The seirena B class floral homeotic mutant of California Poppy (Eschscholzia californica) reveals a function of the enigmatic PI motif in the formation of specific multimeric MADS domain protein complexes

Abstract

The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class-related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences.

Figure 1.

The sei-1 Phenotype and Expression Analysis of Class B Genes in California Poppy. (A) Wild-type flower. (B) Wild-type floral organs. (C) sei-1 flower showing homeotic conversions of petals into sepals and stamens into carpels. (D) sei-1 flower with ectopic sepals peeled away. (E) Overview of the sei-1 floral organs. The arrow indicates the central gynoecium. (F) Amino acid alignment of the deduced wild-type SEI and the mutant sei-1 proteins. Regions of sequence identity are highlighted in gray; the MADS, I, and C domains, and the proposed amphipathic helices of the K domain are indicated by boxes; and the conserved C-terminal PI motif is underlined. The start position of the protein sequence change caused by the genomic DNA insert is marked by an asterisk. (G) Organization of the SEI genomic locus in the wild-type and sei-1 mutant plants. Protein-coding portions are shown as black boxes, 3′UTR as white boxes, and insertion of unknown genomic DNA of California poppy in the sei-1 locus is marked with crosses. The numbers above the exons indicate exon length. The start codon is indicated by a horizontal arrow and the stop codon and polyadenylation (pA) site by vertical arrows. (H) Quantitative PCR on floral buds of all developmental stages (Becker et al., 2005) in wild-type (wt) and sei-1 plants. Wild-type buds of 0 to 1 mm diameter develop through stages 1 to 5 when carpels initiate, 1- to 2-mm buds include stages 6 and 7 when male sporogenic tissue and ovules arise, 2- to 3-mm buds (stage 8) includes stamens with pollen, and after 3-mm buds pass through stage 9 and female meiosis. B class genes and the putative E class gene SEP3 are color coded. Asterisks indicate significant reduction in expression as inferred by analysis of variance (*P < 0.05; **P < 0.01). Error bars show sd, and expression was normalized with ACTIN2 and GAPDH. (I) Expression analysis by quantitative RT-PCR in mature floral organs (stage 10) indicating the relative expression levels of DEF1, DEF2, DEF3, and SEI in the wild type (left) and sei-1 (right). ca, organs with only carpel-like characteristics; gyn, central gynoecium; se, organs with only sepal-like characteristics; se/ca, organs with a mix of sepal and carpel characteristics. Asterisks above the bars indicate a significant decrease of expression in sei-1 when compared with wild-type expression. sei-1 sepals were compared with wild-type petals, and sei-1 carpels were compared with wild-type inner and outer stamens. (J) to (N) In situ hybridization pattern of SEI in longitudinal sections of buds of stage 2 (J), stage 3 (K), stage 4 (L), early stage 5 (M), and late stage 5 (N). Stages are according to Becker et al. (2005). fm, floral meristem; gyn, central gynoecium; pe, petals; pp, petal primordia; se, sepals; st, stamens; stp, stamen primordia. Bars = 100 µm.

Figure 2.

Expression of C Class Genes in the Wild Type and sei-1 Mutants. (A) Quantitative RT-PCR of AG1 and AG2 in floral organs at anthesis in wild-type (wt) and sei-1 plants. Error bars depict sd. Abbreviations are as in Figure 1. (B), (D), and (F) In situ hybridization of combined AG1/2 transcripts in wild-type buds at stage 3 (B), early stage 5 (D), and late stage 5 (F). (C), (E), (G), and (H) AG1/2 in situ hybridization in floral buds of sei-1 plants at stage 3 (C), early stage 5 (E), late stage 5 (G), and stage 7 (H). In (G) and (H), arrows indicate the presence of mosaic organs composed of sepal and carpel-like (se/ca) characteristics. ca, carpels; fm, floral meristem; gyn, central gynoecium; pp, petal primordia; se, sepals; se/ca, sepal-like/carpel-like mosaic organs; st, stamens. Bars = 100 µm.

Figure 3.

Analysis of Interactions of B, C, and E Class Proteins Formed in Planta and in Yeast. (A) B class protein dimerization in Y2H tests quantified with the β-Gal assay. (B) to (I) TriFC experiments showing multimeric complex formation of three B, C, and/or E class proteins that differ between SEI and sei-1 interactions. The two partial YFP fusion constructs and the silent partner are as follows: (B) AG1:YFPN-DEF2:YFPC-SEI, (C) AG1:YFPN-DEF2:YFPC-sei-1, (D) SEP3:YFPN-SEI:YFPC-DEF1, (E) SEP3:YFPN-sei-1:YFPC-DEF1, (F) SEP3:YFPN-SEI:YFPC-DEF2, (G) SEP3:YFPN-sei-1:YFPC-DEF2, (H) AG1:YFPN-SEP3:YFPC-SEI, and (I) AG1:YFPN-SEP3:YFPC-sei-1. The bar length is 50 µm. (J) Y3H analysis of multimer formation of three B, C, and E class proteins. The light-gray columns show interaction strength of the proteins expressed from BD and AD vectors together with the empty ternary vector pTFT1. The black columns show interaction strength when pTFT1 contains the coding sequence as given in the figure. Error bars depict the sd of replicates. Asterisks above the columns indicate significant differences (analysis of variance test; P < 0.05) in reporter gene activation between empty pTFT1 and pTFT1-EcSEP3 interactions, indicating formation of higher order complexes. Arrows indicate absence of multimer formation in cases where SEI was substituted by sei-1 or mSEI proteins. (K) Sequence logo representation of the PI motif from selected PI-like proteins across angiosperms listed in Supplemental Figure 3 online. Numbers refer to the positions of the amino acid within the PI motif alignment. (L) The sequence of the PI motifs of Arabidopsis PI and poppy SEI are shown, and the differing N-terminal amino acids are marked in red and purple. (M) to (O) TriFC multimeric protein interactions of AG1, Ec-SEP3, and the modified SEI protein showing that the five N-terminal amino acids of the PI motif are required for the interaction: (M) AG1:YFPN-SEP3:YFPC-SEI, (N) AG1:YFPN-SEP3:YFPC-sei-1, and (O) AG1:YFPN-SEP3:YFPC-mSEI. The bar length is 50 µm.

Figure 4.

Hypothesis of PI Motif SLiM Evolution. Schematic and highly simplified representation of the phylogeny of seed plants (based on Soltis et al., 2011). Lineage names are given, and the numbers of sequences used for PI motif representation are shown in parentheses. The black rectangle symbolizes the hypothesized SLiM in the PI motif. The SLiM postulated to be required for B-C-E multimer complex formation appeared before the divergence of the Magnoliidae ancestor from the rest of the angiosperm lineages and is indicated by a black star. The PI motif is conserved in sequence and position in representatives of the Magnoliidae, Monocotyledonae, Ranunculales, Asteridae, and many rosids but has been lost in Brassicaceae species shown by a white star, symbolizing the mutation of four amino acid residues. The gray star highlights lineages within the monocots with deviations from the SLiM consensus sequence with positions indicated by asterisks. [See online article for color version of this figure.]