Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na+/H+Antiport Activity of Three Malus

Available online at www. sciencedirect.comJournal of Integrative Agriculture2014, 13(6): 1276- 1283。ScienceDirectJune 2014RESEARCH ARTICLEEffects of NaCl and lso-Osmotic Polyethylene Glycol Stress on Na+/H+Antiport Activity of Three Malus species with Different Salt ToleranceYANG Hong -bing' , DONG Chun-hai, XU Xue feng, WANG Yi2 and HAN Zhen-hai?'Key Lab of Plant Biotechnology in Universities of Shandong/ollege of Life Sciences, Qingdao Agriculural University, Qingdao 266109,P.R.China'Stress Plhysiology and Molecular Biology Laboratory of Fruit Trees, China Agricultural University, Bejing 100193, P.R.ChinaAbstractSalt stress contains osmotic and ionic stress, while iso-osmotic polyethylene glycol (PEG) has only osmotic stress. This studyaimed to compare the different effects on the activity of H-ATPase, proton pump and Na+/H+ antiport in Malus seedlings betweenosmotic and ionic stress. Species of salt tolerant Malus zumi, middle salt tolerant Malus xiaojinensis and salt sensitive Malusbaccata were used as experimental materials. Malus seedlings were treated with NaCl and iso-osmotic PEG stress. The activityof H+-ATPase, proton pump and Na+/H+ antiport of plasmolemma and tonoplast in Malus seedlings were obviously increasedunder salt stress, and those in salt-tolerant species increased more. Under the same NaCl concentration, the activity of H+-ATPase, proton pump and Nat/H+ antiport of plasmolemma and tonoplast in salt-tolerant species were all obviously higher thanthose in salt- sensitive one. Higher Na+/H+ antiport activity of plasmolemma and tonoplast in salt. tolerant species could help toextrude and compartmentalize sodium in roots under salt stress. The ascent rate of activity of H+- ATPase, proton pump and Na+/H+ antiport in Malus seedlings under the three salt concentration stress was all obviously higher than that under the iso-osmoticPEG stress. It indicated that the sodium ion effect had more stimulation on the activity of H*-ATPase, proton pump and Na+/H+antiport in salt-tolerant species, and salt-tolerant species has higher capability of sodium extrusion and compartmentalization inroots and is therefore more salt tolerant.Key words: Malus, NaCl, polyethylene glycol (PEG), Na+/H+ antiport activity, salt toleranceor compartmentalize sodium of cytoplasm under saltstress and enhanced the salt tolerance of cell (Littge andINTRODUCTIONRatajczak 1997; Mitsui et al.2004a, b). Du et al. (2010)cloned a vacuolar Na+/H+ antiporter gene from ZoysiaNa+/H+ antiporters were found from the plasmolemma japonica (ZjNHX1), and found that the vacuolar Na+/as well as the organelle membranes of cells in species of H+ antiporter played an important role in ion homeosta-bacteria and higher plants (Blumwald 2000; Counillonsis and salt tolerance and in Zoysia japonica. Fukudaand Pouyssegur 2000; Padan et al. 2001; Brett et al. et al. (201 1) cloned a vacuolar Na*/H+ antiporter gene2005). Nat exclusion of cell was mainly controlled by(OsNHX1) from rice and found that the expression ofplasmolemma and tonoplast. Through Na+/H+ antiport of OsNHX1 was upregulated by salt stress. The activity ofplasmolemma and tonoplast, cell could extrude sodiumH+-ATPase and proton pump of plasmolemma (WilsonReceived 28 May, 2013 Accepted 7 August, 2013中国煤化工YANG Hong-bing, Tel: +86-532-88030995. E-mail: hbyang@qau.edu.cn; Correspondence HAN Zhen-hai, Tel:.YHCN M H G-edu.cn◎2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(13)60627-9Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na /H* Antiport Activity of Three Malus species with Different1277and Shannon 1995) and tonoplast (Liu et al. 2008) were on salt damage and salt tolerant mechanism in plants.all obviously increased under salt stress; moreover, thatof tonoplast were also increased in some degree underRESULTSwater osmotic stress (Xia et al.2000). Silvaet al. (2010)found that the tonoplast Na+/H+ exchange system ofPopulus euphratica was upregulated and the tonoplastEffects of NaCI and iso-osmotic PEG stressH*-ATPase activity was increased by salt stress. Theon plasmolemma H+-ATPase activity of threeincrease of activity of H+- ATPase and proton pump couldMalus specieshelp to construct the proton electrochemical grads andbecome the basis of Nat/H+ antiport of plasmolemmaTreated with increasing concentration of NaCl andand tonoplast under environmental stress.iso- osmotic PEG, the plasmolemma H+- ATPase activityPeople have found that the tonoplast of CAM plantf M. zumi and M. xiaojinensis was increased (Fig. 1), butMesembryanthemum crsalliumn (Chauhan et al.2000) that of M. baccata was lte increased, especiallyl underand sweet soil plants (Zhang and Blumwald 2001) havethe stress of PEG at 18.8 and 28.5 mmol L'. Under theNa+/H+ antiport activity. And it is believed that the saltsame NaCl concentration, the H+-ATPase activity oftolerance of plants is correlated with the Na+/H+ antiportM. zumi was the highest, followed by M. xiaojinensissactivity. The Na*/H+ antiport activity of plasmolemma and M. baccata, respectively. Bot M. zumi andM. xiao-and tonoplast was found increased under salt stressjinensis increased more than M. baccata under the stress(Allen et al. 1995; Apse et al. 1999).of NaCl at 100 and 150 mmol L and iso-osmotic PEGMalus zumi can maintain normal growth under the sa-(P<0.01). In addition, the H+-ATPase activity of M. xiao-linity of 0.6% of saline alkali soil, which is a salt tolerantjinensis under the stress of NaCl at 150 mmol L' andMalus (Gu et al. 1996). Malus xiaojinensis can bear thethat of M. zumi under the three salt concentration stresssalinity of 0.3% (Zhai et al. 1999), which is a middle saltwas higher than that under the iso-osmotic PEG stress.tolerant Malus; while Malus baccata is a salt sensitiveMalus, and not suitable for saline- alkali soil cultivation(Ma et al. 1992). Salt stress includes osmotic stress andionic stress, while the iso osmotic PEG has only osmoticon tonoplast H+-ATPase activity of three Malusstress. In the present study, we examined the activity ofspeciesH+-ATPase, proton pump and Na*/H+ antiport in Malusseedlings under NaCl and iso-osmotic PEG, and compared As shown in Fig. 2, the tonoplast H+-ATPase activity ofthe effects of the osmotic and ionic stress on Na+/H+ anti-M. zumi increased the most under the stress of NaCl andport activity, which could provide the basis for the study iso-osmotic PEG. The H+- ATPase activity of M. zumi口0 mmolL'昂50 mmolL'霞100 mmolL' a 150 mmolL'00 mmolL'昂18.8 mmolL'图24.5 mmolL'②28.5 mmol L101 A曾20)- 「Malus baccata Malus xiaojinensis Malus zumiFig. 1 Effects of NaCl (A) and iso-osmotic PEG (B) stress on plasmolemma H-ATPase activity.中国煤化工he means+SEof three independent samples (, P<0.05,”", P<0.01, the same as below).MHCNM HG◎2014. CAAS. All rights reserved. Published by Elsevier Ltd.1278YANG Hong-bing etal.and M. xiaojinensis was obviously higher than that of was obviously higher than that of M. baccata under theM. baccata under the stress of NaCl at 100 and 150stress of NaCl at 100 and 150 mmolL' and iso-osmoticmmol L' and iso-osmotic PEG (P<0.01). In addition, the PEG (P<0.01). In addition, the proton pump activityH*-ATPase activity of M. xiaojinensis under the stressof M. baccata under the stress of NaCl at 50 mmolof NaCl at 100 and 150 mmol L1 and that of M. zumi L' and that of M. xiaojinensis and M. zumi under theunder the three salt concentration stress was higher than three salt concentrations was higher than that under thethat under the iso-osmotic PEG stress.iso- osmotic PEG stress.Effects of NaCl and iso-osmotic PEG stress Effects of NaCI and iso-osmotic PEG stress onon plasmolemma proton pump activity of threetonoplast proton pump activity of three MalusMalus speciesspeciesThe plasmolemma proton pump activity of M. bacca-As shown in Fig. 4, the tonoplast proton pump activityta, M. xiaojinensis and M. zumi was increased under of M. xiaojinensis and M. zumi was increased with in-the stress of NaCl at 50, 100 and 150 mmol L' andcreasing concentration of NaCl and iso-osmotic PEG.iso-osmotic PEG (Fig. 3). Under the same NaCl con- Under the same NaCl concentration, the proton pumpcentration, the proton pump activity of M. zumi wasactivity of M. zumi was the highest, followed by thatthe highest, followed by that of M. xiaojinensiss and of M. xiaojinensis and M. baccata, and that of M. zumiM. baccata, and that of M. zumi and M. xiaojinensisand M. xiaojinensis was obviously higher than that of00 mmoIL' B 50 mmolL'图100 mmolL' B 150 mmol L!00 mmol LI日18.8 mmolL'图24.5 mmol L:1盈28.5 mmol L:140] A401 B3030十20 |10-王M. baccata M. xiaojiM. zumiM. baccata M. xiaoiensisFig. 2 Effects of NaCl (A) and iso-osmotic PEG (B) stress on tonoplast H-ATPase activity of three Malus species.口0 mmolL'日50 mmolL'目100 mmolL:! 0 150 mmolL:100mmolL'日18.8 mmolL'日24.5 mmolL:园28.5 mmol L'161 16] B144+122|0f8-6oHM. baccata M. xiaojinensisM. baccata M. siaojinensis M. zumi中国煤化工Fig.3 Effects of NaCl (A) and iso-osmotic PEG (B) stress on plasmolemma proton pump activ.MYHCNM HG◎2014, CAAS. All rights reserved. Published by Elsevier Ltd.Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na*/H* Antiport Activity of Three Malus species with Different1279M. baccata under the stress of NaCl at 100 and 150 of NaCl at 100 and 150 mmol L' and iso-osmotic PEGmmol L' and iso-osmotic PEG (P<0.01). In addition,(P<0.01). In addition, the Na+/H+ antiport activity ofthe proton pump activity of M. baccata under the stress three Malus seedlings under the three salt concentra-of NaCl at 50 mmol L' and that of M. xiaojinensis andtion stress was higher than that under the iso-osmoticM. zumi under the three salt concentrations was higher PEG stress.than that under the iso-osmotic PEG stress.Effects of NaCI and iso-osmotic PEG stressEffects of NaCI and iso-osmotic PEG stress onon tonoplast Na+/H+ antiport activity of threeplasmolemma Na+/H+ antiport activity of three Malus speciesMalus speciesAs shown in Fig. 6, the tonoplast Na+/H+ antiport activityTreated with increasing concentration of NaCl and of M. zumi and M. xiaojinensis was obviously increasediso-osmotic PEG, the plasmolemma Na+/H+ antiport ac-under the stress of NaCl and iso-osmotic PEG (P<0.01).tivity of M. zumi and M. xiaojinensis was increased byUnder the same NaCl concentration, the Na+/H+ antiportseveral folds (Fig. 5), and that of M. baccata increasedactivity of M. zumi was the highest, followed by that ofthe least, especially under the stress of NaCl at 150 M. xiaojinensis and M. baccata, and that of M. baccatammol LI and iso-osmotic PEG. The Na+/H+ antiportincreased relatively less in contrast with that of M. zumiactivity of M. zumi and M. xiaojinensis was obviouslyand M. xiaojinensis under the stress of NaCl at 150higher than that of M. baccata under under the stressmmol L' and iso- osmotic PEG. In addition, the Na+/H+口0 mmolIL:'图50 mmolL'图100 mmolIL'图150 mmol LI口0 mmolL'图18.8 mmolL'图24.5 mmolL' B 28.5 mmol L:I121 A12] B10M. baccata M. xiojinensis M. zumiM. baccata M. xiaojinensisM. zumiFig. 4 Effects of NaCl (A) and iso-osmotic PEG (B) stress on tonoplast proton pump activity of three Malus species.口0 mmolL'园50 mmolL' 8 100 mmol LI 0 150 mmol L'00mmolL:图18.8 mmol L'图24.5 mmol L' 0 28.5 mmol L'0.121 A0.121 B0.1.1-0.08十0.08-旨0.00.06-之里0.0404-0.02-干M. baccata M. xiaojinensis M. zumi中国煤化工Fig. 5 Effects of NaCl (A) and iso-osmotic PEG (B) stress on plasmolemma Na/H+ antiport aMYHCNM HG◎2014. CAAS. All rights reserved. Published by Elsevier Ltd.1280YANG Hong-bing etal.00mmolL+母50mmolL:8100mmoLB150mmolL'00 mmolL+ B 18.8 mmolL:图24.5 mmol L:! B 28.5 mmol L'0.141 A0.141 E0.12+0.10-0.08-0.080.060.06-0.04-0.02-1内M. baccata M. xiaojinensis M. zumiFig. 6 Effects of NaCl (A) and iso -osmotic PEG (B) stress on tonoplast Na+/H+ antiport activity of three Malus species.antiport activity of M. baccata under the stress of NaClNa+/H+ antiport activity of plasmolemma in roots ofat 50 mmol LI and that of M. xiaojinensis and M. zumi Dunalella salina, wheat and Atriplex were all ob-under the three salt concentrations was higher than thatviously increased with increasing concentration ofunder the iso-osmotic PEG stress.NaCl stress (Allen et al. 1995; Blumwald et al.2000).Sodium compartmentalization in vacuole was mainlyDISCUSSIONconcerned with tonoplast Na+/H+ antiport. Fukuda et al.(201 1) found that the expression of OsNHX1 in ricewas upregulated under salt stress. The Na+/H+ antiportSilva et al. (2010) found that the tonoplast H+-ATPaseactivity of tonoplast in leaves of Mesembryanthemumactivity of Populus euphratica was increased under saltcrystallinumn and Peganum harmala were obviouslystress. The H+-ATPase activity of plasmolemma andincreased under high salt concentration stress (Barklatonoplast in three Malus species were all obviouslyet al.1995; Liu et al. 2008) and that in roots of sun-increased under NaCl stress and that in salt-tolerant flower and barley was induced by NaCl (Blumwald etspecies was obviously higher than that in salt sensitiveal. 2000). In this study, the Na+/H+ antiport activityone (Figs. 1 and 2), and that in M. zumi was the highest,of plasmolemma and tonoplast in roots of M. zumithat in M. baccata was the lowest. These results wereand M. xiaojinensis was obviously higher than that ofconsistent with the study of Yang et al. (2002) in wheat. M. baccata under high concentration of NaCl stressHigher H*-ATPase activity under salt stress would be(Figs. 5 and 6). It indicated that the capability of sodiumbeneficial to hydrolyze ATP and to provide much energyextrusion and sodium compartmentalization in roots offor proton transport. The H+-Na* exchange of plasmo-salt-tolerant species was obviously higher than that oflemma and the Na+-H+ exchange of tonoplast needed salt sensitive one.proton grads to provide energy and the Nat/H+ antiportThe H+-ATPase activity of tonoplast in leaves ofwas relation to proton pump activity (Ballesteros et al.Crassula agenten under salt stress was increased1997). The proton pump activity of plasmolemma andmore than that under water stress (Xia et al. 2000).tonoplast in salt-tolerant species was higher than thatExperiments indicated that the activity of H+- ATPase,in salt-sensitive one under NaCl stress (Figs. 3 and 4),proton pump and Na /H+ antiport of plasmolemma andand that in M. zumi and M. xiaojinensis was obviouslytonoplast under the three salt concentration stress washigher than that in M. baccata under high concentration increased more than that under the iso- osmotic PEGof NaCl stress. Higher proton pump activity in salt-tol-stress (Figs. 1-6), and it was especially obvious inerant species would benefit to construct proton grads and M. zumi and M. xiaojinensis of salt-tolerant species. Itprovided enough energy for Na+/H+ antiport.indicated that the sodium ion had greater stimulationSodium extrusion mechanism in plants was mainlyeffect on the activitw 2fH+^ TPoso orntrr pump and中国煤化工concerned with plasmolemma Na+/H+ antiport. The Nat/H+ antiport iYHCNM HG◎2014, CAAS. All rights reserved. Published by EIsevier Ltd.Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na*/H* Antiport Activity of Three Malus species with Different1281CONCLUSIONmmol L, pH=8.0), EDTA or EDTANa, (3 mmol L I), MgSO2(3 mmol L), PVP (0.5% (W/V)), DTT (2 mmol L"), PMSF(0.2 mmol L), glycerol (5% (V/V)), and a certain amount ofThe sodium ion effect had more stimulation on the ac-mannitol to meet the osmotic potential of root tissues withtivity of H+-ATPase, proton pump and Na+/H+ antiport in3 s/times every 10 s. After 8 times of high-speed breakingsalt-tolerant species, and salt-tolerant species has higherand. 4-layer gauze filtering, homogenate was obtained and thencentrifuged with 13 000xg (Beckman SW28) for 15 min tocapability of sodiumextrusion and compartmentalizationremove mitochondria, chloroplasts and other cell debris. Thein roots and is therefore more salt tolerant.obtained supernatant was centrifuged at 80000xg (BeckmanSW28) for 30 min, and the precipitate was carefully stiredwith a pointed fine brush, re-suspended in 5 mL of suspensionMATERIALS AND METHODS(homogenate containing sucrose of 200 mmol L), and mixedwith homogenizer. The membrane vesicle mixtures weresubjected to sucrose gradient solutions of 24% (12 mL):Plant cultivation34% (10 mL): 41% (12 mL) (W/W) sucrose with addition ofHepes-Tris (5 mmol L, pH=6.5) and DTT(1 mmol L) under: (L.) Borkh.centrifugation at 100000xg (Beckman SW28) for 2 h, and themiddle sal-tolerant Malus xiaojinensis Cheng et Jiang andtonoplast vesicles were carefully collected with a sucker at thesalt- tolerant Malus zumi Mats of Malus species were obtainedinterface between 24% (W/W) sucrose and the sample liquid,the plasmolemma vesicles collected between 34% (W/W) andby tssue culture and used in experiments. After roots growing,41% (W/W) sucrose. The vesicles mixtures were diluted withwhich were ventilated continuously. Malus seedlings werea buffer containing EGTA (3 mmol L'), Hepes-Tris (10 mmolplaced in the greenhouse, day/night temperature averagedL', pH=6.5), DTT (1 mmol L ), and PMSF (0.2 mmol L) for26°C/18°C, and day/night relative humidity averaged3-5 times, and centrifuged at 100000xg (Beckman SW55 Ti)50%/65%. Light intensity was about 800 umol m2 s' andfor 30 min. After centrifugation, the precipitate was carefullystired with a pointed fine brush and re-suspended in a storagewas 12 h photoperiod.solution containing glycerol (40%), Hepes-Tris (10 mmolL",pH=6.5), and DTT (2 mmol L). The mixtures were pipettedinPlant material treatmentEppendorf tubes of 0.5 mL, frozen in liquid nitrogen, and keptin ultra-low temperature refrigerator (-80*C). The operationswere conducted under the conditions of 4°C.Malus seedlings were treated with NaCl stress at the heightabout 10 cm. In order to avoid salt shock effect, NaClconcentration was increased to 25 mmol LI per 12 h and the Measurement of H+-ATPase and proton pumpend concentration of NaCl stress was at 50, 100 and 150 mmolactivityL. At the same time, Malus seedlings were treated with iso-osmotic polyethylene glycol (PEG, M,=6 000), and the endconcentration of PEG stress was at 18.8, 24.5 and 28.5 mmolAccording to the method by Fischer Schliebs et al. (1997), theL' (equal to osmotic potential of NaCl under 50, 100 and 150H+-ATPase activity of plasmolemma vesicles was examinedas expression deviation in insulation medium with or withoutmmol L), and the treatment temperature was 25°C.Na,VO, at 50 mmol L', and the tonoplast vesicles H+-ATPaseactivity with or without KNO, at 50 mmol L'. The reactionPlant material choosingsolution (800 μL) consists of Hepes-Tris (30 mmol L,pH=7.5), (NH),MoO, (0.1 mmol L), NaN, (1 mmol L),MgSO, (1 mmol L), Triton X- -100 (0.03% (V/V)), ATPNa,(3The treatment date of the end concentration of NaCl and PEGmmol L) and KCI (50 mmolL'). The mixture of membranestress was at November 21, 201 1, and the young leteral roots vesicles containing membrane protein of 20 μg was incubatedof Malus seedlings were fetched for study after5 d.in a water bath at 37°C for 30 min after addition of the reactionbuffer, and then terminated by adding a solution of 1 mLcontaining SDS (0.1%), H,SO2 (350 mmol L), (NH),MoO,Preparation of plasmolemma and tonoplast(0.5%), and 200 μL of Vc (10%). After color reaction for 40vesiclemin, absorbance value was measured under 750 nm. Eachtreatment was replicated by three times.As described by Yang et al. (2002), total of 20 g materialsThe ATP-dependent rroton p1mn artivitv wac representedwere homogenized in a 40-mL pre-cooling homogenate buffer by a unit time pe中国煤化工uorescence .containing sucrose (200 mmol L), Hepes-Tris or Tris-Mes (50quenching value.iYHC N M H G containing◎2014. CAAS. All rights reserved. Published by Elsevier Ltd.1282YANG Hong-bing etal.sucrose (200 mmol L), KCl (100 mmol LI), NaN, (1 mmolby the vacuolar H+-ATPase in the halophytic plantL'), acridine orange (AO)(5 μumolL), ATPNa, (0.2 mmolL)Mesembryanthemum crystallinumn L. Plant Physiology,and Tricine (20 mmol LI) was adjusted for pH (7.5) with Tris,109, 549-556.added with membrane protein of 40 ug, pre- heated at 28°C forBlumwald E, Aharon G s, Apse M P.2000 Sodium transport in4 min, and added with MgCl, (final concentration 3 mmol LI)plant cells. Biochimica et Biophysica Acta, 1465, 145-151.to start the reaction. A fluorescence spectrofluorometer Blumwald E. 2000. Sodium transport and salt tolerance in(F4500-type, Hitachi) was used to record the maximumplants. Current Opinion in Cell Biology, 12, 431-434.fluorescence value (F) and the fluorescence quenching valueBrett C L, Donowitz M, Rao R.2005. Evolutionary origins of(0F) under excitation light (EX) 495 nm, emission light (EM)eukaryotic sodium/proton exchangers. American Journal540 nm, and slit 2.5 nm. Proton pump activity was expressedof Physiology (Cell Physiology), 288, C223-C239.as“OF/F mg;! protein min!". Each treatment was replicated Chauhan S, Forsthoefel N, Ran Y Q, Quigley F, Nelson D E,three times. .Bohnert H J. 2000. Nat/myo-inositol symporters and Nat/H+ antiport in Mesembryanthemum crysallinum. The PlantJournal, 24, 511-522.Measurement of Na+/H+ antiport activityCounillon L, Pouyssegur J. 2000. The expanding family ofeukaryotic Na+/H+ exchangers. The Journal of BiologicalChemistry, 275, 1-4.DuYH,HeiQ,LiuYX,ZhangH,XuK,XiaT.2010.acceded when proton grads was stable, and the concentration ofIsolation and characterization of a putative vacuolar Nat/EGTA was 3 mmol L, so as to chelated Mg2 and eliminate H+-H+ antiporter gene from Zoysia japonica L. Jourmal ofATPase activity of membrane vesicle. Then sodium gluconatePlant Biology, 53, 251-258.(CH,NaO) was acceded and the end concentration was 20Fischer- Schliebs E, Ball E, Berndt E, Besemfelder Butzmmol L', and noted the fluorescent recovery value(OF). TheE, Binzel M L, Drobny M, Mihlenhoff D, MillerNa+/H+ antiport activity was expressed as“0F'/OF ug ! proteinM L, Rakowski K, Ratajczak R. 1997. Differentialmin!". Each treatment was replicated three times.immunological cross- reactions with antisera against theV-ATPase sub-units of different plant species. BiologicalStatistical analysisChemistry, 378, 1131-1139.Fukuda A, Nakamura A, Hara N, Toki S, Tanaka Y.2011.Molecular and functional analyses of rice NHX-type Na+/Student's t-test was used for comparison between differentH+ antiporter genes. Planta, 233, 175- 188.treatments. A difence was considered to be staistically Gu N L, Zhao H X, MaJ L, Zhou S W. 1996. Adaptation scopesignificant when P<0.05, and especially significant whento saline-alkali soil and application of Malus zumi. JournalP<0.01 (Wang et al.2010).of Tianjin Agricultural College, 3, 48-52. (in Chinese)Liu J X, Hu H B, Zhao G L. 2008. Effects of NaCl stress ontonoplast H+- ATPase and H+-PPase activities in Peganum .Acknowledgementsharmala seedlings. Journal of Desert Research, 28, 274-The authors would like to acknowledge the financial support279. (in Chinese)from the National Natural Science Foundation of ChinaLittge U, Ratajczak R. 1997. 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