Fig 1.
Proteins involved in the eukaryotic nitrate assimilation pathway (NAPs).
The eukaryotic nitrate assimilation pathway and the downstream proteins necessary for the assimilation of ammonium. Abbreviations: NRT2: Nitrate transporter NRT2; EUKNR: assimilatory NAD(P)H:nitrate reductase (EC 1.7.1.1–3); NAD(P)H-NIR: ferredoxin-independent assimilatory nitrite reductase (EC 1.7.1.4); Fd-NIR: ferredoxin-dependent assimilatory nitrite reductase (EC 1.7.7.1); GS: glutamine synthetase (EC 6.3.1.2); GOGAT: Glutamine oxoglutarate aminotransferase (EC 1.4.1.14); GDH: Glutamate dehydrogenase (EC 1.4.1.2). In this article, we focus on the proteins specifically required for the incorporation and reduction of nitrate to ammonium (hereafter abbreviated as NAPs, for “Nitrate Assimilation Proteins”).
Fig 2.
Distribution of NAPs among 172 sampled eukaryotic genomes.
The evolutionary relationships between the sampled species, represented in a cladogram, were constructed from recent bibliographical references (see Materials and methods section). Species names were colored according to the taxonomic groups to which they belong. The presence of each NAP in each taxon is shown with symbols. Black symbols indicate NAP genes that are found within genome clusters of NAP genes. For illustration purposes, some clades of species (e.g. Metazoa) were collapsed into a single terminal leaf. For detailed information about the taxonomic categories and the NAP profiles and NAP cluster status of each species, see Table A in S1 Supporting information. Autotrophic and fungal-like osmotrophic lineages are also indicated (see Table A in S1 Supporting information for information about the nutrient acquisition strategy of each taxon).
Fig 3.
The prokaryotic origins of nrt2, NAD(P)H-nir and Fd-nir shown by phylogenetic analyses.
Schematic representation of the maximum likelihood phylogenetic trees inferred for nrt2, NAD(P)H-nir and Fd-nir, with the aim of reconstructing the origins of the eukaryotic homologs. Prokaryotic sequences were taxonomically characterized following NCBI taxonomic categories. Clades with bacterial sequences belonging to the same taxonomic group were collapsed and colored as indicated in the panel. Similarly, eukaryotic sequences were classified, collapsed and colored according to whether they contain or not a plastid/plastid-related organelle. See S3 Fig, S6 Fig and S7 Fig for the entire representation of phylogenetic trees and Materials and methods section for details on their reconstruction.
Fig 4.
The chimeric origin of euknr shown by sequence-similarity network approach.
Graphical representation of two pre-processed sequence similarity networks constructed from all-vs-all Blast hits between eukaryotic and prokaryotic proteins. Sequences were detected using as queries all eukaryotic EUKNR in (A), and the Cytochrome-b5 (Cyt-b5) regions of Chlamydomonas reinhardtii and Aspergillus nidulans (reference EUKNR sequences) in (C). See Materials and methods section for details on the network pre-processing and construction processes. Each node represents a protein, and each edge represents a Blast hit between two proteins. Proteins were grouped and colored according to their protein domain architecture and protein family information. In (C), we also represented the lowest E-value with which C. reinhardtii aligned with the Cyt-b5 monodomain and the Cyt-b5 multidomain proteins (see Results section). (B) The canonical protein domain architecture of a full-length eukaryotic EUKNR (Pfam domains), with paired lines indicating the gene families from which each domain would have originated (see Results section). Abbreviations: Bact: Bacterial; SUOX: sulfite oxidase; Euk: Eukaryotic; Prot: Protein; EUKNR: eukaryotic nitrate reductase; NADH red: NADH reductase; Cyt-b5: Cytochrome b5-like Heme/Steroid binding Pfam domain; Crei: Chlamydomonas reinhardtii; Anid: Aspergillus nidulans; Oxidored_molyb: Oxidoreductase molybdopterin binding Pfam domain; Mo-co_dimer: Mo-co oxidoreductase dimerization Pfam domain; FAD_binding_6: Ferric reductase NAD binding Pfam domain; NAD_binding_1: Oxidoreductase NAD-binding Pfam domain.
Fig 5.
The evolutionary history of NAPs in eukaryotes.
Simplified representation of the maximum likelihood phylogenetic trees inferred for each NAP (Fd-NIR, NAD(P)H-NIR, NRT2, EUKNR) are shown. Some branches were collapsed into clades (triangles) that represent higher eukaryotic taxonomic groups or groups of species-specific paralogues in the NRT2 phylogeny. Branches and clades were colored according to the taxonomic groups to which they belong (see panel). For the representation of the taxonomic information, only taxonomic categories that are mentioned in the manuscript and that are not indicated by the color code are specified (see Taxonomy panel). For illustration purposes, given the overall poor nodal support of the EUKNR tree, we converted the branches with <90% UFBoot into polytomies (see the draft EUKNR tree in S18 Fig). Taxonomical abbreviations: Chlorarch: Chlorarachniophyta; Chloro: Chlorophyta; Crypto: Cryptophyta; Glauco: Glaucophyta; Ichthyo: Ichthyosporea; Ochro: Ochrophyta; Tereto: Teretosporea.
Fig 6.
NAP clusters in Ichthyosporea and the origins of a putative novel nitrate reductase.
(A) Cluster organization and protein domain architecture of NAP clusters from the ichthyosporeans Creolimax fragrantissima, Sphaeroforma arctica and Phytophthora infestans (Oomycota). Within each cluster each box represents a gene, with the arrowhead indicating its orientation. The Pfam domains predicted for the corresponding protein sequences are represented inside each box (see panel). (B) Schemes showing a simplified representation of the maximum likelihood phylogenetic trees inferred for the N-terminal and C-terminal regions of the putative nitrate reductase identified in C. fragrantissima and S. arctica (CS-pNR, see Results section). For an entire representation of the phylogenetic trees, see S18 Fig and S22 Fig for N-terminal and C-terminal regions, respectively. (C) Schematic representation of the evolutionary origin of CS-pnr, inferred from the phylogenies shown in (B).
Fig 7.
Sphaeroforma arctica culture and qPCR experiments in nitrogen minimal media.
(A) Growth of Sphaeroforma arctica in media with different nitrogen sources (scale bar = 100 μm). (B) S. arctica NAP genes mRNA levels in mL1, mL1 + NaNO3, mL1 + (NH4)2SO4 and mL1 + urea. The y-axis represents copies per copy of ribosomal L13. Results are expressed as the mean ± S.D. of three independent experiments.
Fig 8.
A hypothetical scenario for the evolution of the nitrate assimilation pathway in eukaryotes, based on the current phylogenetic data and in the taxonomic distribution of NAPs.
For each transfer proposed, donor and receptor lineages are indicated; as well as if the transfer is related to the origin of the pathway (1), or if autotrophic (2) or osmotrophic (3) lineages were involved (see the Discussion section ‘HGT and the evolutionary history of NAPs in eukaryotes’). Transfers of NAP genes in clusters are represented with the corresponding NAP symbols surrounded by a square. Branches in red are those where loss of the entire pathway would have occurred, which were parsimoniously inferred from the reconstructed evolutionary history. For the sake of simplicity, some species were collapsed into clades representing higher taxonomic categories. For each species/clade, NAP gene presence/absence (NAP symbols) and their cluster status (symbols colored in black for those NAPs found in the same gene cluster) are indicated. For those clades in which not all the represented species have the same NAP content and cluster status (labeled with *), the most prevalent ones are shown (see Table A in S1 Supporting information for a complete representation of the NAP content and cluster status). Distinct scenarios considering transfers between Stramenopiles and Opisthokonta were evaluated (see S15 Fig and ‘NRT2 and NAD(P)H-NIR’ Results subsection). H1 and H8 scenarios are both the most parsimonious with the number of HGT events among those not rejected with the AU-test. Both scenarios propose a transfer from an ancestral Stramenopiles (either from the lineage leading to Labyrinthulea or from a common ancestor of Stramenopiles) to a common ancestor of Opisthokonta.