PISTILLATA paralogs in Tarenaya hassleriana have diverged in interaction specificity

Abstract

Background: Floral organs are specified by MADS-domain transcription factors that act in a combinatorial manner, as summarized in the (A)BCE model. However, this evolutionarily conserved model is in contrast to a remarkable amount of morphological diversity in flowers. One of the mechanisms suggested to contribute to this diversity is duplication of floral MADS-domain transcription factors. Although gene duplication is often followed by loss of one of the copies, sometimes both copies are retained. If both copies are retained they will initially be redundant, providing freedom for one of the paralogs to change function. Here, we examine the evolutionary fate and functional consequences of a transposition event at the base of the Brassicales that resulted in the duplication of the floral regulator PISTILLATA (PI), using Tarenaya hassleriana (Cleomaceae) as a model system.

Results: The transposition of a genomic region containing a PI gene led to two paralogs which are located at different positions in the genome. The original PI copy is syntenic in position with most angiosperms, whereas the transposed copy is syntenic with the PI genes in Brassicaceae. The two PI paralogs of T. hassleriana have very similar expression patterns. However, they may have diverged in function, as only one of these PI proteins was able to act heterologously in the first whorl of A. thaliana flowers. We also observed differences in protein complex formation between the two paralogs, and the two paralogs exhibit subtle differences in DNA-binding specificity. Sequence analysis indicates that most of the protein sequence divergence between the two T. hassleriana paralogs emerged in a common ancestor of the Cleomaceae and the Brassicaceae.

Conclusions: We found that the PI paralogs in T. hassleriana have similar expression patterns, but may have diverged at the level of protein function. Data suggest that most protein sequence divergence occurred rapidly, prior to the origin of the Brassicaceae and Cleomaceae. It is tempting to speculate that the interaction specificities of the Brassicaceae-specific PI proteins are different compared to the PI found in other angiosperms. This could lead to PI regulating partly different genes in the Brassicaceae, and ultimately might result in change floral in morphology.

Keywords: Cleomaceae; Flower development; Gene duplications; MADS; PISTILLATA; Paralogs; Tarenaya.

Fig. 1

Synteny of both PI paralogs of T. hassleriana. PI paralogs are shown with an orange (Brassicaceae) or red (non-Brassicaeae) trace. Other syntenic genes are linked in grey. a Synteny of ThPI-1 with the Brassicaceae PI orthologs. Shown are the Brassicaceae type I genera Arabidopsis (A. lyrata and A. thaliana) and Capsella (C. rubella and C. grandiflora); the Brassicaceae type II species Eutrema salsugineum, Arabis alpina, Brassica oleraceae (3 paralogs), the basal Brassicaceae Aethionema arabicum and PI-1 of T. hassleriana. b Synteny of ThPI-2 with non-Brassicaceae angiosperms. Shown are 7 rosid species (Medicago truncatula (2 paralogs), Prunus persica, Ricinus communis, T. hassleriana, Theobroma cacao, Citrus sinensis and Vitis vinifera), 3 asterid species (Solanum lycopersicum, Solanum pennellii and Coffea canephora), one Caryophyllales (Beta vulgaris) and the sister species to all other angiosperms, Amborella (A. trichopoda)

Fig. 2

Phylogenetic and sequence analysis of the T. hassleriana B-class genes. a Alignment of PI orthologs from several species. b Maximum-likelihood phylogeny of PI orthologs. The PI orthologs belonging to Brassicaceae species are indicated in beige, the Cleomaceae PI orthologs in blue. c Maximum-likelihood phylogeny showing the position of the ThAP3 paralogs. Alignments for phylogenies were generated with a codon-based DNA-sequence algorithm. d Alignment of ThAP3 paralogs with AtAP3. The MADS-domain, the K-domain and the lineage-specific C-terminal motifs are indicated in A and D. Abbreviations: At = A. thaliana; Al = A. lyrata; Cr = Capsella rubella; Aa =  Aethionema arabicum; Th = T. hassleriana; Gg = G. gynandropsis; Cp = Carica papaya; Tc = Theobroma cacao; Vv = Vitis vinifera; Pt = Populus trichocarpa

Fig. 3

Expression patterns of T. hassleriana B-class genes. Expression patterns of the ThAP3 paralogs (a, e, i, m), ThPI-1 (b, f, j, n) and ThPI-2 (c, g, k, o) as determined by RNA in situ hybridization. Expression was determined in early developmental stages before organ primordia were formed (a-c, i-k) as well as later during organ differentiation (e-g, m-o). Schematics of the different developmental stages and planes are shown in d, h, (longitudinal),L and P, (cross). Br=bract; S=Sepal primordia (green); P/St = whorls giving rise to petals and stamens; P = petal primordia (purple); St = stamen primordia (yellow); C = carpel primordia (orange); Fm = floral meristem. Scale bar = 100 μm

Fig. 4

Expression levels of ThPI-1 and ThPI-2, according to RNA-seq data of mature floral organs. Error bars represent standard deviation. RPKM = reads per kilobase of transcript per million mapped reads. RNA-seq data obtained from [72]

Fig. 5

Heterologous expression of the ThPI paralogs in A. thaliana. a a wildtype (WT) A. thaliana flower. b a 35S::AtPI flower, which shows the phenotype obtained by constitutive expression of the native PI. Note the change in orientation of the first whorl organs. c a 35S::ThPI-1 flower, showing homeotic conversion of sepals to petals. d a 35S::ThPI-2 flower, showing no aberrant phenotype. Top row shows whole flower, bottom row shows dissected sepals (top) and petals (bottom). e AtPI expression levels in 35S::AtPI lines. f ThPI-1 expression levels in 35S::ThPI-1 lines. g ThPI-2 expression levels in 35S::ThPI-2 lines. h ThPI-2 expression levels in additional 35S::ThPI-2 lines. Lines that showed an overexpression phenotype are indicated with an asterisk. Expression measured in leaves, and calculated relative to a reference gene (TIP41). For the expression in e, f and g, Rosette leaves of 6–8 week plants were used. In h, the first developing rosette leaves were used

Fig. 6

DNA-binding protein-complexes formed by T. hassleriana B-class proteins. In (a), homo- and heterodimerization of ThAP3 and ThPI (b) Complexes formed with ThAG, ThSEP3 (Th01528), ThAP3–1 and either of the two ThPI paralogs. The figure shows only the higher order complexes (tetramers); in the right image this part compared to the whole gel is indicated. In (c), EMSA for protein complexes with ThAG and a ThSEP4 paralog (Th21984). (d, e, f) EMSAs testing the formation of a DNA-binding protein complex of the B-class dimers with a ThAP1 paralog (Th13754) and with different ThSEP paralogs: ThSEP3 (Th1528) (d), ThSEP1/2 (Th2854) (e), and ThSEP4 (Th21984) (f). For all experiments, a promoter fragment from the A. thaliana SEP3 promoter was used as probe (Smaczniak et al., 2012a). The control is an empty-vector control, in which no MADS protein production is expected

Fig. 7

DNA-binding specificities as determined by SELEX-seq. a Dotplot comparing the relative affinities between ThPI-1/ThAP3–1 and ThPI-2/ThaP3–1. b Motifs obtained for each of the PI/AP3–1 heterodimers. Motif discovery was performed on the most recurring 40 N sequence for each of the 0.1% k-mers with the highest affinity