1 Introduction

The main sources of nitrogen in plants are ammonium (NH⁴⁺), nitrate (NO³⁻), and amino acids, which are present in the soil as organic and inorganic complexes and compounds (Williams et al., 2001). It is ammonium transport (AMT) that absorbs these sources from the soil. AMT presents over the plasma membrane of root cells and incorporates into glutamine via glutamine synthetase (GS) that is in the cytoplasm and plastids (Kaiser et al., 2002).

Many AMT genes have been identified and cloned from diverse plant species. Furthermore, the biochemistry and molecular biology of AMT in plants has been extensively studied (Loqué & von Wirén, 2004; Schjoerring JK et al., 2002). Previous studies on phylogenetic analyses of the AMT gene family showed that the AMT family could be subdivided into two subfamilies: the AMT1 subfamily and the AMT2 subfamily. There were only one cluster in AMT1 subfamily and two clusters in AMT2 subfamily (Koegel et al., 2013; Loqué & von Wirén, 2004). Several plants’ AMT1 subfamily members have been characterized in yeast and Arabidopsis thaliana, such as  the AtAMT1;1, AtAMT1;2, AtAMT1;3 in Arabidopsis thaliana(Gazzarrini S et al., 1999; Ninnemann O et al., 1994), the LeAMT1;1, LeAMT1;2 and LeAMT1;3 in Lycopersicon esculentum (Lauter FR et al., 1996; von Wiren N et al., 2000b) and the OsAMT1;1, OsAMT1;2, OsAMT1;3 in Oryza sativa (Sonoda Y et al., 2003). Furthermore, the AMT2 subfamily with distinct biochemical features has been identified in several plants including Arabidopsis thaliana (Sohlenkamp C et al., 2002), Oryza sativa (Suenaga A et al., 2003) and Lotus japonicas (Simon-Rosin U et al., 2003.).

Although plant physiologists have studied many of the specific AMT, available comprehensive information of AMT is still limited. For example, it is known that AMT1 cluster genes have a high affinity NH4+ -transport function (Loqué et al., 2006; Yuan L. et al., 2007).  Therefore, it is necessary to make a comprehensive comparison among the plant AMT subfamilies. In this study, we compared the commonness and individuality of AMT, predicted their structural types. In detail, we investigated the AMT families of Arabidopsis thaliana and Oryza sativa, in terms of phylogeny tree, gene and protein information, exon/intron organization, prediction of trans-membrane helices, conserved domain, and subcellular localization.

2 Materials and Methods

We downloaded the Arabidopsis thaliana and Oryza sativa AMT gene sequences form the Gene Database, and protein sequences from the UniProt Database. We used BioEdit (Kaiser et al., 2002) to do Multiple sequence alignment with 60% threshhold for shading. The full-length amino acid sequences of AMTs were aligned with ClustalW in MEGA6 software (Tamura et al., 2013). Then, we use the Neighbor-Joining (NJ) method and Poisson correction model to construct the phylogenetic tree. It carried out 1000 times Bootstrap method.

The exon/intron organization of individual AMT genes was illustrated with the Gene Structure Display Server program (http://gsds1.cbi.pku.edu.cn/index.php) (Guo et al., 2007). The trans-membrane domains in each AMT protein were predicted with TMHMM Server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (L.L. et al., 1998). We used InterPro (http://www.ebi.ac.uk/interpro/) (Alex Mitchell et al., 2015) to discover each AMT protein conserved domain. PSORT II Prediction software (http://psort.hgc.jp/form2.html) (Emanuelsson O. et al., 2007.) was used to make each AMT family gene’s subcellular localization.

3 Results

Phylogenetic Tree Analyses of AMT Genes

A database search with the keywords AMT showed that there were 6 AMT proteins in Arabidopsis thaliana and 10 AMT proteins in Oryza sativa. The result of multiple sequence alignment is shown in Supplement data 1.

Supplement data 1

The detailed information of AMTs was summarized as shown in Table 1. ACCESSION represents the accession number in the UniProt Database; Gene ID represents the Gene ID in the Gene Database; Chromosome represents the gene localization in Chromosome; GI represents the GI in Gene Bank;

Table 1 Database information of members of family of AtAMT and OsAMT.

The distribution of gene on the chromosome is one of the decisive factors of functional features. In this study, the 6 members of Arabidopsis thaliana AMT distributed to number 1, 2, 3 and 4 chromosome, and the 10 members of Oryza sativa AMT distributed to number 1, 2, 3, 4 and 5 chromosome. They all characterize the scattered distribution.

To investigate the evolutionary relationships among Arabidopsis thaliana and Oryza sativa AMT proteins, we used ClustalW to align full-length sequences of the 16 proteins, and constructed a phylogenetic tree with the Neighbor-Joining method using MEGA6 software (Figure 1).

Figure 1 Phylogenetic tree of proteins encoded by AMT genes.

The results showed two subfamily and four clusters. Among the 16 AMT proteins, 8 proteins were in the AMT1 cluster, and the remaining AMT proteins were in AMT2 cluster. Furthermore, AMT genes in O. sativa can be detailed divided into three subclusters. We named each of them includes the initials of the plant species and the cluster number. In cluster 1, they are AtAMT1-1, AtAMT1-2, AtAMT1-3, AtAMT1-4, AtAMT1-5, OsAMT1-1, OsAMT1-2, OsAMT1-3; In cluster 2, they are AtAMT2, OsAMT2-1, OsAMT2-2, OsAMT2-3; In cluster 3, they are OsAMT3-1, OsAMT3-2, OsAMT3-3; In cluster 4, there is only OsAMT4.

Exon/Intron Organization Analyses of AMT Genes.

It has been studied that members of the AMT1 subfamily was mostly intron-free (Salvemini et al., 2001); whereas members of AMT2 subfamily contained some introns in their gene sequences (Suenaga A et al., 2003).

We get the same answer through analyzing the exon/intron structure of the 16 AMT genes in Arabidopsis thaliana and Oryza sativa (Figure 2 A). Genes in the AMT1 cluster all had only 1 exon and null intron, while those in the AMT2 and AMT3 cluster have very different performance. AtAMT2 has 5 exons and 4 introns; OsAMT2-1 and OsAMT2-2 have 3 exons and 2 introns; OsAMT2-3 is the same as cluster1; OsAMT3-1 has 2 exons and 1 introns; OsAMT3-2 has 4 exons and 3 introns; OsAMT3-3 has 3 exons and 2 introns; Only OsAMT4 is in the cluster4, it has 3 exons and 2 introns. Therefore, the substantial differences in gene structure may be resulted from differences in the size of exons and introns among the various genes.

Figure 2 Gene structure (A) and Trans-membrane Helices (B)

Trans-membrane Helices and Conserved Domain Prediction.

We predicted the trans-membrane domains in each AMT protein using TMHMM Server (http://www.cbs.dtu.dk/services/TMHMM/). Details are summarized in Table 2 with visualization in Figure 2. The results indicate that all of them have 5 to 11 trans-membranes, and members in the same subfamily have similar trans-membrane helices (Figure 2 B). In addition, only N-terminus of AtAMT1-4 and AtAMT1-3 are on the cytoplasmic side of the membrane, others are out of the cytoplasmic side, while 15 out of 16 C-terminuses are on the cytoplasmic side, except for OsAMT2-3. The length of encoded proteins ranged from 299 amino acids to 514 amino acids. OsAMT4 has the shortest length, while AtAMT1-2 has the longest one. Interestingly, 14 out of the 16 AMT members has the AMT domain using the InterPro software (http://www.ebi.ac.uk/interpro/), which can provide functional analysis of proteins by classifying them into families and predicting domains and important sites (Table 2).

Table 2 Protein information of members of family of AtAMT and OsAMT.

Subcellular Localization of AMT Genes

Subcellular localization of protein analysis showed that AMT members were mainly localized in plasma membrane, vacuolar, endoplasmic reticulum membrane and Golgi body. Details were as shown in Table 3. 15 out of 16 members were predicted in plasma membrane and only AtAMT1-1 was predicted in endoplasmic reticulum. The results implied that the AMT members were widely distributed in the plant cells.

Table 3 Subcellular localization of AtAMT and OsAMT.

Conclusion

In this paper, we compared and summarized 6 AMTs in Arabidopsis thaliana, and 10 AMTs in Oryza sativa. Through the Phylogenetic Tree analysis, two clusters in Arabidopsis thaliana AMT family and four clusters in Oryza sativa AMT family were classified.

The phylogenetic analysis and gene structure revealed that Genes in cluster 1 were well conserved in terms of exon/intron structure with similar numbers of introns and similar gene lengths. However, there were greater variations in gene structure among the cluster 2 and cluster 3. Further research is needed to investigate the internal mechanism, biologist should make greater efforts.

In AMT gene family, extracellular N-terminus play a role for oligomer stability. We found that the majority of N-terminus of Arabidopsis thaliana and Oryza sativa ammonium transporters were outside the cell, except for AtAMT1-4 and OsAMT1-2. In addition, we analyzed proteins by predicting domains and important sites. These results suggested that the AMT gene family members are well conserved both in terms of gene structure and specific domain of AMT proteins.

Results of Subcellular localization show us that, the vast majority of Arabidopsis thaliana and Oryza sativa AMTs are positioned in plasma membrane. Only AtAMT1-1 is mostly in endoplasmic reticulum. We need biology experiment to prove whether they are located in the predicted location.

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