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. 2017 Jun 29;15(1):52.
doi: 10.1186/s12915-017-0397-z.

Evolution of strigolactone receptors by gradual neo-functionalization of KAI2 paralogues

Rohan Bythell-Douglas  1 , Carl J Rothfels  2 , Dennis W D Stevenson  3 , Sean W Graham  4 , Gane Ka-Shu Wong  5   6   7 , David C Nelson  8 , Tom Bennett  9
Affiliations

Evolution of strigolactone receptors by gradual neo-functionalization of KAI2 paralogues

Rohan Bythell-Douglas et al. BMC Biol. .

Abstract

Background: Strigolactones (SLs) are a class of plant hormones that control many aspects of plant growth. The SL signalling mechanism is homologous to that of karrikins (KARs), smoke-derived compounds that stimulate seed germination. In angiosperms, the SL receptor is an α/β-hydrolase known as DWARF14 (D14); its close homologue, KARRIKIN INSENSITIVE2 (KAI2), functions as a KAR receptor and likely recognizes an uncharacterized, endogenous signal ('KL'). Previous phylogenetic analyses have suggested that the KAI2 lineage is ancestral in land plants, and that canonical D14-type SL receptors only arose in seed plants; this is paradoxical, however, as non-vascular plants synthesize and respond to SLs.

Results: We have used a combination of phylogenetic and structural approaches to re-assess the evolution of the D14/KAI2 family in land plants. We analysed 339 members of the D14/KAI2 family from land plants and charophyte algae. Our phylogenetic analyses show that the divergence between the eu-KAI2 lineage and the DDK (D14/DLK2/KAI2) lineage that includes D14 occurred very early in land plant evolution. We show that eu-KAI2 proteins are highly conserved, and have unique features not found in DDK proteins. Conversely, we show that DDK proteins show considerable sequence and structural variation to each other, and lack clearly definable characteristics. We use homology modelling to show that the earliest members of the DDK lineage structurally resemble KAI2 and that SL receptors in non-seed plants likely do not have D14-like structure. We also show that certain groups of DDK proteins lack the otherwise conserved MORE AXILLARY GROWTH2 (MAX2) interface, and may thus function independently of MAX2, which we show is highly conserved throughout land plant evolution.

Conclusions: Our results suggest that D14-like structure is not required for SL perception, and that SL perception has relatively relaxed structural requirements compared to KAI2-mediated signalling. We suggest that SL perception gradually evolved by neo-functionalization within the DDK lineage, and that the transition from KAI2-like to D14-like protein may have been driven by interactions with protein partners, rather than being required for SL perception per se.

Keywords: Neo-functionalization; Phylogenetics; Strigolactone evolution; Strigolactone signalling.

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Figures

Fig. 1
Fig. 1
The eu-KAI2 and DDK super-clades diverged early in land plant evolution. Codon-level phylogenetic analysis implemented in the Genetic Algorithm for Rapid Likelihood Inference (GARLI) on the whole D14/KAI2 family (339 sequences from 143 species). This analysis was performed using an optimized character set (see Methods). Trees were rooted with charophyte sequences, consistent with contemporary notions of plant organismal phylogeny. Dotted lines indicate alternative positions for the indicated clades that would increase the parsimony of the tree. a Phylogram showing the ‘most likely’ tree from GARLI analysis, labelled to show the high-order relationships between the major clades (as described in Table 1). b Cladogram depicting the phylogenetic tree from (a) in simplified form. Major clades and sub-clades (as listed in Table 1) are collapsed. Numbers associated with internal branches denote maximum likelihood bootstrap support (percent support). M-C-E magnoliids-chloranthales-eudicots
Fig. 2
Fig. 2
The eu-KAI2 and DDK super-clades diverged early in land plant evolution. Nucleotide-level phylogenetic analysis implemented in GARLI on the D14/KAI2 family, minus charophyte and lycophyte KAI2 sequences (296 sequences). Trees were rooted with hornwort KAI2 sequences by comparison with Fig. 1. This analysis was performed using the full-length dataset (780 characters). a Phylogram showing the ‘most likely’ tree from GARLI analysis, labelled to show the high-order relationships between the major clades (as described in Table 1). b Cladogram depicting the phylogenetic tree from (a) in simplified form. Major clades and sub-clades (as listed in Table 1) are collapsed. Numbers associated with internal branches denote maximum likelihood bootstrap support (percent support); values below 50 are indicated by *. M-C magnoliids/chloranthales
Fig. 3
Fig. 3
Reconstruction of D14/KAI2 family evolution. Schematic depicting the complement of D14/KAI2 proteins in major land groups, and their inferred evolutionary origin. Each branch indicates a major land plant group; lycophytes, monilophytes and gymnosperms are further sub-divided into relevant orders/families/etc. The ovals on each branch indicate the core complement of proteins in that group or sub-group and are coloured according to the scheme indicated at the bottom left. Clades which are inferred by parsimony are denoted with a hatched line. Letters and numbers in the ovals denote the clade names as outlined in Table 1. Letters and numbers in the circles indicate clade names. D1 = D14, D2 = DLK2, D3 = DLK3, D4 = DLK4, D23 = DLK23. Circles without symbols at internal branching points represent the minimum inferred D14/KAI2 protein complement in the last common ancestor of each major land plant group
Fig. 4
Fig. 4
Eu-KAI2 proteins have highly conserved structure. Alignment illustrating conservation of primary protein structure in D14/KAI2 proteins. The 265 core positions (numbered) are shown in the alignment, for the whole family (top row), for eu-KAI2 proteins (middle row) and for eu-D14 proteins (bottom row). Positions where the same amino acid is present in >50% of sequences in the clade are denoted by corresponding letter; other positions are denoted by a dash. The colouring of each conserved residue indicates the degree of conservation; pale blue >50%, light blue >70%, mid-blue >90%, dark blue >99%, purple =100%. Structural features are annotated below the alignment. The catalytic triad is indicated by *. MAX2-interacting residues are indicated by m. Predicted alpha helices (based on the crystal structure of AtKAI2 (Protein Data Bank (PDB) code 4HRX1A) are shown by grey bars, predicted beta strands by grey bars with an arrow. The discrete positions in the polypeptide chain where insertions (or deletions) can be tolerated are illustrated with red arrow heads. Residues that are characteristic of eu-KAI2 proteins are underlined in yellow; residues characteristic of eu-D14 are underlined in orange (see Fig. 5)
Fig. 5
Fig. 5
KAI2 and D14 protein characteristics. a We identified well-conserved positions in eu-KAI2 proteins (i.e. >70% of sequences have the same amino acid) in which the amino acid is characteristic of eu-KAI2 proteins (i.e. found in <30% of other sequences). These are listed at the left (position and amino acid). We then tested whether various clades share elements of this structure (i.e. how frequently the same amino acid is found at the same position in that clade). Charophyte and lycophyte KAI2 proteins are a close match, while KAI2B and KAI2E/F proteins from liverworts and mosses respectively have considerable similarity. However, DDK, D14 and DLK2 proteins do not share these characteristics. b We performed the same analysis with eu-D14 proteins, but only identified 7 characteristic residues. We thus extended the search to the combined D14-DLK4 clade and identified another 13 residues characteristic of the wider clade. These are listed at the left (position and amino acid). Very little conservation of these characteristic residues is found in other members of the DDK family
Fig. 6
Fig. 6
Homology models of KAI2 sequences. Models are shown in ribbon representation with the residues that influence the active site cavity shown in stick representation. Cavities are depicted as a transparent surface. Oxygen, nitrogen and sulphur atoms are coloured red, blue and yellow respectively. a The crystal structure of Arabidopsis thaliana KAI2 in complex with karrikin (KAR 1) is shown in navy blue (PDB code 4JYM). Residue numbers correspond to the unified numbering scheme as in Fig. 5; they are –1 relative to those of A. thaliana KAI2. b–d Liverwort KAI2A homology models are shown in royal blue; b Lejeuneaceae sp. c Lunularia cruciata, d Ptilidium pulcherrimum. e–h Liverwort KAI2B models are shown in turquoise; e Riccia berychiana, f Calypogeia fissa, g Lunularia cruciata, h Marchantia polymorpha. i–l Charophyte KAI2 models are shown in purple; i Klebsormidium subtile, j Chara vulgaris, k Coleochaete scutata, l Coleochaete irregularis. m–o Moss KAI2E/F models are shown in green; m Sphagnum recurvatum KAI2E, n Timmia austriaca KAI2F, o Tetraphis pellucida KAI2F
Fig. 7
Fig. 7
Homology models of DDK sequences. Models are shown in ribbon representation with the residues that influence the active site cavity shown in stick representation. Cavities are depicted as a transparent surface. Oxygen, nitrogen and sulphur atoms are coloured red, blue and yellow respectively. a The crystal structure of A. thaliana KAI2 in complex with karrikin (KAR 1) is shown in navy blue (PDB code 4JYM). Residue numbers correspond to the unified numbering scheme as in Fig. 5; they are –1 relative to those of A. thaliana KAI2. b The crystal structure of A. thaliana D14 is shown in red. c, d Lycophyte DDK homology models are shown in olive green; c Selaginella moellendorffii (previously referred to as KAI2b), d Lycopodium annotinum. e–h Monilophyte DDK homology models are shown in lime green; e Osmunda sp. DDKb, f Polypodium amorphum DDKA, g Cystopteris fragilis DDKA, h Asplenium platyneuron DDKB
Fig. 8
Fig. 8
The MAX2 family has a very conservative evolutionary history. Codon-level phylogenetic analysis implemented in GARLI on the whole MAX2 family (57 sequences from 54 species). This analysis was performed using an optimized character set (see Methods). Phylogram showing the ‘most likely’ tree from GARLI analysis, labelled to show the high-order relationships between the major clades (as described in Table 1).Trees were rooted with charophyte sequences, consistent with contemporary notions of plant organismal phylogeny. Numbers associated with internal branches denote maximum likelihood bootstrap support (percent support)
Fig. 9
Fig. 9
Models of D14/KAI2 evolution. BP binding pocket. a Traditional model of land plant evolution, with evolution of the D14/KAI2 family superimposed. A single origin of SL perception in the DDK lineage would be sufficient to explain known patterns of SL sensitivity. b ‘Hornworts-basal’ model of land plant evolution, with evolution of the D14/KAI2 family superimposed. Two independent origins of SL perception in the DDK lineage would be required to explain known patterns of SL sensitivity
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