genomic comparison of p-type atpase ion pumps in · genomic comparison of p-type atpase ion pumps...

Genomic Comparison of P-Type ATPase Ion Pumps inArabidopsis and Rice1

Ivan Baxter, Jason Tchieu, Michael R. Sussman, Marc Boutry, Michael G. Palmgren, Michael Gribskov,Jeffrey F. Harper*, and Kristian B. Axelsen

Department of Cell Biology, Plant Division, The Scripps Research Institute, La Jolla, California 92037 (I.B.,J.F.H.); Department of Biology, University of California, San Diego, La Jolla, California 92093 (J.T., M.G.);Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706 (M.R.S.); Unite de BiochemiePhysiologique, Institut des Sciences de la Vie, Universite Catholique de Louvain, Louvain-La-Neuve,Belgium (M.B.); Plant Physiology and Anatomy Laboratory, Department of Plant Biology, The RoyalVeterinary and Agricultural University, Copenhagen, Denmark (M.G.P., K.B.A.); and SWISS-PROT group,Swiss Institute of Bioinformatics, Geneva, Switzerland (K.B.A.)

Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase geneswere identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draftsequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two differentplant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the sizeof Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexistingrepertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-typeATPases: heavy-metal ATPases (P1B), Ca2�-ATPases (endoplasmic reticulum-type Ca2�-ATPase and autoinhibited Ca2�-ATPase, P2A and P2B), H�-ATPases (autoinhibited H�-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), anda branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potentialorthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intronpositions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known aboutunique and common features of related pumps, clear differences between some members of the calcium pumps indicate thatevolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemicalfunctions.

Ion pumps belonging to the P-type ATPases super-family share a common enzymatic mechanism inwhich ATP hydrolysis is used to transport ionsacross a membrane. The name “P-type” comes from aphosphorylated intermediate that is characteristic ofthe enzyme’s catalytic cycle (Pedersen and Carafoli,1987). Two structures of 3.1 and 2.6 Å resolution of acalcium pump (sarcoplasmic/endoplasmic reticulum[ER] calcium ATPase [SERCA]) have greatly in-creased the understanding of the mechanism of iontransport by P-type ATPases (Toyoshima et al., 2000;Toyoshima and Nomura, 2002). These structures pro-vide the foundation for understanding the mecha-nism of all P-type ATPases. For example, these struc-tures have been used to model P-type proton(Bukrinsky et al., 2001; Kuhlbrandt et al., 2002; Ra-

dresa et al., 2002) and sodium (Ogawa and Toyo-shima, 2002) pumps.

P-type ATPases are found in all three branches oflife and are used to translocate a diverse set of ions,including H�, Na�/K�, H�/K�, Ca2�, heavy metals,and possibly lipids (Axelsen and Palmgren, 1998).The superfamily has been divided into five majorbranches (with 10 sub-branches) that reveal a corre-lation between sequence identity and ion specificity.For example, all the heavy-metal pumps from bacte-ria, plants, and humans share significant sequencesimilarities and cluster together as the P1B subfamily(Palmgren and Axelsen, 1998). Thus, the ion specific-ities characteristic of the different branches of P-typeATPases appear to have evolved very early.

The most P-type ATPases yet identified in a singleorganism have been found in the completed genomesequence of Arabidopsis. Following the 45 pumpsidentified in the initial genome release (Axelsen andPalmgren, 2001), an additional P-type ATPase wasidentified in further releases (P-type ATPase database;http://biobase.dk/�axe/Patbase.html) giving a to-tal of 46 P-type ATPases in this model plant. Incomparison, Brewer’s yeast (Saccharomyces cerevisiae)has only 16 (Catty et al., 1997), fission yeast (Schizo-saccharomyces pombe) has 14, Caenhorabditis elegans has

1 Work is supported in part by the Department of Energy (grantno. DOE DE–FG03–94ER20152 to J.F.H.), Syngenta (grant toJ.F.H.), by the National Science Foundation (grant no. DBI 0077378to J.F.H. and M.G.), and by Human Frontiers Science Program(grant no. RG0268 to J.F.H., M.R.S., M.B., and M.G.P.).

* Corresponding author; e-mail [email protected]; fax858 –784 –9840.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.103.021923.

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21, fruitfly (Drosophila melanogaster) has 14, Synecho-cystis sp. PCC6803 has 9, and human (Homo sapiens)has 36 (P-type ATPase database; http://biobase.dk/�axe/Patbase.html).

The apparent expansion in numbers of P-typeATPases in Arabidopsis has at least three potentialexplanations. First, multiple isoforms may haveevolved different modes of regulation and activity toprovide specific cell types with unique biochemicalproperties. Second, multiple isoforms may haveevolved under the control of different tissue-specificpromoters to provide the appropriate levels of ex-pression in specific tissues. Third, some of the mul-tiple isoforms may represent functionally redundantduplications.

The recent completion of two shotgun sequences ofthe rice (Oryza sativa) genome (Goff et al., 2002; Yu etal., 2002) provides the first opportunity to comparethe full complement of P-type ATPases from a mono-cot with that of a dicot, Arabidopsis. Here, we reportthe identification of 43 members of the P-type AT-Pase superfamily in rice. The observation of similarlylarge families in both rice and Arabidopsis suggeststhat both dicots and monocots have evolved with alarge preexisting repertoire of P-type ATPases. Al-though the unique and redundant functions of thesemultiple pumps are largely unknown, comparison ofthe full complement of genes from two differentplants allows for the grouping of similar pumps. Ourgenomic comparison of sequence identities and in-tron positions suggests that a common angiospermancestor harbored a core set of at least 23 P-typeATPases, providing the archetypal representativesfor 23 clusters of P-type ATPases currently present inrice and Arabidopsis.

RESULTS AND DISCUSSION

Forty-Three Rice P-Type ATPase Genes

Our analysis identified 43 members of the P-typeATPase family in rice (Fig. 1; Supplemental Table A;supplementary data can be viewed at www.plant-physiol.org). This is three fewer than found in Ara-bidopsis. To ensure that all possible P-type ATPasegenes were identified, multiple independent se-quences were searched in several different ways. Wesearched both the Beijing Genomics Institute (Yu etal., 2002) and Syngenta (Goff et al., 2002) rice genomesequences, in addition to the partial sequences de-posited at The Institute for Genome Research (byDecember 16, 2002). We searched each data set foreither (a) sequences with overall sequence identity toknown plant P-type ATPases or (b) sequences thatmatched one of five DKTGTXX motifs (where XX isLT, IT, VT, MT, or II), a sequence that is found inevery known P-type ATPase (Axelsen and Palmgren,1998). Because one additional Arabidopsis isoformwas identified in subsequent releases of the “com-plete” Arabidopsis genome, it is possible that addi-

tional rice isoforms may be identified in future ver-sions of the rice genome sequence.

The Expansion of the Rice Genome Did NotIncrease the Number of P-Type ATPases

The rice genome is approximately 3.5 times the sizeof Arabidopsis (430 Mb compared with 120 Mb; Goffet al., 2002; Yu et al., 2002). This size difference hasbeen attributed to (a) a 1.25- to 2.5-fold increase in thenumber of genes (32,000–55,000 in rice versus 26,000in Arabidopsis), (b) an increase in intergenic dis-tances, and (c) an increase in intron size and number.Of these variables, the only difference we confirmedfor the rice P-type ATPase genes was a 2-fold in-crease in the average size of introns (356 to 164 bp).

In the context of a possible 2.5-fold increase in thenumber of rice genes, it is noteworthy that the num-ber of P-type ATPases did not show a parallel expan-sion. This same observation has been noted for 15other gene families (Banuelos et al., 2002; Goff et al.,2002; Baumberger et al., 2003; Jasinski et al., 2003),raising a question about the function of the addi-tional 6,000 to 29,000 rice-specific genes. At present,the expectation is that much of the increased numberof genes is due to “over-predictions,” because manyof the novel rice genes are not supported by ex-pressed sequence tag (EST) data and do not showsimilarity to known proteins in any organisms, in-cluding corn (Zea mays; Yu et al., 2002). Because ofuncertainties in the accurate prediction of rice genemodels, we did not attempt to evaluate the intragenicdistances surrounding the rice P-type ATPases.

In contrast to a general expectation of more intronsin rice genes compared with Arabidopsis (Yu et al.,2002), we observed the same average number (13)and highly similar positions within potential or-thologs (Figs. 2–7). Thus, the approximate numberand organization of P-type ATPases appear to havebeen maintained during the evolution of rice andArabidopsis.

Twenty-Three Clusters of the P-Type ATPases in theMonocot-Dicot Ancestor

Of the 10 subfamilies of P-type ATPases identifiedto date, six are present in both rice and Arabidopsis:P1B (heavy-metal ATPases [HMA]), P2A (endoplasmicreticulum [ER]-type calcium ATPase [ECA]), P2B (au-toinhibited calcium ATPase [ACA]), P3A (autoinhib-ited H� ATPase [AHA]), P4 (putative aminophospho-lipid ATPase [ALA]), and P5 (P-type ATPase, type 5,P5; Fig. 1). Absent from both organisms are the P1A(K�), P2C (Na�/K�), P2D (Na� or Ca2�), and P3B(Mg2�) subfamilies.

We further divided the six subfamilies of plantP-type ATPases into 23 clusters, based on proteinsequence similiarities, with members of each clusterpresent in each organism (Figs. 2–7). For ECAs (P2A)

Arabidopsis and Rice P-Type ATPases

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and AHAs (P3A), a similar delineation of clusters haspreviously been proposed (Pittman et al., 1999;Arango et al., 2003). For genes in all 23 clusters, aninspection of intron positions provides strong corrob-oration of clustering patterns (see Figs. 2–7). Within acluster, there is a striking conservation of intronnumbers and positions; within each cluster, an aver-age of 82% of all intron positions are conserved. Incontrast, intron patterns are highly dissimilar be-tween clusters. Together this suggests that (a) theangiosperm ancestor had at least 23 P-type ATPases,(b) these 23 P-type ATPases had been present for aconsiderable time period before the split, and (c) thegene structure has been relatively stable since thesplit from the common angiosperm ancestor.

In total, we identified only three putative ATPase-like proteins that have features suggesting a dysfunc-tional ATPase or a pseudogene. Two were previouslyreported for Arabidopsis (At5g53010; Fig. 4; andAt4g11730; Fig. 5) with similarity to ACA and AHApumps, respectively (Axelsen and Palmgren, 2001),whereas a third was found here in rice (PlantsT no.64568; Fig. 6) with similarity to ALA pumps. Al-though these putative proteins have regions of strongidentity to P-type ATPases, they lack one or moreregions considered essential for activity. Becausenone of these ATPase-like genes has a correspondinghomolog in the other species, they either representpsuedogenes or encode unique, species-specificATPase-like proteins.

Three Rice Genes Have Arabidopsis-Like GC Profiles

It has been observed that most monocot genes(with experimentally confirmed gene models) have anegative gradient of GC content starting with thestart codon (Wong et al., 2002). This phenomenon hasbeen observed in multiple monocots and is absent inall dicots studied. The change in GC content ismainly a function of the wobble position of monocotcodons. As a result, the 5� end of monocot genes canhave up to 25% higher GC content than the 3� end(Yu et al., 2002). These gradients have not been re-ported in Arabidopsis, which has an average GCcontent of 43% throughout the genic regions (Yu etal., 2002).

Figure 1. A, Phylogenetic tree showing 23 clusters of Arabidopsisand rice P-type ATPases. The tree was constructed by aligning full-length sequences with ClustalW, and then the Phylip programs prot-

dist and fitch were used to create the tree. The major branches of thephylogenetic tree are named according to (Axelsen and Palmgren,1998). B, Overview of plant P-type ATPases showing topology dif-ferences and ion specificities. Branches are designated from P1B toP5, with protein names underneath. The putative transported ions(substrates) are indicated. Boxes, Transmembrane segments; blackcircles, regulatory regions containing autoinhibitory sequences;white circles, HMA domains; black squares, CC dipeptide domains;white squares, poly-His domains. Cluster numbers correspond to thenumbers found in the phylogenetic tree in A. For each cluster, thenumber of sequences in rice and Arabidopsis is indicated. If asubcellular location is known for a member of that group, it is notedin the right column. PM, Plasma membrane; Vac, vacuole.

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The majority of the rice P-type ATPases (33 of 43genes) have 5� GC gradients (Fig. 8). Of the Arabi-dopsis P-type ATPases, only AtHMA5, whose genemodel has yet to be confirmed by a cDNA, has a

small gradient. Interestingly, three genes, OsAHA9,OsALA9, and OsALA10, have flat GC profiles similarto Arabidopsis genes. Although the potential func-tions of these gradients in transcription and transla-tion remain unknown, it is clear that since the mono-cot/dicot divergence, either the monocots haveevolved these gradients or the dicots have lost them.Within the context of the rice P-type ATPases, thethree genes with “flat-GC profiles” present an inter-esting opportunity to study this phenomenon fromthe perspective of regulatory function and genomeevolution.

HMA, P1B

There are eight P1B ATPases in rice just like inArabidopsis. The P1B ATPases are divided into six

Figure 3. A, Phylogenetic tree of the P2A subfamily revealing twoclusters. The tree was constructed from full-length protein sequences.Numbers denote clusters. Branches leading to the rice isoforms arethicker, and the rice isforms are preceded by a bullet. All bootstrapvalues are greater than 90/100. B, Distributions of introns and exonsin the P2A subfamily arranged in clusters. The phylogenetic tree usedto define the clusters is taken from A but is shown in a different view.The genes are aligned around the phosphorylation site, [DKTGT],marked by a star. Phylogenetic tree branch lengths are not to scale,but are included to show groupings.

Figure 2. A, Phylogenetic tree of the P1B subfamily revealing sixclusters. The tree was constructed from full-length protein sequences.Numbers denote clusters. Branches leading to the rice isoforms arethicker and the rice isforms are preceded by a bullet. The bootstrapvalues for OsHMA2 and OsHMA3 having a separate branch were51/100 so they were collapsed to the nearest node. All other boot-strap values are greater than 98/100. B, Distributions of introns andexons in the P1B subfamily arranged in clusters. The phylogenetic treeused to define the clusters is taken from A but is shown in a differentview. The genes are aligned around the phosphorylation site, [DK-TGT], marked by a star. Phylogenetic tree branch lengths are not toscale but are included to show groupings.





clusters, based on sequence alignments and intronpositions (Fig. 2). Seven Arabidopsis P1B ATPaseswere previously identified (Axelsen and Palmgren,2001), but the completion of the Arabidopsis se-quencing revealed an eighth gene, AtHMA8, which ismost identical (43%) to PAA1/AtHMA6. It was pre-viously observed that Arabidopsis underwent anexpansion in numbers of P1B ATPases compared withother eukaryotic organisms, which only have one ortwo (Axelsen and Palmgren, 2001). The parallel dis-covery of eight P1B ATPases in rice suggests that thisexpansion was important for the evolution of angio-sperms (for a recent review of heavy-metal transportin plants, see Williams et al., 2000).

Clusters 1 and 2 are possible Zn2�/Co2�/Cd2�/Pb2� ATPases, whereas isoforms in clusters 3 to 6 arebelieved to be Cu�/Ag� ATPases (Axelsen andPalmgren, 2001). Although pumps within clustersshare at least 50% identity, the identity between thefarthest clusters is less than 27%, giving the HMA(P1B) subfamily the distinction of the most diversesubfamily of plant pumps.

Several heavy-metal-binding motifs have beenidentified in the plant HMAs, including heavy-metal-associated domains, CC dipeptide motifs, and poly-His motifs (Fig. 1). One key difference between clus-ters is the kind, numbers, and location of heavy-metal-binding motifs. For example, pumps fromclusters 1 and 2 lack the N-terminal heavy-metal-associated domains found in pumps from clusters 3to 6. Instead, they have a poly-His motif (cluster 1) ora CC dipeptide (cluster2) in the N-terminal domain.

HMA Regulation

No regulatory mechanisms have been elucidatedfor the plant HMAs. However, the presence of longC- and N-terminal cytoplasmic tails containingheavy-metal-binding motifs point to these regions aspotentially significant for ion sensing or regulation.

HMA Phenotypes

There are two examples of experimental evidencethat support the predicted substrate specificity ofplant HMAs: RAN1/AtHMA7 (cluster 4) and PAA1/AtHMA6 (cluster 5), both of which are putative Cu�/Ag�-pumps. First, experiments by Hirayama et al.(1999) looking for mutants that show ethylene phe-notypes in response to trans-cyclooctene, an ethyleneantagonist, have implicated RAN1/AtHMA7 inethylene-signaling pathways. They showed that the“ethylene-related” phenotype could be rescued byexcess Cu and that the plant pump could comple-ment a mutation in yeast of ccc2�, a yeast copperATPase. Second, experiments by Shikanai, Pilon, andNiyogi have shown PAA1/HMA6 mutants have re-stricted electron transport between the PS and PQ1proteins of the photosynthetic apparatus, less holo-

Figure 4. A, Phylogenetic tree of the P2B subfamily revealing fourclusters. The tree was constructed from full-length protein sequences.Numbers denote clusters. Branches leading to the rice isoforms arethicker, and the rice isoforms are preceded by a bullet. All bootstrapvalues are greater than 71/100. An ACA-like protein is noted inbrackets. B, Distributions of introns and exons in the P2B subfamilyarranged in clusters. The phylogenetic tree used to define the clustersis taken from A but is shown in a different view. The genes arealigned around the phosphorylation site, [DKTGT], marked by a star.Phylogenetic tree branch lengths are not to scale but are included toshow groupings. An ACA-like protein is listed by its ArabidopsisGenome Institute (AGI) number.

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plastocyanin in the thylakoid lumen, and less holo-Cu/ZnSOD in the chloroplast stroma (T. Shikanai,M. Pilon, and K. Niyogi, personal communication).Cu2� levels in chloroplasts isolated from PAA1 mu-tant plants are lower, whereas the levels in wholeleaves are the same as in wild-type. The electrontransport defect is rescued by Cu2� feeding. Never-theless, a direct biochemical test of Cu2� transport byRAN1/AtHMA7 and PAA1/AtHMA6 has not beenreported.

HMA Subcellular Localization

Very little is known about the subcellular locationsfor members of clusters 1, 3, 4, and 6. However, incluster 2, AtHMA2 has been localized to the plasmamembrane by green fluorescent protein fusions andimmunodetection in membrane fractionation with ananti-HMA2 antiserum (Y. Wang and J.F. Harper, un-published data). In cluster 5, PAA1/AtHMA6 hasbeen localized to the chloroplast by fusing anN-terminal peptide to a passenger protein and show-ing chloroplast import (T. Shikanai, M. Pilon, and K.Niyogi, unpublished data). The PAA1/AtHMA6phenotypes suggest that it is localized to the chloro-plast envelope.

ECA, P2A

The P2A ATPases include the well-characterizedmammalian SERCA Ca2�-ATPases (Carafoli andBrini, 2000; Geisler et al., 2000a; Sze et al., 2000).Three members of this subfamily are found in the ricegenome, compared with four in Arabidopsis. The P2AATPases are divided into two clusters, based on se-quence alignments and intron positions (Fig. 3).Within cluster 1, AtECA2 may be special, because ithas lost three of seven introns and has diverged fromthe other pumps in its cluster (Fig. 3). Experiments inyeast suggest that ECAs can transport Ca2�, Mn2�,and Zn2� (Wu et al., 2002).

The previously reported gene model for OsECA1(Chen et al., 1997) has been changed here as a resultof comparisons with other homologs. The gene Os-ECA1 was first identified as a gibberillic acid-induced gene in the aleurone layer (Chen et al., 1997).The new version (PlantsT no. 64529) has been cor-rected to match the updated rice genomic sequenceas well as its most similar Arabidopsis homolog,AtECA3.

ECA Regulation

In mammals, the P2A pumps are regulated by phos-pholambans. Neither phospholambans nor other reg-

Figure 5. A, Phylogenetic tree of the P3A subfamily revealing fiveclusters. The tree was constructed from full-length protein sequences.Numbers denote clusters. Branches leading to the rice isoforms arethicker, and the rice isoforms are preceded by a bullet. The branch-ing of OsAHA5 and 7 and AtAHA7 is ambiguous (bootstrap values24/100), so the branches were collapsed to a common node. Allother values are greater than 73/100. AHA-like protein is noted inbrackets. B, Distributions of introns and exons in the P3A subfamilyarranged in clusters. The phylogenetic tree used to define the clustersis taken from A but is shown in a different view. The genes are

aligned around the phosphorylation site, [DKTGT], marked by a star.The position is marked on the scale below. Phylogenetic tree branchlengths are not to scale but are included to show groupings. AnAHA-like protein is listed by its AGI number. OsAHA10� is missinga C-terminal autoinhibitor domain.





ulators of P2A proteins have been identified in plants,and the phospholamban-binding site on humanSERCA2 is not conserved in the plant ECA proteins.

ECA Phenotypes

Plants with a transfer DNA disruption of AtECA1grow poorly on low Ca2� or high Mn2� concentra-tions in the media and have reduced ability to growroot hairs when grown on high Mn2� (Wu et al.,2002).

ECA Subcellular Localization

The only localization experiments reported for anArabidopsis ECA confirmed an ER location forAtECA1 (Hong et al., 1999). This is consistent with asimilar ER location for the closest related animalhomologs (SERCAs). Nevertheless, one report of atomato (Lycopersicon esculentum) ECA isoform locatedat the plasma membrane (Ferrol and Bennett, 1996)suggests that the location of every ECA isoformneeds to be experimentally verified in an isoform-specific fashion.

ACA, P2B

The plant P2B ATPases are related to the mam-malian plasma membrane calmodulin-stimulatedATPases, but differ in having an N-terminal ratherthan a C-terminal autoinhibitor (Carafoli and Brini,2000; Geisler et al., 2000; Sze et al., 2000). There are 10P2B ATPases in Arabidopsis and 11 in rice. The P2BATPases are divided into four clusters, based onsequence alignments and intron positions (Fig. 4).Cluster 3 is special because it is the only cluster ofP-type ATPases that harbors intron-less genes. In con-trast, genes in cluster 4 have as many as 33 introns.

ACA Regulation

In plants, the four best characterized P2B ATPaseshave been shown to have an N-terminal, calmodulin-regulated autoinhibitory domain (i.e. AtACA2,AtACA4, AtACA8, and cauliflower [Brassica oleracea]BCA1; Malmstrom et al., 1997; Harper et al., 1998;Bonza et al., 2000; Geisler et al., 2000b). When Ca2�/calmodulin is bound, the ATPases are stimulated (forrecent reviews, see Geisler et al., 2000a; Sze et al.,

Figure 6. A, Phylogenetic tree of the P4 subfamily revealing fiveclusters. The tree was constructed from full-length protein sequences.Numbers denote clusters. Branches leading to the rice isoforms arethicker, and the rice isoforms are preceded by a bullet. ALA-like geneis in brackets near the appropriate cluster. The bootstrap values for

OsALA4 and OsALA5 having a separate branch were 43/100 so theywere collapsed to the nearest node. The rest of the bootstrap valueswere greater than 76/100. B, Distributions of introns and exons in theP4 subfamily arranged in clusters. The phylogenetic tree used todefine the clusters is taken from A but is shown in a different view.The genes are aligned around the phosphorylation site, [DKTGT],marked by a star. Phylogenetic tree branch lengths are not to scalebut are included to show groupings. An ALA-like gene is noted by itsPlantsT number.

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2000). However, the extrapolation that all membersof this subfamily are regulated in a similar way wasbrought into question by an in silico analysis of clus-ter 3 (OsACA3, AtACA12, and 13). When cluster 3pumps were aligned with other ACAs, they showeda marked divergence in the putative calmodulin-binding region (Supplemental Data D). In addition,they showed a substitution of Asp to Asn in theotherwise conserved motif SDY[R/K/Q]Q juxta-posed to transmembrane 2 on the cytosolic surface. Asubstitution of Asp to Asn in AtACA2 has beenshown to render this isoform calmodulin indepen-dent (i.e. constitutively active; Curran et al., 2000).Thus, isoform-specific differences in regulation areexpected among the ACAs.

ACA Phenotypes

Although no studies of plant lines harboring P2Bgenes have been published, a disruption of everycalcium pump has been obtained (J.F. Harper andM.G. Palmgren, unpublished data) from large inser-tional mutant collections (Young et al., 2001; Sessionset al., 2002). Thus, we anticipate great advances inour understanding of the role of the ACAs in plantsin the near future.

ACA Subcellular Localization

In contrast to animals, where the analogous P2Bpumps are located only in the plasma membrane,plants appear to have ACA pumps in multiple sub-cellular locations. AtACA2, a member of cluster 1,has been localized to the ER (Harper et al., 1998).AtACA4, a member of cluster 2, has been localized tothe vacuole (Geisler et al., 2000b). AtACA8, a memberof cluster 4, has been shown to reside in the plasmamembrane (Bonza et al., 2000). A representative ofcluster 3 has not yet been characterized.

AHA, P3A

The P3A H�-ATPases are not found in animal sys-tems. Arabidopsis has 11 while rice has 10. Of the sixsubfamilies of P-type ATPases, the P3A branch showsthe least divergence. The P3A ATPases are dividedinto five clusters, based on sequence alignments andintron positions (Fig. 5). Although the bootstrap val-ues that delineate clusters 2 and 4 are quite low, thisbranch is further supported by a similar clusteringanalysis that included additional tobacco (Nicotianatabacum) genes (Arango et al., 2003).

AHA Regulation

P3A ATPases have been subjected to substantialbiochemical characterization, because they energizethe plasma membrane and are likely to be under tightregulation to be able to quickly respond to the cells’needs (Palmgren, 2001). Differences in biochemicalactivity between isoforms when expressed in Brew-er’s yeast have been noted for ATPases from Arabi-dopsis and tobacco. The C terminus has been shownto harbor an autoinhibitory region as well as other

Figure 7. Distributions of introns and exons in the P5 subfamilyforming a single cluster. The genes are aligned around the phosphor-ylation site, [DKTGT], marked by a star.

Figure 8. GC gradient analysis reveals three Arabidopsis-like profiles in rice. GC content of DNA sequences was calculatedusing an in-house program called GeneGC (http://plantst.sdsc.edu/plantst/html/geneGC.shtml). The GC content was calcu-lated in all of the full-length P-type ATPases in rice and Arabidopsis using a 121-bp sliding window that moved in steps of51 bp. The GC content of the Arabidopsis sequences varies between 40% and 50%. The average of all the Arabidopsis genesis shown in B for comparison. All of the rice genes with 5� gradients are shown in A. The genes in B do not have 5� gradientsand were classified as having high GC content or Arabidopsis-like “normal” GC content. Only the averages of the genes withhigh GC content are shown. Genes with unusual GC patterns have different markers.





regulatory sites. The C-terminal regions (after thefinal transmembrane helix) are the most divergentregions of the proteins (36% identical versus 73% forthe protein core). The C termini bind 14-3-3 proteinsas a part of the activation mechanism, a binding thatdepends upon phosphorylation of the conservedpenultimate Thr (for recent reviews, see Palmgren,2001; Arango et al., 2003). Interestingly, OsAHA10 ispredicted to be missing the entire C-terminal do-main, which would make it the only constitutivelyactive proton pump found in plants (Arango et al.,2003). Although there are ESTs indicating thatOsAHA10 is expressed, a sequence confirmation of a“truncated end” has not been established.

AHA Phenotypes

Genetic disruption and RNA interference havebeen carried out on the members of cluster 1 in bothArabidopsis and tobacco. In Arabidopsis, nondomi-nant, double disruptions of AtAHA4 and AtAHA11(the only Arabidopsis genes in the cluster) have noapparent effect on the health of the plant (I. Baxterand J.F. Harper, unpublished data). RNA interfer-ence of the three tobacco genes from this cluster alsohad no observable effect on plant development (F.Gevaudant, J. Kanczewska, and M. Boutry, unpub-lished data). Unexpectedly, a transfer DNA insertionin AtAHA4, which segregates as a semidominantmutation, confers salt sensitivity (Vitart et al., 2001).

Genetic disruption of several isoforms from cluster2 shows deleterious effects on the health of theplants. RNA interference of PMA4 in tobacco causesdefects in development, sugar transport, and malesterility (Zhao et al., 2000). The double knockout ofAtAHA1 and 2 is embryonic lethal, and AtAHA3 isessential for male gametogenesis (M.R. Sussman, un-published data). At present, all biochemical, geneticdisruption, and RNAi studies of proton pumps inplants have been performed with members of clus-ters 1 and 2. Evidence for similar or unique functionsof pumps from cluster 3, 4, or 5 still needs to beexplored.

AHA Subcellular Localization and Tissue Specificity

The plasma membrane proton pump has long beenconsidered a marker of the plasma membrane, basedon immunocytology and membrane fractionation(DeWitt et al., 1996). Nevertheless, a PM targetingdestination has been confirmed for only two of the11 Arabidopsis isoforms using an isoform-specificepitope tagging strategy. Thus, the potential of anendomembrane targeting location for one of the un-characterized isoforms remains a formal possibility.

The favored rationale to explain the presence of somany isoforms is the need to express differentamounts of pump in different tissues. Tissue-specificexpression patterns have been observed for different

isoforms in multiple plant species, including Arabi-dopsis and tobacco (Arango et al., 2003). For exam-ple, AtAHA10 has been localized to the developingseed (Harper et al., 1994). AtAHA4 is expressed in theroot endodermis, in flowers, and in maturing siliques(Vitart et al., 2001). AHA2 is expressed predomi-nantly but not exclusively in the root epidermis (J.F.Harper, unpublished data). AtAHA9 is expressed inanthers (Houlne and Boutry, 1994). ESTs of OsAHA10have been found in the rice panicle at floweringstage. However, potential orthologous isoforms fromtobacco and Arabidopsis do not always show thesame tissue-specific pattern of expression, suggestingthat the distinction between clusters is not related totissue specificity (Arango et al., 2003).

Putative ALA, P4

This large subfamily of divergent ATPases, whichis only found in eukaryotes, includes 12 and 10 genesin Arabidopsis and rice, respectively. The P4 ATPasesare divided into five clusters, based on sequencealignments and intron positions (Fig. 6). The previ-ously reported gene model for AtALA3 has beenchanged based on the similarity with its ortholog,OsALA8. The donor site of the second-to-last exonhas been moved four nucleotides upstream, resultingin the last exon being read in another frame andmaking the protein 90 amino acids longer. The P4ATPases have been implicated in aminophospholipidflipping. However, it is not clear if the flipase activityis the primary function of P4 ATPases or an indirecteffect of its activity.

ALA Phenotypes

The subfamily is relatively uncharacterized; onlyone isoform in plants, AtALA1, has been studied(Gomes et al., 2000). Antisense suppression ofAtALA1 demonstrated a role in cold tolerance ofArabidopsis plants (Gomes et al., 2000). Work onother P4 ATPases showed that the yeast DRS2 proteinis involved in formation of clathrin-coated vesicles(Gall et al., 2002), whereas human ATP8B1 is in-volved in transport of conjugated bile acids (Bull etal., 1998).

Unknown (P5, P5)

There is a single member of the P5 ATPases in eachorganism; the proteins are 77% identical (Fig. 7). TheP5 ATPases are the least studied of the P-type ATPasesubfamilies. The only protein to have been studied insome detail is SPF1/COD1, one of two members inBrewer’s yeast (Cronin et al., 2002; Vashist et al.,2002). The studies have given no clear idea of ionspecificity, but they do suggest a functional connec-tion to lipid biogenesis and pathogen response.

Baxter et al.




CONCLUSIONS

The current draft sequences of the rice genomehave enabled the first comprehensive comparison ofP-type ATPase genes in two plant species. Eventhough the respective pumps and their genes areremarkably similar, the analysis of the rice sequenceshas done more than confirm the previous findings inArabidopsis. The genomic comparison has increasedour knowledge in five ways. (a) Because the majorityof the 89 P-type ATPase genes in these two organ-isms have not been cloned as full-length cDNAs, wehave been able to correct and verify predicted genemodels, such as the change described here forAtALA3 based on similarity to OsALA8. (b) Compar-ison of protein sequences and intron organizationfrom the two plant species has confirmed that at least23 ATPases evolved very early in evolution beforethe divergence of dicots and monocots. (c) The ob-servation that no cluster was lost in Arabidopsis orrice suggests that all 23 archetypal clusters play animportant role for plant survival at the evolutionaryscale. The observation that knockout or RNAi ofsome clusters (as discussed above) has little impactmight simply indicate that these clusters are notessential in tested growth conditions but that theypresumably confer some advantage to the plantin nature. (d) Of the three ATPase-like proteins inArabidopsis or rice, none are conserved in bothorganisms, suggesting that they represent pseudo-genes or have species-specific functions. (e) Withinsome clusters, the number of genes varies betweenrice and Arabidopsis. The greatest expansion wasin ALA cluster 3, with one isoform in rice and four inArabidopsis. This example of gene duplication ordeletion may reflect gene specialization and species-specific differences in transport activities. The mostchallenging task ahead is to understand the con-served and species-specific functions of all 89different rice and Arabidopsis P-type ATPases.

MATERIALS AND METHODS

Identification

To identify all P-type ATPases in the rice genome, we searched threedifferent databases: (a) the Rice Annotated Protein Database at The Institutefor Genome Research (including all sequences predicted from the Interna-tional Rice Genome Sequencing Project), (b) the Syngenta Rice GenomicSequence of Lotus subsp. japonica (Goff et al., 2002; http://www.tmri.org),and (c) the Bejing Genomics Institute 93–11 cultivar genomic sequences ofLotus subsp. Indica (downloaded May 27, 2002; http://btn.genomics.org.cn/rice). Both the Syngenta and the Bejing Genomics Institute sequences werereported to cover more than 90% of known rice genes or Unigene cluster atthe time of analysis (Goff et al., 2002; Yu et al., 2002). If the contigs from thethree sequencing projects are evenly distributed over the genome, wesearched more than 99% of the genome for P-type ATPases.

Contigs that contained P-type ATPase genes were identified by TBlastN(Altschul et al., 1990) searches using either the 46 Arabidopsis P-typeATPases or the six currently known variations of the DKTGTXX motif thatare conserved in all known P-type ATPases (http://biobase.dk/�axe/Pat-base.html). Forty-eight occurrences of the DKTGTXX motifs were found inthe Syngenta genomic sequence and 56 in the Chinese genomic sequence. A

nonredundant set of 43 rice P-type ATPases was identified after makinggene model predictions.

Editing

Gene models (open reading frames) were predicted by submitting 8,000bp of genomic sequence encompassing each potential gene to Genemark(http://opal.biology.gatech.edu/GeneMark/; M. Borodovsky and A.Lukashin, unpublished data). In some cases, longer sequences were exam-ined to ensure the inclusion of the 5� or 3� end of the genes. The predictedcoding sequences were aligned with the closest Arabidopsis P-type ATPasehomolog using ClustalW (Thompson et al., 1994). The candidate rice genesthat either did not have any recognizable open reading frames or coded forproteins other than P-type ATPases were eliminated. Sequences that ap-peared to be bacterial in nature, including several putative Mg2� ATPases(P3B), were also eliminated from the analyses. The sequences in question arebeing stored in a separate database on the PlantsT Web site (http://plantst.sdsc.edu). Possible pseudogenes were removed from analyses whereappropriate.

The remaining 43 sequences were edited by hand, using the closestArabidopsis homolog as a template, to improve intron/exon predictions.Although Genemark was useful as a starting point for creating gene models,this program correctly predicted only one of the rice genes, which is inagreement with the predicted accuracy of gene model prediction programs(Pertea and Salzberg, 2002). Although most gene models were based entirelyon the Syngenta japonica sequence, in a few cases we combined sequencingdata from both the Syngenta and Chinese sequences as well as from ESTdata to make complete predictions. The final gene models are deposited inGenBank (http://www.ncbi.nlm.nih.gov) and PlantsT (http://plantst.sdsc.edu) databases. Updated annotations to our predicted genes can besubmitted to the PlantsT Web site.

Analysis

Phylogenetic analyses were performed using the Phylip package (Phy-logeny Inference Package, v3.2, Department of Genetics, University ofWashington, Seattle). Initial alignments were done with ClustalW using allof the rice and Arabidopsis P-type ATPases. The segments conserved amongall P-type ATPases (Axelsen and Palmgren, 1998) were extracted using theinitial alignments with the Arabidopsis isoforms (http://biobase.dk/�axe/Patbase.html). Alignments using the conserved sequences produced thesame phylogenetic trees as alignments using full-length sequences, so weused full-length sequences for all analysis. After alignment with ClustalW,bootstrapping was performed using the Phylip programs seqboot, protdist,and consense. Subsequently, the proteins were divided up into subfamilyand alignments, and bootstrapping analysis was done on each subfamilyindividually (subfamily alignments are included as Supplemental DataB–G). Similarity was calculated using the protdist program from Phylipv3.6a3 (Department of Genetics, University of Washington, Seattle). Introns,exons, and gene lengths were calculated by comparing the cDNA sequenceto the genomic sequence with the Sim4 program (Florea et al., 1998). Aprogram was written to calculate the GC content along the length of a gene(GeneGC, http://plantst.sdsc.edu/plantst/html/geneGC.shtml). The GCcontent across the genes was calculated using a 129-bp sliding window thatmoved in 51-bp steps.

Received February 9, 2003; returned for revision February 25, 2003; acceptedFebruary 25, 2003.

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CORRECTION

Vol. 132: 618–628, 2003

Baxter I., Tchieu J., SussmanM.R., Boutry M., PalmgrenM.G., Gribskov M., Harper J.F., andAxelsen K.B. Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice.

The authors regret the omission of a ninth heavy-metal ATPase in rice (Oryza sativa),OsHMA9. The correct number of P-type ATPases in rice is therefore 44, not 43. OsHMA9 isalmost identical to OsHMA6 in cluster 4 (Figure 2). In Figure 2A, OsHMA9 would beextremely close to OsHMA6, and, in Figure 2B, it has the same intron pattern as OsHMA6.The protein and RNA sequences for OsHMA9 were included in the original supplementalfiles (Supplemental Tables I and K). The DNA sequence for OsHMA9 and the other riceP-type ATPases is now included as Supplemental Table L. The authors wish to thank GynAn for pointing out this error.

Plant Physiology, July 2005, Vol. 138, p. 1807, www.plantphysiol.org � 2005 American Society of Plant Biologists 1807

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