In silico characterization of a nitrate reductase gene family and analysis of the predicted proteins from the moss Physcomitrella patens

Abstract

Assimilatory nitrate reductase (NR; EC 1.7.1.1-3) catalyzes the reduction of nitrate to nitrite. This enzyme has a conserved structure common to fungi, algae and plants. However, some differences in the amino acid sequence between plant and algal NR suggest that the activity regulation mechanisms have changed during plant evolution. Since only NRs from angiosperms have been studied, the search and analysis of NR genes and proteins from the moss Physcomitrella patens, a basal land plant, was performed to widen the knowledge of land plant NR structure. A family of three nr genes, named ppnia1;1, ppnia1;2 and ppnia2, was localized in the P. patens genome. The predicted proteins are canonical NRs with the conserved domains Molybdene-Cytochorme b -Cytochrome b reductase and possess 20 amino acid residues important for the enzymatic function conserved in plant and algal NRs. Interestingly, moss NRs lack a consensus sequence, common to angiosperm NRs, that is a target for posttranslational regulation. A phylogenetic tree with embryophyte and green algae NR sequences was constructed and P. patens NRs localized at the base of embryophyte NR evolution. The data presented here suggest that bryophytes and vascular plants have different systems to regulate NR activity.

Keywords: Physcomitrella patens; basal embryophytes; nitrate reductase; posttranslational regulation.

Figure 1.

Map of the Physcomitrella patens nitrate reductase gene family. Scaffolds correspond to the P. patens genome database (http://www.cosmoss.org/). Arrows show the translational orientation of the genes. Gene length includes the 5′and 3′untranslated regions. Map not drawn to scale.

Figure 2.

Amino acid sequence comparison between P. patens NR and selected vascular plant NR. Underlined sections indicate the protein domains: MoCo, solid line; Cyt b, dotted line and Cyt b reductase, dashed line. Conserved amino acid residues important for NR function are underlined and in bold type. Gray shaded sequence indicates the binding site for 14–3-3 proteins, conserved in vascular plants. Note that P. patens NRs lack that sequence and have E instead of S in the phosphorylation site. Sequences were obtained from the GenBank database using the following accession numbers: Nicotiana benthamiana nia1 (BAE46746.1); Cucumis sativus nia2 (ADN96689.1); Arabidopsis thaliana nia1 (NP_177899.1); Zea mays nia1 (AAD38068.1); Selaginella moellendorffii nia1 (XP_002972481.1); Physcomitrella patens nia1;1 (BAE19754.1) and nia1;2 (BAE19755.1). Amino acid numbering corresponds to the N. benthamiana sequence.

Figure 3.

Phylogenetic tree of algae and land plants NR amino acid sequences. Only full NR sequences were included in this analysis, the GenBank accession numbers used are indicated in Table 2. The evolutionary tree was constructed with the MEGA5 software. Bootstrap analysis of 1000 replications was performed on the trees inferred from the neighbor-joining method. Bootstrap values are noted in the nodes, those below 50% are not shown.