Figure 1.
Table with total number of B3 protein per species and diagram of domain organization of the B3 family based on their protein sequences.
A – The identification of all proteins containing the B3 domain was conducted using the INTERPRO code of the B3 in respective species web site. Complete information of the genes and domains are in tables S1, S2, S3 and S4. B – Diagram of the domains organization of selected B3 proteins of each family. All selected proteins are from Arabidopsis, contain the typical domains found in each respective family and have been studied before: ABI3, At3g24650 (ABI3); HSI, At2g30470 (HSI2/VAL); ARF, At1g59750 (ARF1); RAV, At1g13260 (RAV1); REM, At3g18990 (VRN1). Proteins of REM family have one to 11 B3 domains. The domains are shown in scale and solid lines denote protein sequences. Bar – 100 amino acids.
Figure 2.
Phylogeny of B3 domains sequences.
A, Rooted in ABI3/HSI Neighbor-joining tree of all five B3 families based on the B3 domain amino acid sequences of moss. B, Rooted in REM I class Neighbor-joining tree of all B3 domains members of REM family from Arabidopsis. C, Rooted in REM I class Neighbor-joining tree of all B3 domains member of REM family from poplar. D, Rooted in REM I class Neighbor-joining tree of all B3 domain member of REM family from rice. A, B, C and D - bootstrap values from 1,000 replicates. The scale bar represents a 0.1 estimated aminoacid substitution per residue. Bootstrap values >40 are shown. The color chosen for each group of homologous is used to indicate the B3 domain types in figures 3 and 4. E, Alignment of the COBBLER-derived B3 domain sequences from A. thaliana proteins. REM* represent an alignment with all B3 domain from REM X A, REM VIII A and REM VII A types. All of them have one common point with 50% of boostrap in Figure 2B. The black bar and white bars represent predicted α-helix and β-sheet regions, respectively, within the B3 domain [37]. The red line underneath of sequences indicates residues of the embedded COBBLER consensus block. Black Stars represent amino acid residues that make direct contact with DNA in the RAV protein [37]. Meaningful similarities are indicated by color bars.
Figure 3.
Phylogenetic relationships among Arabidopsis, poplar, rice and P. patens B3 protein sequences of the REM family.
A, Unrooted Neighbor-joining consensus tree of the entire amino acid sequences of Arabidopsis, poplar, rice and P. patens REM I class proteins. B, Unrooted Neighbor-joining consensus tree of the entire amino acid sequences of rice and P. patens REM III class proteins. C, Unrooted Neighbor-joining consensus tree of the entire amino acid sequences of Arabidopsis and rice REM V class proteins. D, Unrooted Neighbor-joining consensus tree of the entire amino acid sequences of Arabidopsis and poplar REM VI class proteins. E, Unrooted Neighbor-joining consensus tree of the entire amino acid sequences of Arabidopsis, poplar and rice REM VIII class proteins divided in specific groups. PtREM17 was excluded from this analysis because the sequence is short and do not makes any common alignment. F, Unrooted Neighbor-joining consensus tree of the entire amino acid sequences of poplar REM XI class proteins. G, Unrooted Neighbor-joining tree of all B3 domains members of REM VIII class from Arabidopsis, poplar and rice showing two groups well supported (REM VIII A and REM VIII B). Additionally, REM VIII B was classified in other specific groups as supported in Figure 3E and other analysis (see text). The number and type of B3 domain classified in Figure 2 is represented for each gene with different colours. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Bootstrap values >40 are shown. The scale bar represents a 0.05 estimated amino acid substitution per residue.
Figure 4.
Phylogenetic relationships and exon/intron structure with B3 domain localization in each gene for REM family of Arabidopsis and rice.
A, Unrooted Neighbor-joining tree of the entire amino acid sequences of Arabidopsis REM I and REM VI proteins. B, Unrooted Neighbor-joining tree of the entire amino acid sequences of Arabidopsis REM VII proteins. C, Unrooted Neighbor-joining tree of the entire amino acid sequences of Arabidopsis REM VIII proteins. D, Unrooted Neighbor-joining tree of entire amino acid sequences of Arabidopsis REM IX proteins. E, Unrooted Neighbor-joining tree of the entire amino acid sequences of rice REM I proteins. F, Unrooted Neighbor-joining tree of the entire amino acid sequences of rice REM III proteins. G, Unrooted Neighbor-joining tree of the entire amino acid sequences of rice REM V proteins. H, Unrooted Neighbor-joining tree of entire amino acid sequences of rice REM VIII proteins. I, Unrooted Neighbor-joining tree of entire amino acid sequences of rice REM XIII proteins. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Bootstrap values >40 are shown. The scale bar represents a 0.1 estimated amino acid substitution per residue. The phylogenetic tree and exon/intron structure with domain localization of each B3 domain type are shown. Each colored box represent one B3 type domain, as indicate in the figure. The different colours of REM B3 domains per protein were based on different class found in phylogenetic analysis of REM B3 domains showed in Figure 2B, 2C, 2D. MIPS Arabidopsis thaliana and Orysa sativa database was used for exon/intron structure information [87]. As it is not possible to construct phylogenetic tree with less than 4 genes, specific classes with few members of classes were not showed in this figure such as AtREM X and OsREM XII.
Figure 5.
The locations of the B3 superfamily genes on the Arabidopsis chromosomes.
The chromosome number is indicated at the top of each chromosome. The chromosomal positions of the B3 genes are indicated by their locus identifier. Families are shown with a box in the corresponding gene (see legends). Duplicated B3 genes identified by our study are designated by numbers in open circles. B3 genes that participated of the most recent duplicated segmental region studied by Blanc et al. [38] are identified by a number in solid black circles. B3 genes that suffered duplication according to our study and that were considered ancient duplicated genes studied by Blanc et al. [38] were marked by a black star.
Figure 6.
Alignment of amino acid sequences and exon/intron structures of B3 domain from a representative member of all A. thaliana and P. patens groups.
Gaps are indicated by dashes. Underlined letters denote splicing sites. Bold and regular letter were used to help visualization of different exons.
Figure 7.
Phylogenetic relationship and exon/intron structure of B3 domains from ABI3 and HSI family in C. reinhardtii, V. carteri, P. patens, rice, poplar and Arabidopsis.
A, Unrooted Neighbor-joining tree of the B3 domain from C. reinhardtii, V. carteri, P. patens, rice, poplar and Arabidopsis. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Bootstrap values >50 are shown. The scale bar represents a 0.05 estimated amino acid substitution per residue. B, Alignment of amino acid sequences and exon/intron structures of B3 domains from all C. reinhardtii, V. carteri, P. patens, rice, poplar and Arabidopsis species. Gaps are indicated by dashes. Underlined letters denote splicing sites. Bold and regular letters were used to help visualization of different exons.
Figure 8.
B3 domain modelling of RAV1, ABI3, ARF17 and six distinct REM domain types (REM3, VRN1/REM5, REM22 and REM37).
These proteins belong to different B3 families and contain most of the types of B3 domains identified by our phylogenetic analysis. The NMR solution structures for the Arabidopsis protein REM14 (residues 1 to 102; PDB ID 1YEL) or RAV1 (residues 182 to 295; PDB ID 1WID) were used as template. The REM proteins evaluated contain identical structure to RAV1, although they present poor sequence conservation in the two loops (between β strand 1 and 2 and between β strand 4 and 5) that are proposed, in the model, to interact with the DNA (arrows). In addition, the loops are also shorter when compared with member of RAV, ARF, ABI3 and HSI families.
Figure 9.
Schematic representation of the phylogeny of the B3 superfamily during plant evolution.
Circles with a number inside denote large-scale duplication events: 1, ∼143–220 MYA [52]; 2, ∼66–109 MYA [50], [51]; 3, ∼50–60 MYA [39], [50]; 4, ∼30–60 MYA [53], ∼24–40 MYA [38]; and 6, ∼8–13 MYA [40]. Solid black circles denote recent tandem duplication for B3 genes discovered in this study. The solid black squares, family or class names denote origin of the B3 families during evolution.