Conserved and diversified gene families of monovalent cation/h(+) antiporters from algae to flowering plants

Abstract

All organisms have evolved strategies to regulate ion and pH homeostasis in response to developmental and environmental cues. One strategy is mediated by monovalent cation-proton antiporters (CPA) that are classified in two superfamilies. Many CPA1 genes from bacteria, fungi, metazoa, and plants have been functionally characterized; though roles of plant CPA2 genes encoding K(+)-efflux antiporter (KEA) and cation/H(+) exchanger (CHX) families are largely unknown. Phylogenetic analysis showed that three clades of the CPA1 Na(+)-H(+) exchanger (NHX) family have been conserved from single-celled algae to Arabidopsis. These are (i) plasma membrane-bound SOS1/AtNHX7 that share ancestry with prokaryote NhaP, (ii) endosomal AtNHX5/6 that is part of the eukaryote Intracellular-NHE clade, and (iii) a vacuolar NHX clade (AtNHX1-4) specific to plants. Early diversification of KEA genes possibly from an ancestral cyanobacterium gene is suggested by three types seen in all plants. Intriguingly, CHX genes diversified from three to four members in one subclade of early land plants to 28 genes in eight subclades of Arabidopsis. Homologs from Spirogyra or Physcomitrella share high similarity with AtCHX20, suggesting that guard cell-specific AtCHX20 and its closest relatives are founders of the family, and pollen-expressed CHX genes appeared later in monocots and early eudicots. AtCHX proteins mediate K(+) transport and pH homeostasis, and have been localized to intracellular and plasma membrane. Thus KEA genes are conserved from green algae to angiosperms, and their presence in red algae and secondary endosymbionts suggest a role in plastids. In contrast, AtNHX1-4 subtype evolved in plant cells to handle ion homeostasis of vacuoles. The great diversity of CHX genes in land plants compared to metazoa, fungi, or algae would imply a significant role of ion and pH homeostasis at dynamic endomembranes in the vegetative and reproductive success of flowering plants.

Keywords: cargo sorting; cation homeostasis; dynamic endomembrane; pH homeostasis; protein; secretory system.

Figure 1

Protein phylogeny of cation–proton antiporters shows the evolutionary history of CPA1 and CPA2 families in plants. The conserved Pfam 00999 domains from diverse organisms were aligned by MUSCLE and the evolutionary history of the sequences were determined by RAxML, maximum likelihood using the Jones–Taylor–Thornton (JTT) model with a bootstrap of 500. (Final ML optimization likelihood = −104241.644676). Both N and C ends were trimmed, so aligned TM domain for proteins was ~400 residues, corresponding to AtCHX17 residue 33–426. Organisms are color-coded as follows: bacteria (magenta), Cyanobacteria (dark-blue), protist: Dictyostelium (red), fungi (brown), algae (green), and plants (dark green). NhaP and NhaA genes from ancestral bacteria likely gave rise to eukaryote CPA1 (NhaP and NHX) and CPA2 (KEA and CHX) families, respectively. Abbreviated protein names and organisms are defined in Table S1 in Supplementary Material. Unrooted rectangular tree is shown in Figure S1 in Supplementary Material.

Figure 2

Diversification of CPA1 genes into three clades preceded the evolution of early land plants. The C-tails were trimmed to maximize alignment, so AtNHX1 sequence included residues 1–448. Tree was generated with maximum likelihood of RAxML using the Jones–Taylor–Thornton (JTT) model and tested by bootstrap of 500. (Final ML optimization likelihood = −30244.238457). Single-celled alga (Cre), moss (Ppa), and club moss (Smo) NHX members were present in each of three clades, NhaP/SOS1, endosomal AtNHX5/6, and vacuolar AtNHX1–4. Organisms and proteins are defined in Table S2 in Supplementary Material, and color-coded as bacteria (magenta), Cyanobacteria (dark-blue), fungi (brown), protist (red), green algae (green), early land plants, and angiosperms (dark green). The “vacuolar” AtNHX1–4 clade is specific to plants.

Figure 3

Phylogenetic tree shows plant KEAs originated from ancestral genes in Cyanobacteria. The conserved Pfam 00999 domains in KEA sequences from diverse organisms were aligned by MUSCLE and the evolutionary history of the genes were determined by maximum likelihood using the Jones–Taylor–Thornton (JTT) model with a Bootstrap of 100. The percentage of trees in which the associated sequences clustered together is shown next to the branches. The analysis involved 66 amino acid sequences. All positions containing gaps and missing data were eliminated, resulting in a total of 307 positions in the final dataset. Branch colors refer to bacteria (pink), Cyanobacteria (blue), red alga (red), metazoa (brown), and plants (green). Organisms and accession numbers of protein sequences are listed in Table S3 in Supplementary Material.

Figure 4

Alignment of AtKEA sequences with their bacterial homologs – AtKEA sequences were aligned using MUSCLE (Edgar, 2004) with their closest prokaryotic homologs: E. coli KefC and Cyanobacterial sequence SynjbKEAII (Q2JI45 Table S3 in Supplementary Material). The soluble N-terminal domain of AtKEA1 was removed. Conserved residues are shown in orange. Approximate positions of possible transmembrane helices are shown above the alignment. Secondary structure of the KTN domain is indicated as well as residues that were shown to be involved in Glutathione-binding in the KefC KTN structure. A short cytoplasmic regulatory loop in the E. coli KefC sequence as well as the conserved residues in the Arabidopsis sequences are shown in magenta. The residues corresponding to the proton binding residues in the structure of the bacterial NhaA sequence are shown in red. The Rossman fold glycine motif is shown in green. Below the alignment the two specific clade Ib inserts in different plants are shown.

Figure 5

K⁺-efflux antiporter diversified into three types in early photosynthetic eukaryotes. The analysis involved 97 amino acid sequences. The tree includes all proteins found in A. thaliana, G. max, O. sativa, Z. mays, S. moellendorffii, P. patens, C. reinhardtii, and V. carteri, as well as one representative sequence for each group from the remaining plant species listed in Table S3 in Supplementary Material. All positions containing gaps and missing data were eliminated. There were a total of 152 (335 excluding incomplete sequences GmaKEA11 and RcoKEA6) positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Branch colors refer to dicots (green), monocot (dark-blue), early land plants (orange–red), and green algae (light blue). Species and accession numbers of protein sequences are listed in Table S3 in Supplementary Material.

Figure 6

Distinct protein domains of three KEA clades. The relative length and position of the KTN and Na_H exchanger domain in the three KEA clades is shown graphically using information obtained with the Simple Modular Architecture Research Tool (Smart, http://smart.embl-heidelberg.de/). All plant clade I proteins contain a KTN domain. In the clade II group this domain is lost, making it the shortest KEA proteins. The clade Ia proteins all have acquired a long N-terminal domain, typically containing predicted coiled coil structures. The C-terminus of the clade Ib proteins is slightly longer as compared to the other sequences. Some green algal clade II proteins have a split Na_H domain, which might be a result of erroneous gene predictions. The transmembrane helix predictions are very variable, and especially unsuccessful for the clade Ib group of proteins. The transmembrane helices shown in Figure 2 are numbered from 1 to 12 in KefC.

Figure 7

CHX homologs diversified from moss to flowering plants. Full-length CHX proteins from 15 species were aligned and then analyzed by maximum likelihood. Spirogyra (Spra), moss (Ppa), and club moss (Smo; green) CHXs clustered with AtCHX20 (subclade IVa). Both monocot (blue) and dicot (black) plants had orthologs of AtCHX20, AtCHX16–19 (IVb), AtCHX28 (I), AtCHX1/2 (I), and AtCHX15 (IVc). Corn, sorghum, or rice had two to four genes encoding OsCHX6 homologs in a monocot cluster near AtCHX24/25. Most dicots had additional CHX homologs that clustered with AtCHX24/25 (V), 26/27, and 13/14 (III). AtCHX3–12 proteins (subclade II) are specific to A. thaliana and A. lyrata. A cluster of CHX resulting from multiple gene duplications is specific to Medicago truncatula (Mtr). See summary in Table 5. Species, protein accession numbers and protein properties are described in Table S4 in Supplementary Material, and unrooted rectangular tree is in Figure S4 in Supplementary Material.