Phylogenetic relationships within cation transporter families of Arabidopsis

Abstract

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

Figure 1

Overview of Arabidopsis K⁺ transporters. A tree of all K⁺ transporters from Arabidopsis has five major branches: a, KUP/HAK/KT transporters (13 genes); b, Trk/HKT transporters (Na⁺ transporter; one gene); c, KCO (2P/4TM) K⁺ channels (six genes); d, Shaker-type (1P/6TM) K⁺ channels (nine genes); and e, K⁺/H⁺ antiporter homologs (six genes). Predicted membrane topologies for each branch are shown. The apparent absence of K⁺ channels of the 2P/8TM family is remarkable as is the diversity in the AtKUP/HAK/KT transporters. Proteins for which a complete cDNA sequence is available are indicated by bold letters and lines. Arabidopsis Genome Initiative (AGI) genome codes are given except for AtKUP3 = AtKUP4, AtHAK5, AtHKT1, GORK, KAT2, and AKT2 (GenBank accession nos.) because of errors in the sequences predicted by AGI. Programs used were HMMTOP (Tusnady and Simon, 1998) for topology predictions of the KEA and AtKUP/HAK/KT families, ClustalX (Thompson et al., 1997) for alignments, and graphical output produced by Treeview (Page, 1996). Values indicate the number of times of 1,000 bootstraps that each branch topology was found during bootstrap analysis.

Figure 2

Phylogenetic tree of Arabidopsis K⁺ channels. A non-rooted tree reflects the structural and functional properties of Arabidopsis K⁺ channels. The two major branches are the 2P/4TM-type and the 1P/6TM (Shaker)-type channels, as depicted by the sketches. For KAT1 the proposed topology has been confirmed experimentally (Uozumi et al., 1998). The 1P/6TM (Shaker-type) channels are further subdivided into the depolarization-activated SKOR and GORK and the KATs and AKTs. All the 1P/6TM channels possess a putative cyclic nucleotide-binding site (CNB), and AKT channels also have an ankyrin repeat consensus site (AR; see sketches). P-loops are labeled with asterisks. Proteins for which a complete cDNA sequence is available are indicated by bold letters and lines. Programs used were pfscan (http://www.isrec.isb-sib.ch/software/PFSCAN form.html) for motif searches, ClustalX (Thompson et al., 1997) for alignments, and Treeview (Page, 1996) for graphical output. Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 3

Phylogenetic tree of Arabidopsis KUP/HAK/KT (POT) transporters. Proteins for which the complete cDNA has been sequenced are printed in bold and are marked with bold lines. We are using the name AtKUP/HAK/KT except for four cases with conflicting numbers: The names AtKT5 (Quintero and Blatt, 1997) and AtHAK5 (Rubio et al., 2000) have been given to different genes, whereas AtKUP3 (Kim et al., 1998) and AtKT4 (Quintero and Blatt, 1997) have been used for the same gene, and so were AtKUP4 and AtKT3. Furthermore, AtKUP4 = AtKT3 corresponds to the TRH1 gene (Rigas et al., 2001). Programs used were ClustalX (Thompson et al., 1997) for alignments and Treeview (Page, 1996) for graphical output. Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 4

Phylogenetic tree of Arabidopsis cation antiporters. Members of the families CPA1, CPA2, CaCA, NhaD, and CCC are presented (http://www.biology.ucsd.edu/∼ipaulsen/transport/) with homologous protein sequences from the yeast Saccharomyces cerevisiae. Gene names, accession nos., and family assignment are shown for each Arabidopsis sequence. Alignments of full-length sequences were performed using ClustalW (Thompson et al., 1994). The tree was constructed using the neighbor joining function of Paup 4.0 (Swofford, 1998). Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 5

Phylogenetic tree of the Arabidopsis CNGC transporters. Entries in the Munich Information Center for Protein Sequences Arabidopsis database (MATDB) were compared with available corresponding cDNA entries in the National Center for Biotechnology Information database, to minimize errors for each predicted protein sequence. By default, the MATDB predicted protein sequences were used for final alignment. Exceptions are: accession no. AAF97331.1 for At1g01340, accession no. CAB40128.1 for At2g46430, accession no. AAF73129.1 for At3g17690, and accession no. AAF73128.1 for At3g17700. Bold lines indicate that protein sequences predicted from cDNAs are available. Final protein alignment, tree drawing, and bootstrap analysis were done with ClustalX (Higgins and Sharp, 1988), and the tree was drawn using Treeview. CNGCs 1 through 6 have already been named in the literature (Köhler et al., 1999). To generate a uniform nomenclature, ACBK1, CNBT1, and CNBT2 are assigned the names CNGC10, 20, and 19, respectively, and the remaining genes are also assigned systematic names. Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 6

Phylogenetic tree of plant CDF transporters. The phylogenetic tree of the CE protein family was drawn using PHYLIP (Felsenstein, 1989) after alignment of the sequences with CLUSTAL W (Thompson et al., 1994). For ZAT (AtMTP1; At2g46800), TgMTP1, TgMTP2, TgMTP3, TmMTP1, TaMTP1, BjMTP1, and the yeast sequences ZRC1 (gi no. 736309) and COT1 (gi no. 171263), the protein sequences were predicted from cDNAs, and these branches of the tree are in bold. For AtMTPa1 (At3g61940), AtMTPa2 (At3g58810), AtMTPb (At2g29410), AtMTPc1 (At2g47830), AtMTPc2 (At3g12100), AtMTPc3 (At3g58060), and AtMTPc4 (At1g51610) protein sequences were translated from the ORFs predicted from genomic sequences, and these branches are represented by thin lines. Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 7

Phylogenetic tree of Arabidopsis NRAMP transporters. The phylogenetic tree of AtNRAMP protein sequences was drawn using Treeview program after alignment of the sequences with ClustalX program. For AtNRAMP1, 2, 3, 4, and EIN2 protein sequences predicted from cDNA translation were used (AAF36535, AAD41078, AAF13278, AAF13279, and AAD41077, bold lines). For AtNRAMP5 and 6, protein sequences translated from the ORFs predicted from genomic sequences (CAB37464 and AAF18493, thin lines) were used because cDNA sequences are not available for these genes. Note that for EIN2, only the sequence of the NRAMP homologous domain of the protein was taken into account to construct the tree. Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 8

Phylogenetic tree of Arabidopsis ZIP transporters. Gene names and accession nos. are shown for each Arabidopsis sequence. Proteins for which a full-length cDNA is available are indicated by bold letters and lines. Alignments of full-length sequences were performed using ClustalW (Higgins and Sharp, 1988). The tree and bootstrap analyses were performed using the neighbor-joining algorithm implemented in MEGA version 2.0 (Kumar et al., 2000). Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis.

Figure 9

Chromosome positions of genes for selected cation transport families. Genes were arranged on chromosomes according to their locations in the genomic sequence (i.e. not the genetic map). Each chromosome is identified by its number and shown three times (black bar followed by two copies in gray). Each of the six gene families is separately mapped with all members aligned in a single column, as labeled at the top.