Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants

Abstract

Background: Nitrogen uptake, reallocation within the plant, and between subcellular compartments involves ammonium, nitrate and peptide transporters. Ammonium transporters are separated into two distinct families (AMT1 and AMT2), each comprised of five members on average in angiosperms. Nitrate transporters also form two discrete families (NRT1 and NRT2), with angiosperms having four NRT2s, on average. NRT1s share an evolutionary history with peptide transporters (PTRs). The NRT1/PTR family in land plants usually has more than 50 members and contains also members with distinct activities, such as glucosinolate and abscisic acid transport.

Results: Phylogenetic reconstructions of each family across 20 land plant species with available genome sequences were supplemented with subcellular localization and transmembrane topology predictions. This revealed that both AMT families diverged prior to the separation of bryophytes and vascular plants forming two distinct clans, designated as supergroups, each. Ten supergroups were identified for the NRT1/PTR family. It is apparent that nitrate and peptide transport within the NRT1/PTR family is polyphyletic, that is, nitrate and/or peptide transport likely evolved multiple times within land plants. The NRT2 family separated into two distinct clans early in vascular plant evolution. Subsequent duplications occurring prior to the eudicot/monocot separation led to the existence of two AMT1, six AMT2, 31 NRT1/PTR, and two NRT2 clans, designated as groups.

Conclusion: Phylogenetic separation of groups suggests functional divergence within the angiosperms for each family. Distinct groups within the NRT1/PTR family appear to separate peptide and nitrate transport activities as well as other activities contained within the family, for example nitrite transport. Conversely, distinct activities, such as abscisic acid and glucosinolate transport, appear to have recently evolved from nitrate transporters.

Figure 1

NRT1/PTR phylogeny. Unrooted maximum likelihood phylogenetic reconstruction of the NRT1/PTR families in plants and a set of 24 non-plant sequences identified as best GenBank BLAST hits using representative members from each supergroup as query. Taxonomic groups are colored such that blue refers to eudicots, red to monocots, green to chlorophytes, yellow to bryophytes, and orange to lycophytes. Percent Bootstrap values from 1,000 replicates are given for central branches only up to the branches defining supergroups. The approximate location of functionally characterized transporters discussed is indicated (NRT: nitrate transporter, GTR: glucosinolate transporter, PTR: peptide transporter, NiTR: nitrite (NO₂^-) transporter, NAXT: nitrate excretion transporter, and AIT: abscisic acid (ABA) transporter). *Note that supergroup F is paraphyletic; the containing clades have been combined owing to poor bootstrap support separating them. For detailed phylogenies of each superfamily and group definitions see Additional file 2. For database accession numbers of the 1,101 protein sequences included see Additional file 1.

Figure 2

Maximum Likelihood phylogenetic reconstruction of the NRT2 family. Taxonomic groups are colored as described in Figure  1. A) Phylogram of the NRT2 family including 81 plant sequences only. Chlorophyte sequences were used to root the tree. Bootstrap values of the ML analysis are given and corresponding bootstrap values (>75%) from a distance neighbor joining and a parsimony analysis are indicated as green and red stars, respectively (within groups, bootstrap values >75% are displayed as stars only for all three analyses). Subcellular localization predictions are indicated as colored boxes framing gene identifiers and transmembrane topology predictions are given as numbers to the right of the gene identifier. A classification system is indicated as group labels to the right (E = eudicot, M = monocot). B) Unrooted phylogeny of the NRT2 family including twelve non-plant sequences identified as best GenBank BLAST hits using representative members from each plant group. Bootstrap values are given for central branches only up to the branch defining a group. Approximate locations of functionally characterized members discussed in the text are indicated.For database accession numbers of the 93 protein sequences included, see Additional file 1.

Figure 3

Maximum Likelihood phylogenetic reconstruction of the AMT1 family. Taxonomic groups are colored as in Figure  1. A) Rooted phylogram of the AMT1 family in plants only. Bootstrap values of the ML analysis are given and corresponding bootstrap values (>75%) from a distance neighbor joining and a parsimony analysis are indicated as green and red stars, respectively (within groups, bootstrap values >75% are displayed as stars only for all three analyses). Subcellular localization predictions, transmembrane topology predictions, and a classification system is indicated as described in Figure  2. B) Unrooted phylogeny of the AMT1 family including 21 non-plant sequences identified as best GenBank BLAST hits using representative members from each plant group. Bootstrap values are given for central branches only up to the branch defining a group.

Figure 4

Maximum Likelihood phylogenetic reconstruction of the AMT2 family. Taxonomic groups are colored as described in Figure  1. A) Phylogram of the AMT2 family including plant sequences only supplemented with ML bootstrap values (NJ and parsimony bootstrap values (>75%) indicated as blue and red stars), subcellular localization predictions, topology predictions, and proposed classification system. The root of this tree was defined through the analysis shown in B). B) Unrooted phylogeny of the AMT2 family including eleven most similar non-plant sequences present in GenBank. Color-coding as described above. For database accession numbers of the protein sequences included, see Additional file 1.

Figure 5

In silico expression profile of putative AMTs and NRTs. Microarray data were obtained from The Bio-Array Resource for Plant Biology [81] for all family members from A. thaliana, O. sativa, and P. trichocarpa. Note, that AMT3s and AMT4s are members of the AMT2 family.