The role of bZIP transcription factors in green plant evolution: adaptive features emerging from four founder genes

Abstract

Background: Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are regulators of many central developmental and physiological processes including photomorphogenesis, leaf and seed formation, energy homeostasis, and abiotic and biotic stress responses. Here we performed a comprehensive phylogenetic analysis of bZIP genes from algae, mosses, ferns, gymnosperms and angiosperms.

Methodology/principal findings: We identified 13 groups of bZIP homologues in angiosperms, three more than known before, that represent 34 Possible Groups of Orthologues (PoGOs). The 34 PoGOs may correspond to the complete set of ancestral angiosperm bZIP genes that participated in the diversification of flowering plants. Homologous genes dedicated to seed-related processes and ABA-mediated stress responses originated in the common ancestor of seed plants, and three groups of homologues emerged in the angiosperm lineage, of which one group plays a role in optimizing the use of energy.

Conclusions/significance: Our data suggest that the ancestor of green plants possessed four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments.

Figure 1. Phylogeny of bZIP transcription factors in green plants.

(A) Model of angiosperm bZIP evolution with two large clades, one including groups A, D, F, G and J, and the other including groups B, C, E, H, I and L. Sister groups B and K, E and L, D and F, and G and J, respectively, were defined based on bootstrap support of >50%. The position of Group S could not be clearly defined. (B) Consensus tree inferred from NJ analyses of bryophyte and algal bZIP sequences. This tree reveals new evolutionary relationships among green plant bZIPs, which were not observed when the complete ViridiZIP set was analyzed. Group C appears to be related to two other groups (cI and cII) and members of these three groups are orthologues of OtbZIP5, constituting the Group Proto-C. Group b was identified as a sister group of Group B and genes of both groups are orthologous to the algal OtbZIP3 gene, forming the Group Proto-B. Groups Proto-B and H have a common ancestral origin. Similarly, Groups G and J diverged from the same ancestor and are both orthologous to the algal gene OtbZIP2. Finally, Groups E and I show a sisterhood relation but no ancestral link to a bZIP from algae could be established. (C) Tree inferred from NJ analyses of the ViridiZIP data set (bZIPs from algae to angiosperms). This tree indicates that Group S probably originated from Proto-C, and Group K from Proto-B. Tree topology and functional data support these hypotheses. Bootstrap values were calculated from NJ analyses. Red, values obtained with p-distances and, black, with PAM matrix.

Figure 2. Motifs conserved in angiosperm bZIPs.

A summary of the motif sequences is given in Table S2. Arrows indicate intron positions conserved among most members of each group. Representative bZIP sizes and positions of conserved motifs are shown. (*) Group A has two motifs (A1 and A2), that are important putative kinase phosphorylation sites involved in ABA responses. Both motifs appear to be conserved in most members of this group of homologues, except for OsbZIP8, 13, 14 and 15, and PtrbZIP5 and 10, which lack motif A1. The same sequences and also PtrbZIP9 lack motif A2. Due to the lack of complete sequences, no structures are shown for Groups AL, GP, b, cI and cII.

Figure 3. Classification of bZIPs from Arabidopsis, black cottonwood and rice.

Thirteen groups of homologues (A to L, and S) were defined through NJ phylogenetic analyses with the bZARP set (Figures S1 and S3). The organization into Possible Groups of Orthologues (PoGOs) was done by more refined NJ phylogenetic analyses inside each group of homologues, including also sequences from other eudicots and monocots. The alignment used for these analyses corresponds to a concatenated sequence of the group-specific conserved motifs identified employing MEME (http://meme.sdsc.edu/meme/website/intro.html; Figure 2). (*) Represents genes that lack group-wise conserved motifs, thus they were included inside a PoGO according to their best hit to another bZIP. Because the relation of AtbZIP72, PtrbZIP37, 81, 82 and 89 could not be clarified, they were not included in any of the groups of homologous or orthologous genes. One Possible Group of Paralogues (PoGP I1) was found in Arabidopsis. Column ‘Gene code’ provides the gene identifiers for Arabidopsis, black cottonwood and rice bZIP sequences taken from TAIR (http://www.arabidopsis.org/), JGI (http://www.jgi.doe.gov/) or TIGR (http://www.tigr.org/), respectively. ‘Synonym’ indicates published and often cites names of bZIP genes. The GenBank accession numbers of nucleotide sequences are given.

Figure 4. Global Phylogeny of bZIPs in green plants.

This tree is a consensus of NJ analyses with p-distance performed with the ViridiZIP set. Bootstrap values in yellow were calculated from NJ analysis (PAM matrices, and with 44 and 60 amino acid alignments; only the highest bootstrap values are shown). Bootstrap values in red were calculated from ML analyses using the JTT+Γ evolutionary model (either with 44 or 60 amino acid alignments; only the highest bootstrap values are shown). GPX, GPI, GPII, GPIII, and GPIV indicate putative gymnosperm specific groups. Each group of homologues is colored following the same colour scheme used in Tables I and SV. The center of the tree depicts a typical bZIP dimer bound to DNA, representing the conserved bZIP domain (GCN4 from Saccharomyces cerevisiae; Protein Data Bank entry 2DGC).

Figure 5. Phylogenetic profile and structure of bZIPs in green plants.

Groups E, L and I belong to the same branch as Groups Proto-B, Proto-C and H but their exact position is not clear (Figure 1A). Similarly, Groups A, D and F do not have a clear position, though they belong to the same branch as Groups G and J (Figure 1A). The relation of Groups AL and GP to the other groups could not be established. bZIPs of the species studied here were grouped at the level of higher taxa, i.e., algae (represented by C. reinhardtii and O. tauri); bryophytes (P. patens); gymnosperms (P. glauca and P. taeda), and angiosperms (O. sativa, A. thaliana and P. trichocarpa). Solid boxes indicate that at least one bZIP was found for a given group of homologues in the respective taxon. Squared boxes indicate that homologous bZIP sequences were not yet observed in gymnosperms, possibly due to sampling limitations. Notably, however, sequences of the respective groups are conserved in bryophytes and angiosperms. Dashed lines with brackets shown in Groups Proto-B and Proto-C indicate that there is an orthologous bZIP in at least one of the algal species, although it does not strictly belong to any of the homologous groups. The half lines present in G and J indicate the presence of common orthologues in algae. Groups AL, GP, K, L and S appear to be lineage specific.

Figure 6. Most parsimonious model explaining the emergence of the four green plant founder bZIP genes.

The four founder genes (in Groups G+J, Proto-C, Proto-B and H) are derived from a unique ancestral gene common to all eukaryotes. Groups Proto-B and Proto-C most likely derived from a multifunctional UPR/oxidative stress gene. Groups Proto-B and H are sister groups and their relationship to Group Proto-C was found by analyzing angiosperm bZIPs (Figure 1A). Group G+J is the ancestral group of a large set of bZIP genes included in Groups A, D and F, but the ancestral function played by this group is still largely unknown.