased levels ofnitrosylated proteins al. 2004), (Abatand lowtemperature al. on, cellular localization on, cellularlocalization Pisum This articleisprotected bycopyright.Allrightsreserved. Accepted Article findings, 120 m m (20-180 ofNaCl concentrations varying at seedlings sunflower by tolerance supplemented in Hoagland medium is based on our earl m 120 with seedlings of sunflower A treatment solution. nutrient Hoagland half-strength soaked germination paper in plastic in distilledand sterilized thenimbibed seeds were work is focused on the analysis of roots and cotyledons of 2-day old seedling only so as tomonitor under running tap water for 1 hr. Seed sterilization sterilization hr. under running Seed waterfor1 tap ( seeds Sunflower Seed germination and treatments and methods Materials of saltstress to theimpact differential termsofThese patternsin S- expression m metabolic exhibited significant proteins and other chaperones transport, proteins,defense-related, regulatory proteins, metabolism, cytoskeletal primary proteins of many of S-nitrosylation, extent ofthe Interms undertaken. seedlings was sunflower ofthe analysis comparative signaling, a abiotic stress role ofnitricthe in tounderstand In in order seedling androots oxide was compared cotyledons. m a roots seedling in wasalsoanalyzed correlation to known role Inviewofthecrucial level. proteome scavenging enzymes,current our objective istound fromour previous publi available strong evidences sunflower seedlings tolerance by (Si heme oxygenase and (Kaur and Bhatla 2016) reported crucial dismutase roleofthe superoxide cotyle theseedling by salt stress distance sensingof mechanisms both tolerance sunflower seedlings, in decadehave furtherover thepast we crop, oilseed important inthis germination seed with associated mechanisms signaling of the understanding deeper 2011) and andprotease (Sadegipour Bhatla 2003, Va pattern (hypocotyl lengthand root growth) -80 were at harvested and stored according to David et al. (2010). Cotyledon and root tissues from seedlings showing uniform growth 120 m with by Hoagland solution supplementing wasprovided treatment Salt provided. stress leads to seedling death. So we are working at a concentration at which the seedling is able to tolerate salt survives though itshows significant reduction ingrowth. Any further increase NaClin concentration M ) stress-induced modulation of) stress-induced nitrosothiol cont M has been adoptedbeen for allhas further experiment Helianthus annuus and itslong-distance sensing. in coordination with endogenous NO. Thus, on the basis of these on thebasis NO.Thus, withendogenous in coordination trays. Seedlings were grown in dark at 25°C and were irrigated with with wereirrigated and dark at25°C in grown were Seedlings trays. , var. KBSH 54) andkept detergent aliquid were washedwith nd cotyledons in response to salt stress. NaCl (120 NaCl(120 tosalt stress. response nd cotyledons in cations on NO crosstalk with the above stated ROS ROS stated above with the crosstalk NO on cations was done using 0.005% mercuric chloride (HgCl mercuric chloride 0.005% using was done be played by nitrite by accumulation inthe beplayed its tissue, ngh and Bhatla 2016) in modulating2016) ngh andBhatla in saltstress water forhthereafterwhich 2 were sown on pre- erstandmolecular further these interactionsat the ent andS-nitrosoglutath undertaken critical analysis of various salt stress stress salt various of analysis critical undertaken (Arora and Bhatla 2015), glutathione reductase reductase Bhatla glutathione 2015), (Arora and nitrosylation have discussedbeen reference with statuscontroland saltstress ofS-nitrosylation in as a rapid response to salt and also in long in long also salt and to response asarapid odulation in the extent of theextent their S-nitrosylation. in odulation ndana Bhatla andIn our2006). continuation of dons. In this context, we have very recently very have we context, In this dons. ier investigationson theextent of salt stress s since at this concentrationthesince seedling atthis s o C until furtherC use. Present ione reductase (GSNOR) (GSNOR) reductase ione M ). Based on these earlier Basedonthese ). M M NaCl NaCl 2 ) This articleisprotected bycopyright.Allrightsreserved. Accepted Article membrane at a current of 400 mA for 1 hat4°Ctoac for mA a currentof 400 at membrane subjec was gel The min. 15 4°C for atleast at methanol) The homogenate was centrifuged at 10000 was centrifuged homogenate The out of the cassette andwash cassette out ofthe was taken the gel electrophoresis, hrs.After for2 and150V 0.5 h 100V for on h, 0.5 75V for performed at Tetr Miniprotean using of proteins forseparation gel (reducing agent) and 1 m Acetone precipitation of proteins was undertaken to eliminate residual MMTS. 1 m (methylmethanethiosulphonate),2.5% SDSand incubated at 50ºC for 20min with frequentvortexing. m 25 with treated were samples protein All controls. as negative served with 1 ml extraction buffer (50 m (50 buffer ml extraction 1 with Munich,Germany). 500 mg rootcotyledon and tissueswere powdered in liquidmixedandnitrogen determin was nitrosothiol and nitrite of amount The contents and of nitrite nitrosothiol Determination cotyledons). to roots from (transcellular, response signaling distance solution in ofthemolecules NaCl vicinity in the both application) ofsalt hrs 48 (within response signaling as rapid expected changes biochemical various 1 Protein was quantified according to Bradford (1976) and homogenizedin grindingmedium m (50 Lindermayr etal. (2005).Cotyledonandroot tissues ground were tofinepowder with liquid nitrogen 1 in (prepared 5% sulfanilamide with pre-treated was homogenate The acid. andacetic iodide potassium iodine, containing solution Trijodide with filled andnitrosothiol measurements, content respectively, wereinjected into thereactionvessel which was containing 0.06 0.06 containing EDTA, 0.1m The acetone precipitated biotin-labelled protei proteins S-nitrosylated biotin-labelled of detection for analysis blot Western Western blot analysis orwere purified byaffinity chromatography. ascorbate. excess biotinand remove to again done was of proteins precipitation Acetone for hr. 1 37ºC at and incubated thoroughly mixed centrifugation twice at 10,000 at10,000 twice centrifugation S-nitrosylated proteins isol were S-nitrosylated Analysis of S-nitrosylated proteinsby Biotin Switch Technique (BST) ofconcentrations sodium nitrite. known using wasgenerated curve Standard of curve nitrite. standard using and areas peak integrating by contents SNO and nitrite quantify to used was Software(v3.2) NO Analysis Sievers content. . Samples were then treated with 250 µ M neocuproine and 1% SDS, pH- 7.7). Protei pH-7.7). SDS, 1% and neocuproine M Tris, pH 6.8, 10% (v/v) glycerol, 0.008% Tris, pH 10% glycerol, 6.8, (v/v) M ed in transfer buffer (0.025 buffer (0.025 ed in transfer biotin-HPDP (to biotinylate the proteins) were then added and samples were samples and added then were proteins) the biotinylate (to biotin-HPDP g 20for mineach at 4 ated and purified usin M Tris-HCl pH 7.5, 10 m 10 7.5, pH Tris-HCl The biotin-labelled S-nitrosylated proteins were then subjected to to subjected then were proteins S-nitrosylated biotin-labelled The M GSNO for GSNO min 30dark. Samples atRTintreated with H ns were resuspendedHENS bufferm in (25 g M for 20 min for20 at4°Cobtainto total solubleprotein (TSP). Tris-HCl, 1 m M ed using Sievers’ Nitric OxideAnalyzer (NOA 280i, (i.e., and roots) also the effect ofover long NaCl 0 a Cell(Biorad,Hercules, CA).Electrophoresiswas and protein and concentration mg.mlwas adjusted to1 HCl)to remove nitrite for measurement of SNO C. 100 µl and 200 µl of the homogenate for nitrite nitrite for homogenate of the µl 200 and µl 100 C. M hieve complete transfer of proteins (as judged by by proteins (asjudged transferof hieve complete g BiotinSwitchTechni Tris, 0.192 Tris, ted to Western onto blot NC of transfer proteins n was mixed with non-reducing Laemmli buffer buffer Laemmli with non-reducing mixed n was (w/v) bromophenol (w/v) bromophenol on blue 10% and loaded M M NEM, m 2.5 NEM, EDTA, 0.1m M glycine, 0.1% (w/v) SDS and 20% (w/v)SDSand20% 0.1% glycine, M EDTA) and followed by by followed and EDTA) M que according (BST), to

Neocuproine pH-7.8). M sodium ascorbate M HEPES,m 1 M MMTS MMTS 2 M O -

This articleisprotected bycopyright.Allrightsreserved. Accepted Article beads attached to biotinylated proteins were washed twice with 20 volumes of washing buffer (25 m 100 at centrifuged was sample protein The shaking. 1–2 at for RT incubated mixture thehwith protein gentle to added was then protein slurry/mg agarose HEPES pH 7.7, 0.5% Triton-X-100, 1 m proteins resuspended in HENS bufferwere mixed with 2 volumes of neutralization buffer (25 m HEPES pH 7.7, 1 pH 7.7, HEPES m The biotinylatedproteins werepurified affinityby proteins of biotinylated forpurification chromatography Affinity water. MilliQ 60% ACN. The washed samples were air dr m with50 washedthree were min times andsamples RT in dark. The excess IAAand DTT were removed proteins. The reduced proteins were then incubated with 25 m m 5 min. furthe by gel cubeswere thendehydrated min. The 10 for water with then and (ACN) acetonitrile 60% in min 10 for washing by destained were then pieces gel and cubes into gelwascut The digestion. in-gel to subjected were lanes Complete Blue. Brilliant on12% SDS-PAGE gel.The gel with resolvedproteins was then fixed and stained with Coomassie loaded was each sample of µg 30 and assay protein BCA using wasquantified Protein kit. clean-up Gel according to Merl (2012)et al. and von Toerne etal ofsamplesmassThe preparation and spectrometric proteins S-nitrosylated of analysis Proteomic analysis. further until -20ºC at stored was proteins) nitrosylated S- (containing supernatant and the centrifuged was sample The buffer. neutralization in mercaptoethanol 20-30 for incubating by were eluted matrix the to bound proteins 100 the with min proteins, m milliQ water) for 10–15 min. Once for 10–15milliQ water) in 10ml (1 dissolved tablet Fast Sigma for 5minpreparedBCIP/NBT using freshly anddeveloped each orbital shaker.Thereafter, d a in MO) Louis, St. Sigma-Aldrich, from obtained antibody (anti-biotin enzyme phosphatase alkaline with conjugated biotin against antibody monoclonal Tweenin 20 TBS]for 2hrsatRT. The membrane 0.2% buffer BSA, blocking [3% in was then incubated blot with transferred The proteins Ponceau staining). (Sigma Aldrich, St. Louis, MO) in 50 m 50 in MO) Louis, St. Aldrich, (Sigma TFA solution for30min. The supernatants containing the elutedpeptides werethen driedin speedvac obtainedthen was transferred toanew tube andremaining gelpieces were incubated with99.9%/0.1% so supernatant The shaking. minwith gentle 15 for incubating and TFA ACN/0.1% 60% using gel left were overnight was solutions and ABC added the M DTT was then added and gel pieces were inc pieces were gel thenaddedand was DTT M EDTA,600 m the membranethe was washed inwash buffer (0.2% Tween-20 inTBS)three times the desirable color intensity wasobtai colorintensity desirable the M M NaCl, 0.5% Triton-X-100). After washing out non-specific M ABC was added and incubated for 10 min. Further, 25 m 25 Further, min. 10 for and ABC was added incubated EDTA, 100 100 EDTA, m ied at 37ºC for 15 min and 0.01 µg.µl 0.01 and min 15 for 37ºC at ied chromatography according chromatography ilution of 1:10000 in blocking buffer, at 4°C on an on at4°C buffer, blocking in 1:10000 of ilution r incubating with 100% acetonitrile (ACN) for 10 10 for (ACN) 100%acetonitrile with incubating r g M . (2013).. The proteinswere first purified using 1D by incubating the samples with 100% ACN for 10 100% ACNfor with thesamples incubating by for 5 minutes and supernatant was removed. The wasminutes removed. andsupernatant 5 for ammonium bicarbonate (ABC), 100% ACNand was then incubated overnight with anti-mouse with anti-mouse was thenincubated overnight at 37ºC. proteinsThe were then eluted from the analysis of S-nitrosylated proteins was done done was proteins of S-nitrosylated analysis M ubated 10 at60ºCfor NaCl). 30 NaCl). M iodoacetamidesolution for 15min at ned, the membrane was placed in in placed was membrane ned, the μ l of equilibrated neutravidin neutravidin l of equilibrated to Sell etal to Sell (2008). The min to reduce the min the toreduce -1 trypsin solution solution trypsin M

β M M M -

This articleisprotected bycopyright.Allrightsreserved. Accepted Article Miniprotean Tetra Cell (Biorad, Hercules, (Biorad, Cell Tetra 0.1 in Miniprotean soaked was proteins CA).resolved with gel The vertical native polyacrylamide gel at 4°C (conditions: 75V for minutes,20 150 V for 2 h) using andquantified Proteinextracted as describedabove wa analysed for thedisappearance of theNADH fluorescen nm) and (340 radiation ultraviolet to were exposed gels and was removed paper the filter min, 10 After m 3 prepared freshly in soaked strips paper filter with werecovered andgels drained was sodium phosphate buffer, pH 7.4, containing 2 m of S-nitrosog analysis for PAGE Native and stored at -20 at and stored and 0.1 %formic acid in98 % ACN (B) at a min flow rate300nl of Waltham, MA) with a non-linear 170 min gradient using 5 % ACN in 0.1 % formic acid in water (A) reversedby phase chromatography operated on nano-HPLC (Ultimate 3000,Dionex, ThermoScientific, loaded onto the peptides column and weretrap elut on XL Scientific,Waltham,a (Thermo LTQ-Orbitrap performed was analysis LC-MS/MS agitation. under min 30 for atRT incubated and TFA ACN/0.5% m containingadded50 µgprotein was concentration measuredwas using reag Bradford 10centrifugedobtainedat then was 000 m 0.1 glycerol, 10% and tissues fromextracted root cotyledon monitoring the rate of NADH oxidation in the pr GSNO reductase activity wasmeasured spectrophotomet assay activity reductase (GSNOR) S-nitrosoglutathione software. Scaffold using displayed were results used and was database Swiss-Prot identification, peptide maximum) MS spectrum was acquired in the Orbitrap with amass range from 200 to 1500 Da. For half full-width 000 (60 high-resolution a analysis, fragment During charged. doubly at least were and were selected for fragmentationin the linear iontrap equilibration for 15min to starting conditions. From the MS prescan, the 10most abundant peptide ions 140min: 14.5-90% A, 140–145 min: 90% A - 95 % B, 145–150min: 95 % B and followed by transfer buffer buffer [0.025 transfer Hercules,(Biorad, Cell Tetra Miniprotean using in washed was then proteins resolved CA). with gel The Hercules, gels (Biorad, precast on12% loaded V 150 for 2 h) minutes, 20 for 75V CA) (conditions: Protein wasextracted and describedquantified as (GSNOR) reductase S-nitrosoglutathione of analysis blot Western NADH (6.22m recorded for 5minutes at340nm. Enzyme activity wa M NADH and 0.5 m M -1 0 C until further analysis. The pre-fractionated dried samples were dissolved in 2% in dissolved were samples dried pre-fractionated The analysis. further until C cm M M -1 M EDTA, 0.2% Triton X-100 and protease inhibitorcocktail).The homogenate ). ). Tris, 0.192 0.192 Tris, EDTA. Reaction was started by adding 0.5 m to reactionmixture m containing 20 M glycine, 0.1% (w/v) SDS and 20% methanol] at 4°C for at least 4°C for at least at methanol] 20% and SDS (w/v) 0.1% glycine, g lutathione reductase (1 g each) in1ml buffer (0.1 extraction for 20 min for20 at 4 abovewas constituted in reducingbuffer Laemmli and M ed aftered 5min andseparated onan column analytical esenceof GSNO at 340nm. Briefly, proteinwas ent (Bradford 1976). Aliquot from each sample sample each from Aliquot 1976). (Bradford ent NADH,min,inan ice-bath. for15 buffer Excess if they exceeded an intens s calculatedmolar using extinction coefficient for s loaded for single dimension separation on a 6% a on separation dimension single for s loaded ce due to GSNOR activity ce due toGSNOR activity MA). Samples wereand injected automatically rically accordingrically to Sakamoto etal(2002) by ˚ C to get total soluble protein (TSP). Protein Protein protein (TSP). soluble total C toget (GSNOR) activity (GSNOR)activity -1 . Thegradient settingswere: 5– M M Tris-HCl buffer (pH 8.0), 0.2 0.2 8.0), buffer Tris-HCl (pH GSNO and absorbance was was and absorbance GSNO ity of at least 200counts (Chaki et (Chaki al.2011b). M Tris-HCl pH 7.5, pH7.5, Tris-HCl M GSNO. GSNO. M

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein was added to a reaction mixture containing 0.1 containing mixture to areaction added was protein desalting columns. was quant Protein GSNO 250µ and toobtain total soluble proteins (TSPs). The supernatant obtained was treatedwith 10m of enzyme activity was defined as the amount of enzyme required to reduce 1 m 1 reduce to required enzyme of amount as the was defined activity of enzyme homogenates obtained after centrifugation at 10 000 (0.1 medium ingrinding were homogenized seedlings etiolated from harvested g) (1 roots and mg) (500 Cotyledons modifications. some with (1984), al. et Hossain to according Spectrophotometric estimation ofmonodehydroascorbate reductase (MDHAR) activity was done Monodehydroascorbate reducta medium (0.1 grinding in homogenized and nitrogen liquid with powder fine to ground g)were (1 roots and mg) (500 et al. (2005) by estimating the increase in absorbance due to reduction of NAD + to NADH. Cotyledons phosphateGlyceraldehyde-3- dehydrogenase (GAPDH Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) activity assay 10–30 min. for water) MilliQ ml in10 dissolved tablet Fast (1Sigma BCIP/NBT prepared freshly using developed me the Finally, shaker. an orbital antibody, Sigma-AldrichChemicals Pvt. Ltd., St. Loui min each and incubated insecondary antibody (antirabbit IgG conjugated toalkaline phosphatase shaker. Thereafter,membrane waswashed the in wash orbital on an at4°C overnight for buffer, blocking in of 1:2000 a dilution in Sweden) Vännäs, Agrisera, from obtained antibody (anti-GSNOR GSNOR against antibody polyclonal arabbit with incubated in PBS, 20 Tween 0.2% BSA, (3% buffer blocking in was incubated then proteins transferred with the blot Subsequently membrane. NC onto proteins min.15 Thegel was subjected to a current ofmA 400 for 1 h4 at min. Enzyme activity was calculated using extinction coefficient of 6.22 m of6.22 coefficient extinction using was calculated activity Enzyme min. by additionby of 50 µlof 10m and the reactionglyceraldehyde-3-phosphate volume 250 µ 250 min. Enzyme activity wascalculated activity min. Enzyme additionof 1 U ofascorbate oxidase and thereafter,monitoring the change in absorbanceat 340nm for 1 M decrease in absorbancedue to oxid obtai proteins (TSPs) estima was content Protein columns. desalting 10 Tris-HCl(pH 7.0), 2.5 m M GSNO and 250 µ 250 and GSNO M Tris-HCl, pH 7.5). The homogenates were then centrifuged at 10 000 000 at10 centrifuged were then homogenates The 7.5). pH Tris-HCl, M GSH for 20 minGSH20 at forRT. Residual DTT and GSNO wasremoved usingPD-10 ned frommeasuring respectivesamples by analyzedwere forMDHARactivity M GSHmin for 20 at RT. Residual DTT and GSNO was removed using PD- M M of NAD of NAD ascorbate, 0.2 m ascorbate,0.2 se (MDHAR)activityassay mbrane was washed in wash buffer three times for 5 min and 5 for threetimes each buffer wash mbranewashed in was ation of NADH at 340 nm. Themixture reactionml) (1 0.1 contained using extinctioncoef ified according to Bradford (1976). + and the change in absorbance was monitored at340 nm for 1 M NAPH and sample. The reaction was initiated by was initiated The reaction andsample. NAPH g ted according to ted according Bradford pH 7.4) for 2 hrs at RT. The membrane was then then was membrane The RT. hrsat for 2 pH 7.4) was adjusted wasreaction to950µl.The initiated was for20min withat 4ºCwere treated 10m buffer (0.2%Tween-20 5 forin TBS) threetimes s, MO) (1:2500 in wash buffer) for1 haton RT ) activity was measured was) accordingactivity toLindermayr ficient ofm 6.22 M Ti-C, 0 µ 50 Tris-HCl, ˚ C toachievecomplete transfer of Each aliquot containing 150µg aliquot Each M M

-1 -1 M cm cm M M arsenate, 100 µg. ml µg. 100 arsenate, (1976). Total soluble Tris-HCl, pH7.0). The NAD -1 -1 for NADH. One unit for NADH. One unit One unit forNADH. g for 20minat 4°C M + min min DTT, µ 250 DTT, -1 at25°C. M DTT, DTT, M -1

This articleisprotected bycopyright.Allrightsreserved. Accepted Article cotyledons both from spectrophotometric and zymographic analysis (Fig. 2). However, treatment with 10 10 with However, treatment 2). (Fig. analysis and zymographic spectrophotometric from both cotyledons ( is higher in GSNORactivity revealed that activity electrons from NADH. Spectrophotometric and zy m fold higher than seedling cotyledons (control conditions). Roots derived from seedlings subjected to 120 3- to be was found roots seedling from derived homogenates the in content nitrosothiol terms In absolute stress simila exhibit cotyledons Roots and nitriteandroots content in seedling to compared as nitrite content in accumulation.comp Thus,as sensitivity of roots was alsomuch higher than that of cotyledons in terms of its impact on nitrite saltstress Additionally, seedlings. control in the content nitrite higher 23-fold almost had Roots roots. general,tissues were1). In observed cotyledons (Fig. presence of 120 m Analysis of2-day old, dark-grown sunflower seedling cotyledonsand roots raisedin theabsence or accumulation Seedling roots exhibitmuchhigher Results wasof as defined enzymeactivity the amo thrice. thrice. ** and 0.01 < **p < 0.05, (*p of significance levels effectsANOVA. Theone-way of treat using analyzed Inc, Chicago, was used IL) for st were changes (salt-treated/control) 2-fold log and normalized were samples treated salt and control both in intensities protein S-nitroso The analysis Statistical 25°C. reduction of GSNO (a reservoir of NO) to oxidized glutathione (GSSG) and ammonium (NH and ammonium (GSSG) glutathione of to oxidized NO) (areservoir GSNO of reduction m of 120 presence or absence in the grown and roots cotyledons analyz were abundance protein its and activity GSNOR m 120 accumulation in response to salt stress. Thus, it is evident that both the tissue systems exhibit almost similar sensitivity for nitrosothiol control). of (72% stress to salt response in content nitrosothiol in increase asubstantial exhibited also ∼ M 50%) in enzyme activity was evident upon salt NaCl stress exhibited further increase in nitrosothiol content by 61% (Fig. 1). Seedling cotyledons

M NaCl reversibly inhibits GSNOR activity activity GSNOR inhibits reversibly NaCl M

NaCl showed striking differences in the co in the differences striking showed NaCl ared to the respective ared tothe salt controls, respective 27%sumToup, salt incotyledons. calculated before comparison. SPSS 22.0 statistical program 22.0 (SPSS SPSS statistical comparison. before calculated cotyledons may be considered ani considered bemay cotyledons atisticalanalysis ofthe data. Effectsofvarious treatmentswere r sensitivity for accumulation of nitrosothiols in response to salt salt to response in ofnitrosothiols accumulation for sensitivity r sensitivity to salt stress than cotyledons in term of nitrite cotyledons intermof stress than nitrite tosalt sensitivity unt of enzyme required to oxidize 1 m *p < 0.001). All experiments were performed at least least at wereperformed experiments All 0.001). < *p cotyledons than in roots. A significant reduction reduction in significant than roots. A cotyledons in sunflower seedling roots and cotyledonsand in sunflower seedlingroots to be low accumulators of nitrite in with contrast treatmentboth insunflower seedling roots and mographic (native-PAGE) analysis of GSNOR analysis ofGSNOR (native-PAGE) mographic ments were accordingly categorized into various various into categorized ments accordingly were ed in 2-day old, dark-grown sunflower seedlings ntent of nitrite in the respective control control the respective in ofnitrite ntent stressed roots exhi roots stressed ndex/markernitrosative of stress. stress-sensitized accumulation of of accumulation stress-sensitized M NaCl. GSNOR catalyzes the the catalyzes GSNOR NaCl. bited 71.5% increase M NADH min NADH 4 + ) using -1 at This articleisprotected bycopyright.Allrightsreserved. Accepted Article m than in roots thereby highlighting its role in long role inlong its highlighting thereby roots than in evident that S-nitrosylation of cytosolic proteins is a stronger biomarker of stress in seedling cotyledons bot in treatment asaresponseGSNO to nitrosylation salt stress and GSNO, proteins indicated in the highlighted zone clearly showed a downregulation of S- to response in heads arrow with highlighted bands protein most in evident was S-nitrosylation enhanced Although than cotyledons. in noteworthy was less proteins ofsoluble of S-nitrosylation extent the roots, µ with 250 treatment upon enhanced which is further homogenates Compared tocontrolNaCl), (- (+ subjecting the10 000 m 120 of orpresence absence in the 2 days for dark in seedlings sunflower Raising GSNO and stress by salt sunflower in proteins ofcytosolic S-nitrosylation S-nitrosylation. by regulated is enzyme reveals that further roots, and cotyledons unaltered. Restorprotein remained treatment (Fig. 2). This showsthat salt stress modula interestingly showed nodifference protein cont in the of GSNO activity catalytic the impairing in involved is or sulfenation, asS-nitrosylation such modification, oxidative ofreversible kind some that suggesting proteins (18%), 9% each in thecategory of regulato metabolic other by followed metabolism carbohydrate of category the to belonged (37%) S-nitrosylation miscellaneous proteins. In sunflower seedling cotyledons, highest number of proteins having undergone prot regulatory proteins, defense-related chaperones, functional categories namely, those belonging to carbohydratemetabolism, othermetabolic proteins, 8 into grouped were proteins These 2). 1, (Table S-nitrosylated be to found were cotyledons and roots impact of salt stress on S-nitrosylation of cytosolic proteins.A total of 61 proteins both from seedling the study to analysis LC-MS/MS to subjected were obtained so eluates The proteins. S-nitrosylated 10 and 000 m 120 (-/+ seedlings sunflower old 2-day from derived roots and Cotyledons roots and cotyledons seedling in trends opposite reveals proteins of S-nitrosylated analysis LC-MS/MS proteins. of S-nitrosylated detection nitrosylation ofproteins. These results show that biotin switch method specificis a techniquefor devoid of ascorbate served as negative controls and these homogenates, therefore, did not exhibit any S- sunflowerseedling cotyledons as molecular masses. Both salt stress and GSNO application brought about enhanced S-nitrosylation in of varying proteins of cytosolic of S-nitrosylation extent ofthe upregulation ageneral revealed GSNO M DTTagent)the ef (areversed reducing inhibitory g supernatant was subjected to biotinylation followed by affinity chromatography to purify purify to chromatography affinity by followed biotinylation to was subjected supernatant g supernatant fromthe homogenatesof seedling roots and cotyledons to 250µ is evident from the indicated (arrow ) NaCl condition (salt stress) led to enhanced S-nitrosylation in the the in enhanced S-nitrosylation led to (salt stress) ) NaClcondition ation of GSNOR activity of(upation GSNORactivity to distance signaling. Samples Samples signaling. distance ry proteins, chaperones and defense-related proteins proteins defense-related and chaperones ry proteins, R under saltstress conditions. Westernblot analysis tes the activity of GSNOR. However, the amount of of amount the However, GSNOR. of activity the tes seedlingroots and cotyledons is modulated both ent under salt stressconditions as well asafter DTT eins, transport proteins, cy proteins, transport eins, hNaCl (-)conditions (Fig. (+) NaCl and 3). It is fect of salt treatment on enzyme activity, thereby thereby onenzyme activity, fect ofsalt treatment M of GSNO. In the caseGSNO.the ofseedling ofIn 80%) with DTT both in seedling heads) 10 proteinbands (Fig. 3). M treated with GSH and those NaCl)were homogenized toskeletal proteins and toskeletal proteins M NaCl andalso M

This articleisprotected bycopyright.Allrightsreserved. Accepted Article activity in seedling cotyledons (Fig. 5). In (Fig. cotyledons inseedling activity suggesting thereby activity inenzyme to reduction Nitrite isconsidered as a biomarker of metaboli NO upon treatment withm 10 be restored and could wasreversible saltstress under activity of enzyme the inhibition that observed activitiesrevealed thatthe Spectrophotometric analysis of GAPDH and MDHAR activity in sunflower seedling cotyledons and cotyledons roots seedling reg together anddenitrosylation S-nitrosylation (Fig. 4). 17are unique to cotyledons, 4 are unique to roots whereas 40 arecommon toboth the tissue systems S-nitrosylation, by regulated being proteins Ofthe total 3). (Table roots in S-nitrosylation by modulated areboth specific androots,8 cotyle cotyledons to nitrosylated proteinsare being reported for the first time in plant systems ofwhich 9 werecommon in 18 S- that noted further It was categories. ofdifferent ofproteins S-nitrosylation of proportion seedling cotyledons (Fig. 4). Thus,both the tissue systems respond in a similar manner in terms of the in observed that to similar large and by remained roots seedling in proteins S-nitrosylated of distribution relative The 4). (Fig. respectively proteins, cytoskeletal and transport to belonged 3% and 5% and intracellular reservoirs and carriers of nitric oxide in plants. Information on modulation of nitrosothiol ofnitrosothiol modulation on Information plants. in oxide of nitric carriers and reservoirs intracellular S-nitrosothi 2016). al. et (Kuruthukulangarakoola been nitrite in havealsoreported of (Yam pH acidic at non-enzymatically or (NR) reductase by the of nitrate action enzymatically either oxide to nitric promote its reduction ofnitrite levels variousabiotic stress factors, including salinity (Zio nitrosative stress. An increase in the levels of nitrite has been observed in citrus plants subjected to stress and NO donor treatment. Thus, nitrite accumulation serves as a biomarker of salt and/or in level nitrite Furthermore, cotyledons. in roots than demonstrat have observations Present 2006). Stremlau of membrane plasma intheroot activity NOR) (Jasid et al. 2006). NO synthesis has also been reported by the action of nitrite-NO oxidoreductase (Ni- homogenates of (Bethke etal.2004), leaves intobacco andcell fromnitrite in acidic conditions has been demonstrated in the apoplast of aleurone layer in barley levels in various plant organs and tissues (Salgado etal.2013). Thenon-enzymatic production ofNO MDHARactivity increasedin response to 120m Discussion Discussion 5). (Fig. homogenates incontrol activity enzyme of restoration Arabidopsis thaliana M of both the enzymes decrease in the presence of 120 m 120 of presence in the decrease enzymes the of both DTT (reducing agent). Treatment of homogenates with 250 µ (Modolo et al. 2006) and in the chloroplasts of soybean leaves leaves soybean of chloroplasts and in the 2006) al. et (Modolo case ofsunflower seedling Arabidopsis M ulate GAPDH and MDHAR activity in sunflower Nicotiana tabaccum sm, thereby necessitating the thereby sm, gas etal. 2013). Itbeen has suspensions (Planchetal. et 2005), in the leaf ols major particularly GSNO, actas (RSNOs), dons and only 1 (calreticulin) was reported be to NaClTreatment stress. m 10 with the tissue is enhanced both in response to NaCl the role of S-nitrosylation in regulating enzyme enzyme in regulating of S-nitrosylation role the ed preferential nitriteed preferential asaki 2000,Rockel et al. leaves fromNO-fumigated plants (Stöhr et al. 2001, Stöhr and and Stöhr 2001, etal. (Stöhr rootshowever, GAPDHand accumulation seedling in suggested that elevated suggested thatelevated quantification of nitrite nitrite quantification of 2002). Elevated levels M NaCl. was alsoIt M M DTT led to GSNO led led GSNO This articleisprotected bycopyright.Allrightsreserved. Accepted Article Arabidopsis thaliana Arabidopsis regulated positively by the modulation le of cellular Signaling network bycontrolled 2006). Barroso etal. mediate may of RSNOs, accumulation to leading activity, GSNOR of a downregulation that earlier suggested been ithasalso observations, present from evident As response. stress salt long-distance as a cotyledons as in well as NaCl) inducer, stress the with contact direct in are (which roots in both observations present the through evident bothenhanced NO/nitrite level anddownregulation of indicate that ingeneral plant tissuesmaintain a relatively higher levelof SNOs. This canbeattributed to statedearlier reportstogether with present observationson sunflower seedling roots and cotyledons showntobe downregulated inpeaplants in response to cadmiumstress (Barroso al. et 2006). Theabove- been have expression gene and its activity GSNOR 2006). et al. (Barroso pea and 2002) al. et (Sakamoto GSNOR-mediated reduction of inGSNO hasbeendemonstrated tobacco (Diaz et al. 2003), 1.1.1.284). It catalyzes the1.1.1.284). It NADH-depende EC reductase; (S-nitrosoglutathione ofGSNOR activity the by regulated is cells plant in ofGSNO level NO. (GSH) and The glutathione between of reaction as aresult is formed GSNO (S-nitrosoglutathione) importanceofbiomolecule this instressconditions. as comparedto NO incellsystemsand participatein signal transduction pathway thusindicating the NO with (Kuruth increases after fumigation rapidly also show 2.5 times higher RSNO levels than control plants suggesting that the endogenous level ofNO stress. salinity to response inplant of RSNOs role suggesting contentm upon120RSNO total wounding and low temperature (Corpas et al.2008). Pr Similar observationshave also been reported fromthe leavesof peaplants inresponse tomechanical demonstrated to elevate total nitrosothiol content by resistan disease regulate to shown content in response to abioticor biotic stress (Let content in responsespecific toa stressislimitedas modulation of GSNOR activity without any no withoutany of modulation GSNOR activity obser present contrast, In levels. protein GSNOR GS diverse changes in highlighted investigations of abiotic in stressconditions avariety and to alsoin response demonstrated the significance activity mo ofGSNOR and ofof light) on a (heat, cold, injury have the impact variety stress factors al.(2014) et Kubienova al (Chaki et activity GSNOR difference in show tothe pathogen different sensitivity with Two2007). cultivarsof sunflower show enhancedresistance against activity low GSNOR (Feechan et2005). al. In this context transgenic plants of M NaCl treatment root inboth seedling and cotyledon tissues thereby ce in plants (Feechan et al.20 et plants (Feechan in ce nitrosative stress in plants stress (Airaki et nitrosative in plants al. 2011, Lee et al. 2008, nt reduction of GSNO to GSSG and NH GSNO toGSSGof and nt reduction teworthy changes in GSNOR protein levels. levels. protein GSNOR in changes teworthy ukulangarakoola et al. 2016). SNOs aremore stable errier etal. 2012a). Nitrosothiol content has been NOR activity as well as the parallel changes in parallel changes asthe in as well NOR activity . 2009b). Detailed investigations undertaken by by undertaken investigations Detailed 2009b). . compared to compared that availa 2-fold inoliveplants (Valderrama etal.2007). vations highlight the impact of salt stress on stress ofsalt impact the highlight vations vels of S-nitrosothiols and GSNOR activity in and GSNORactivity S-nitrosothiols of vels dulation both duringnormal plant development esentobservations also indicate an increasein GSNOR activity in response to stress. This is This inis stress. responseto activity GSNOR salicylic acid has also been observed to be be to observed been hasalso acid salicylic Peronospora Arabidopsis Cucumis 05). Salinity stresshas been

parasitica and plants fumigated withNO plantsfumigated Plasmopara ble on alterations inNO on alterations ble Arabidopsis Pisum 4 + (Rusterucci et al. . In the recent past, past, recent the In .

sativum

halstedii Arabidopsis exhibiting . These These . also also

This articleisprotected bycopyright.Allrightsreserved. Accepted Article long-term salt treatment (Cam treatment salt long-term and short to response in of S-nitrosylation extent inthe adecrease showed proteins mitochondrial pea of proteins in the homogenates from 2009).stress NaCl beenhas reportedto bring minor about changes in theextent of S-nitrosylation of al. et (Tanou salinity to in response plants citrus in observed also was of S-nitrosylation pattern Similar 2009). and Deswal (Abat of8proteins denitrosylation and 9 proteins atleast of S-nitrosylation enhanced denitrosylation may beassociatedwithfastupregulatio may denitrosylation et 2006,activity (Lindermayr etal. Romero-Puertas the inhibit enzymeproteins isoftenobserved to ac getting S-nitrosylated ordenitrosylated willshowenha lines.above-stated pathwaysfrom onexpected is the enhanced observed the roots, and seedlingcotyledons cycle and thesein glyoxylate gluconeogenesis. Since photores toglycolysis, roots). enzymes belong These metabolismundergo S-nitrosylation (upregulationin nitrosylated proteins in sunflower seedlings shows th in responseto changes in thecellular redox environment (Singh1995).al. et LC-MS/MS analysis of S- NO (Vanzo etal. 2014). Furthermore, non-enzymatic de have andbeen recently classified as denitrosylases reductase thioredoxin/thioredoxin and GSNO reductase that noted be may it context, this In proteins. different of denitrosylation also and S-nitrosylation enhanced both to lead can stress that indicate thus response to varied abiotic stress conditions (Ziogas etal.2013). Present observations and earlier reports of ofS-nitrosoproteome modulation differential observed enrichment of S-nitrosylation of several proteins in response to ozone stress. In the recent past, etal. (2014) analyzed the abundance Vanzo stress. salt of irrespective treatment GSNO to inresponse downregulation showed zone indicated GSNOtreatment was,however, lessthan that inseedling cotyledons. Interestingly proteins in the and stress salt to response in of S-nitrosylation upregulation showing homogenates root from proteins GSNO treatment as well tosaltstress in response nitrosylated proteins in seedling of S- analysis Immunoblot S-nitrosylation. oftheir extent and pattern the in differences noteworthy cytosolicproteins from sunflowerseedling and roots cotyledons in response tosaltstress highlight Present proteomic analysispertains to under for abiotic proteomicmaterial stress analysis analysisrespectivethefurther of may enzymes. be It stress in seedling roots. This view, however, ne

investigations on deciphering the qualitative and quantitative variations in the S-nitrosylation of of S-nitrosylation in the variations quantitative and qualitative the deciphering on investigations Arabidopsis ejo et al. 2013). Similar observations were observations Similar 2013). ejo etal. cotyledons consistently shows accumula consistently cotyledons Arabidopsis of S-nitrosylated proteins in callus and leaf extracts from poplar and and poplar from extracts leaf and callus in proteins of S-nitrosylated , 2. Mostof the cellvolumein cotyledon tissue is filled with oil cell suspensions (Fares et al eds to be proved throughfurther beeds proved detailed biochemical to Brassica juncea these enzymes might be themselvesthese enzymesregulated alsoby tivity whiletivity may denitrosylation promote theenzyme al. 2007, Palmieri etal.2010,Yun2011).Thus, It is, however, notclearwhichupon however, oftheseenzymesIt is, seedlingand downregulation cotyledons inseedling at many enzymes belonging to primary carbohydrate enzymesprimary to belonging many at resolved by electrophoresis. by Theresolved of numberzones of on two accounts: 1. ofMost the available data on n of various metabolic proc noted that sunflower is an unconventional research is anunconventional sunflower noted that nced or reduced enzyme activity. S-nitrosylation of ondark-grown havebeenundertaken vestigations or decreased S-nitrosylation of various enzymesdecreased various or S-nitrosylation of piration, photosynthesis, acidpiration, photosynthesis, tricarboxylic cycle, nitrosylation of proteins has also been reported by low temperature revealed both both revealed low temperature by also recorded in recorded also tion in at least 10 protein zonesprotein 10 tion inatleast . 2011). . Furthermore, analysis esses required to combat esses required combat to citrus plants in in plants citrus This articleisprotected bycopyright.Allrightsreserved. Accepted Article phosphoglyceratekinase has earlier beenreported in and isomerase phosphate triose (GAPDH), dehydrogenase 3-phosphate glyceraldehyde of S-nitrosylation seedlings. stressed salt in balance energy and metabolites various of concentration response tosalt stress observed inpresent the work suggests thatnitric oxidecanmodulate the pathway, functions togenerate primarily re Zaffagnini et al. 2013). Phosphoribulokinase (PRK), an enzyme of the reductive pentose phosphate shown to GAPDH hasbeen in of cytosolic nitrosylation various nuclear eventssuch as in involved is enzyme This 2005). etal. Lindermayr 1995, Whorton and (Padgett ofprotein the center such modificationa can affect protein function by reversib dependent S-nitrosylation exhibits GAPDH S-nitrosylation/denitrosylation in sunflower seedling cotyledons and roots in response to salt stress are stress salt to response in and roots cotyledons seedling sunflower in S-nitrosylation/denitrosylation In the category of “ stimuli. ofstress otherkind any or treatment or NOdonor stress salt to in response differentially areregulated pathways metabolic plant in various involved enzymes different that suggests proteins plant various in proteins of S-nitrosylation on reports nitrosylation(Ortega-Galisteo 2012). etal. Present results along withsupporting dataprevious from S- by be to inhibited shown earlier been have aconitase and dehydrogenase malate Both work). nitrosylation pattern accompanyingsalt stresssunflower in seedlingcotyledonsand roots (present itsS- of modulation and it shows NADH through in generation ATP isinvolved dehydrogenase Malate stress. salt upon S-nitrosylation by be regulated to found also were and aconitase, dehydrogenase S-nitrosylationet(Abat al. 2008, Deswaland Abat 2009). cycle,malateof TCAsuch Enzymesas 2003). RuBisCO andtriose phosphate isomerase have Substantial S-nitrosylation ofa variety of enzymes associated with 2009, Abat et al. 2008, Tanou etal. 2012, Vanzo etal. 2014). inproteins are agreement withearlier reports (Marcus that be noted further may It stress. salt conditions of routes andcatabolic biosynthetic various carbohydrate upregulation ofS-nitrosylation highlights the metaboli Keeping thesefactsinview proteins. and signaling monolayer impregnatedbodies w encasedinaphospholipid modulation of enzyme activity and degradation of the of the degradation and activity of enzyme modulation residue in this subunitof RuBisCO residingadjacent to theactive site is believed toplay a role in the its large subunit in of S-nitrosylation to exhibit been earlier reported has cycle, Calvin-Benson of enzyme the key RuBisCo, above-stated its toaffect islikely work) present phosphate fornucleotidebiosynthesis. Thus,S-nitrosylationof such anenzymeobserved (as in the other metabolic proteins metabolic other Arabidopsis in response to NO donor treatment (Lindermayr et al. 2005). The Cys RNA transport, DNA replication and transcription and 1999).S- DNA RNA transport, transcription (Sirover replication ”,present observationson themodulation of the extent of ducing equivalents (NADPH) ducing equivalents a functions. As also observedinthefunctions. work, present the noteworthy large number of enzymes exhibiting large number thenoteworthy et al. 2003, Lindermayr et al. 2005, Abat and Deswal 2005,and 2003,Lindermayr etal.Abat et al. le modulation of enzyme activity, indicating how how indicating activity, enzyme of modulation le the inactivationofcysteine residueinactive the c tendency of the seedling cotyledons to modulate modulate to cotyledons the seedling of c tendency protein thereby altering it altering thereby protein most of these observations on S-nitrosylation of S-nitrosylation on observations these of most been characterizedby and showntobe inhibited Arabidopsis thaliana to enable the seedling to survivetheharsh seedlingto in toenablethe hibit its catalytic activity (Holtgrefe et 2008, et al. (Holtgrefe activity catalytic its hibit ith a variety of intrinsic andextrinsic ofintrinsic structural ith avariety carbohydrate metabolism carbohydrate (Lindermayr et al. 2005). al. (Lindermayr et s turnover (Marcus et al. turnover (Marcuset s nd provides ribose 5- in This articleisprotected bycopyright.Allrightsreserved. Accepted Article Differential status of S-nitrosylation of various ROS-scavenging enzymes suggests the role of these ofthese role the suggests enzymes ROS-scavenging ofvarious of S-nitrosylation status Differential 2014). S-nitrosylation of MDAR under salinity stress al. et (Begara-Morales activity ofAPX in reduction results of S-nitrosylation blocking In contrast, enhanced its activity thereby promotingH thereby activity enhanced its (Begara-Morales et al. 2014). These investigators APXbeen has found tobe S-nitrosylated by salt stress leading to enhanced activity of the enzyme stre tosalt in response translation and transcription essential components of this pathway and they have been reported to be induced at the level of both are enzymes these Both cycle. ascorbate-glutathione in the role critical a plays S-nitrosylation that possible be may it and roots, cotyledons seedling sunflower and MDAR in ofAPX status nitrosylation of S- modulation the on observations present of view II. In Prx and AOX Mn-SOD, of regulation stre salinity to subjected pea plants that reported et (Tanou Mn-SOD and Fe-SOD of of S-nitrosylation modulation to leads saltstress plants, citrus stress. In salinity after 14days ofpea proteins mitochondrial in the peroxiredoxin and Mn-SOD of S-nitrosylation reported (2013) et al. Camejo context, Inthis (upregulation). cotyledons seedling in and catalase peroxiredoxin 2-cys peroxidase, L-ascorbate sunflower includes phenylalanineammonia lyase and Mn-SOD inseedling roots (denitrosylation) and of modulation exhibiting proteins related defense other seedling in cotyledonsth stress NaCl combat to mechanisms distance long v/s (roots) salt with contact direct in salt stress in tolerating beplaying might proteins two these role significant the highlights observation This roots. seedling in denitrosylation and cotyledons in S-nitrosylation enhanced exhibit and systems tissue the both to common are which two the are D1 annexin and reductase monodehydroascorbate although systems, tissue the both in S-nitrosylation by modulated be to been found have enzymes ROS-scavenging known well denitrosylation in seedling roots in resp Calreticulin is a new report from the present wo and Hsp 90are alsoavailable (Lindermayr et al.20 prot mitochondrial the from of Hsp-90 nitrosylation includeHsp90-1, endoplasmin homolog and luminalbinding protein. Camejoet(2013) al. reported S- oppositemanner. The common proteins getting modulated in this category in both the tissue systems chaperones mediateto a crosstalk between NOandethylene. A significantmodulation of S-nitrosylation of likely are cycle methylMet of enzymes the associated or SAM synthetase of S-nitrosylation and plants suchcomponents flavonoids, as lignin andDNA.SAMis methylMet cycle whichprovides activatedmethyl in synthetase adenosylmethionine S- and synthase methionine of adenosylhomocysteinase, S-nitrosylation with conformity in in response to salt stressevident is insunflower seedling cotyledons and roots, though in Arabidopsis onse to salt stress. In the category theof category onse tosaltstress.In 2 O 2 turnover, causing enhanced re enhanced causing turnover, (Lindermayr et al. 2005). These enzymes are part of of part are enzymes These 2005). al. et (Lindermayr ss exhibitedpost-transcriptional andpost-translational rough changes in ROS scavenging mechanisms.ROS scavengingrough changes The in ss (Begara-Morales etal. 2014; 2015). Furthermore, eins. Similar reports onS-nitrosylation of Hsp 70 rk under the category of category under the rk reported that S-nitrosylation of APX at Cys-32 Cys-32 ofAPXat S-nitrosylation that reported groups as SAM for methylation of various cell cell various of methylation for asSAM groups has earlier been found to inhibit enzyme activity. activity. enzyme has been inhibit earlier found to 05, Molassiotis et al. 2010, Fares et al. 2011). the extent of S-nitrosylation/ denitrosylation in in denitrosylation of extent S-nitrosylation/ the al. 2009). al. Furthermore, Martiet al.(2011) also the precursor for ethylene biosynthesis in biosynthesis precursorethylene for also the sistance to oxidative stress. stress. oxidative to sistance defense-related proteins defense-related chaperones exhibiting exhibiting chaperones , This articleisprotected bycopyright.Allrightsreserved. Accepted Article Quite a few novel proteins have also been identified as identified been also have proteins novel a few Quite nitrosylation might affectloading withof vesicles response tostress factors. conformationalAny ch in signals distance long rapid of intransfer filaments of cytoskeleton the role signify cotyledons and roots seedling sunflower in proteins cytoskeletal of these patterns S-nitrosylation of trends alpha2 in sunflowerseedling cotyledons and roots, respectively, in response tosalt stress. Theopposite tubulin and actin-2 of downregulation and up- both highlights work Present etal.2005). (Lindermayr in in S-nitrosylation variations undergo to reported been earlier have beta tubulin seedlings.A variety of in S-nitrosylation for astargets factors and initiation elongation different identified (2005) al. et Lindermayr synthesis. protein in oxide of nitric proteins regulatory enzymes in regulating cellular redoxhomoestasis. Changes inthe S-nitrosylation statusvarious of in plants. Stress canlead plants. Stress in of GSNORactivity, Downregulation leading to accumu both enhanced NO/nitrite level and downregulation of GSNOR activity in response to stress (Fig. 2,3). relative maintain a tissuesplant general, conditions. In participateinsignal transduction pathway thus indicating the importanceof this biomolecule instress more SNOs are stress. to salinity plant response in ofRSNOs role the suggest also observations Present plants. in oxide ofnitric carriers and reservoirs major intracellular as act GSNO, particularly (RSNOs), S-nitrosothiols stress. nitrosative and/or salt To sum up,present observations have demonstrated that nitrite accumulation serves as a biomarker of seedlings. sunflower in stress salt of sensing distance long during activities enzyme regulating by denitrosylation and S-nitrosylation both of role the highlight an ea mightact as to increase activity inenzyme Denitrosyla of the both enzymes. activities promotes m 120 of presence the in roots seedling in proteins of denitrosylation However, data. MS/MS insunf activity and MDHAR GAPDH of inhibition to be S-nitrosylated in sunflower seedling roots found were which enzymes regulatory important two are (MDHAR) reductase monodehydroascorbate Palmieri et al. 2010). Glyceraldehy etal. 2007, Belenghi 2007, et al. Romero-Puertas 2006, etal. (Lindermayr activity enzyme promotes denitrosylation while activity enzyme of inhibition to leads often S-nitrosylation that demonstrated been has It enzymes. various of activities the regulate together S-nitrosylation/denitrosylation such asendoplasmin chaperones and synthase citrate protein, storage seed globulin 11S synthase, malate D, phospholipase aquaporin, pectinesterases, calreticulin, calmodulin, oleosins, dikinase, phosphate pyruvate These include observed in the present work as a response to salt stress suggests the possible role cytoskeletal proteins to both enhanced S-nitrosylation and also denitrosylation of different proteins. proteins. of different denitrosylation and also S-nitrosylation enhanced both to homolog,chaperone protein ClpB1. Arabidopsis such as actin, actin polymerizing factor, tubulin alpha and and alpha tubulin factor, polymerizing actin as actin, such de-3-phosphate dehydrogenase (GAPDH) and (GAPDH)and dehydrogenase de-3-phosphate variousmetabolites and their long distancetransport. anges in the components of cytoskeleton duecytoskeleton in toS- angescomponents of the and cotyledons (presentwork). stress-induced Salt stable as compared to NOinand cell systems rly response to salt stress. These results together together results These stress. salt to response rly tion of proteins in sunflower seedling roots leading leading roots seedling insunflower proteins of tion in line with present observation on sunflower sunflower on observation present with in line ly higher level of SNOs. This can be attributed to to attributed canbe This ofSNOs. level higher ly targets of targets S-nitrosylatio lower seedling cotyledons correlates with with LC- correlates cotyledons lower seedling lation of RSNOs, may mediate nitrosative stress stress nitrosative mediate may ofRSNOs, lation n insunflower seedlings. Arabidopsis M NaCl

This articleisprotected bycopyright.Allrightsreserved. Accepted Article FEBS J Kalanchoe pinnata activity. carboxylase juncea The authors have no conflicts of interest to declare. Disclosures providing financialassistance. ofto Delhi University Authors grateful are Acknowledgements UniversityGermanofService AcademicExchange Delhiand by (DAAD). supported waswork This Funding survive in theharsh conditions of saltstress. cotyledonsmodulate to various carbohydrate biosynthetic catabolic and routesenable to theseedling to seedling of the tendency metabolic the highlights ofS-nitrosylation upregulation exhibiting enzymes various metabolic processes required Thus,denitrosylatio promote themay enzyme activity. while denitrosylation activity enzyme the to inhibit observed often is ofproteins S-nitrosylation activity. or enzyme enhanced will show reduced ordenitrosylated S-nitrosylated getting upon enzymes of these cotyledons and downregulation in seedling roots, Fig. belonging to primary carbohydratemetabolism undergo S-nitrosylation (upregulation seedlingin in proteins ofS-nitrosylated analysis LC-MS/MS environment. redox cellular the in changes to response in been reported also has proteins of denitrosylation non-enzymatic Furthermore, andbeen classified thes recently denitrosylases as In this context mayit be notedthatGSNO reductase and thioredoxin/thioredoxinreductase have 10.1080/15592324.2015.1071753. cotyledons asan and early long distance signaling response to NaClstress. Plant SignalBehav. doi modulation of superoxide dismutase (FeSOD and Cu/Z organs LC-ES/MS. by Plant Cell Physiol. 52:2006–2015. of S-nitrosoglutathione quantification and Detection ( pepper in (GSNO) Arora D., and Bhatla S.C. (2015) Nitric oxide triggers a concentration-dependent differential differential a concentration-dependent triggers oxide Nitric (2015) S.C. and Bhatla D., Arora (2011) F.J. and Corpas, J.M. Palma, J.B., Barroso, M., Leterrier, L., Sánchez-Moreno, M., Airaki, Abat, J.K., Mattoo, A.K. and Deswal, R. (2008) S-nitrosylated proteins of a medicinal CAM plant plant CAM medicinal a of proteins S-nitrosylated (2008) R. Deswal, and A.K. Mattoo, J.K., Abat, Abat, J.K. Deswal, R. and (2009) Differen References by low temperature: change in S-nitrosylation of in S-nitrosylation change by low temperature: 275: 2862–2872. -ribulose-1,5-bisphosphate carboxylase/oxygenaseactivity targeted for inhibition. Proteomics 9: 4368–4380. to combat stress in seedling roots.The noteworthy large number of and German Academic Exchange Service (DAAD) for e enzymes might themselvesbeenzymes NO. alsoregulatede by sunflower seedlings has shown that many enzymes enzymes many that shown has seedlings sunflower tialmodulation of S-nitrosoproteome of 6). Further work is required to demonstrate which nSOD) activity inactivity sunflowernSOD) and seedling roots Rubisco is responsible for the inactivation of its n may benassociatedwith fastupregulation of Capsicum annuum Capsicum L.) plant plant L.) Brassica This articleisprotected bycopyright.Allrightsreserved. Accepted Article carbonylation.6: PLoS e20714.One desiccation toleranceof recalcitrant plants? on in going what's nitrosylation: etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 210: 215- 210: Planta plants. in responses light-inducible three elongation, hypocotyl and inhibits etiolation, pea plants under cadmium stress. J. Exp. Bot in reductase ofS-nitrosoglutathione expression and of S-nitrosoglutathione Localization etal. (2006) A., proteins. J. J. Proteomics proteins. in changes Salinity-induced F. (2013) Sevilla, and A. Jiménez, 248-254. 72: Biochem. Anal. binding. protein-dye of principle the utilizing protein of Cell tissues. Plant mechanisms andcellularNat. functions. Rev.Mol.Cell Biol Biophys Biochim. regulation. 221. nitrosylation of a critical cysteine residue. J. Biol. J. ofChem residue. cysteine a critical nitrosylation ofvan activity F.(2007)Metacaspase and Breusegem, 527–538. 65: Bot. Exp. J. and S-nitrosylation. nitration tyrosine by (APX) peroxidase ascorbate of cytosolic Dual regulation (2014) et al. J., Jaramillo, 5983–5996. 66: Bot. Exp. J. S-nitrosylation. and nitration by reductase M.N., etal. 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J. roots. plant in oxide ofnitric roles possible and Formation (2006) S. Stremlau, and hr, C. cus on nitrate reductase and S-nitrosoglutathione reductase in stress-related stress-related in reductase S-nitrosoglutathione and reductase nitrate cus on ‐ based signaling and associated post associated and signaling based ras, A., A., ras, Fernandez-Ocana, ionof nitric oxide from nitrite. Planta 212 lower seedling cotyledons. Nitric Oxide, 53: 54-64. 53:54-64. Oxide, Nitric cotyledons. seedling lower and Rockel, P. (2001)and Rockel,P. e and iron modulate heme oxygenase activity a as activity oxygenase heme modulate iron e and Diamantidis, G., Fotopoulos, V. and Molassiotis, Chaki, M., Luque, F., et al. al. et F., Luque, M., Chaki, A plasma-membrane-bound ‐ translational modifications ‐ nitrosylated proteins in : 835–841. This articleisprotected bycopyright.Allrightsreserved. Accepted Article dehydrogenase from Arabidopsis thaliana. J. Biol. Chem. 288: 22777-22789. glyceraldehyde-3-phosphate of cytoplasmic denitrosylation and ofnitrosylation Mechanisms (2013) optimized rapid workflow for SRM-based quantification. J. Proteome Res. 12: 1331-1343. 1331-1343. 12: Res. J. Proteome quantification. forSRM-based workflow rapid optimized co levels protein andplasma Psp Mbl2, Apoe, (2013) of NADPH oxidase regulates cell deathin Sci. 355:1477-1488. Biol. B. Lond. R. Soc. Trans. Philos. vivo. in photoinhibition in species nitrogen active of involvement usingprotoplasts anovel fluorescence imaging method. Plant 49: Physiol Biochem 230-234. 10.4161/psb.5.10.12159 doi: induced adventitious rooting5(10): in1163-1166. sunflower. Plant Behav. Signal. Arabidopsis. Front. Plant Sci. 4:430. in defense responses and developmental multiple control inthat plants proteins cysteine-rich number, GAPDHby nitric oxide inresponse (2010) Regulation of Nicotiana tab emerging redox-basedpost-translational modifi 89:433-450. acclimation incitrusplants.Biol. Mol. Plant (2015) ofsodium hydrosulfide during drought andsodiumnitroprussideprimingmolecules Roles as antioxidant activities in Stylosanthesguianensis. J. Exp. Bot. 56: 3223-3228. Zaffagnini, M., Morisse, S., Bedhomme, M., Marchand, C.H., Festa, M., Rouhier, N., et al. Zhou, B., Guo, Z., Xing, J. and Huang, B. (2005) Nitric oxide is involved in abscisic acid-induced acid-induced in abscisic is involved Nitricoxide B. (2005) J. and Huang, Xing, Z., B., Guo, Zhou, Yamasaki, H. (2000) Nitrite-dependent nitric oxide production pathway: implications for al. et A., J.,Kulik, Dahan, A., Anielska-Mazur, J., Astier, M., Bucholc, I., Wawer, an S-nitrosylation: (2006) G.J. Loake, and Yun,Wang, Y., B.W., Kwon, E., Hong,Yoon, J.K., J. von Toerne, C., Kahle, M., Schäfer, A., Ispiryan, R., Blindert, M., Hrabe deAngelis, al.M., et Yun, B.W., Feechan, A., Yin, M., Saidi, N.B., Le Bihan, T., Yu, M., et al. (2011) S-nitrosylation S-nitrosylation (2011) et al. Yu,M., T., Bihan, Le N.B., Saidi, Yin, M., A., Feechan, B.W., Yun, in and on bodies oil activity lipoxygenase of (2011) Localization S.C. Bhatla, and M.K. Yadav, of specificsteps auxin- A. andBhatla, S., David, modulates S.C. (2010)Yadav, oxide Nitric Xu, S., Guerra, D., U.Lee, andVierling, E. (2013) S-nitrosoglutathione reductasesare low-copy Ziogas, V., Tanou, G., Belghazi, M., Filippou, P., Grigorios,P., M., Fotopoulos, Ziogas, V.,Belghazi, Filippou, V., Tanou, D.andMolassiotis, G., A. acum osmotic stress-activated osmotic protei acum to salinity. to salinity. Biochem. J. 429: 73–83. plant immunity. Nature 478: 264–268. 264–268. 478: Nature immunity. plant cation in plants. J. Exp. Bot. 57: 1777-1784. rrelate with diabetic phenotype in NZO Mice: An n kinase and its cellular partner kinase cellular and its n This articleisprotected bycopyright.Allrightsreserved. Accepted Article phosphoglycerat bisphosphoglyce hydratase Aconitate Carbohydratemetabolism change refers to protein abundance in salt-stressed dar old, (2-day seedlings sunflower supernatant) of I Table dehydrogenase 3-phosphate Glyceraldehyde- 48 P42896 Enolase 57 Q9LXS6 synthaseCitrate e mutase rate-independent 2,3- dehydrogenase1 Alcohol Protein Poemc nlss L-SM) f -irsltd p S-nitrosylated of (LC-MS/MS) analysis Proteomic :

Accession 450 98 Q42560 163 41 P14673 281 37 P25861 61 P35494 no. no. kDa (56.3) RGNDEVMAR FQR (84.6) SENAVQANMELE VDLACMR (57.3) VLLQDFTGVPAV (77) (77) FGVTEFVNPK ALELR (91.5) ALELR (91.5) AAVPSGASTGIYE (85.5) VGLATGQIK SGETEDTFIADLS VR (96.3) VNQIGSVTESIEA R (58.7) LYDPGYLNTAPV (88.6) SSICYIDGDEGILR VR (98) VR (98) VPTVDVSVVDLT (69.9) AGIALNDHFVK HK(43.8) GWDAQVLGEAP LDQLQLLIK (64.7) (94.1) GIDAQVASGGGR Peptide sequence (Ionscore) cotyledons over control ones. gon sbetd o 2 m NC srs. Fold stress. NaCl mM 120 to subjected grown) k oen fo te oyeos (10,000g cotyledons the from roteins GO Biological GO Biological Tricarboxylic Tricarboxylic Glycolysis Oxidation- acid cycle acid cycle Glycolysis Glycolysis reduction process process

change 10,11 14,92 15,03 Fold Fold 5,95 7,62 8,79 change Log 2 Log fold fold 3 3 3 3 3 4 4 3 This articleisprotected bycopyright.Allrightsreserved. Accepted Article dehydrogenase Isocitrate -QFR 5 NNALVP Pentose NPNLASLVVDPD 53 Q9FFR3 6- carboxykinase uvate Phosphoenolpyr 1 enhancer protein evolving Oxygen- 65 P08216 Malate synthase dehydrogenase Malate 64 P49297 Isocitrate lyase 527 47 P50217 406 74 P42066 28 P85194 36 O48905 (70.2) LIDDMVAYALK ADELK (79.5) EHYLNTEEFIDAV IGINGFGR(58.6) (65.9) MIINALNSGAK R (94.8) YGVELDGDGLGV (100.1) MNEILYELR ATVLIETLPAVFQ (60.2) WSGANYYDR GK(103.8) TVQGGITSTAAM MK (109.9) MK (109.9) EMVILGTQYAGE HTR(81.6) FGTVLENVVFDE (62.2) TTLSTDHNR PGGER (60.2) DGIDYAAVTVQL PAGGR (60.8) GGSTGYDNAVAL VSSDGTIK (90.2) LTYTLDEIEGPLE (86.8) LDLTAEELTEEK (65.1) LNVQVSDVK (104.7) MELVDAAFPLLK Photosynthesis Gluconeogenes Tricarboxylic Tricarboxylic Tricarboxylic Glyoxylate acid cycle acid cycle acidcycle cycle is is 10,15 13,82 18,44 22,19 7,95 7,77 6,31 3 3 3 4 3 3 4 3 4 This articleisprotected bycopyright.Allrightsreserved. Accepted Article phosphoglucona bisphosphatase bisphosphate bisphosphate phosphate dehydrogenase te nase Phosphoribuloki Triosephosphate P48493 21 ALLNETNEFVGD Glycolysis ALLNETNEFVGD 21 P48493 Triosephosphate 80 O20250 Transketolase 1,7- Sedoheptulose- ansferase 1 hydroxymethyltr Serine chainsmall carboxylase Ribulose large chain carboxylase Ribulose dikinase Pyruvate 232 45 P26302 425 42 P46285 57 Q9SZJ5 20 P08705 54 P45738 105 Q42736 (79.5) LPANLVQAQR FAK(63.9) K (60.8) (60.8) K ANDFDLMYEQV EVTTGPLC (100.7) TPGHPENFETPGI (94.6) VTTTIGFGSPNK (44.4) ATFDNPDYDK IVK (37.1) YTGGMVPDVNQI MEK(69.5) LDESTGYIDYDQ (39.8) MGTPALTSR TQLAK (69.4) KYETLSYLPPLTE EYPQAWIR (47.6) SPGYYDGR (49.2) IIGFDNVR (52.8) FIEK(76.9) EITLGFVDLLRDD (81.7) DLATEGNEIIR (95.7) K FEFQAMDTLDTD (52.8) AALDLLLPYQR R (50.2) DMMDIEFTVQEN LTAR (96.4) VMANADTPNDA phosphate cycle Photosynthesis Photorespiratio Photosynthesis Photosynthesis phosphate phosphate phosphate Reductive Reductive Reductive pathway pentose- pentose- pentose- fixation, fixation, fixation, Carbon Carbon cycle cycle n 16,51 5,54 1,82 5,29 4,58 3,58 9,76 7,46 4 4 2 1 1 2 2 2 3 3 This articleisprotected bycopyright.Allrightsreserved. Accepted Article ysteinase Adenosylhomoc Othermetabolic proteins isomerase e e methyltransferas homocysteine mate-- opteroyltrigluta methyltetrahydr 5- e reductoisomeras Ketol-acid carboxylase Biotin e 2 aminotransferas Alanine kinase 4 Adenylate 312 53 P32112 462 85 Q42662 63 O82043 58 O04983 60 Q9LDV4 27 Q08480 NAGVPTVPGSDG GLR (91.7) GLR(91.7) VASPAQAQEVHA (71.8) ATDVMIAGK (62.7) K VAVVCGYGDVG (64.3) DSAAVFAWK LVHGEPER (57.6) VVKLQEELDIDV (50.2) K GNASVPAMEMT (57.1) YLFAGVVDGR (68.9) FALESFWDGK (40) VNLAGHEEYIVR GSSSFNEAR (59.4) (52.5) LLQSTEEAVR (41.8) HIEFQVLADK (65.2) ATGAYSHSQGIK (122.1) QVLAEENQR ALVVINPGNPTG DEAMK(96.5) GELVSDDLVVGII GFILDGFPR (47.8) R (90.7) VLNFAIDDSILEE K (83.9) (83.9) K

homocysteine biosynthesis biosynthesis biosynthesis Aminoacid biosynthetic Amino acid Amino Nucleotide Fatty acid Fattyacid metabolic L-alanine catabolic process process process L- 13,49 20,78 12,98 7,86 5,68 5,23

3 3 4 3 3 4 4 2

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein ClpB1 protein protein protein 2 protein 90-1 Chaperone isomerase cis-trans Peptidyl prolyl Luminal binding Heat shock homolog Endoplasmin Chaperones dehydrogenase1 UDP-glucose6- nine synthase adenosylmethio S- alpha1 PhospholipaseD

P42730 101 101 P42730 18 P21569 9Z1 53 Q9FZE1 207 73 P24067 81 P27323 93 P35016 41 P49613 92 Q43007 NNPVLIGEPGVG ER (65.1) (65.1) ER VQLDSQPEEIDNL (55.7) TAVVEGLAQR (49.8) K GR (108.3) GR (108.3) VFFDMTVGGAPA (46.2) AADLTYWESAAR (38.4) LPIYEPGLEDVVK IETVGGVMTK DILLLDVAPLTLG (72.9)EK KENEGEVEEVDE (62.2) ELISNSSDALDK (54.1) ADLVNNLGTIAR (65.1) SGTSAFVEK (70.2) LGIIEDAANR (81) ELISNASDALDK VTNDKIAA (66.5) VHTVLISTQHDET (89.6) K TNMVMVFGEITT EIDR (93.1) AYLPVQELLNGE (60.5) VLMLVWDDR

adenosylmethio Protein Protein folding Protein Protein folding Protein folding Lipidcatabolic biosynthetic biosynthetic glucuronate Binding of of Binding unfolding process process process Protein Protein protein protein UDP- nine nine S- 14,66 21,39 3,88 8,73 7,38 7,74 8,45 6,87

2 2 4 3 3 4 4 3 3 3

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein S3a protein protein 1subunit protein peroxiredoxin peroxidase ribosomal40S 36 Q9SYT0 Annexin D1 alpha T-complex 14-3-3-like Regulatory proteins reductase corbate Monodehydroas 2-Cys 57 P45739 Catalase L-ascorbate Defense-related proteins 418 30 P49198 59 P28769 632 29 O65352 47 Q42711 28 Q6ER94 28 Q05431 (50.8) ELSNDFER (94.1) TGTDEGALTR TEVIGA (46.6) LDRPAEEAAPEA EDHSYR (62.2) VFEVSLADLQND AAELLK (80.3) EVGDGTTSVVIV (68.8) VLVELAELQDR (89.1) (89.1) DAGVIAGLNVAR (87.4) (56.9) DEEIDYFPSR VFFIR (104.1) EGNFDIVGNNFP (49.6) APGVQTPVIVR QAFDEAIAELDTL IEER(120) VVAAADGGEELT (48.5) K EAVAPYERPALS (74.1) K EIDDADQLVEAL DVTK(54.9) SGGLGDLNYPLIS IGR (77.1) EGVIQHSTINNLA IHIALR (49.7) FDAEQAHGANSG

Protein Protein folding Abiotic stressAbiotic Translation biotic biotic stress Abiotic and catabolism catabolism Oxidation- Signaling Hydrogen Hydrogen assembly reduction response response peroxide peroxide process 10,08 13,17 4,72 6,42 5,31 5,18 5,00 8,18

2 2 3 2 3 3 2 4 2 3

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein CBSX3 protein alphasubunit ATP synthase containing CBS domain- PIP1-3 PIP1-3 Aquaporin betasubunit ATP synthase Transportproteins 4A-3 initiation factor Eukaryotic 2factor Elongation 120 55 P18260 23 Q9LEV3 073 31 Q08733 59 Q1KXV2 47 Q9CAI7 94 Q9ASR1 NIFR (64.3) NIFR(64.3) NMSVIAHVDHGK (87) (87) TAIAIDTILNQK GR (88) ISNFYTNFQVDEI (62.3) AAELTTLLESR (64.1) VGDIMTEENK (57.3) (57.3) DSTLIMQLLR GEDSYK (101.3) SLGAAIIYNK (44.8) QPIGTSAQTDK (67.8) VPVGGATLGR GMDVIDTGAPLS SEEDR(80.5) ELQDIIAILGLDEL DVNEQDVLLFVD GK (96.7) GLDVIQQAQSGT R (95.4) MFVLDEADEMLS (68.4) K DSVVAGFQWAS (64.5) EGALAEENMR (65) (110.4) IAQEVAGDVR STLTDSLVAAAGI

ATP synthesis, homeostasis ion transport ion Translation Translation hydrolysis/ Cell Cell redox transport transport synthesis Water ATP 10,94 10,77 16,07 4,59 6,88 5,63

2 2 3 3 3 3 2 4

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein G3 protein 17 P04464 Calmodulin 42 Q96292 Actin-2 Cytoskeletalproteins deaminase Porphobilinogen 34 P83218 Pectinesterase 19 P29529 Oleosin seedstorage globulin 11S homolog cycle 48 protein Cell division Miscellaneousproteins chain alpha-2Tubulin 436 41 Q43316 56 P19084 90 P54774 50 Q6VAG0 NAPSIIFIDEIDSIA EEFVK (86.5) EADVDGDGQINY (61.5) DQDGFISAAELR (44.2) EITALAPSSMK (53.3) DAYVGDEAQSK (71.1) AGFAGDDAPR (61.8) (73.2) TDPNQNTGIVIQK (60) VGSDLSAFYR VK(63.1) QTAGSVPESLDY (72.6) K GTLQDAGEYAGQ (49.2) (49.2) ILSQPLADIGGK VIK (92.6) EGQVVVIPQNFA R (109) TNDNAMIANLAG DNELR(115.9) VQIVDNQGNSVF (67) EDLAALEK(48.2) EDAANNFAR DIGGLENVK (67) ETVVEVPNVSWE (78.2) PK

Storage protein Storage protein streaming, cell based process biosynthesis Microtubule modification Cytoplasmic Calcium ion Chlorophyll Cell cycle Cell Cell Cell wall binding growth 14,63 11,91 18,95 4,32 5,92 5,26 8,35 4,93

2 2 3 4 2 2 3 4 4 2

This articleisprotected bycopyright.Allrightsreserved. Accepted Article

R R (96.6) GSPLALAQAYET This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein abundance in salt-stressed roots over contr phosphate hydratase Aconitate Carbohydratemetabolism TableII of sunflowerofseedling dark-grown)(2-dayold, subje aaesnhs P81 6 AVITPVQTricarboxylic ATVLIETLPAVFQ 65 P08216 Malate synthase dehydrogenase Malate 64 P49297 Isocitrate lyase dehydrogenase Isocitrate dehydrogenase Glyceraldehyde-3- dehydrogenase 1 Alcohol Protein :Proteomic analysis (LC-MS/MS)of S-nitrosylated p Accession 450 98 Q42560 495 36 O48905 527 47 P50217 37 P25861 FGVTEFVNPK (77) 41 P14673 no. no. kDa (56.3) RGNDEVMAR FQR (84.6) SENAVQANMELE DLACMR (57.3) VLLQDFTGVPAVV (86.8) LDLTAEELTEEK (65.1) LNVQVSDVK (104.7) MELVDAAFPLLK (60.2) (60.2) WSGANYYDR (103.8) K TVQGGITSTAAMG (70.2) LIDDMVAYALK ADELK (79.5) EHYLNTEEFIDAV (58.6) IGINGFGR R (98) VPTVDVSVVDLTV (69.9) AGIALNDHFVK Peptide sequence (Ionscore) ol ones. cted to 120 mM NaCl stress.changeNaClFoldtorefers mM 120 to cted roteins from theroots (10,000g supernatant) Tricarboxylic Tricarboxylic Tricarboxylic Biological Biological Glyoxylate Oxidation- acid cycle acid cycle Glycolysis acidcycle process reduction reduction process process GO cycle change Fold Fold 0,34 0,14 0,06 0,19 0,15 0,09 0,32 change Log 2 Log fold fold -2 -2 -3 -3 -4 -2 -3 -3 -2 This articleisprotected bycopyright.Allrightsreserved. Accepted Article bisphosphate phosphogluconate enhancer1 protein Oxygen-evolving rnktls O05 80 O20250 Transketolase sferase 1 hydroxymethyltran Serine chain carboxylase large Ribulose se Phosphoribulokina kinase Phosphoglycerate dehydrogenase 6- 9F3 53 Q9FFR3 9Z5 57 Q9SZJ5 491 50 Q42961 814 28 P85194 478 54 P45738 45 P26302 NPNLASLVVDPDF GGER (60.2) DGIDYAAVTVQLP PAGGR (60.8) GGSTGYDNAVAL SSDGTIK (90.2) LTYTLDEIEGPLEV (65.9) MIINALNSGAK R (94.8) YGVELDGDGLGV (100.1) MNEILYELR (78.9) ELDYLVGAVSNPK DK(57.7) GVSLLLPSDVVIA VTTGPLC (100.7) TPGHPENFETPGIE (94.6) VTTTIGFGSPNK (69.5)EK LDESTGYIDYDQM MGTPALTSR (39.8) FIEK(76.9) EITLGFVDLLRDD (81.7) DLATEGNEIIR (95.7) K FEFQAMDTLDTD (60.8) ANDFDLMYEQVK (79.5) LPANLVQAQR AK(63.9) phosphatecycle Photorespiratio Photosynthesis Photosynthesis acid cycle Glycolysis phosphate phosphate Reductive Reductive pentose- pentose- pathway pathway fixation, fixation, Pentose Carbon cycle n n 0,13 0,20 0,18 0,33 0,23 0,26 0,04 -3 -3 -2 -2 -2 -2 -2 -2 -5 This articleisprotected bycopyright.Allrightsreserved. Accepted Article einase Adenosylhomocyst isomerase Triosephosphate alpha1 PhospholipaseD transisomerase Peptidyl cis-prolyl methyltransferase homocysteine eroyltriglutamate-- methyltetrahydropt 5- reductoisomerase Ketol-acid 58 O04983 carboxylase Biotin aminotransferase 2 Alanine Othermetabolic proteins Q9LDV 407 92 Q43007 85 Q42662 63 O82043 312 53 P32112 21 P48493 259 18 P21569 4 60 NAGVPTVPGSDGL (71.8) ATDVMIAGK (62.7) VAVVCGYGDVGK (64.3) DSAAVFAWK GLR(91.7) VASPAQAQEVHA (83.9) ALLNETNEFVGDK AYLPVQELLNGEE (60.5) VLMLVWDDR VHGEPER(57.6) VVKLQEELDIDVL (50.2) GNASVPAMEMTK (57.1) YLFAGVVDGR (68.9) FALESFWDGK (40) VNLAGHEEYIVR GSSSFNEAR(59.4) (65.2) ATGAYSHSQGIK VLAEENQR (122.1) ALVVINPGNPTGQ GR (108.3) GR (108.3) VFFDMTVGGAPA LQSTEEAVR (52.5) (41.8) HIEFQVLADK L-homocysteine Lipid catabolic Potein Potein folding biosynthesis biosynthesis biosynthesis biosynthetic Amino Amino acid Amino acid Glycolysis Fatty acid L-alanine catabolic process process process process process 0,38 0,07 0,14 0,07 0,20 0,26 0,18 0,59 -1 -1 -4 -3 -4 -4 -2 -2 -2 -1 This articleisprotected bycopyright.Allrightsreserved. Accepted Article bate reductase protein 2 nei 1 9Y0 36 Q9SYT0 Annexin D1 48 Q9ZPP1 Calreticulin e synthase adenosylmethionin S- ammonia-lyase Phenylalanine Monodehydroascor Defense-related proteins Luminal binding 90-1 Heat shock protein homolog Endoplasmin Chaperones 008 72 O04058 471 47 Q42711 463 41 P49613 233 81 P27323 93 P35016 207 73 P24067 (50.8) ELSNDFER (94.1) TGTDEGALTR KPEGYDDIPK (47.7 (55.6) LLSGDVDQK GIQTSEDYR (52.5) VTNDKIAA (66.5) VHTVLISTQHDET (89.6) TNMVMVFGEITTK IDR(93.1) R R (87.2) EINSVNDNPLINVS (48.5) EAVAPYERPALSK (74.1) EIDDADQLVEALK (72.9) K KENEGEVEEVDEE (62.2) ELISNSSDALDK (54.1) ADLVNNLGTIAR (65.1) SGTSAFVEK (70.2) LGIIEDAANR(81) ELISNASDALDK (89.1) (89.1) DAGVIAGLNVAR (87.4) ETVGGVMTK DILLLDVAPLTLGI

Cellular oxidantCellular adenosylmethio Protein Protein folding Protein Protein folding Protein folding Cinnamic acid detoxification biosynthetic biosynthetic Oxidation- Binding of of Binding assembly reduction reduction process process process process protein protein nine S-

0,07 0,07 0,08 0,10 0,04 0,07 0,06 0,11

-4 -4 -4 -4 -3 -3 -5 -4 -4 -3

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein S3a protein subunit alphasubunit ATP synthase 29 O65352 14-3-3-likeprotein dismutase(Mn) Superoxide subunit betasubunit ATP synthase Transportproteins 94 Q9ASR1 Elongation 2factor ribosomal40S Regulatory proteins Q1KXV Q9LYK 120 55 P18260 418 30 P49198 8 2 27 59 NIFR (64.3) NIFR(64.3) NMSVIAHVDHGK TK (64.2)TK LVVETTANQDPLV (87) (87) TAIAIDTILNQK R (88) ISNFYTNFQVDEIG (62.3) AAELTTLLESR (57.3) DSTLIMQLLR GEDSYK(101.3) QAFDEAIAELDTL TAEAVDILK (38.6) process process TAEAVDILK (38.6) PVGGATLGR (67.8) GMDVIDTGAPLSV SEEDR(80.5) ELQDIIAILGLDEL DVNEQDVLLFVD (68.4) DSVVAGFQWASK (64.5) EGALAEENMR (65) (110.4) AQEVAGDVR STLTDSLVAAAGII EVIGA (46.6) EVIGA(46.6) LDRPAEEAAPEAT DHSYR (62.2) VFEVSLADLQNDE

hydrolysis/synt ATPsynthesis, ion transport ion Translation Translation Superoxide metabolic Signaling process process hesis ATP

0,12 0,10 0,07 0,07 0,30 0,12

-3 -3 -3 -4 -4 -4 -2 -3

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein 48 protein 48 amdln 044 17 P04464 Calmodulin Actin-2 31 Q08733 PIP1-3Aquaporin deaminase Porphobilinogen 34 P83218 Pectinesterase G3storage protein globulin 11Sseed homolog Cell cycledivision Miscellaneousproteins chain alpha-2Tubulin Cytoskeletalproteins Q6VAG 436 41 Q43316 922 42 Q96292 104 56 P19084 90 P54774 0 50 NELR (115.9) NELR(115.9) NAPSIIFIDEIDSIAP EEFVK (86.5) EADVDGDGQINY (61.5) DQDGFISAAELR (44.2) EITALAPSSMK (53.3) DAYVGDEAQSK (71.1) AGFAGDDAPR (61.8) SLGAAIIYNK (44.8) QPIGTSAQTDK (96.6) GSPLALAQAYETR (49.2) ILSQPLADIGGK (67) EDLAALEK (48.2) EDAANNFAR (73.2) TDPNQNTGIVIQK VGSDLSAFYR (60) IK (92.6) EGQVVVIPQNFAV R (109) TNDNAMIANLAG VQIVDNQGNSVFD DIGGLENVK(67) ETVVEVPNVSWE (78.2) K

Water transport Storage protein streaming, cell based process based process modification Cytoplasmic biosynthesis Microtubule Calciumion Chlorophyll Chlorophyll Cell cycle Cell Cell wall Cell binding binding growth 0,17 0,09 0,19 0,11 0,31 0,24 0,47 0,15

-3 -3 -3 -2 -3 -3 -2 -2 -1 -3

This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein G3 protein synthaseCitrate cotyledonsOnly Malate synthase Both cotyledons rootsand Protein response to salt stress III Table itncroyae Chloroplast carboxylase Biotin Phospholipase D alpha 1 Cell membrane 214 214 Cell membrane PhospholipasealphaD 1 nolsi ooo Endoplasmic reticulum Endoplasminhomolog qaoi I13 elmmrn 10 VFYIVMQ 140 Cell membrane PIP1-3Aquaporin subunit alphasubunit T-complex1 protein Adenylate kinase Pectinesterase reductoisomerase Ketol-acid hprn rti lB Cytoplasm, nucleus, ChaperoneClpB1 protein dehydrogenase UDP-glucose6- B oancnann Mitochondria CBS domain-containing dikinase Pyruvate phosphate globulin 11Sseed storage Calmodulin Nvl -irsltd rtis n ufoe seedli sunflower S-nitrosylated in proteins Novel : Peroxisome localization Subcellular (lumen) Cytoplasm Chloroplast chloroplast ***MVKI 5 Cytoplasm, nucleus Chloroplast Protein Protein body mebrane Glyoxysome Cytoplasm,cell Cytoplasm Cell Cell wall

Position Position Sequence GPS-SNO (using 1.0) S-nitrosylationPrediction of sites

312 312 293 741 741 675 675 400 400 579 579 386 742 742 311 380 380 312 312 154 154 324 324 3 KGMYKEA 533 110 110 4 SAAISRG 147 7 AAVVLQD 170 6 AIQPSVF 36 21 32 8 ------g oyeos n ros nw eot) in reports) (new roots cotyledons and ng NGVEETI NRLANTP APVRSSI VRPELRQ KNYEPHR DLATMET HAEHEMN CMEILYE QRPESLE TMEVARD RALHDAL GVDVYTA CPSLSSS GKVPLIN MAAVTSS LARGQLR GTAVNIQ RQQQQNQ C C C C C C C C C C C C C C C QGRKIHA QGRKIHA C C C YIDGDEG YIDGDEG SRSESTQ SRSESTQ CIGAGYV C C C TMLHPGY TMLHPGY C C SMKFKVN SMKFKVN VQKVNRI AKTSMSS C YEDVASG YEDVASG SN***** SN***** MVFGNMG MVFGNMG VVVTSKY VVVTSKY LGAICGA WEDIFDA IGATTLE STAISAS STAISAS IVKRTLE MPEAYRE VMREVRK VMREVRK AGAVGAL DIHARRP STAAARH QLQNIEA KMFTRQC KMFTRQC This articleisprotected bycopyright.Allrightsreserved. Accepted Article protein CBSX3 protein

Oleosin Oil body membrane - - - - body Oil membrane Oleosin Calreticulin rootsOnly (lumen) Endoplasmic reticulum - - - -

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