Sodium hydrosulfide induces systemic thermotolerance to strawberry plants through transcriptional regulation of heat shock proteins and aquaporin

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1 Christou et l. BMC Plnt Biology 2014, 14:42 RESEARCH ARTICLE Open Aess Soium hyrosulfie inues systemi thermotolerne to strwerry plnts through trnsriptionl regultion of het shok proteins n quporin Anstsis Christou 1,3, Pngiot Filippou 2, George A Mngnris 2 n Vsileios Fotopoulos 2* Astrt Bkgroun: Temperture extremes represent n importnt limiting ftor to plnt growth n proutivity. The present stuy evlute the effet of hyroponi pretretment of strwerry (Frgri x nnss v. Cmros ) roots with n H 2 S onor, soium hyrosulfie (NHS; 100 μm for 48 h), on the response of plnts to ute het shok tretment (42 C, 8 h). Results: Het stress-inue phenotypi mge ws meliorte in NHS-pretrete plnts, whih mnge to preserve higher mximum photohemil PSII quntum yiels thn stresse plnts. Apprent mitigting effets of H 2 S pretretment were registere regring oxitive n nitrostive seonry stress, sine mlonilehye (MDA), H 2 O 2 n nitri oxie (NO) were quntifie in lower mounts thn in het-stresse plnts. In ition, NHS pretretment preserve sorte/glutthione homeostsis, s eviene y lower ASC n GSH pool reox isturnes n enhne trnsription of ASC (GDH) n GSH iosyntheti enzymes (GS, GCS), 8 h fter het stress imposition. Furthermore, NHS root pretretment resulte in inution of gene expression levels of n rry of protetive moleules, suh s enzymti ntioxints (APX, CAT, MnSOD, GR), het shok proteins (HSP70, HSP80, HSP90) n quporins (PIP). Conlusion: Overll, we propose tht H 2 S root pretretment tivtes oorinte network of het shok efense-relte pthwys t trnsriptionl level n systemilly protets strwerry plnts from het shok-inue mge. Keywors: Asori i, Het shok proteins, Hyrogen sulfie, Nitrostive stress, Oxitive stress, Priming, Thermotolerne, Frgri x nnss Bkgroun Threts of limte hnge n glol wrming rener het stress generl onern for the griulturl setor worlwie [1]. Plnts expose to high tempertures my experiene severe ellulr injury tht my le to ell eth within short perio [2]. The primry trgets of het shok injury in plnts re photosynthesis [3], wter sttus [4], ron ssimiltion proesses [5] n memrne stility [6]. At the ellulr level, het stress results to protein enturtion n ggregtion, inrese * Corresponene: vssilis.fotopoulos@ut..y 2 Deprtment of Agriulturl Sienes, Biotehnology n Foo Siene, Cyprus University of Tehnology, 3603 Lemesos, Cyprus Full list of uthor informtion is ville t the en of the rtile fluiity of memrne lipis, intivtion of enzymes in hloroplst lmell n mitohonri, inhiition of protein synthesis n seonry oxitive stress through the proution of retive oxygen speies (ROS) [7]. Consequently, plnts mnifest ifferent mehnisms for pttion n protetion in elevte tempertures. The initil het stress signl, proly pereive s the inrese of plsmlemm lipi ilyer fluiity [8], triggers ownstrem signling proesses for trnsriptionl regultion [9]. Up-regultion of mitogen tivte protein kinse (MAPK) trnsution pthwy through the inution of C 2+ influx [10], ROS signling n hormonl tivtion, s well s het shok protein (HSP)/ 2014 Christou et l.; liensee BioMe Centrl Lt. This is n Open Aess rtile istriute uner the terms of the Cretive Commons Attriution Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie the originl work is properly ite. The Cretive Commons Puli Domin Deition wiver ( pplies to the t me ville in this rtile, unless otherwise stte.

2 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 2 of 11 hperone signl trnsution pthwys, seems to e the key plyers in plnt trnsriptionl regultion uner het stress [11]. As result, mjor thermotolerne mehnisms, suh s the inution of ntioxint mhinery, umultion of het shok proteins, osmolytes n seonry metolite justments, re tivte, riving to ellulr homeostsis n repiring of mge proteins n memrnes [12]. Extensive plnt reeing efforts n more reent trnsgeni pprohes hve lrgely vlite tht het stress tolerne is multigeni trit [13]. In ition, enefits from trnsgeni pprohes hve een limite n hve not le to gronomilly improve rops for het tolerne uner fiel onitions [14]. Thus, onsierle ttention hs een evote in lleviting the etrimentl effets of high tempertures in plnts through the exogenous pplition of vrious priming gents. Exogenously pplie lium [7], sori i [15], sisi i [16], sliyli i [17], H 2 O 2 n NO [18] mnge to enhne thermotolerne of trete plnts. Furthermore, see pretretment with H 2 O 2 improve het tolerne of whet seelings through the llevition of oxitive mge n the up-regultion of stress proteins [19]. Sulfur-ontining efense ompouns (SDCs) re ruil for the survivl of plnts uner ioti n ioti stress [20]. Reent eviene revele the role of H 2 Sin orhestrting plnt responses to environmentl stimuli [21,22]. More preisely, exogenous pplition of H 2 S onor soium hyrosulfie (NHS) mnge to llevite hevy metl toxiity in germinting whet [23], uumer [24] n rley seelings [25]. Exogenous pplition of NHS ws foun to promote osmoti stress tolerne in sweet potto [26], soyen seelings [27] n strwerry plnts [28]. Furthermore, H 2 S promote root orgnogenesis [29] n ws lso foun to e involve in gur ell signling [30]. Reent reports showe tht NHS pretretment signifintly inrese het tolerne in too suspension ulture ells [31] n mize seelings [32,33], respetively. However, whether H 2 S priming oul trnsriptionlly inue systemi tivtion of plnt efense mehnisms for proviing tolerne to susequent exposure of het-sensitive soft fruit rop suh s strwerry remins lrgely unexplore. In the present stuy we hypothesize tht trnsient pre-exposure of strwerry plnt roots to H 2 S my inue systemi thermotolerne to susequent exposure of plnts to het shok tretment (42 C, 8 h). Therefore, the effets of root pretretment with H 2 S onor NHS on severl key omponents of stress tolerne mehnisms in the leves of strwerry plnts were investigte following omine physiologil, iohemil n moleulr pproh. As fr s is known, this is the first stuy eling with the employment of H 2 S for the protetion of fruit rop from temperture extremes, s well s the first report to implite the trnsriptionl regultion of HSPs n quporins in the response. Results Phenotypi oservtions Exposure of strwerry plnts to 42 C for 8 h resulte in mil wilting n lef urling (Figure 1C), while NHS root pretretment prior to stress exposure exhiite ovious mitigting effet, s eviene y the onservtion of plnt lef turgor n struture (Figure 1D). Nonstresse NHS-trete plnts (Figure 1B) isplye similr phenotype with ontrol plnts (Figure 1A), verifying the non-toxi effets of NHS t the onentrtion pplie. Hyrogen sulfie ontent Soium hyrosulfie root pretretment resulte in signifintly higher solute H 2 S ontent in strwerry leves ompre with ontrol smples, thus verifying its sttus s n H 2 S onor (t not shown). Het stress use mrke moultion in H 2 S lef ontent, mnifeste y signifint inrese fter 1, 4 n 8 h of exposure to 42 C ompre with ontrol smples. A signifint inrese in H 2 S ontent ws lso reore in NHS-pretrete plnts fter 1 h exposure to het stress, grully lowering to ontrol levels therefter (Figure 2). Chlorophyll fluoresene Apprent negtive effets of het stress on F v /F m rtio of strwerry plnts were registere; signifint reution on F v/ F m rtio ws mesure in plnts sujete to het stress for 8 h. Nevertheless, root pretretment with NHS prior to het exposure enle strwerry plnts sujete for 8 h to het shok tretment to mintin higher quntum effiieny of photosystem II ompre with plnts iretly expose to het stress (Figure 3). Cellulr mge effets Het stress enhne memrne mge, resulting in inrese MDA ontent (Figure 4). MDA ontent ws oule within 1 h of exposure to 42 C, following n inresing pttern therefter. Inversely, NHS root pretretment prior to stress exposure mnge to mitigte the levels of lipi peroxition, vlite y the lower MDA ontent ompre with non-pretrete stresse plnts. Hyrogen peroxie n nitri oxie ontent Progressive het shok use mrke inrese in oth H 2 O 2 n NO ontent, rehing mximum levels fter 8 h (Figure 5). However, lower rtes of H 2 O 2 umultion were reore in NHS-pretrete plnts susequently

3 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 3 of 11 A B C D Figure 1 Phenotypi effets of H 2 S onor NHS (100 μm) on strwerry plnts expose to het shok tretment (42 C) for 8 h. {(A) Control: pretrete with H 2 O n sujete to 23 C for 8 h, (B) NHS: pretrete with NHS n sujete to 23 C for 8 h, (C) Het: pretrete with H 2 O n sujete to 42 C for 8 h n (D) NHS Het: pretrete with NHS n sujete to 42 C for 8 h}. Re rrows inite wilte, urle leves. expose to progressive het shok tretment, leit signifintly higher thn ontrol, unstresse plnts (Figure 5A). In turn, no signifint moultion in NO ontent ws reore in NHS-trete plnts expose to het tretment, ompre with ontrol smples (Figure 5B). ASC n GSH ontent/reox stte Exposure to elevte temperture for 8 h resulte in signifint inrese in oth totl sorte n totl glutthione ontent, while NHS root pretretment prior to het exposure resulte to greter inrese in oth ntioxints pools. The inrese is likely ttriute to the inrese of oth reue n oxiize forms of sorte n glutthione. More preisely, het stress resulte in non-signifint inrese in ASC ontent, while signifint inrese in GSH lef ontent ws reore. NHS pretretment prior to het exposure resulte in greter inrese of oth reue forms (Figure 6A n D). On the other hn, sustntil inrese in oth DHA n GSSG ontent ws registere fter het exposure for 8 h. Interestingly, root pretretment with NHS mnge to meliorte further oxition of oth ntioxint moleules s eviene y the lower levels of DHA n GSSG ontent (Figure 6B n E). Overll, NHS root pretretment mnge to signifintly llevite oth sorte n glutthione reox stte isturnes ompre with non-pretrete stresse plnts (Figure 6C n F). In ition, totl sorte n glutthione pools of NHS-trete unstresse plnts were mintine in similr levels to those oserve in ontrol smples. Reltive H 2 S ontent 0.5 Control NHS Het NHS Exposure time to 42 o C (h) Figure 2 Effet of H 2 S onor NHS (100 μm) on H 2 S ontent of strwerry leves expose to het shok tretment (42 C) for 8 h, reltively to ontent t time point 0 h. Tretment ronyms re esrie in Figure 1 ption. Dt re mens ± SE of three replitions. Brs with ifferent letters re signifintly ifferent t p < 5. Het

4 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 4 of 11 F v /F m Control NHS Tretments Gene expression levels The reltive expression rtio of iverse set of speifi genes involve in ntioxint mhinery, ellulr reox regultion, signl trnsution n protein struture stility, ssye y quntittive rel-time RT-PCR, is presente in Figure 7. These inlue key enzymti ntioxint (ytosoli sorte peroxise, APX; tlse, CAT; mngnese superoxie ismutse, MnSOD; glutthione reutse, GR), sorte n glutthione iosynthesis ( L -gltose ehyrogense, GDH; glutmylysteine synthetse, GCS; glutthione synthetse, GS), NO iosynthesis (nitrte reutse, NR), trnsription ftor (ehyrtion-responsive element ining ftor, DREB), het shok proteins (HSP70, HSP80, HSP90) n quporin (PIP) genes. Overll, het stress lone or in omintion with NHS root pretretment signifintly Het NHS Het Figure 3 Effet of H 2 S onor NHS (100 μm) on hlorophyll fluoresene ( Fv / Fm ) of strwerry leves expose to 42 C for 8 h. Tretment ronyms re esrie in Figure 1 ption. Dt re mens ± SE of three replitions. Brs with ifferent letters re signifintly ifferent t p <5. Reltive MDA ontent Control NHS Het NHS Het Exposure time to 42 C (h) Figure 4 Effet of H 2 S onor NHS (100 μm) on mlonilehye (MDA) ontent of strwerry plnt lef tissue expose to het shok tretment (42 C) for 8 h, reltively to ontent t time point 0 h. Tretment ronyms re esrie in Figure 1 ption. Dt re mens ± SE of three replitions. Brs with ifferent letters re signifintly ifferent t p < 5. inue mrna expression levels of most exmine genes, eing highly epenent on the urtion of het exposure. The min trens oserve were the overll low levels of regultion of ll genes exmine in plnts trete solely with NHS, ompre with ontrol smples, exept for GDH 8 h fter the en of root inution (Figure 7). In turn, 1 h of het stress imposition revele signifint up-regultion of most genes exmine (with the exeptions of GR, NR, GCS n DREB), gretly meliorte fter progressive het stress exposure. Furthermore, het tretment use the rpi umultion of high levels of APX, PIP, HSP80 n HSP90 trnsripts s erly s 1 h fter het stress imposition (see Aitionl file 1: Tle S1). Nevertheless, the expression pttern of most genes exmine in non-trete plnt suffering het stress for 8 h ws generlly similr to respetive ontrols. Interestingly, the protetive effet of NHS root pretretment on het stress tolerne in terms of trnsriptionl inution of efense-relte genes ws reore 4 h fter stress imposition, rehing mximl inution levels for most genes exmine (exept for CAT, NR, GS n DREB) fter 8 h of het stress (Figure 7). Conversely, no signifint tivtion of efense-relte moleulr mhinery ws reore 1 h fter stress imposition in NHSpretrete n susequently stresse plnts. Disussion Het stress ffets ro spetrum of ellulr omponents n metolism, often using irreversile mge to plnt growth n evelopment. Despite the onstnt vnes eing me towrs unerstning plnt responses to tempertures extremes n improving thermotolerne, exmples of plnts with het tolerne through onventionl reeing n trnsgeni pprohes re limite n onense into lortory onitions [14]. Therefore, ompouns tht might result in the mitigtion of high temperture etrimentl effets oul potentilly e of gret importne. We hve reently postulte the priming effet of H 2 S in the llevition of slinity n non-ioni osmoti stress [28]. In the urrent stuy, n rry of physiologil, iohemil n moleulr pprohes provie novel eviene tht root pretretment with H 2 S onor NHS enhne thermotolerne of strwerry plnts susequently expose to temperture extremes, supporting the notion tht H 2 Sis key signling moleule in plnts. This seems to e of pivotl importne for strwerry ultivtion, sine strwerry growth n proutivity re foun to e gretly ffete y temperture extremes [34]. Furthermore, this stuy revele tht the role of NHS in lleviting het shok stress oul e ttriute to H 2 S, s the levels of enogenous H 2 S inrese following NHS pretretment n susequent stress imposition, in orne

5 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 5 of 11 (A) 4.0 Reltive H 2 O 2 ontent 3.0 (B) Reltive NO ontent Control 0.5 NHS Het NHS Het Exposure time to 42 C (h) Figure 5 Effet of H 2 S onor NHS (100 μm) on hyrogen peroxie (H 2 O 2 ) (A) n nitri oxie (NO) (B) ontent of strwerry plnt lef tissue expose to het shok tretment (42 C) for 8 h, reltively to ontent t time point 0 h. Tretment ronyms re esrie in Figure 1 ption. Dt re mens ± SE of three replitions. Brs with ifferent letters re signifintly ifferent t p < 5. with similr finings y Zhng et l. [27]. Importntly, Zhng et l. [26,29] reporte tht only H 2 S, n no other N + - or sulfur-ontining ompouns relese from NHS, hve protetive role uring ioti stresses, while similr finings were provie y Li et l. [32] in mize plnts uner het stress following n H 2 S inhiitor pproh. Photosystem II (PSII) is the most thermlly sensitive omponent of photosynthesis n its tivity is gretly reue or even prtilly stoppe uner high tempertures [35]. Our results re in greement with previous finings highlighting tht mximl photohemil effiieny (F v /F m ) in leves of severl plnt speies ws reue uner het stress onitions [7,36]. However, NHS pplition results in the llevition of het stress injuries to PSII tivity, s eviene y the higher F v /F m rtio preserve in pre-trete stresse strwerry plnts ompre with untrete stresse plnts (Figure 3). Furthermore, in orer to verify the role of NHS root pretretment in lleviting het shok erive oxitive mge, lipi peroxition n H 2 O 2 lef ontent were ssye. Results showe tht NHS mitigte het stress-inue MDA n H 2 O 2 ontent inrese in strwerry leves, thus suggesting tht oxitive mge to memrnes n other ell omponents ws reverse following H 2 S tretment (Figures 4 n 5). Our finings further support previous results showing tht NHS pretretment resulte in lower ontent of oth MDA n H 2 O 2 in stresse plnts. More preisely, NHS resulte to the onservtion of MDA n H 2 O 2 ontent inrese ginst osmoti stress [26,27], or uner high luminum onentrtion [23]. In ition, we reently provie eviene tht NHS root pretretment resulte in lower levels of MDA n H 2 O 2 in strwerry plnts suffering ioni n non-ioni osmoti stress [28]. Rpi (within 1 h) umultion of H 2 O 2 in het-stresse plnts lone, my suggest role for H 2 O 2 in triggering the erly expression of het-shok proteins in stresse plnts, s previously reporte [37,38]. Our finings re further supporte from the trnsript levels of mjor enzymti ntioxints (APX, CAT, MnSOD, GR), whih were foun to e inue in NHS-pretrete plnts in omprison with untrete ones, fter 4 n 8 h of exposure t 42 C. Trnsript levels re in line with previous

6 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 6 of 11 (A) (D) Reltive ASC ontent Control NHS Het NHS Het Reltive GSH ontent 0.5 (B) Reltive DHA ontent (C) (E) Reltive GSSG ontent (F) Reltive ASC reox stte 0.5 Reltive GSH reox stte Figure 6 Effet of H 2 S onor NHS (100 μm) on sorte n glutthione pool n reox stte of strwerry plnt lef tissue expose to het shok tretment (42 C) for 8 h, reltively to ontent t time point 0 h. {reue sorte (ASC) (A), ehyrosorte (DHA) (B), sorte reox stte (C), reue glutthione (GSH) (D), oxiize glutthione (GSSG) (E) n glutthione reox stte (F)}. Tretment ronyms re esrie in Figure 1 ption. Dt re mens ± SE of three replitions. oservtions in NHS-pretrete strwerry plnts expose to NCl n PEG-6000 tretment [28], s well s with enhne ntioxint enzymti tivities in NHStrete plnts uner mium [39], het [32] n rought [27] stress, s well s in NHS-trete sltstresse germinting sees [40], thus renering the pprent inution of ntioxint enzymes of prime importne for the enhne tolerne oserve. Besies ROS, NO n other NO-erive prouts, umultively lle retive nitrogen speies (RNS), my lso e overproue uner ioti stress onitions, using seonry nitrostive stress in plnts [41,42]. NO hs lso een shown to t in prllel with other signling moleules for regulting mny iologil proesses, inluing responses to ioti stresses [43-45]. Results inite tht NHS pretretment mnge to sustin NO ontent in levels similr to ontrol, s supporte y the non-signifint moultion of NR reltive expression ompre with ontrol smples. In ontrst, strwerry plnts experiening het shok tretment

7 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 7 of h 1 h 4 h 8 h Antioxints ASC/GSH iosynthesis Het Shok Proteins Aquporin Trnsription ftor RNS iosynthesis APX CAT GR Mn-SOD GDH GCS GS HSP70 HSP80 HSP90 PIP DREB NR > <-7.5 Fol hnge Figure 7 Het mp showing temporl expression pttern of selete genes ssoite with enzymti ntioxints, RNS iosynthesis, reox homeostsis, het shok proteins n quporins in leves of strwerry plnts uner non-stress n het stress onitions. Following root pre-tretment with 100 μμ NHS, the experimentl plnts were grown either uner norml (23 C) or extreme temperture (42 C) onitions for 8 h, s esrie in Figure 1. Reltive mrna unne ws evlute y rel-time RT-PCR using three iologil repets. Up-regultion is inite in green; own-regultion is inite in re. Digonl otte lines represent sttistilly signifint vlues t p < 5. A sle of olor intensity is presente s legen. Atul reltive expression t, otine from 3 inepenent replites, re shown in Aitionl file 1: Tle S1. exhiite mrke inrese in lef NO ontent, espite similr NR expression levels with ontrol smples. Suh negtive orreltion oul e ttriute to feek inhiition of NR [46], possily ue to NO toxiity [47]. However, the inrese in NO ontent in het-stresse plnts my e the key ftor riving to the rpi umultion of HSP trnsripts oserve. Xun et l. [48] reporte tht NO ts upstrem of AtCM3 in thermotolerne for the stimultion of DNA-ining tivity of het shok trnsription ftors n the umultion of HSPs. Interestingly, reent finings y Li et l. [32] suggeste tht H 2 S my e ownstrem signl moleule in NO-inue het tolerne of mize seelings, further supporting the interply etween retive speies towrs inue tolerne. Besie their prtiiption in oxitive proesses tht my le to ell mge, ROS prtiipte in reox sttese sensing mehnisms tht re tivte or mplifie in response to environmentl stimuli [49,50]. In prllel with ROS etoxifition, sorte n glutthione re moleules with regultory role, sine they prtiipte in the reox signling of the plnt ell uner ioti stress onitions [51]. In reent yers, severl stuies hve strengthene the notion tht high ASC/DHA n/ or GSH/GSSG rtios, sustine y inrese ASC n GSH or iminution of DHA n GSSG ellulr proution, my e the key element for enhne tolerne uring ioti stress exposure [52]. In the urrent stuy, we showe tht trnsient root exposure to H 2 S onor NHS prior to stress exposure mnge to sustin higher rtios of ASC/DHA n GSH/GSSG, ompre with non-pretrete het stresse plnts, s eviene y higher ASC n GSH reox sttes. Het stress resulte in iret n progressive inrese of reue n oxiize glutthione n oxiize sorte. However, NHS pretretment prior to het exposure resulte in n itionl inrese of reue sorte n glutthione n the iminution of their oxiize forms. The inue expression of GDH n GCS provies support for these oservtions, sine these enzymes ontriute to ASC n GSH iosynthesis n reox homeostsis, respetively. Our results re in greement with those of Shn et l. [53,54], who reporte tht the oserve inue tolerne in NHS-pretrete whet seelings uner wter n opper stress ws ttriute to the inrese tivity of enzymti ntioxints n ASC (Lgltono-1,4-ltone ehyrogense; GlLDH) n GSH iosynthesis (GCS) enzyme tivities, s well s to the inrese ontents of ASC, GSH, totl sorte n totl glutthione, in omprison with untrete stresse seelings. Chnges in genotypi expression leing to inrese synthesis of het shok proteins (HSPs) is known to e n erly n importnt ptive strtegy in ells tht re sujete to ll types of stresses [55]. The HSPs, rnging in moleulr mss from out 10 to 200 kd, hve een foun to umulte in gret mounts uring het stress in vrious ellulr strutures, suh s ell wll, hloroplsts, riosomes n mitohonri [2,56]. Their role in mintining ellulr homeostsis uner het stress, minly y ssisting the

8 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 8 of 11 orret foling of stress-umulte misfole proteins, preventing their ggregtion n promoting proteolyti egrtion of misfole or enture proteins, s well s in prtiipting in signl trnsution, hs een reently reviewe [57]. In the urrent stuy, HSPs ppere to e sustntilly umulte in ells immeitely fter het stress exposure, sine HSP70, HSP80 n HSP90 expression levels were foun to e signifintly inue, ompre with ontrol smples, s erly s fter 1 h of exposure to 42 C. The inrese expression levels of the exmine HSPs my e ttriute to the rpi umultion of H 2 O 2 n NO, whih funtion s signling moleules for the proution of HSPs, s lso previously reporte [37,38,48]. Inution of HSPs in stresse plnts ws lowere fter prolonge exposure to het tretment. Our results re in greement with previously reporte finings, se on trnsriptomi n proteomi nlyses, highlighting the importne of the erly umultion of HSPs for the quisition of thermotolerne in plnts experiening temperture extremes [57,58]. The role of HSP70 in Ariopsis thlin het shok responses n thermotolerne hs reently een eluite [59]. On the ontrry, NHS tretment prior to het stress inue the up-regultion of HSPs only fter 4 h of exposure to het shok onitions, proviing eviene for their possile ontriution to the oserve mitigtion of het stress evstting effets, me pprent fter 4 h of exposure to 42 C. The lower H 2 O 2 n NO ontents uring the erly stges of het exposure in NHS-pretrete plnts n e ttriute to lte umultion of HSPs (fter 4 h of stress imposition), sine oth tive moleules funtion in prllel with HSPs iosynthesis. In turn, limtion of Aloe ver plnts to less severe temperture extremes resulte in elevte expression of HSPs [60]. The moleulr n funtionl hrteriztion of quporins (PIPs), lss of memrne proteins tht filitte wter iffusion ross ell memrnes, hs revele the signifine of their regultion in response to verse environmentl stimuli [61,62]. In plnts, quporins re lolize in unne in the plsmlemm n the vuolr memrne [63]. Reent stuies she light to the possile role of quporins in ioti stress tolerne. Ayi et l. [62] onfirme the role of PIP1 n PIP2 in osmoti n slinity stress tolerne in urum whet. Furthermore, Iglesis-Aost et l. [64] reporte eline in PIP1 n PIP2 trnsript unne in the roots of rooli plnts uner inresing temperture, while Chen n Aror [65] highlighte the role of quporins (SoPIP2;1 n SoδTIP) uring the reovery of spinh leves from reversile freeze-thw injury. In the urrent stuy, gene expression nlysis revele tht PIP h the sme expression pttern s HSPs, suggesting tht het exposure use the erly inution (fter 1 h) of PIP expression, whih ws eliminte fter prolonge het stress. On the other hn, NHS pretretment-inue PIP up-regultion ws pprent fter 4 h exposure t 42 C (see Aitionl file 1: Tle S1). Conlusion A oorinte, trnsient inution of ntioxints, HSPs n quporin gene expression ws registere when plnts were expose to het shok tretment, whih ws eeslte s stress imposition progresse. The erly (1 h) n trnsient up-regultion of efense-relte genes in plnts expose iretly to het stress onitions seems to provie inequte signl for trnsriptionl regultion of efense pthwys, leing to wek het stress responses. On the ontrry, NHS root priming emonstrte elye (fter 4 h) ut prolonge (mximize fter 8 h) trnsriptionl tivtion of efense responses, resulting in quire thermotolerne vi the suffiient proution of protetive moleules suh s HSPs n ntioxints. The energyonsuming oorinte orhestrtion of severl inepenent pthwys is most likely fesile through inrese photosyntheti pity in NHS-trete plnts [66]. Overll, t reporte herein provie novel informtion for the improvement of rop tolerne to het stress n lens itionl support to the suggeste role of H 2 Sinplntresponses to environmentl stimuli. The urrent stte of knowlege in efining the ontriution of H 2 S in plnt tolerne mehnisms to ioti stress wrrnts further investigtion, inluing the potentil pplition of syntheti inhiitors of H 2 S iosynthesis (e.g [67]). Methos Plnt growth n stress tretments Forty-eight strwerry (v. Cmros ) plnts were grown in pet in greenhouse for six months n susequently trnsferre n grown hyroponilly in ontinuously erte hlf-strength Hogln nutrient solution in growth room with 16 h photoperio (250 μmol m -2 s -1 ), 23 C/ 20 C y/night temperture n 65% reltive humiity. After one week, roots of one hlf of the plnts were inute in eionize wter ontining n H 2 Sonor,soium hyrosulfie (NHS; 100 μm for 48 h; hnge every 12 h). At the en of the inution perio, plnts were trnsferre to hlf-strength Hogln nutrient solution. As result, plnts either pretrete or not with NHS n grown hyroponilly in ontinuously erte hlfstrength Hogln nutrient solution were simultneously expose (0 h, stress imposition) or not to elevte temperture tretment (42 C) for 8 h. Overll, strwerry plnts were sujete to 4 tretments, s presente in etil in Figure 1 n esrie shemtilly in Aitionl file 2: Figure S1. Eh tretment ws inepenently run in triplite, n eh replite onsiste of 4 iniviul plnts. Fully expne leves were smple immeitely fter the imposition of het stress tretment (0 h) n fter 1, 4 n 8 h of exposure to 42 C. Leves were flsh-frozen

9 Christou et l. BMC Plnt Biology 2014, 14:42 Pge 9 of 11 in liqui nitrogen n store t -80 C, unless otherwise stte. Physiologil n iohemil mesurements The rtio of vrile fluoresene to mximum fluoresene (F v/ F m ), representing the mximum photohemil effiieny of photosystem II (PSII), ws etermine using portle hlorophyll fluorometer (OptiSi OS-30p Chlorophyll Fluorometer, Opti-Sienes In, USA). Leves were inute in rk for 1 h prior to mesurements. The omprtive rtes of lipi peroxition in strwerry leves were ssye in terms of MDA ontent oring to Heth n Pker [68]. Retive speies quntifition Nitri oxie ontent ws iniretly ssye y mesuring nitrite (NO 2 ), stle n non-voltile rekown prout of NO reution, vi the Griess retion, s esrie y Zhou et l. [69]. Lef H 2 O 2 ontent ws ssye s esrie y Loreto n Velikov [70], while H 2 S ontent ws etermine following the methoology esrie y Nshef et l. [71]. Desriptions of ll retive speies quntifition protools followe n e foun in [28]. ASC n GSH ontent/reox stte Reue sorte (ASC) n ehyrosorte (oxiize sorte; DHA) were mesure oring to Foyer et l. [72]. Reox stte of sorte ws expresse s the rtio of ASC to totl sorte (ASC/ASC + DHA). The levels of reue glutthione (GSH) n oxiize glutthione (GSSG) were ssye s esrie y Griffith [73], while the glutthione reox stte ws expresse s the rtio of GSH to totl glutthione (GSH/ GSH + GSSG). RNA isoltion, DNA synthesis n gene expression nlysis Totl RNA from strwerry leves ws isolte following the protool esrie y [74]. The integrity of totl RNA ws heke spetrophotometrilly (A260/ A280) using NnoDrop Spetrophotometer ND-1000 (Lteh Interntionl Lt, Rigmer, UK), followe y gel eletrophoresis. For first strn DNA synthesis, 1 μg of totl RNA ws reverse-trnsrie using the Primesript 1 st Strn Synthesis kit, oring to mnufturer s instrutions (Tkr Bio In., Jpn). Quntittive reltime RT-PCR ws performe in finl volume of 10 μl, ontining 4 μl of ten-fol ilute first strn DNA, 0.5 μl of eh of the gene speifi primers (10 pm) n 5 μl of KAPA SYBR FAST qpcr supermix (Tkr Bio In). The initil enturtion stge ws t 95 C for 3 min, followe y 40 yles of mplifition (95 C for 30 s, T C for 45 s, n 72 C for 45 s) n finl elongtion stge t 72 C for 5 min. Gene mplifition yle ws followe y melting urve run, rrying out 61 yles with 0.5 C inrement etween 65 C - 95 C. PCR retions of eh tretment were performe in triplite with n iq5 rel-time PCR etetion system (Bio- R Lortories, In., Cliforni, USA). Fol hnge in RNA expression ws estimte using threshol yles. The housekeeping referene gene use ws 18S (T = 46 C) [75]. The sttistil nlysis of qrt-pcr results (pirwise fixe rellotion rnomiztion test) ws performe using the REST softwre, oring to Pfffl et l. [76]. The list of gene-speifi primers use is presente in Aitionl file 3: Tle S2. Sttistil nlysis Sttistil nlysis ws rrie out using the softwre pkge SPSS v17.0 (SPSS In., Chigo, USA) n the omprison of verges of eh tretment ws se on the nlysis of vrine (One-Wy ANOVA) oring to Dunn s multiple rnge test t signifine level 5% (P 5). Aitionl files Aitionl file 1: Tle S1. Effets of H 2 S onor NHS on the reltive mrna expression (fol hnge) of enzymti ntioxints, het shok proteins, quporins n enzymes involve in RNS iosynthesis, reox homeostsis n trnsription regultion, in leves of strwerry plnts uner non-stress n het shok onitions ompre with ontrols, s etermine y qrt-pcr. Aitionl file 2: Figure S1. Shemti representtion of the experimentl esign. Aitionl file 3: Tle S2. Oligonuleoties use s primers for rel-time RT-PCR. Arevitions APX: Cytosoli sorte peroxise; ASC: Reue sorte; CAT: Ctlse; DREB: Dehyrtion-responsive element ining ftor; GCS: Glutmylysteine synthetse; GDH: L-gltose ehyrogense; GR: Glutthione reutse; GS: Glutthione synthetse; GSH: Reue glutthione; H 2 S: Hyrogen sulfie; HSPs: Het shok proteins; MnSOD: Mngnese superoxie ismutse; NHS: Soium hyrosulfie; NO: Nitri oxie; NR: Nitrte reutse; PIP: Aquporin; RNS: Retive nitrogen speies; ROS: Retive oxygen speies. Competing interests The uthors elre tht they hve no ompeting interests. Authors ontriutions AC, GAM n VF esigne the stuy, while VF lso esigne the oligonuleotie primers use herein. AC performe the experiments n took re of the plnts. AC n PF rrie out the lortory work n t nlysis. AC, GAM n VF wrote the mnusript n prepre the figures. All uthors re n pprove the mnusript. Aknowlegements The uthors woul like to thnk Ms. Chrystll Antoniou n Ms. Ann Mlynrzyk for exellent tehnil ssistne, s well s Ms. Dine Aulh for lnguge eiting. This work ws prtly supporte y Cyprus University of Tehnology Internl Grnt EX032 to VF. The uthors woul lso like to knowlege support y the CUT Open Aess Author Fun.

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Nulei Ais Res 2002, 30:e36. oi: / Cite this rtile s: Christou et l.: Soium hyrosulfie inues systemi thermotolerne to strwerry plnts through trnsriptionl regultion of het shok proteins n quporin. BMC Plnt Biology :42. Sumit your next mnusript to BioMe Centrl n tke full vntge of: Convenient online sumission Thorough peer review No spe onstrints or olor figure hrges Immeite pulition on eptne Inlusion in PuMe, CAS, Sopus n Google Sholr Reserh whih is freely ville for reistriution Sumit your mnusript t