Proc. Natl. Acad. Sci. USAVol. 89, pp. 9205-9209, October 1992Plant Biology

Higher plant Ca2+-ATPase: Primary structure and regulation ofmRNA abundance by salt

(tomato/cation transport/salinity)

LARRY E. WIMMERS, NICHOLAS N. EWING, AND ALAN B. BENNETTMann Laboratory, Department of Vegetable Crops, University of California, Davis, CA 95616

Communicated by Emanuel P. Epstein, May 26, 1992 (received for review February 20, 1992)

ABSTRACT Calcium-dependent regulatory mechanismsparticipate in diverse developmentally, hormonally, and envi-ronmentally regulated processes, with the precise control ofcytosolic Ca2+ concentration being critical to such mechanisms.In plant cells, P-type Ca2+-ATPases localized in the plasmamembrane and the endoplasmic reticulum are thought to playa central role in regulating cytoplasmic Ca2+ concentrations.Ca2+-ATPase activity has been identified in isolated plant cellmembranes, but the protein has not been characterized at themolecular level. We have isolated a partial-length cDNA(LCA1) and a complete genomic clone (gLCA13) encoding aputative endoplasmic reticulum-localized Ca2+-ATPase in to-mato. The deduced amino acid sequence specifies a protein(Lycopersicon Ca2+-ATPase) of 1048 amino acids with a mo-lecular mass of 116 kDa, eight probable transmembrane do-mains, and all of the highly conserved functional domainscommon to P-type cation-translocating ATPases. In addition,the protein shares -50% amino acid sequence identity withanimal sarcoplasmic/endoplasmic reticulum Ca2+-ATPasesbut <30% identity with other P-type ATPases. Genomic DNAblot hybridization analysis indicates that the LycopersiconCa2+-ATPase is encoded by a single gene. RNA blot hybrid-ization analysis indicates the presence of three transcript sizesin root tissue and a single, much less abundant, transcript inleaves. Lycopersicon Ca2+-ATPase mRNA levels increase dra-matically upon a 1-day exposure to 50 mM NaCl. Thus thisreport describes the primary structure of a higher-plant Ca2+-ATPase and the regulation of its mRNA abundance by saltstress.

The regulation of cytosolic Ca2+ levels is widely recognizedas a central element of cellular regulatory processes ineukaryotes and is essential to the proposed role for Ca2+ asa second messenger. Active Ca2+ transport maintains lowlevels of cytosolic Ca2+ and in this way interacts with Ca2+release channels to affect regulated modulation of cytosolicCa2+. In plants, transient changes in cytosolic Ca2+ arethought to participate in many signal transduction pathways,including phytochrome responses (1), gravity perception (2),and stomatal function (3, 4). The alteration of cytosolic Ca2+levels has also been proposed to be a primary insult in bothchilling and salt stress of plant cells (5-7).

Active Ca2+ transport from the cytosol is catalyzed byhigh-affinity Ca2+-ATPases localized in the plasma mem-brane (PM) and sarcoplasmic/endoplasmic reticulum (SER)and in plant cells by a lower-affinity Ca2+/H+ antiportlocalized in the vacuolar membrane (refs. 8 and 9 andreferences therein). High-affinity Ca2+-ATPase-driven trans-port is proposed to play the most significant role in modu-lating cytosolic Ca2+ levels in the physiological range (8, 10,11).

Ca2+-ATPases have been purified, and their respectivegenes have been cloned from a wide variety of eukaryoticcells and are probably ubiquitous. In animals, Ca2+-ATPaseson both the SER and PM belong to the superfamily of P-typeion-translocating ATPases and have been well-characterized.The properties of both enzymes have been reviewed (11-13);similarities include formation of a phosphorylated reactionintermediate, inhibition by vanadate, requirement for Mg2+,and high substrate specificity for MgATP. In addition, bothfunction as dimers in the membrane, maintain Ca2+ gradientsof 1:1000 or greater, and exhibit a Kca of 0.1-1.0 AM at thecytosolic side. Nevertheless, animal SER and PM Ca2+-ATPases can be distinguished by several criteria. The SERCa2+-ATPase has a molecular mass of 110 kDa, and thePM Ca2+-ATPase has a molecular mass of 140 kDa. The ratioof Ca2+ transported per ATP hydrolyzed is 1 for the PMCa2+-ATPase and 2 for the SER Ca2+-ATPase, and the PMCa2+-ATPase is stimulated by Ca2+/calmodulin whereas theSER Ca2+-ATPase is not. Overall sequence identity betweenthe PM and SER Ca2+-ATPases is relatively low, beingsimilar to that observed between functionally divergentP-type ATPases such as the Na+/K+- and H+-ATPases.

Plant PM and ER Ca2+-ATPases are also P-type ATPases.Both are inhibited by vanadate, have a Kca of <1 ,uM, requireMg2+, and have a high affinity for MgATP (9, 10, 14-16),although the PM Ca2+-ATPase can also utilize other Mg-nucleotide triphosphates (15). The PM Ca2+-ATPase has anapparent molecular mass of 140 kDa but probably functionsas a dimer (17). However, conflicting reports concerningmolecular mass and calmodulin sensitivity of Ca2+-ATPasesin plant ER and PM preparations indicate the possibility ofspecies and/or organ differences (8, 18). In spite of the majorimportance ascribed to Ca2+-ATPases and their intensivestudy at the biochemical and physiological levels, higher-plant Ca2+-ATPases have not been purified or characterizedat the molecular level.The objectives of our research have been to determine the

structure and critically assess the function of higher-plantCa2+-ATPases in signal transduction and in responses toenvironmental stresses such as chilling and salinity. To thisend, we have isolated cDNA and genomic clones putativelyencoding a higher-plant ER-localized Ca2+-ATPase, Lyco-persicon Ca2+-ATPase (LCA). Here we describe the primarysequence of this Ca2+-ATPase,* its genetic complexity, andmRNA abundance under normal and high-salt conditions.

MATERIALS AND METHODScDNA Cloning. A tomato-root cDNA library comprised of

2.5 x 105 individual transformants was screened with a

Abbreviations: PM, plasma membrane; SER, sarcoplasmic/endoplasmic reticulum; LCA, Lycopersicon Ca2+-ATPase; Tm, melt-ing temperature.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. M%324).

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degenerate oligonucleotide [5'-GGIGCA(G)TCG(A)TTIAC-ICCA(G)TCICCIGTCAT-3'] corresponding to the highlyconserved amino acid sequence of the nucleotide bindingdomain found in all P-type ATPases (MTGDGVNDAP), asdescribed (19).Genomic Cloning. A tomato genomic library in A Charon 35

(ref. 20; kindly supplied by R. L. Fischer, University ofCalifornia, Berkeley) was screened with the LCA1 cDNAlabeled by random priming with [32P]dCTP (21) at conditionscorresponding to a melting temperature (Tm) of -20'C.RNA-Based Polymerase Chain Reaction (PCR) Amplifica-

tion. PCRs (22) were performed using, as template, first-strand cDNA synthesized from tomato-root poly(A) RNA,random-hexamer primers, and reverse transcriptase (Super-script RT; BRL). Reactions used two gene-specific primersflanking suspected intron locations. Reactions were cycled 30times at 940C for 2 min, 550C for 1 min (40C below thepredicted Tm for each primer), and 720C for 1 min. Eachreaction yielded a single product as determined by agarosegel electrophoresis. Reactions were desalted using Ul-trafree-MC microultrafiltration devices (Millipore) and li-gated into the plasmid PCR1000 (Clontech) for sequencing.

Sequencing. All sequencing was done by the dideoxynu-cleotide method (23). Both strands of the entire LCA1 cDNAand 10.3 kilobases (kb) of a single A clone containing theentire LCA1 gene were sequenced. Intron junctions wereconfirmed by sequencing PCR-amplified cDNA fragmentsspanning suspected introns. Sequence analysis was carriedout using PC-GENE computer software (IntelliGenetics).Genomic DNA Blot Hybridization Analysis. Tomato ge-

nomic DNA (10 jig) was digested with the indicated restric-tion enzymes, fractionated by agarose gel electrophoresis,and blotted to nylon membranes as described (19). Mem-branes were hybridized with random-primed LCA1 cDNA(final stringency of Tm -25°C). Genomic DNA and RNA (seebelow) blots were autoradiographed at -80°C using pre-flashed Kodak XAR-5 film and an intensifying screen(Cronex; DuPont).RNA Blot Hybridization Analysis. Root and leaf tissue was

removed from 10-day-old tomato seedlings grown hydropon-ically in lx Hoagland's solution (24). For salt treatments,NaCl was added to 50 mM after 10 days and plants wereharvested 24 hr later. Total RNA was isolated by the methodof Cathala et al. (25) and poly(A) RNA was purified byoligo(dT) affinity chromatography (26). Poly(A) RNA sam-ples (5 ,g) were size-fractionated by formaldehyde/agarosegel electrophoresis and transferred to nitrocellulose by cap-illary blotting. Hybridizations with random-primed LCA1and LHA2 cDNA probes were performed at 42°C as de-scribed (19). Final washes were done at a stringency of Tm-10°C. mRNA levels were quantified by optical densitome-try of autoradiographic images within the linear responserange using the Bioimage analysis system (Millipore).

RESULTSIsolation of cDNA and Genomic Clones. Screening of 2.5 x

105 colonies of a tomato-root cDNA library with the nucle-otide binding domain oligonucleotide yielded 10 stronglyhybridizing clones. Cross-hybridization between selectedprobes identified three classes of cDNAs. Nucleotide se-quence analysis indicated that a 1.8-kb cDNA was a partial-length cDNA encoding a polypeptide with extensive se-quence similarity to animal SER Ca2+-ATPases. This cDNAis referred to as LCA1 (Lycopersicon Ca2+-ATPase 1) byfollowing nomenclature suggested previously (27). In addi-tion to LCA1, two other classes of cDNAs were identifiedthat encode the PM H+-ATPases LHA1 and LHA2 asdescribed (19). Attempts to isolate a full-length LCA1 cDNA

clone from several tomato cDNA libraries using a 5' fragmentof the LCA1 cDNA were unsuccessful.The LCA1 cDNA was used to screen 7.5 x 105 plaques of

a tomato genomic library. Twenty-seven hybridizing plaqueswere purified. Restriction enzyme mapping indicated thatthese represented overlapping clones of a single genomiclocus. One clone (gLCA13) contained a single 14-kb genomicfragment that spans the entire LCA coding sequence (Fig. 1).

Sequence Analysis. The nucleotide sequences of LCA1 and10.3 kb of gLCA13 were determined, and an open readingframe encoding a putative Ca2+-ATPase was deduced (here-after referred to as LCA). The open reading frame is inter-rupted by six introns (Fig. 1). Three introns were identifiedby alignment with the partial-length cDNA, but three otherpotential introns resided in regions not within the clonedLCA1 cDNA. These intron junctions were confirmed bysequence analysis of cDNA fragments amplified by a RNA-based PCR using primers flanking each of the possibleintrons. Thus, introns shown in Fig. 1 were experimentallydetermined by alignment of the gLCA13 sequence withsequences of either the LCA1 cDNA or PCR-amplifiedcDNA fragments.The predicted amino acid sequence of LCA is shown in

Fig. 2. The location of the translation initiation codon (ATG)was predicted by alignment with SER Ca2+-ATPase se-quences and was the first ATG within 25 base pairs of thetermination codons in all three reading frames. This initiationcodon corresponds to a predicted open reading frame encod-ing a polypeptide with 1048 amino acid residues and amolecular mass of 116 kDa. The deduced amino acid se-quence contains all of the highly conserved domains commonto P-type ATPases (Fig. 2). Hydropathy analysis (Fig. 3)predicts eight transmembrane segments and a large cytoplas-mic loop between amino acids 450 and 750, consistent withthe structural model of other P-type ATPases (28).The deduced amino acid sequence of LCA was compared

to other P-type ATPases. The greatest homology is seen withthe SER Ca2+-ATPases of animals. Overall amino acididentity of LCA with animal SER Ca2+-ATPases is 50% (28,32-38), 27-35% with three putative yeast Ca2+-ATPases (39,40), and 25-31% with all of the other P-type ATPases (19, 27,41-56), including animal PM Ca2+-ATPases (57, 58) (Table 1).In addition to the highly conserved functional domains of allP-type ATPases, large regions of relatively high amino acidsequence identity (>60%) are seen between LCA and theSER Ca2+-ATPase, but not between LCA and other P-typeATPases (regions A, B, and C; Fig. 2). The amino acididentity between LCA and animal SER Ca2+-ATPases withinthe combined 250 residues of these regions is 70% andbetween LCA and all other P-type ATPases is 18-29%.Regions A, B, and C overlap five of the eight putative

transmembrane domains (Fig. 3) and include the high-affinityCa2+ binding site. Site-directed mutagenesis of individualamino acids in a rabbit SER Ca2+-ATPase has revealed that11 amino acid residues that reside within these transmem-

kB 1 2 3 4 5 6 7

gLA1 I U 1Icc 'a F0 0 C

gLCA1 3 rw -w MzI1

LCA1l

8 9 10

wo- IwI w

11 A l

FIG. 1. Structure of the tomato Ca2+-ATPase gene. The se-quenced portion of genomic clone gLCA13 is shown with the map ofrestriction enzyme sites and the location of the cDNA probe (cross-hatched box) used in genomic DNA blot analysis. Exons are repre-sented by solid boxes. The location and exon composition of thepartial-length cDNA LCA1 are shown below the map of gLCA13.

11 / Xl

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SERCA1 HE ------AAHSKSTEECLAYFGVSET --------TGLTPDOVKRHLEKYGNNELPAEEGKSLWELVIEQFE-DLLVRILLLAACISFVLAWF ---EEGEETITAFVEPFVI 94LCA NEEKP--FPAWSUSVDOCLKEYOVKLE --------KGLSTYEVDKRRERYGLNELEKEKGKPLRLVLEQFD-DTLVKILLGAAFISFVLAYVNQDETGESGFEAYVEPLVI 101LHA1 KAEKPEVLDAVLKETVD-LEN I PI EEVFENLRCTREGLTATAAQERLS I FGYNKLEEKKESKFLKFL- -GFNUPLSWVNEAAAI1AIALA ---NG0GKPPDWQDFVG- - - I 103

*--- * * ** * * * * * ** ** * *A

* -*-**-******** * *0*0*0 *0 ****** ********0* * *** ***0***0*0*

SERCA1 LLILIANA2I0VWERNAENAIEALKEYEPE6RADRKSVRIKARDIVPDIVEVAVGDKVPADIRILSIKSTTLRVD6SILTGESVSVIKHTEPVPDPRAVNODKKN20LCA LUWI LVLNAIV"JWESNAEKALEALKEESAVLRDGYL-VPDFPAKELVPD IVELRVGDKVPRVATLKSSTLRVEOSSLTGESMPVTKSTDFLATDDCELQAKEN 212LHAl ITLL I INSTISFIEENNAGNAAAALNARLAPAVLRDGKWDEED--ASVLVPGDI ISIKLGDI IPADARL--LEGDPLKIDOSALTGESLPVTKGPG-0------------ 198

A- -I-** *** ** **** ****- 0* ******** ** 0*** * * **** **

SERCAl MLFSGTNIAAGKALGIVATTGVSTEIGKIRDQN--AATEaDKTPLQQKLDEFGEOLSKVISLICVAVWLINIGNFNDPVHGGSUIRG -------AIYYFKIA--VALAVAAI 307LCA 3VFAGTTVV2GSCIClV2TGCTE2IGIOROIDAS2EESDTPLKKKLDEFGNRLTFAIGVVCLV*AINYYFLSWVVDDWPSDFRFSFEKCAYYFKIA--VAUVAAI322LHA1 GVYSGSTCKQGEIEAVVIATGVHTFFGKAAHLVDSTN ---------- QVGNFQKVLT-AIGNFCIC--SIAVGI IE! VM--------YPIQNRKYRPGIDNLLVLLIGGI 289

*** **-***0***** 0** ****** *0**0***** ****************- * * 0* ** * * * ***

SERCAl PEGLPAVI TTCLALGTRRN IAIVRSLPSVETLGCTSVICSDKTGTLTTNQMSVC1MFI IDICWGDFCSLNEFSITGSTYAPEGEVLKNDKPIRSOQFDGLVELAT I CAL 419LCA PEGLPSVI TTCLALGTRIMANAIVRKLQSVETLGCTTVICSDKTGTLTTNQNSVSEFFTLGRKTTA-CRV- -FGVEGTTYDPKDGGIHNCCMDA- -MLLLKEI CAI 429LHA1 PIAMPTVLSVTMAIGSHRLAOQGAITKRNTAIEEMAGNDVLCSDKTGTLTLHKLTVDKALI --------------EVFACGIDADTVLMAARASRIEN------- 374

------------ Ti ------------0*0 * 0**** ***000 * * **

SERCAl CNDSSLDFNETKGVYEKVGEATETALTTLVEKMNVFMTEVRNLSKVERANACNSVIRQLMK ------------KEFTLEFSRDRKSMSVYCSPAASSRMVGNKMFVKGAPE 519LCA CNDAGV- FCDGR- LFKATGLPTEAALKVLVEKGVPDSKARCKI RDAQI VSSYLIDRNTVKLGCCDWWNKSKRVATLEFDRVRKS4GVI VREPNGS-----HRLLVKGAFE 534LhA1 ----------------------QDAIDTAIVGMLADPKEARAGIREIHF----------------------------LPFNPTDKRTALTYLDGEGKNHRVS-----KGAPE 431

0* * 0* * *0* * .*0 00000* -- *- *00

SERCA1 GVIDRCNYVRVGTTR-VPHTGPVKEKILSVIKEWGTGRDTLRCLALATRD---------TPPKREENVLDDSSRFNEYETDLTFVGWGMLDPPRKEVMGSSILCRDAGIRV 622LCA SLLERSTYVuLABYSTVPLDESC-RQ- -LL L LmLESS*GLRCLGLAY*DDLG00 LS3YYAATHa0*UWIN6DPCIYfILVFGWGIRDIPPREEVHRAVIIDCRRAGIK1 64"LHA1 0ILN------LAHNKS-DIERRVHTVIDKF--AERGLRSLGVAYQE--------VPEGRKESA----------GGPFIALLPLFDPPRHDSAETIRRALNLGVNV 511

* ** *0** *0 ** *- - - - -ITI- - - - - - - -- - - -- - - - TV+V- --*0*0***** ** * * ** * **0*0* 0*0******* ************** **

SERCA1 IITGDNKGTAIAICRRIGI - - -FGENEEVADRAYTGREFDDLPLAEOREACRRAC--CFARVEPSHKSKIIVEYLOSYDEITAMTGDGVNDAPALKKAEIGIA G-SGTAV 727LCA MVITGDNKSTAEAVCREIQL ----FSNGENLRGSSFTGKEFMAFSSOQQI E I LSODGGKVFSRAEPRHKQE IVRLKEGEGIVA4TGDGVNDAPALKLAD I GIAMGI TGTEV 752LHAIK1JTCDOLAIGKETGRRLGMGTNMYPSSALLGQTK--DESIAALPIDELIEK ----ADGFAGVFPEHNYEIVKRLQAAKHICGMTGDGVNDAPALKKADIGIAVD-DATDA 615

0*** 0 0 * * ** ***00*0*********_~~~~~-_- -

Vl

* *00*00*** * * *****0*00*0*0*****0**** *0*0** 0*0***0*** 0*00*0** 0 000 **. **0* *

SERCA1 AKTASE4VLADDNFSTIVAAVEEGRAIYNNKOFIRYLISSNVGEWCI FLTAALGLPEALIPQLLWIVLVTDGLPATALGFNPPLDINDRPPRSPKEPLISGW ---- LF 835LCA AKEASDNVLADOHFSTIVSAVAEGRSIYNIAF I RYNI SSNVGEVI SI FLTAVLGIPECLIPLLWNLVTDGPPATALGFNPADVDIMQKPPRKNTDALINHSW --- VF 860LHA1 ARSASDIVLTEPGLSVIISAVLTSRAIFQRMNYTIYAVSITI-RIVLGFMLLALIW.FDFPPFNVLIIAILDG----TIMTISKD--------- RVKPSPLPDSWKLAEIF 714

****** ** * *00* 0* 0 *.**0 ** *

******0*0** * *0 0 0*0 00 00*0***0**** 0*0**

SERCA1 FRYMAIGGYVGAATVGAAAUUF------------ HYAEDGPGVTYHOLTHFMQCTE----DHPHFEGLD--------CE FEAPE- -PHTMALSVLVTIENCNALHSLSEHO 921LCA FRYNVIGSYVGIATVGI FIVWYTOASF ---LGINIVSDGHTLVELSOLRUGECSTWTHFTVSPFKAGNRLITFSDPCEYFTVGKVKAMTLSLSVLVAIEMFNSLNALSEDN 969LHA1 TTGWLGGYLAITVIFFWMAYKTNFFPRI FGVSTL-EKTATDDFRKL --------- ASAIYLQVSTISQALI FVTRSRSWSFVERPGLLLVFAFFVA-QLVATLIAVYANW 815

***0 * 0 0 0 0 * 0 *

SERCA1 SLMRMPP----WVNILLGSICLSMSLHFLILYVDP----------LPHIFKL ------- KALDLTQW------LUVLKISLPVIG -----LDEILKFIAR ----NL---- 993LCA SL I KMPP----RMPULLVAMSLSFALHSVILYVPF--------- LADIFGI ------- VPLSLVEW-U----LLVILLSAPVIL----- IDEVLKFVGRRRRRTKLK --- 1046LHAI SFMAIEGIGGIWLYNIVYIPLDLIKFLIRYALSGKADLVLEORIAFTRKKDFGELRELHARTLNGLPKIFSETTUFNELNLAEEAKRRAEIARLR 927

* * 0 0 0 0 0 * 0 0 **C

SERCA1 ---------------------------EG 995LCA ---------------------------AA 1048LHA1 ELHTLKGIVESVVKLKGLDIETIQSYTV956

brane domains are essential to Ca2+ binding (59). All 11 of complete conservation of Ithese residues are conserved in LCA. This high degree of suggest that LCA is funconservation of the 11 functional amino acid residues is not ATPases. It is therefore likcseen in any other P-type ATPase. For example, Na+/K+- Ca2+-ATPase that is locali;ATPases share 8 of the putative Ca2+-binding residues and Genomic DNA Blot HybriH+-ATPases (including tomato LHA1) share only 3 identical number of genes likelyresidues. Overall, the high degree of sequence identity of ATPases in tomato a 600-bESER Ca2+-ATPase-specific domains A, B, and C and the the LCA1 cDNA was used

DNA (Fig. 4). The probe fr50 A B C40 '1 2 3 4 5 6 7 8 Table 1. Amino acid sequenc

P-type ATPases30 -

-D 20-

10 P-type ATPaseIi ~~~~~~~~~~~~~~AnimalSER Ca2+-ATPase2.0-... ....PM Ca3+-ATPase

'O%-lo YeastZ:~~~~FPMR1-20 ~~~~~~~~~~PMR2

-30rCTA3

200 400 600 800 1000Amino Acid Number

FIG. 3. Hydropathic profile of the deduced LCA amino acidsequence determined by the method of Kyte and Doolittle (30) witha window of 15 residues. Areas below horizontal bars indicatedomains of high homology unique to LCA and the SER Ca2+-ATPases as displayed in Fig. 2. Numbers 1-8 indicate transmem-brane domains predicted by the method of Rao and Argos (31).

Plant H+-ATPaseYeast H+-ATPaseH+/K+-ATPaseNa+/K+-ATPase

FIG. 2. Deduced amino acidsequence of LCA, aligned with arabbit fast-twitch muscle SERCa2+-ATPase (SERCAl) (28) anda tomato plasma-membrane H+-ATPase (LHA1) (19). Asterisksindicate identical residues be-tween LCA and SERCA1 (above)and LCA and LHA1 (below).Dashed underlines indicate areasof homology shared with allP-type ATPases. Solid lines indi-cate areas of high homology (60%oor greater) unique to LCA andSER Ca2+-ATPases. Alignmentwas done by the method of Myersand Miller (29) using the struc-ture-genetic matrix, an open gapcost of 5, and a unit gap cost of 3.

11 functional amino acid residuesctionally related to SER Ca2+-ely that gLCA13 encodes a P-typeized in the ER membrane.idization Analysis. To estimate theto encode ER-localized Ca2+-ase-pair restriction fragment fromto probe blots of tomato genomicragment comprised a region of the

ce comparison of LCA and several

% identity SequencesDomains compared,

Overall A + B + C no.

49-51 70 1127-30 24-29 3

35 24 127 25 127 27 1

25-26 18-21 425-27 18 228 23 3

29-31 21-27 11

Percentage of identical residues determined in alignments asdescribed in Fig. 2. All sequences are deduced from cloned genesequences: animal SER Ca2+ (28, 32-38); PM Ca2+ (57, 58); yeastPMR1, PMR2, and CTA3 (39, 40); plant H+ (19, 27, 43, 44, 51, 53);yeast H+ (52, 53); H+/K+ (47, 48, 55); and Na+/K+ (41, 42, 45, 46,49, 50, 54, 57) -ATPases.

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23.1 -

9.4 -6.5 -

4.3 --

2.3 -2.0 -

FIG. 4. Gel-blot hybridizationanalysis of LCA genomic DNA.Each lane was loaded with 10 .gof tomato genomic DNA digestedwith the indicated restriction en-zymes. Blots were hybridizedwith a 32P-labeled 600-base-paircDNA fragment and washed atmoderate stringency [65°C, 2xstandard saline citrate (SSC)].Molecular sizes in kb are indi-cated.

gene devoid of introns and of restriction sites used to digestthe genomic DNA (Fig. 1). At moderate stringency (Tm-25°C), the probe hybridized to a single genomic restrictionfragment in each digest. The size of each hybridizing frag-ment corresponded to the size predicted by restriction en-zyme analysis ofgLCA13 (Fig. 1). Thus these results suggestthat LCA is encoded by a single gene in tomato.RNA Blot Hybridization Analysis. To characterize the

structure and abundance ofLCA mRNA, gel blots of poly(A)RNA isolated from roots and leaves oftomato seedlings wereprobed with LCA1 at high stringency (Tm -100C). In tomatotissues grown under low-salt conditions (1x Hoagland'ssolution), the cDNA hybridized to transcripts of 3.5, 4.2, and6.5 kb in roots and to a single less-abundant 4.2-kb transcriptin leaves (Fig. 5). Because RNA gel blots were probed athigher stringency than genomic DNA gel blots, it is likely thatthe multiple transcripts were derived from a single LCA gene.LCA mRNA was estimated to be 9-fold less abundant inleaves than in roots.LCA and LHA1 mRNA levels in roots and leaves were also

assayed by RNA gel-blot hybridization analysis of poly(A)RNA isolated from tissues grown in the absence ofadded saltor after a 1-day exposure to 50mM NaCl. Relative to controlplants, the level of LCA mRNA was significantly enhancedin leaves (310%) and, to a lesser extent, in roots (180%o) aftersalt exposure (Fig. 6). In contrast, the level of LHA2 mRNAwas not significantly affected by salt treatment in eitherleaves or roots (Fig. 6).

o 0u c

9.5 -7.5 -

4.41-W.

2.4FIG. 5. Gel-blot analysis of

LCA RNA. Each lane was loadedwith 5 ,g of poly(A) RNA isolatedfrom the seedling organ indicated.Blots were probed with the LCA1cDNA and washed at high strin-gency (60°C, 0.2x SSC). Molecu-lar sizes in kb are indicated.

1.4 -

DISCUSSIONgLCA13 encodes a P-type ion-translocating ATPase puta-tively identified as a Ca2 -ATPase that is likely to be local-ized in the ER. The deduced LCA amino acid sequenceincludes all of the highly conserved functional domains andpredicted transmembrane structure common to all P-typeATPases. Its proposed identification as an ER-localizedCa2+-ATPase rests on the relatively high degree of sequencesimilarity between LCA and SER Ca2+-ATPases. Overallsequence comparisons indicate that the LCA polypeptide issignificantly more similar to animal SER Ca2+-ATPases thanto all other P-type ATPases (Table 1). Indeed, outside of thehighly conserved functional domains common to all P-typeATPases, little sequence similarity is seen between thededuced LCA amino acid sequence and any other P-typeATPase, including animal PM Ca2+-ATPases and higherplant H+-ATPases. Thus, the deduced LCA amino acidsequence is more highly related to the SER Ca2+-ATPasesfrom distant evolutionary sources than to H+-ATPases fromthe same species, tomato (19). It should be noted thatrelatively little sequence similarity is seen between LCA andthree putative yeast Ca2+-ATPases. The subcellular localiza-tion of these enzymes, however, has not been firmly estab-lished, and one (PMR2) may actually be a Na+-ATPase (60).The LCA and SER Ca2+-ATPases share several large areas

of high sequence conservation outside of the P-type ATPaseconserved functional domains. These areas encompass fiveofthe eight putative transmembrane segments (segments 1, 2,5, 7, and 8). Three of these transmembrane sequences arethought to contain high-affinity calcium binding domains.Mutation of 11 residues in these segments abolishes Ca2+-dependent phosphorylation and Ca2+ transport in an animalSER Ca2+-ATPase (59). All 11 of these residues are con-served in LCA. It is interesting to note that these samedomains are not conserved in PM Ca2+-ATPases. The highlevel of sequence conservation over these transmembranesegments suggests that these regions specify a subset ofCa2+-ATPases and that this subset may be defined by itssubcellular location, the ER.The animal PM Ca2+-ATPase is calmodulin-stimulated;

binding calmodulin at a site near the C terminus in an areapredicted to form an amphiphilic a-helix localized in thecytoplasmic domain (61, 62). A similar domain near the Cterminus is seen in the putative Ca2+-ATPase encoded by theyeast PMR2 and CTA3 genes (39). The animal SER Ca2+-ATPase is not calmodulin-stimulated and lacks the conservedcalmodulin binding motif of the PM Ca2+-ATPases. The

Probe: LCA1 LHA2

o +o

+

9.5 -

7.5 -

4.4- _

2.4 - n-1 p.2.4-

1.4 -

FIG. 6. Gel-blot analysis ofLCA and LHA2 mRNA abun-dance in plants exposed to 0 or 50mM NaCl. Each lane was loadedwith 5 ,ug of poly(A) RNA isolatedfrom leaves of seedlings grownhydroponically in 1x Hoagland'ssolution or exposed to 50 mMNaCI for 1 day. Blots were hybrid-ized to LCA1 cDNA and washedat high stringency (60°C, 0.2xSSC). Data shown represent themore conservative of two experi-ments with similar results. Molec-ular sizes in kb are indicated.

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Proc. Natl. Acad. Sci. USA 89 (1992) 9209

deduced LCA amino acid sequence does not contain prob-able amphiphilic a-helices in the C-terminal 100 amino acids,suggesting that the LCA Ca2+-ATPase is not calmodulin-stimulated. There are conflicting reports as to whether higherplant ER and PM Ca2+-ATPases are calmodulin-stimulated(2, 8, 18).Based on genomic DNA gel-blot analysis, there appears to

be a single gene encoding SER-like Ca2+-ATPases in bothtomato and Drosophila (59), whereas in mammals the SERCa2+-ATPases are encoded by three genes (32, 62). AlthoughLCA appears to be encoded by a single gene, mRNA levelsvary in different tissues and the presence of multiple tran-scripts suggests the possibility of differential mRNA splicingthat could yield tissue-specific gene products. Tissue speci-ficity of differentially spliced mRNAs has been observed forthe slow twitch/cardiac muscle SER Ca2+-ATPase(SERCA2) (28). One of the SERCA2 isoforms is seen only inslow twitch and cardiac muscle, whereas another is seen ina variety of tissues including brain, kidney, and stomach.Accumulation ofLCA rftRNA in response to salt exposure

appears to be a relatively specific response, in that mRNAsencoding H+-ATPases did not show a similar response. Ourobservation is consistent with a current model that salt stressperturbs intracellular Ca2+ levels (6). In concurrence withthis model, the increase in LCA mRNA may represent aresponse to enhance the capacity for intracellular Ca2+sequestration. This interpretation implies that enhancedCa2+-ATPase gene expression and enhanced Ca2+-ATPaselevels represent an adaptive response to salinity. Becausegene transfer in tomato is relatively straightforward, cloningofthe putative Ca2+-ATPase gene presents the opportunity todirectly test this hypothesis in transgenic plants with elevatedor reduced levels of LCA gene expression.

We thank J. Durnan, S. Fujimoto, and K. Toenjes for technicalassistance and K. Green for manuscript preparation. This work wassupported by National Science Foundation Grant DMB87-16112 andL.E.W. was supported by a National Science Foundation Postdoc-toral Research Fellowship in Plant Biology.

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