Received 28 Jun. 2003 Accepted 28 Aug. 2003Supported by the National Natural Science Foundation of China (30070069, 30270793), the Hi-Tech Research and Development (863)Program of China (2002AA629080) and the Shandong Provincial Natural Science Foundation (Z2000D01).

* Author for correspondence. Fax: +86 (0)531 6180107; E-mail:<bswang@sdnu.edu.cn>.

http://www.chineseplantscience.com

Cloning and Expression Analysis of the B Subunit of V-H+-ATPase inLeaves of Halophyte Suaeda salsa Under Salt Stress

LI Ping-Hua, WANG Zeng-Lan, ZHANG Hui, WANG Bao-Shan*

(College of Life Sciences, Shandong Normal University, Jinan 250014, China)

Abstract: For salinity stress tolerance in plants, the vacuolar type H+-ATPase (V-H+-ATPase) is of primeimportance in establishing an electrochemical H+-gradient across tonoplast that energizes sodiumsequestration into the central vacuole. In this paper, the sequence of a cDNA encoding the B subunit ofthe vacuolar-type H+-ATPase from Suaeda salsa L., a plant that can survive in seawater was reported. TheB subunit cDNA is 1 974 nucleotides long and include a 18 bp poly(A+) tail together with a complete 1 470bp coding region for a 489 amino acid with a conservative ATP binding site and a predicted molecular massof 54.29 kD. Northern and Western blotting analyses indicated that the expression of B subunit wassignificantly up-regulated by NaCl treatment. Moreover, the expressions of B subunit were coordinatedwith c subunit of V-H+-ATPase at transcript and translation levels under NaCl stress. The increased V-H+-ATPase subunit amounts and activity of Suaeda salsa provide the energy for the compartmentation ofsodium in response to salinity.Key words: B subunit; c subunit; NaCl stress; Suaeda salsa ; V-H+-ATPase

Soil salinity is a major constraint to food productionbecause it limits crop yield and restricts the use of landpreviously uncultivated (Flowers and Yeo, 1995). For plantadaptation to excess sodium chloride concentration，lowNa+ influx, high Na+ efflux and the vacuolar Na+ accumula-tion are the main strategies. The vacuolar sodium seques-tration is mediated by a secondary active Na+/H+ antiportat the tonoplast (Barkla et al., 1995; Apse et al., 1999) and itis energized by a proton motive force established by thevacuolar H+-ATPase (EC 3.6.1.3) and H+-PPase (EC 3.6.1.1)(Dietz et al., 2001).

The V-H+-ATPase is a large, multimeric enzyme com-posed of a hydrophilic V1 complex on the cytosolic face ofthe tonoplast and a membrane-bound V0 complex (Lüttgeand Ratajczak, 1997; Sze et al., 1999). The V1 domain thatfunctions in ATP hydrolysis is composed of eight subunits(A-H). The A subunit that is characterized as a catalyticsubunit of ATP hydrolysis and subunit B that seems as anon-catalytic regulate subunit of ATP hydrolysis are thetwo most important subunits in the V1 domain. The V0 do-main that is in charge of proton transportation is composedof five subunits (c, c', c'', a and d). The V-H+-ATPase isimportant not only as a “house-keeping enzyme” to main-tain cytosolic ion homeostasis and cellular metabolism, butalso as an “eco-enzyme” which functions as a stress re-sponse enzyme undergoing moderate changes in expres-sion of subunits and modulations of enzyme activity under

the environmental stress (Ratajczak, 2000).In plant, cDNAs encoding the subunit B of V-H+-AT-

Pase were reported for Mesembryanthemum crystallinum,Arabidopsis thaliana, Oryza sativa, Citrus unshiu and Nic-otiana tabacum. Although some useful information aboutthe expression of V-H+-ATPase subunit B under salt stresswas gathered, most of these information was at the proteinlevel and more information about regulation of V-H+-AT-Pase subunit B at transcription and translation levels undersalt stress was needed to understand the salt-tolerant mecha-nism of plants.

Suaeda salsa is a C3 halophyte that could accumulateNa+ in the vacuole to keep the plant surviving in seawater.Our previous results showed that the increase in V-ATPaseactivity of S. salsa under NaCl stress is not obtained bystructural changes of the enzyme, but by an increase in itsprotein amount (Wang et al., 2001). As a further step to-wards understanding the molecular events leading to vacu-olar salt sequestration and salt tolerance, a cDNA encodingB subunit of V-H+-ATPase from a cDNA library of S. salsawas isolated and the transcription and translation regula-tion of this gene under salt stress was analyzed.

1 Materials and Methods1.1 Plant material

Seeds of halophyte plant Suaeda salsa L. (collected fromthe Yellow River Delta, Shandong Province, China) were

Acta Botanica Sinica植 物 学 报 2004, 46 (1): 93-99

Acta Botanica Sinica 植物学报 Vol.46 No.1 200494

germinated in acid-washed sand. Seedlings were grown inHoangland solution (mmol/L): 5 Ca(NO3)2, 1 KH2PO4, 2MgSO4, 5 KNO3, 0.2 Fe-EDTA, 2.5× 10-4 H3BO3，5×10-4 H2MoO4， 5 × 10-4 CuSO4， 2 × 10-3 ZnSO4，2×10-3 MnCl2 under 16 h light /8 h darkness, a temperatureof 25-35 ℃/20-25 ℃, a relative humidity of 60%/80%, anda proton flux density of 600 µmol.m-2.s-1.

Six-week-old seedlings were treated with 0 (control), 100and 400 mmol/L NaCl. The final salinity level was achievedby raising 50 mmol/L NaCl every 12 h in order to avoidosmotic shock.1.2 cDNA library construction

Seedlings of S. salsa were treated with 400 mmol/L NaClfor 48 h; aerial part tissue was collected and ground underliquid nitrogen using a mortar and pestle. Total RNA wasextracted using RNAgent (Promega), poly(A+) RNA wasselected with Messagemaker kit (Gibco, BRL). First-strandcDNA synthesis was carried out by an oligo-dT linker-primerwith an XhoⅠ cloning site. The 5' end of each cDNA wasligated to an adaptor with an EcoRⅠ-compatible overhang.cDNA was ligated undirectionally into the EcoR Ⅰ andXhoⅠ sites of the λ -ZAP express vector (Stratagene) ,packaged in vitro, and amplified. The amplified library rep-resents approximately 106 recombinants.1.3 Cloning and sequence analysis

The phage library was converted to the plasmid form bymass excision according to the protocol described byStratagene. The obtained phagemid of the library was usedto infect Escherichia coli strain XLOLR. The E. coli wasgrown for 45 min and then plated at low density on mediumcontaining Luria-Bertani broth, tetracycline (10 mg/L), andkanamycin (25 mg/L). Cultured in 37 ℃ overnight, indi-vidual colonies were selected randomly for plasmid DNApurification and sequencing.

Sequencing reactions contained the standard T3 se-quencing primer (5'-ATTAACCCTCACTAA AGGGAA-3'),and thus read into the presumed 5' end of the cDNA, T7primer (5'-TAATACGACT CACTATAGGG-3') situated atopposite ends of the inserts. Double-strand sequencing ofplasmid was performed on an Automated Sequencer (PE-Applied Biosystems). Sequences were analyzed usingDNASIS software, and databank searches were conductedthrough the BLAST program.1.4 Membrane vesicle isolation

Tonoplast-enriched membrane vesicles were isolatedaccording to the method of Wang et al. (2000) and Mandalaand Taiz (1985) with modifications. Leaves were washedwith cold deionized water and homogenized in an extrac-tion medium (50 mmol/L Tricine-Tris (pH 7.8), 3 mmol/L

EGTA, 3 mmol/L MgSO4, 0.5% (V/V) PVP, 2 mmol/L DTT,0.2 mmol/L PMSF, 5% (V/V) glycerol and mannitol whichhad the same osmotic potential with the leaves). Two mL ofthe medium were used for each 2 g of fresh material. Thehomogenate was filtered through four layers of cheese-cloth and centrifuged at 10 000g for 20 min (Backman 45Ti).The supernatant was loaded on a 0:25% (W/W) sucrosegradient solution (5 mmol/L Hepes adjusted to pH 7.5 withTris, 1 mmol/L DTT) and centrifuged at 100 000 g for 2 h(Backman SW 40Ti). The vesicles located at the 0:25%(W/W) sucrose interface (tonoplast-enriched vesicles) werecarefully collected and diluted 3-4 folds with a dilutionbuffer (3 mmol/L MgSO4, 10 mmol/L Hepes-Tris pH 7.5, 1mmol/L DTT) and centrifuged at 100 000g for 30 min (Backman45Ti). The pellets were suspended in a storage buffer (40%(V/V) glycerol, 2 mmol/L DTT, 10 mmol/L Hepes, adjusted topH 7.5 with Tris). The tonoplast-enriched vesicles were fro-zen in liquid nitrogen and stored at –75 ℃ for further use. Allsteps of the procedure were performed at 2-4 ℃.1.5 Measurement of protein content

Protein content was determined as Bradford (1976) us-ing bovine serum album as a protein standard.1.6 V-H+-ATPase activity assay

V-H+-ATPase hydrolysis activity of tonoplast-enrichedmembrane vesicles were calculated from the amount of in-organic phosphate released in the absence and presenceof 50 mmol/L KNO3. Enzyme reactions were run at 37 ℃ for30 min with a protein concentration of 5 µg in a assay me-dium containing 30 mmol/L Tris/Mes, pH 7.5, 0.1 mmol/L(NH4) 4 MoO4, 1 mmol/L NaN3, 1 mmol/L MgSO4, 0.03%(V/V) TritionX-100, 50 mmol/L KCl, 3 mmol/L ATPNa2. Inor-ganic phosphate was assayed using the method of Lin andMorales (1977).1.7 RNA extraction and Northern blotting analysis

Total RNA of leaves of S. salas was isolated byguanidinium thioisocyanate extraction (Chomczynski andSacci, 1987). RNA was quantified by optical density at 260nm, and the concentration was confirmed by electrophore-sis on an RNA formaldehyde gel (Sambrook et al., 1989).Total RNA was loaded on 1.2% (W/V) agarose denaturingformaldenhyde gel. It was then transferred to Hybond-N+

membrane. In order to affirm uniformity in loading for RNAblots, RNA was stained by ethidium bromide. RNA wascross-linked to the membrane by 254 nm UV irradiation.RNA Northern blot hybridization was performed as de-scribed by Sambrook et al. (1989). For high stringency inthe presence of 50% (W/V) formamide, A 32P-labeled DNAprobe (3' non-coding region) was prepared using a randomprimer labeled kit (Random Primers Systems; TaKaRa,

95 LI Ping-Hua et al.: Analysis of V-H+-ATPase B Subunit

Japan). Hybridization and washes were carried out at 65 ℃.After drying the blots, autoradiography of the filters wasobtained on Kodak Xar-5 X-ray films using an intensifyingscreen at –70 ℃.1.8 SDS-PAGE and Western blotting analysis

Tonoplast protein was extracted by 30% (V/V) ethanol/70% (V/V) acetone. Sodium-dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) was performed as describedin Ratajczak (1994) on slab gels containing 12% (W/V)acrylamide and by using the Lammli buffer system (Laemmli,1970). For Western blotting analysis, proteins were trans-ferred electronically from acrylamide gels to ImmoblionHybond-P membranes under conditions previously de-scribed (Fischer-schliebs et al., 1997). After blocking freeprotein binding sites for 1 h in 1% (W/V) fat free milk powerdissolved in Tris buffered saline (TBS), the membranes wereincubated with the antisera ATP95 against the V-H+-AT-Pase holoenzyme of Kalanchoe daigremontiana (Fischer-schliebs et al., 2000). An alkaline phosphatase-coupledgoat-anti rabbit IgG was used as a secondary antibody.Immunostaining was performed using nitroblue tetrazoliumby formation of an indigo dye-precipitate with antigen-coupled alkaline phosphate (Chow et al., 1990).

2 Results2.1 Isolation and sequencing of the subunit B of V-H+-ATPase from S. salsa

The salt-stressed cDNA library of halophyte S. salsawas sequenced as expressed sequence tags (EST). Fromthe ESTs, we isolated a cDNA clone (BE859205) that hadhigh homology with V-H+-ATPase B subunit of Arabidopsis.To gain insight into the possible biochemical and physi-ological functions of the V-H+-ATPase subunit B in S. salsa,sequence of the subunit B was determined. By sequencingusing T3 and T7 primers, we obtained full sequence of thecDNA (GenBank accession number AY231438). The insertfragment was 1 974 bp with 1 470 bp open reading frame, a 32bp 5' un-translated region and a 472 bp non-coding 3' region.The deduced amino acid was 489 amino acids with a pre-dicted molecular weight of 54.29 kD. The start codon ATGwas found in a likely initiation sequence GATAATGGG simi-lar to the consensus initiation sequence AACAATGGC inplants (Lütcke et al., 1987) and two putative polyadenylationsignal consensus sequences (AATAAA) (Rothnie et al.,1994) were present in the 3' un-translated end (from 1 583 to1 588 and from 1 869 to 1 874) of the cDNA.2.2 Homology analysis of amino acid sequence of V-H+-ATPase B subunit in S. salsa

Amino acid sequence alignment analysis suggested that

the V-H+-ATPase B subunit of S. salsa had high homologywith other reported V-H+-ATPase subunit B in higher plants,lower plants and insects. It shared 93%, 92%, 92%, 83%,78%, 77%, 77% amino acid identity with those of Mesem-bryanthemum crystallinum, Arabidopsis thaliana, Oryzasativa, Acetabularia acetabulum, Cyanidium caldarium,Drosophila melanogaster, Aedes aegypti, respectively andthere was a conservative ATP binding site of “324-SGSIT-328”.

The cluster results in Fig.1 suggested that the V-H+-ATPase subunit B coming from higher plants was clus-tered into a group; the V-H+-ATPase subunit B coming frominsects D. melanogaster and A. aegypti was clusted into agroup; A. acetabulum and C. caldarium were between thehigher plants and insects as lower plants. The data indictedthat the evolution of V-H+-ATPase subunit B was accom-panied with the biological evolution. In higher plants, theV-H+-ATPase subunit B from salt-tolerant plants S. salsaand M. crystallinum was in one branch and V-H+-ATPasesubunit B from glycophytes A. thaliana and O. sativa wasin another branch, which indicated an adaptation of V-AT-Pase B subunit to environment stress during evolution.

Fig.1. Amino acid sequence cluster analysis of subunit B of V-H+-ATPase in Suaeda salsa with related sequences from Mesem-bryanthemum crystallinum, Arabidopsis thaliana, Oryza sativa,Acetabularia acetabulum, Cyanidium caldarium, Drosophilamelanogaster and Aedes aegypti. Sequence identification num-bers from the National Center for Biotechnology Information aregi: 26986106， 17065080, 14626084, 1303677, 7436110, 8810and 4680480, respectively.

2.3 Effect of NaCl on the activity of tonoplast V-H+-AT-Pase in the leaves of S. salsa seedlings

The activity of the leaf V-H+-ATPase was significantlyinduced by NaCl stress (Fig.2). The activities were 2.16-and 2.58-fold higher than that of the control under 100 and400 mmol/L NaCl treatment for 2 d, respectively.

Acta Botanica Sinica 植物学报 Vol.46 No.1 200496

2.4 Changes in mRNA levels for the subunit B fromleaves of S. salsa in response to salt stress

In order to investigate transcript inducibility of V-H+-ATPase B subunit in leaves of S. salsa during NaCl stress,Northern blot hybridization was carried out with total RNAisolated from plant leaves stressed by the addition of 400mmol/L NaCl for 12, 24, 48,72, 96 and 120 h.

Following NaCl addition, amounts of subunit B tran-scripts in leaves increased for up to 48 h and then decreased(Fig.3). From 72 to 120 h, the mRNA levels of B subunit insalt-stressed leaves were still higher than those of controls.

Fig.2. Effect of NaCl on the activities of leaf tonoplast V-H+-ATPase of Suaeda salsa under NaCl treatment. Data are means(n=15) of three independent experiments ± SE.

Fig.3. Northern blotting analysis of transcript levels for V-H+-ATPase B subunit under 400 mmol/L NaCl stress from 0 to 120h. Thirty µg of total RNA per lane was loaded on a 1.2% (W/V)agarose denaturing formaldenhyde gel. In a lower panel, equalloading of RNA was verified on the gel by ethidium bromidestaining of the agarose gel.

Fig.5. Western blotting analysis of tonoplast vesicle prepara-tions isolated from Suaeda salsa leaves exposed to 0, 100 and 400mmol/L NaCl treatment for 2 d. Immunstaining was performedusing antisreum ATP95. Letters on the left margin indicated theposition of subunits B and c, and letters on the right marginshowed the molecular weight. Lane 1, control; lane 2, 100mmol/L NaCl treatment; lane 3, 400 mmol/L NaCl treatment.Fifteen µg protein was added per pocket.

Fig.4. Northern blotting analysis of transcript levels for sub-units B and c of V-H+-ATPase from Suaeda salsa under 100 and400 mmol/L NaCl stress for 2 d. Twenty µg of total RNA per lanewere loaded on a 1.2% (W/V) agarose denaturing formaldenhydegel. In a lower panel, equal loading of RNA was verified on the gelby ethidium bromide staining of the agarose gel.

2.6 Effect of NaCl treatment on the subunit amounts of V-H+-ATPase in leaves of S. salsa

Western blotting analysis with the antiserum ATP95 wasdirected against the V-H+-ATPase holoenzyme of K.daigremintiana cross-reacted with four polypeptides(Fig.5). It revealed a significant increase of protein of 54 kDsubunit B and 16 kD subunit c under NaCl stress comparedto that of the control and the protein was increased withthe increase of NaCl concentration.

2.5 Coordinate of subunits B and c of V-H+-ATPase fromS. salsa

cDNA encoding c subunit (BE240898) of V-H+-ATPasewas also isolated from cDNA library of S. salsa. In order toinvestigate the possible relationship of subunits of V-H+-ATPase, Northern blotting analysis was performed usingsubunit c cDNA as probe. The markedly induced mRNAincreases of both subunit B and c were detected after addi-tion of 100 and 400 mmol/L NaCl (Fig. 4).

97 LI Ping-Hua et al.: Analysis of V-H+-ATPase B Subunit

3 DiscussionV-H+-ATPase B subunit belongs to a conserved AT-

Pase family and Gogarten et al. (1989) concluded that V-H+-ATPase subunits could be useful molecular markers forear ly evolut ion. Our results a lso indicated thatV-H+-ATPase B subunit is a conservative subunit and itsevolution was accompanied with the biological evolution.For plants, the great divergence in amino acid sequencesof V-H+-ATPase subunit B apparents in the amino and car-boxyl termini indicates that these regions are more subjectto evolutionary changes and methods such as point muta-tion should be used to discern the importance of theseamino acids divergence. While, the amino acid sequence ofV-H+-ATPase subunit B from halophyte S.salsa had thehighest identity with that of M. crystallinum, which is alsoa halophilic species as its growth rate is maximal at moder-ate salt concentrations (Tsiantis et al., 1996), indicatingthat V-H+-ATPase subunit B plays an important role in plantsadapted to salinity soil and its evolution is also accompa-nied by environmental adaptation.

S. salsa can accumulate more than 1 mol/L Na+ in leavesand the vacuolar compartmentation of Na+ is the main strat-egy for it to cope with salinity (Wang et al., 2001). VacuolarNa+ sequestration is known to depend on the activityof tonoplast Na+/H+ antiport that is mainly energizedby V-H+-ATPase on the tonoplast. Indeed, the tonoplastNa+/H+ antiport activity of S. salsa is significantly increasedunder NaCl stress (Wang, unpublished data) and at thesame time, the activity of V-H+-ATPase is also significantlyincreased (Fig.2).

The activity of V-H+-ATPase is often regulated by themodulating of enzyme structure or the changing in expres-sion of subunits (Ratajczak, 2000). In order to discern thepossible modulation mechanism of V-H+-ATPase activityof S. salsa under NaCl stress, the expression of B subunitof V-H+-ATPase was analyzed. The results in Fig.3 indi-cated that the transcript of V-H+-ATPase subunit B wasobviously up-regulated by NaCl stress. Under 400 mmol/LNaCl stress, the mRNA of B subunit reached the maximallevel during 48 h NaCl treatment and kept higher than thatof the control during the next 2 d, which indicated a dy-namic change of B subunit under salt stress. The mRNAlevel of B subunit was also increased with the increase ofNaCl concentration (Fig.4).

The V-H+-ATPase subunit B is an important non-cata-lytic ATP binding subunit located in the V1 domain. While,proton-translocation of V-H+-ATPase is mainly charged bythe V0 domain subunit c. Is the NaCl-induced transcript

activation of subunit B in V1 domain accompanied by thechange of subunit c in V0 domain? The expression of V-H+-ATPase subunit c of S. salsa was also analyzed to resolvethis question. Figure 4 suggests that the expression of sub-unit c was also increased with the increase of NaClconcentration. It also indicated a cooperative relationshipbetween subunits B and c of V-H+-ATPase from S. salsa.Western blotting analysis with the antiserum ATP95 re-vealed a significant increase of protein amounts of the V-H+-ATPase subunits B and c of S. slasa under 100 and 400mmol/L NaCl treatment, which gave some other evidencefor a salt-induced coordinate up-regulation of V-H+-ATPasesubunits at translation level. The coordinated salt-inducedincrease of subunits B and c of V-H+-ATPase from S. salsaat transcription and translation levels indicated an increaseof V-H+-ATPase holoenzyme amounts, which maybe thereason for the increase of V-H+-ATPase activity of S. salsaunder salt stress. The salt-induced increase of V-H+-AT-Pase activity energies the Na+/H+ antiport that finally causedthe Na+ compartmentation of S. salsa.

The salt-induced expression of V-H+-ATPase subunit Bwas reported in halotolerant plants such as common iceplants (Dietz et al., 2001) and sugar beet (Kirsch et al.,1996). While for glycophytes, salt-reduced protein amountof V-H+-ATPase subunit B was reported in wheat (Wang etal., 2000) and the protein amount of subunit B in pea wasnot changed by sodium chloride exposure (Yu et al., 2001).The former data and our results suggested that salt stressaffected V-H+-ATPase subunit B expression differently inglycophytes and halophytes.

Our results showed that the expression of V-ATPase Bsubunit from leaves of S. salsa was significantly up-regu-lated and was coordinated with subunit c at transcriptionand translation level under NaCl stress. The increased V-H+-ATPase amounts had a close relationship with the ac-tivity of V-H+-ATPase, which provided the energy for thecompartmentalization of sodium in response to salinity.More information will be needed to study the structure,function and regulation of V-H+-ATPase B subunit, whichcould be important to discern the real role of subunit Bcontributed to V-H+-ATPase and the composition and re-action mode of V-H+-ATPase in response to environmentalstimuli, and further display the salt-tolerant mechanism ofhalophytes such as S. salsa.

Acknowledgements: The authors thank Professor Dr.Ulrich LÜTTGE (Darmstadt University, Germany) for pro-viding an antiserum ATP95 directed against the V-H+-AT-Pase holoenzyme of Kalanchoe daigremontiana.

Acta Botanica Sinica 植物学报 Vol.46 No.1 200498

References:

Apse M P, Aharon G S, Snedden W A, Blumwald E. 1999. Salttolerance conferred by overexpression of a vacuolar Na+ /H+

antiport in Arabidopsis. Science, 285:1256–1258.Barkla B J, Zingarelli L, Blumwald E, Smith J A C. 1995. Tono-

plast Na+/H+ antiport activity and its energization by the vacu-olar H+-ATPase in the halophytic plant Mesembryanthemumcrystallinum L. Plant Physiol, 109:549–556.

Bradford M M. 1976. A rapid and sensitive method for thequantitation of mocrogram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem, 72:248-254.

Chomczynski P, Sacci N. 1987. Single-step method of RNA iso-lation by acid guanidinium thiocyanate-phenol-chlorofromextraction. Anal Biochem, 162:156-159.

Chow W S, Ball M C, Anderson J M. 1990. Growth and photo-synthetic responses of spinach to salinity: implication of K+

nutrition for salt tolerance. Aust J Plant Physiol, 17:563-587.Dietz K J, Arbinger B. 1996. cDNA sequence and expression of

SuE of the vacuolar H+-ATPase in the inducible crassulaceanacid metabolism plant Mesembryanthemum crystallinum.Biochim Biophys Acta, 1281:134–138.

Dietz K J, Tavakoli N, Kluge C, Mimura T, Sharma S S, Harris GC, Chardonnens A N, Golldack D. 2001. Significance of the V-ATPase for the adaptation to stressful growth conditions andits regulation on the molecular and biochemical level. J ExpBot, 52:1969-1980.

Fischer-schliebs E, Ball E, Berndt E, Besemfelder B E, Binzel ML, Drobny M, Mühlenhoff D, Müller M L, Rakowski K,Ratajczak R. 1997. Differential immunological cross-reactionswith antisera against the V-H+-ATPase of Kalanchoedaigremontiana reveal structural differences of V-H+-ATPasesubunits of different plant species. Biol Chem, 278:1131-1139.

Fischer-schliebs E, Martina D, Erika B, Rafaol R, Lüttge U. 2000.Variation in nitrate nutrition leads to changes in the perfor-mance on V-ATPase and immunological differences of proteo-lipid subunit in tobacco (Nicotiana tabacum) leaves. Aust JPlant Physiol, 27:639-648.

Flowers T J, Yeo A R. 1995. Breeding for salinity resistance incrop plants: where next? Aust J Plant Physiol, 22:875-884.

Gogarten J P, Kibak H, Dittrich P, Taiz L, Bowman E J, ManolsonM F, Poole R J, Date T, Oshima T, Konishi J, Denda K,Yoshida M. 1989. Evolution of the vacuolar H+-ATPase: im-plication for the origin of eukaryotes. Proc Natl Acad SciUSA, 86:6661-6665.

Kirsch M, Zhigang A, Viereck R, Löw R, Rausch T. 1996. Saltstress induces an increased expression of V-type H+-ATPasein mature sugar beet leaves. Plant Mol Biol, 32:543-547.

Laemmli U K. 1970. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature, 222:680-685.

Lin T, Morales M F. 1977. Application of a one step procedurefor measuring inorganic phosphate in the presence of proteins:the actomyosin ATPase system. Anal Biochem, 77:10-17.

Löw R, Rockel B, Kirsch M, Ratajczak R, Hortensteiner S,Martinoia E, Lüttge U, Rausch T. 1996. Early salt stresseffects on the differential expression of vacuolar H+-ATPasegenes in roots and leaves of Mesembryanthemum crystallinum.Plant Physiol, 110:259–265.

Lütcke H A, Chow K C, Mickel F S, Moss K A, Kern H F,Scheele G A.1987. Selection of AUG initiation codons differsin plants and animals. EMBO J, 6:43-48.

Lüttge U, Ratajczak R. 1997. The physiology, biochemistry andmolecular biology of the plant vacuolar ATPase. Adv Bot Res,25:253–296

Mandala M, Taiz L. 1985. Partial purification of a tonoplastATPase from corn coleoptiles. Plant Physiol, 78:327-333.

Ratajczak R. 1994. The non-ionic detergent Brij 58 conserves thestructure of the tonoplast H+-ATPase of the Mesembryanthe-mum crystallinum during solubilization and purification. BotActa, 107:201-209.

Ratajczak R. 2000. Structure, function and regulation of the plantvacuolar H+-translocating ATPase. Biochim Biophys Acta,1465:17–36.

Rothnie H M, Reid J, Hohn T. 1994. The contribution ofAAUAAA and the upstream element UUUGUA to the effi-

ciency of mRNA 3'-end formation in plants. EMBO J, 13:

2200-2210.Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a

Laboratory Manual. 2nd ed. New York: Cold Spring HarborLaboratory Press. 363-371.

Sze H, Li X, Palmgren M G. 1999. Energization of plant cellmembranes by H+-pumping ATPases: regulation andbiosynthesis. Plant Cell, 11:677–690.

Tsiantis M S, Bartholomew D M, Smith J A C. 1996. Salt regula-tion of transcript levels for the c subunit of a leaf vacuolar H+-ATPase in the halophyte Mesembryanthemum crystallinum.Plant J, 9:729–736.

Wang B S, Lüttge U, Ratajczak R. 2001, Effects of salt treatmentand osmotic stress on V-ATPase and V-PPase in leaves of thehalophyte Suaeda salsa. J Exp Bot, 52:2355-2365.

Wang B S, Ratajczak R, Zhang J H. 2000. Activity, amount andsubunit composition of vacuolar-type H+-ATPase and H+-PPase in wheat roots under severe NaCl stress. J Plant Physiol,157:109-116.

Yu H-F, Chen J, Wang X-C. 2001. Effect of salt stress on theactivity and amount of tonoplast H+-ATPase from pea roots.Acta Bot Sin , 43: 586-591.

(Managing editor: ZHAO Li-Hui)