Proteomic analysis of glucohexaose induced resistance to downy mildew in Cucumis sativus

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1 AJCS 7(9): (2013) ISSN: Proteomic nlysis of glucohexose induced resistnce to downy mildew in Cucumis stivus Yuhn HA 1,#, Chunfei WU 1,#, Dwei ZHA 1, Leen Thung 3, Yng YU 1, Hiyn FAN 1,2,* 1 College of Bioscience nd Biotechnology, Shenyng Agriculturl University, Shenyng , PR Chin 2 Key Lortory of Protected Horticulture of Ministry of Eduction, Shenyng Agriculturl University, Shenyng , PR Chin 3 School of Food nd Agriculture Science, The University of Queenslnd, Brisne 4072, Austrli *Corresponding uthor: hyfn74@163.com #Co-first uthors with equl contriutions Astrct Glucohexose, s one of synthetic oligoscchrides, induces the resistnce response to protect plnts from pthogen infection y inducing the systemic cquired resistnce-like (SAR-like) response. To study the moleculr mechnism of glucohexose induced resistnce, we investigte the physiologicl, iochemicl nd proteomic chnges fter glucohexose tretment. The results shows cucumer plnts hd the highest protection level of 66.79% 48 h fter the third times of 10 μg ml -1 glucohexose tretment. Significnt increses in chlorophyll, photosynthetic rte, solule sugr, leve dry weight nd H 2 2 were oserved fter glucohexose tretment. Eighteen up-regulted proteins were identified y MALDI-TF/TF in glucohexose-treted plnts, predicted to e involved in photosynthesis, photorespirtion, oxidtive urst, trnscriptionl regultion, signl trnsduction nd pthogen defense processes. The identifiction of up-regulted proteins involved in photosynthetic processes is significnt finding which suggests tht oost in metolites is required for reprtition of resources towrds defense mechnisms. The proteins which responded to glucohexose lso included those ssocited with oxidtive urst response, such s APX nd isocitrte dehydrogense. More comprehensive studies out the link etween the moleculr mechnisms regulted y RS medited photosynthesis nd cucumer induced resistnce y glucohexose, re necessry in the future to roden our understnding of induced resistnce in plnts. Keywords: glucohexose; cucumer; induced resistnce; 2-DE; MALDI-TF/TF. Arevition: APX_cytosolic sco rte peroxidse; BRs_Brssinosteroids; G_glycolte oxidse; H 2 2 _hydrogen peroxide; HPR_NADH-dependent hydroxypyruvte reductse; HR_hypersensitive defense response; ICDH_NADP(+)-dependent isocitrte dehydrogense; PKC_protein kinse C; RS_rective oxygen species; RBP_RNA inding protein; RuBPCse_riulose isphosphte croxylse; shsp_ssmll het shock proteins. Introduction Cucumer (Cucumis stivus L.) is n importnt vegetle crop grown worldwide. Cucumer downy mildew, cused y Pseudoperonospor cuensis, is n importnt disese in most cucumer production res. It dversely ffects plnt growth nd cuses severe yield losses. Sprying the plnts with vrious chemicls, including oligoscchrides, induces resistnce to the fungus infection y ctivting pthogenesis-relted proteins. ligoscchrides re elieved to e elicitors of the plnt s nturl defense mechnisms. When pthogens such s cteri, fungi, nd viruses chllenge plnt cell, it develops complex defense mechnisms. Numerous studies hve shown tht the cell wll of plnts cn e digested into oligoscchrides y specific enzymes (Fry et l., 1993). These oligoscchrides ct s signling molecules to induce erly defense systems (Montesno et l., 2003). Glucohexose is synthetic oligoscchride which ws synthesized y Reserch Center for Eco-Environmentl Science, Chinese Acdemy of Science. The Structurl formuls showed in Fig. 1. Previously, we detected the ctivity of glucohexose nd nlyzed its resistnce to some plnts. The study hd shown tht glucohexose cn protect plnts from vrious pthogen, such s Erysiphe cichorcerum, Pseudomons syringe pv. Lchrymns, Botrytis cinere nd Xnthomons cmpestris pv. Vesictori, nd protects plnts from pthogen infection y inducing the systemic cquired resistnce-like (SAR-like) response (Li et l., 2001). To dte, only few studies investigted oligoscchrides elicitors. The existence of homologous elicitor-inding sites within plnt txonomic fmily my provide preliminry evidence for puttive evolutionry reltionships in pthogen perception mechnisms mong plnts (Cosio et l., 1996). A 75kD protein identified y ffinity leling represents functionl receptor for n N-ctylchitooligosccride elicitor (Ito et l., 1997). Highly conserved signling elements lso pper to e employed in elicitor signl trnsduction, such s G-proteins, clcium trnsients, ion chnnels, rective oxygen species, nitric oxide, protein kinses nd phosphtses, cyclic GMP, cyclic ADP riose nd ftty cid derivtives (Scheel, 1998). In ddition, ion-relted chnges specificlly in the efflux of N + nd K + ions might e closely ssocited with the signl trnsduction system for defense responses t the tissue level (Amno et l., 1997). Finlly, expression in soyen of yest cdna encoding the cinnmte 4-hydroxylse P450 induced the glyceollin iosynthesis pthwy (Schopter et l., 1998). Thus, lthough genetic 1242

2 studies hve identified some importnt components of resistnce pthwys, the mechnisms involved in the glucohexose induced SAR-like response in cucumer remin uncler. Bsed on the concept nd experimentl evidence tht mrna levels do not lwys correspond with protein levels (Gygi et l., 1999; Ideker et l., 2001), nd proteins ultimtely crry out the functions, so proteomic nlysis through protein profiling ecomes eqully importnt to understnd how genes/proteins re regulted. Plnt proteomics s tool to compre protein chnges hs rpidly developed over the lst severl yers nd hs provided lrge mount of informtion out the individul proteins involved in specific iologicl responses. However, there is very limited numer of pulished proteomic studies on cucumers (C. stivus L.) including: cucumer lef proteomics in response to Acienzolr-S-Methyl (ASM) (Sligrm et l., 2008); trichoderm-treted plnts ecoming more resistnt to pthogen ttcks (Segrr et l., 2007); the responses of cucumer seedlings to slt stress (Du et l., 2010); β-mylse isozymes in dehydrted cucumer cotyledons (Todk et l., 2007); phloem exudtes (Wlz wt l., 2004) nd xylem sp (Buhtz et l.,2004); nd the response of cucumer seedlings to cucumer powdery mildew fungus (Fn et l., 2009; Fn et l., 2009). As result, severl confirmed nd puttive defense-relted proteins were identified. To dte, however, no proteomic studies hve een undertken to revel protein undnce regulted y glucohexose. Here we investigted the effect of glucohexose induction on cucumer resistnce ginst P. cuensis. Differentil proteomics of the C. stivus L. cv. Shndong mici lef re reported to revel the metolic reprogrmming following glucohexose induced tretment nd discuss how it prevents dmge in cucumer plnts fter pthogen inocultion. We lso report the physiologicl nd iochemicl chnges in cucumer plnts s response to fungus infection. Results nd discussion Reltive efficcy of glucohexose medited resistnce to P. cuensis in cucumer Firstly, the reltive efficcy of the glucohexose tretments to protect cucumer from fungl infection ws determined. The dt showed tht the numer of glucohexose sprys hd significnt influence on cucumer resistnce to P. cuensis. As shown in Tle 1, the reltive efficcy of the glucohexose induced protective mechnisms grdully incresed with the numer of glucohexose tretments. The reltive efficcy of the tretment reched mximum of 65.40% fter three tretments with 10 μg.ml -1 glucohexose. As shown in Tle 2, the degree of resistnce lso incresed with the length of time etween tretments. In the first investigtion, the reltive efficcy of seven dy tretment intervls reched mximum of 66.49%, which ws higher thn tht for two or five dy tretment intervls (41.4% nd 20.64%, respectively). In the second investigtion, two dys fter the first investigtion, the reltive efficcy of the seven dy tretment intervls remined t 64.33%, ut the reltive efficcies of the two nd five dy tretment intervls declined to 19.28% nd 29.69%, respectively, implying tht the time intervl etween glucohexose pplictions hs strong effect on cucumer resistnce. The effect of the time elpsed etween tretment nd inocultion on induced resistnce ginst P.cuensis is shown in Fig. 2. The result shows tht cucumer plnts hd the highest protection level of 66.79% 48 h fter the lst glucohexose tretment. The level of resistnce declined grdully fter 48h, nd protection ginst P. cuensis ws reduced to only 11.11% t 144h fter the finl glucohexose tretment. The present study investigted the reltive efficcy of inducing resistnce to downy mildew fungus infection in cucumer y glucohexose tretment. This results indicted tht the concentrtion of glucohexose, the numer of glucohexose sprys nd the length of time etween tretments hve significnt influence on cucumer resistnce to P. cuensis. And the level of resistnce decline grdully followed the time elpsed fter glucohexose tretment. The most effective condition is three tretments with 10 μg.ml -1 glucohexose nd seven dy tretment intervls, we use this condition for studies elow. Physiologicl nd iochemicl chnges in response to glucohexose tretment In this study, significnt increse in totl chlorophyll content ws oserved in the glucohexose treted plnts (Tle 3), nd the photosynthetic rte ws lwys significntly higher thn the control (Fig.3). A drmtic decline in chlorophyll nd photosynthetic ctivity were descried for severl plnt species infected y oomycetes cusing downy mildew (Ingrm, 1981; Lindenthl et l., 2005), nd the chlorophyll content of cucumer ws positively correlted with resistnce to downy mildew (Shrm et l., 2004). Thus, tretment with 10 μg.ml -1 glucohexose ppers to elevte chlorophyll contents nd photosynthetic rte to compenste for the decrese cused y fungus infection. There ws no significnt chnge in seedling height, however, leve dry weight incresed in the induced compred to control plnts (Tle 3). Lower concentrtions of solule sugrs in downy mildew infected leves hve lso een reported (Mndl et l., 2009; Yun, 1993). An increse in solule crohydrte contents ws noted in glucohexose treted plnts (Tle 3). Thus, tretment with glucohexose prior to fungus inocultion my prevent decreses in the solule crohydrte content. These results indicte the potentil to enhnce tolernce of cucumer plnts to P. cuensis through induction of nturl resistnce metolites. Chnges in protein undnce in response to glucohexose tretment To elucidte the proteomic response to glucohexose tretment in downy mildew susceptile C. cucumis plnts, comprtive study of the cucumer lef proteome of un-inoculted nd 10 μg.ml -1 glucohexose treted plnts (proteins isolted 48 h fter tretment) ws performed. Representtive 2-DE gels re shown in Fig. 3. ver 800 protein spots per gel were reveled for ech tretment nd 45 protein spots showed expression chnges (>+2-fold or < 2-fold). Twenty five up-regulted spots were nlyzed y MALDI-TF/TF MS. Among these proteins, 18 were well mtched to proteins in the NCBInr dtse (Fig.4, Tle 4). Spots which showed up t the sme reltive positions in different gels were regrded s the sme protein. The differentilly expressed proteins identified could e divided into six functionl groups sed on homologies, consisting of proteins involved in photosynthesis (protein 3, 7, 12, 13, 15, 2, 9, 14 nd 16), photorespirtion (protein 2, 8, 11 nd 14), oxidtive urst (protein 4 nd 10), trnscriptionl regultion (protein 6 nd 18), signl trnsduction (protein 17) nd other pthogenesis-relted processes (protein 1 nd 5). Among the proteins identified, four re involved in light- 1243

3 Tle 1. The effect of the numer of glucohexose tretments on induced resistnce ginst P. cuensis Tretment Numer of First investigtion Second investigtion sprys Disese index Reltive efficcy* Disese index Reltive efficcy* Control μg.mL ±3.35c ± ± ± ±7.69e ±6.16d 1μg.mL ±3.61c ± ± ± ±3.86d ±4.58c Cucumer seedlings were treted once, twice, or three times with 1 μg.ml -1 nd 10 μg.ml -1 glucohexose, with 7 dys etween tretments. Deionized wter ws used to spry control plnts. *Dt re the men of independent mesurements of three replictes ± stndrd devition (SD). Vlues followed y different letters re significntly different t 0.05% level. H H H H H H H H H H H H H H H H H H H H Fig 1. Structure pttern of glucohexose. dependent rections of photosynthesis, including ATP synthse (protein 3), Chlorophyll - inding protein (protein 7 nd 12), oxygen-evolving complex protein (protein 15) nd Chromoplst-specific crotenoid-ssocited protein (protein 13). The ccumultion of these four proteins suggests tht glucohexose tretment up-regultes photosynthetic ctivity in cucumer leves (Fig.3). The chlorophyll - inding proteins re involved in hrvesting light energy nd trnsferring it to photochemicl rection centers (Green et l., 1991). The oxygen-evolving complex, long with mngnese, chloride nd clcium, ppers to form the simplest structure, involved in the photooxidtion of wter during the light rections of photosynthesis (Ghnotkis et l., 1987). The chromoplst-specific crotenoid-ssocited protein is component of the pigment complex in chromoplsts which is involved in chromoplst iogenesis nd crotenoid iosynthesis. During the first stges of photosynthesis, light energy is converted into chemicl energy, in the form of the energy-crriers ATP nd NADPH (Smirr et l., 1993). The ccumultion of Chlorophyll - inding proteins, oxygen-evolving complex proteins nd Chromoplst-specific crotenoid-ssocited proteins would enhnce the light rection nd trnsfer more photon energy to chemicl energy. The increse in ATP synthse my e n indiction tht the light rection hs een up-regulted to produce more energy to counter pthogen-induced stress. Three proteins were found to e involved in the drk rection of photosynthesis, including riulose isphosphte croxylse (RuBPCse; protein 2, 14), sedoheptulose-1,7-isphosphtse (protein 9) nd chloroplst ltex ldolse-like protein (protein 16). These proteins were up-regulted 48 h fter glucohexose tretment (Fig.3). RuBPCse is the primry enzyme of the cron fixtion pthwy in the chloroplst of C3 plnts. Sedoheptulose-1,7-isphosphtse nd the chloroplst ltex ldolse-like protein re two importnt enzymes in the Clvin cycle. Up-regultion of these three proteins my oost cron dioxide fixtion nd increse the plnt s cpcity to resist pthogen invsion. Downy mildew disese reduces chlorophyll sustntilly, resulting in severe disruptions to photosynthesis in cucumer nd significnt metolic chnges following P. cuensis infection hve een reported (Sun et l., 2009; Shrm et l., 2004). In the study, mintining reltively high chlorophyll content in glucohexose-treted plnts could ensure tht plnts cn efficiently cpture nd convert light energy, nd then improve photosynthetic rte. Proteomic nlysis of glucohexose treted plnts showed enhncement of severl proteins involved in the light nd drk rections of photosynthesis in the chloroplst. The revivl of the photosynthetic mchinery could e ensured even fter the initil chlorophyll degrdtion cused y the fungus infection. Rective xygen Species (RS) re continuously produced s yproducts of eroic metolism or in response to iotic nd iotic stresses. Furthermore, they re considerly more undnt t the site of ttempted invsion during the erly stges of plnt defense rections which include the oxidtive urst (Nnd et l., 2010). The extrcellulr hydrogen peroxide (H 2 2 ) generted during oxidtive urst, hs een shown to ply n essentil role in signling the development of the hypersensitive defense response (HR). Two processes induce rective oxygen genertion, eroic metolism nd oxidtion of NADPH. Protein 4, identified s n isocitrte dehydrogense in this study, is n importnt source of NADPH. Recently, study showed tht isocitrte dehydrogense ctivity incresed fter pthogen invsion (Cozzone, 2005). ne of the primry functions of NADP(+)-dependent isocitrte dehydrogense (ICDH) is the control of cytosolic nd mitochondril redox lnce nd the cellulr defense ginst oxidtive dmge 1244

4 Tle 2. The effect of intervls of 10μg.mL -1 glucohexose on cucumer resistnce ginst P. cuensis First investigtion Second investigtion Tretment Disese index Reltive efficcy* Disese index Reltive efficcy* 2d ± ±4.06 5d ±7.04c ±5.47c 7d ±7.57d ±4.46d Control Cucumer seedlings were spryed three times with 10 μg.ml -1 glucohexose with either two, five, or seven dys etween tretments. Deionized wter ws used to spry control plnts.* Dt re the men of independent mesurements of three replictes ± stndrd devition (SD). Vlues followed y different letters re significntly different t 0.05% level. d d c Fig 2. The effect of the time elpsed from finl tretment to inocultion on induced resistnce ginst P.cuensis. Cucumer seedlings were spryed with 10 μg.ml -1 glucohexose every 7 dys for 21 dys. Then, t 24 h, 48 h, 72 h, 96 h, 120 h nd 144 h fter the lst tretment, cucumer leves were chllenged with P. cuensis. Dt re the men of three independent mesurements ± stndrd devition shown y verticl error rs. (Lee et l., 2001). Protein 10, identified s cytosolic scorte peroxidse (APX), is the cytosolic H 2 2 -scvenging enzyme. In chloroplsts, H 2 2 is reduced y APX using scorte s n electron donor. xidized scorte is then eliminted y monodehydroscorte reductse in series of ctlytic rections known s the Hlliwell-Asd pthwy (Dvletov et l., 2005). In our study, the level of APX nd isocitrte dehydrogense incresed considerly following glucohexose tretment. Thus the production nd the scvenging of RS pper to e one of the importnt events which occur during glucohexose-induced systemic cquired resistnce in cucumer. To verify our conjecture tht the oxidtive urst is one of the mjor events which occurs during glucohexose-induced responses in cucumer plnts. The result shows tht H 2 2 concentrtion incresed intensively 5h fter glucohexose tretment (Fig.5). This result suggested glucohexose cn induce oxidtive urst in cucumer leves which looks worthy of further reserch. Two proteins involved in photorespirtion: NADHdependent hydroxypyruvte reductse (HPR; protein 8) nd glycolte oxidse (G; protein 11), were identified. HPR nd G re key enzymes in photorespirtion. Photorespirtion occurs when the C 2 levels inside lef ecome low, it enles the plnt to recruit C 2 molecules lost during photosynthesis (Somerville et l., 1980; Somerville, 2001). It is widely ccepted tht photosynthesis influences wide rnge of processes from ioenergetics, photosystem II function, nd cron metolism to nitrogen ssimiltion nd respirtion. In prticulr, H 2 2 minly comes from the photorespirtory pthwy in photosynthetic cells, photorespirtion mkes key contriution to cellulr redox homeostsis. Formerly H 2 2 ws considered s toxic molecule which cn cuses oxidtive stress, ut now it is recognized s mjor signling molecule in plnt stress responses, prticulrly fungus infection (Moreno et l., 2005; Foyer et l., 2005). Chlorophyll dmge cused y pthogenic ttck could e prevented y glucohexose tretment s stted ove, nd possily metolic reprogrmming in chloroplst led to chnges in redox stte. The hypothesis is supported from the result tht HPR nd glycolte oxidse which re involved in photorespirtion, showed up-regulted in glucohexose treted plnts. It hs een reported tht photorespirtion plys protective role ginst iotic stresses (Fujit et l., 2006; Noctor et l., 2002). Bsed on the dt presented here, we suggest tht some peroxisoml photorespirtory enzymes my e induced y glucohexose nd my provide protection ginst disese. Protein 6 ws identified s RNA inding protein (RBP). RBPs ply mjor roles in post-trnscriptionl control of RNAs, which is mjor wy to regulte ptterns of gene expression during development (Lee et l., 2006). Although it ws not cler which gene encoded the RNA inding protein identified, it my ctivte the trnscription of n importnt gene involved in defense responses in cucumer. Protein 18 hs 100% identity with retrotrnsposon protein identified s puttive polyprotein. Plnt retrotrnsposons re lrgely quiescent during development ut re ctivted y vrious stresses including pthogen ttck, wounding nd under cell culture conditions (Wessler, 1996), it suggests tht retrotrnsposons could e stress-induced genertors of genomic diversity. In this study, we found tht the retrotrns- 1245

5 Time Tle 3. Effects of glucohexose on height, lef dry weight, totl chlorophyll nd solule crohydrte of cucumer seedlings Solule crohydrte Height Dry weight Totl chlorophyll Tretment cm* % chnge g cm -2 * % chnge mg g -1 fw* % chnge mg g 1 fw* % chnge Control 9.85± ± ± ± d glucohexose 9.93± ± ± ± Control 15.58± ± ± ± d 15.82± ± glucohexose 0.063± ±0.08 Control 24.95± ± ± ± d glucohexose 25.00± ± ± ± The first leves of cucumer seedlings were treted with 10 μg.ml -1 glucohexose three times every seven dys. Experiments were conducted fter 7d of ech glucohexose tretment. 1-7d : the 7 th dy fter the first tretment, 2-7d: the 7 th dy fter the second tretment, 3-7d: the 7 th dy fter the third tretment. *Dt re the men of independent mesurements of three replictes ± stndrd devition (SD). Vlues followed y different letters re significntly different t 0.05% level. -1 ṣ -2 lṃ o m µ 2 C control glucohexose Fig 3. Effects of glucohexose on the rtes of photosynthesis in cucumer seedlings. The first leves of cucumer seedlings were spryed with 10 μg.ml -1 glucohexose every seven Time dys fter for glucohexose 21 dys. Experiments tretment were conducted fter 1d nd 7d of ech glucohexose tretment. 1-1d: the 1 st dy fter the first tretment, 1-7d : the 7 th dy fter the first tretment, 2-1d : the 1 st dy fter the second tretment, 2-7d: the 7 th dy fter the second tretment, 3-1d : the 1 st dy fter the third tretment, 3-7d: the 7 th dy fter the third tretment. Dt re the men of three independent mesurements ± stndrd devition shown y verticl error rs. poson protein identified ws enhnced 48 h fter glucohexose tretment when defense-relted genes might strt to e expressed. Protein 17 ppers to e protein kinse C (PKC) due to the presence of this functionl domin. PKC is fmily of enzymes involved in controlling the function of other proteins through the phosphoryltion of hydroxyl groups on their serine nd threonine mino cid residues (Stone et l., 1990). An increse in the level of PKC following glucohexose tretment suggests tht it is plying role in controlling resistnt proteins in the plnt through post-trnsltionl modifiction. Protein 1 ws identified s lef senescence protein. An overlp in gene expression etween plnt defense responses nd the lef senescence processes hs een reported (Quirino et l., 1999). ur results suggest tht if lef senescence proteins re up-regulted s result of glucohexose tretment, then the plnt defense response might hve een simultneously ctivted through cross-tlks with pthwys involved in senescence. Protein 5 ws the hypotheticl protein si_19374, nd it hd high similrity (97%) with chloroplst het shock protein 70 (gi ). Smll het shock proteins (shsps) re diverse group of het induced proteins tht re especilly undnt in plnts nd re known to e induced in response to short-term stress. HSP70 nd relted genes re involved in protein folding, modultion of signl trnsducers nd controlling the iologicl ctivity of folded regultory proteins, fcilitting refolding nd proteolytic degrdtion of non-ntive proteins. HSP70 my ply key role in disese resistnce y cting s co-chperones ssocited with stiliztion of Rx protein levels (Lee et l., 2000). ur findings suggest tht high undnce of proteins relted to light-dependent rection nd drk rection of photosynthesis provides the plnt with incresed primry metolites to rellocte to secondry metolism, it is sme s the report on SA induced resistnce to Mungen Yellow Mosic Indi Virus in Vign mungo (Kundu et l., 2011). The undnce of RS relted proteins, s oserved in this study, demonstrtes tht the SAR my e induced y RS genertion fter glucohexose tretment. Involvement of photorespirtion in glucohexose medited resistnce to downy mildew is indicted y the undnce of HPR nd glycolte oxidse. H 2 2 is generted s y-product of photorespirtion nd is recognized s signling molecule to trigger defense response. High undnce of severl proteins 1246

6 Score NM/SC c Tle 4. Glucohexose induced cucumer lef proteins identified y MALDI MS nd MS/MS nlysis. Spot No. NCBI Accession Protein Nme [Species] MW/ pi MW/ pi No. theor. oser. 1 gi hypotheticl protein LC_s11g / / /16 [ryz stiv Jponic Group] 2 gi riulose-1,5-isphosphte croxylse/oxygense lrge suunit [Borneosicyos simplex] 51370/ / /44 3 gi ATP synthse suunit lph,chloroplstic 55405/ / /40 [Cucumis stivus] 4 gi isocitrte dehydrogense [Cucumis stivus] 46432/ / /39 5 gi hypotheticl protein si_19374 [ryz 69347/ / /12 stiv Indic Group] 6 gi RNA inding protein, puttive [Ricinus 65723/ / /30 communis] 7 gi LHCII type I chlorophyll /-inding 27950/ / /27 protein [Vign rdit] 8 gi NADH-dependent hydroxypyruvte 41908/ / /52 reductse [Cucumis stivus] 9 gi sedoheptulose-1,7-isphosphtse 42532/ / /40 [Cucumis stivus] 10 gi cytosolic scorte peroxidse[cucumis 27549/ / /45 stivus] 11 gi glycolte oxidse [Pnx ginseng] 20607/ / /39 12 gi LHCII type I CAB / / /100 [Populus euphrtic] 13 gi Chromoplst-specific crotenoid / / /28 ssocited protein [Cucumis stivus] 14 gi Riulose isphosphte croxylse smll 21392/ / /51 chin 15 gi oxygen-evolving complex protein 1 [ryz 26603/ / /34 stiv] 16 gi chloroplst ltex ldolse-like protein 34016/ / /40 [Mnihot esculent] 17 gi predicted protein 48954/ / /27 [Populus trichocrp] 18 gi puttive polyprotein [ryz stiv Jponic Group] / / /11 Numers correspond to the 2-DE gels shown in Figs. 3. Theoreticl nd oserved MW(kD) nd pi vlues. c Numer of mss vlues mtched(nm) nd the percentge of sequence coverge (SC). involved in signl trnsduction, trnscriptionl regultion nd plnt defense responses my hve contriuted further to reduce the susceptiility of glucohexose treted cucumer plnts compred to the untreted plnts. verll, our results suggest tht the enhncement of photosynthesis nd production nd scvenging of RS re the mjor events which occur during glucohexose induced systemic resistnce cquired in cucumer. H 2 2 hs een found to enhnce plnt photosynthesis (zki et l., 2009), elevted H 2 2 levels resulting from enhnced NADPH oxidse ctivity re involved in the BR-induced stress tolernce nd H 2 2 plys key role in BRs induced photosynthesis (Jing et l., 2012). It is possile tht glucohexose cn ctivte the continuous production of H 2 2, nd glucohexose-induced H 2 2 my induce chnges in expression of photosynthetic genes. Further investigtions of the link etween the moleculr mechnisms tht RS medited photosynthesis regulte nd glucohexose induced resistnce could roden our understnding of induced resistnce in plnts. Mterils nd methods Mterils C. stivus L. cv. Shndong mici, cucumer cultivr known for its susceptiility to P. cuensis, ws grown in humus pots (Pet moss: vermiculite=1:2) in the greenhouse for ll experiments. Glucohexose ws synthesized t the Reserch Center for Eco-Environmentl Science, Chinese Acdemy of Science. Following glucohexose tretment, plnts were inoculted with P. cuensis, the most importnt pthologicl fungus of cucumer. Glucohexose tretments To determine the effect of the numer of glucohexose tretments on cucumer resistnce, smples consisting of 30 one-month-old seedlings were treted once, twice, or three times with 1 μg.ml -1 nd 10 μg.ml -1 glucohexose, with 7 dys etween tretments. 1247

7 Fig 4. Representtive 2-DE gels nd close-up views of gel regions showing differences in protein undnce following glucohexose tretment compred to the control. Identified glucohexose induced proteins re indicted y circles nd numers. These numers on the 2D-gel correspond to the spot numer in Tle

8 The whole plnt ws spryed until runoff ws seen. Deionized wter ws used to spry control plnts. n the 2nd dy fter the finl tretment, cucumer leves were chllenged with suspension of conidi/ml of P. cuensis. The first disese index ws determined when the control groups were oviously infected nd second investigtion ws crried out two dys lter. To determine the effect of the intervl etween two glucohexose tretments on resistnce, 30 one-month-old seedlings were spryed three times with 10 μg.ml -1 glucohexose with either two, five, or seven dys etween tretments. n the 2 nd dy fter the finl tretment, cucumer leves were chllenged s ove. To investigte the time post-tretment when glucohexose confers the most resistnce, 30 one-month-old seedlings were spryed with 10 μg.ml -1 glucohexose every 7 dys for 21 dys. Then, t 1d, 2d, 3d, 4d, 5d nd 6dy fter the lst tretment, cucumer leves were chllenged s ove. Disese symptoms The disese progression of cucumer downy mildew ws estimted on the sis of the severity of lef scs s follows: Grde 0: no scs oserved; Grde 1: less thn 1/10 of lef infected; Grde 3: 1/10-1/4 of lef infected; Grde 5: 1/4-1/2 of lef infected; Grde 7:1/2-3/4 of lef infected; Grde 9: more thn 3/4 of lef infected. Disese index= 100 (no. of disesed leves of ech grde disese grde) / (totl no. leves 9) Reltive efficcy (%)= (disese index of control disese index of treted plnt) / disese index of control 100 Physiologicl nd iochemicl experiments Physiologicl nd iochemicl experiments were conducted one nd seven dys fter ech glucohexose tretment, the first leves were used for experiments. Totl chlorophyll, chlorophyll nd chlorophyll were determined spectrophotometriclly. Leves were extrcted in ethnol nd cetone (v/v=1:1) nd centrifuged t 4000 rpm for 10 min. After suitle dilution, the extinction coefficient of the superntnt ws mesured t wvelengths of 645 nd 663 nm using Hitchi U-3900 spectrophotometer (Jpnese). The solule crohydrte content ws determined in the queous solution with nthrone sulfuric cid regent. The rtes of photosynthetic were determined using Beckmn 865 Anlyzer (USA). The intrcellulr ccumultion of H 2 2 ws determined using the fluorescent proes H 2 DCFDA. Briefly, freshly cut, cucumer lef epidermis were incuted with solution contining Tris uffer (10mM Tris, 50mM KCl, ph 7.2). nd 50 mm H 2 DCFDA. The tissues were incuted for 20 min t room temperture in the drk nd fluorescence ws mesured with excittion wvelength 488 nm nd emission wvelength 525 nm y fluorescence spectrophotometer. Control groups were sujected to the sme mnipultion, except for the tretment with lef epidermis. Protein extrction Cucumer leves were treted every seven dys with 10 μg.ml -1 glucohexose for 21 dys. The first leves were used for protein extrction 48 h fter the finl tretment. Cucumer leves were ground in liquid nitrogen in prechilled mortr, nd then finely ground powder ws collected in 50 ml Flcon tue. 5 ml of 2% w/v DTT ws dded to 1 g of ground tissue. The homogente ws Fig 5. Mesurement of H 2 2 fter glucohexose tretment in cucumer leves. P mens the leves treted with 10μg.mL -1 glucohexose nd CK is the control group. The x-xis represents time fter tretment of glucohexose to the leves. Dt re the men of three independent mesurements ± stndrd devition shown y verticl error rs. centrifuged for 30 min t 1000 rpm nd the superntnt ws removed to new tue. The proteins in the superntnt were precipitted with cold 80% cetone t -20 for 12 hours, then centrifuged t rpm for 30 min. The pellet of precipitted proteins nd deris ws wshed three times with 5 ml of cold 80% cetone. After centrifugtion t rpm for 20 min, pellets were ir dried nd stored t -80. Two-dimensionl gel electrophoresis nd Imge nlysis 1 ml of lysis uffer (9.5 M Ure, 2 mol L -1 Thioure, 1% TBP, 1% DTT, 4% CHAPS, 1% Cock til, 0.001% Bromophenol lue, 2% IPG uffer) ws dded to the 400 mg pellets. The protein concentrtion of the finl superntnt ws mesured using BSA s the stndrd ccording to the Brdford method. Smples were used to rehydrte IEF strips, 24 cm in length with ph rnges of 4-7. Rehydrtion ws done in rehydrtion try t 22 under lyer of minerl oil. Strips were then ssemled onto IEF nd focused for 1 h t 250 V, 1 h t 500 V, 1 h t 1000 V, 4 h t V, nd then holding t 10000V until Vh ws reched. The current ws monitored nd did not exceed 50mA/strip throughout the run. Before loding in the second dimension the strips were equilirted for 2 10 min in 2 120mL equilirtion uffer contining 50 mm Tris-HCl (ph 8.8), 6 M Ure, 30% Glycerol, 2% SDS. DTT (10 mg/ml) ws dded to the first equilirted uffer nd iodocetmide (5 mg/ml) ws dded to the second equilirtion. For the second dimension, the strips were loded onto second dimension 12.5% SDS-PAGE gels. The gels were silver-stined ccording to Yn et l. (2000) with modifictions. The silver-stined gels were digitlized with UMAX Power Look 2100XL (Mxium Tech.) nd nlyzed using PDQuest Advnced 2-D Anlysis softwre, version 8.0.1(Bio-Rd). nly spots present in ech of the three replicte gels of oth groups were considered for susequent nlysis. Quntity chnges were estimted for the spots etween inner limit (±2-fold) nd outer limits (>+2-fold or < 2-fold). Selected protein spots were sujected to in-gel digestion for identifiction y MALDI-TF MS nd MS/MS. 1249

9 MALDI-TF MS nd MS/MS nlysis nd dtse serch Interesting spots were excised from gels with pipette tips nd plced in Eppendorf tues nd wshed three times with milliq wter. Then the gel ws wshed with 100 μl of 100 mm NH 4 HC 3 for 5 min, followed y 100 μl of Acetonitrile for 10 min, until n opque suspension ws formed. After ir drying t for min, the gel plugs were rehydrted with 10 ng/μl of trypin solution in digest uffer contining 50 mm NH 4 HN 3. Peptides were extrcted with 20 μl of 100 mm NH 4 HC 3, followed y 20 μl of 50% ACN/0.5% TFA t lest twice nd once with 10 μl of ACN. The extrcted peptides were purified using ZipTip C18-microcolumns (Millipore), ccording to the mnufcturer s instructions. Protein MS ws conducted on 4700 MALDI-TF mss spectrometer (Applied Biosystems). The digested protein smples were mixed (1:1 v/v) with sturted solution of recrystllized CHCA mtrix dissolved in 0.1% TFA/50%, then spotted on MALDI plte. Dt Interprettion nd utomted dtse serching were crried out using the GPS Explorer Softwre (Applied Biosystems) nd the MASCT progrm (Mtrix) respectively. Comined MS-MS/MS serches were conducted with the following criteri: NCBInr dtse ( ll entries; MS/MS mss tolernce of 0.15 D; Crmidomethyl-Cys (vrile modifiction). Clcultion y the softwre ws used s criteri for correct identifiction. Sttisticl nlysis All these experiments were repeted three times, nd 30 plnts were used ech tretment. Its result descrie y verge men±stndrd error. Tukey s test ws used for testing the men divergence etween tretments y using the SPSS sttisticl softwre pckge. Conclusion The present study investigted the reltive efficcy of inducing resistnce to infection y downy mildew fungus in cucumer y tretment with the glucohexose. Tretment of glucohexose 48 h prior to inocultion with P. cuensis decresed disese symptoms in concentrtion nd intervl-dependnt mnner. We oserved tht glucohexose tretment induced chnges in energy metolism with increses in chlorophyll levels nd the rtes of photosynthesis s well s the solule crohydrte contents of leves. Comprtive nlysis of the C. stivus L. cv. Shndong mici lef proteome etween non-inoculted control plnts nd 10 μg.ml -1 glucohexose treted plnts showed reltive chnges in undnces of severl proteins. The predicted functionl nnottions of the identified proteins suggested their involvement in photosynthesis, photorespirtion, oxidtive urst, trnscriptionl regultion, signl trnsduction nd pthogen defense processes. xidtive urst nd photosynthesis relted proteins indicted importnt moleculr mechnisms need further investigtion. Acknowledgements This work ws supported y the Ntionl Nture Science Foundtion of Chin (Nos ), the University utstnding Young Scholrs Growth Project of Lioning Province (LJQ ) nd the Science nd Technology Progrm of Shenyng City (F ). 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