B-function expression in the flower center underlies the homeotic phenotype of Lacandonia schismatica (Triuridaceae)

Abstract

Spontaneous homeotic transformations have been described in natural populations of both plants and animals, but little is known about the molecular-genetic mechanisms underlying these processes in plants. In the ABC model of floral organ identity in Arabidopsis thaliana, the B- and C-functions are necessary for stamen morphogenesis, and C alone is required for carpel identity. We provide ABC model-based molecular-genetic evidence that explains the unique inside-out homeotic floral organ arrangement of the monocotyledonous mycoheterotroph species Lacandonia schismatica (Triuridaceae) from Mexico. Whereas a quarter million flowering plant species bear central carpels surrounded by stamens, L. schismatica stamens occur in the center of the flower and are surrounded by carpels. The simplest explanation for this is that the B-function is displaced toward the flower center. Our analyses of the spatio-temporal pattern of B- and C-function gene expression are consistent with this hypothesis. The hypothesis is further supported by conservation between the B-function genes of L. schismatica and Arabidopsis, as the former are able to rescue stamens in Arabidopsis transgenic complementation lines, and Ls-AP3 and Ls-PI are able to interact with each other and with the corresponding Arabidopsis B-function proteins in yeast. Thus, relatively simple molecular modifications may underlie important morphological shifts in natural populations of extant plant taxa.

Figure 1.

L. schismatica MADS Box B and C Orthologs. (A) B and C MADS domain protein sequences of diverse angiosperms. L. schismatica orthologs possess diagnostic C-terminal motifs characteristic of each linage. (B) Bayesian phylogenetic analysis (AG, left; B genes, right) corroborate the orthology of the L. schismatica genes (AG in red; AP3 and PI in blue) to C and B genes of Arabidopsis, respectively. Trees are supported with high posterior probabilities showed for the main branches. The L. schismatica sequences were nested within monocots. The MADS box has been excluded from this analysis to align domains with the most informative sites. Gymnosperm genes were used as outgroups. Larger alignments confirmed these results (see Supplemental Figure 1 online). [See online article for color version of this figure.]

Figure 2.

In Situ Localization of L. schismatica B Gene (Ls-AP3 and Ls-PI) mRNA. (A) to (E) Longitudinal sections of L. schismatica flower buds hybridized with Ls-AP3 ([A] to [C]) and Ls-PI ([D] and [E]) probes. Expression of Ls-AP3 is restricted to the apical zone (central zone in a cross section) of very early flower buds, where stamen primordia (yellow arrowhead in [A]) later arise and mature stamens will develop, while Ls-PI is expressed in a broader domain during the early stages of floral development and is then restricted to regions where both stamen (yellow arrowheads) and carpel (red asterisks) primordia develop. Ls-AP3 expression is shown in four stages of floral development in (A) to (C), and Ls-PI expression is shown for three stages in (D) and (E). (F) to (J) Light fields of (A) to (E), respectively, for structural references. (K) to (M) Scanning electron micrographs of three developmental stages of L. schismatica flower development for structural reference. Note that the form of the expression domain of Ls-AP3 in (A) coincides with the form of the region within the compound primordia where stamen primordia will arise (K) (yellow line). (N) Enlarged image of Ls-PI expression in the young meristem shown in (D). (O) In situ hybridization with an Ls-AP3 sense probe. b, bract; c, carpel; s, stamen; t, tepal organs. Bars = 100 μm. [See online article for color version of this figure.]

Figure 3.

In Situ Localization of C Gene (Ls-AG and Ls-STK) mRNA in L. schismatica Flower. (A) and (B) Longitudinal section of a young bud and of a noncentral section of an older meristem, respectively, hybridized with an antisense Ls-AG probe. Signal is observed in a pattern similar to that of Ls-PI, with a broad pattern during the early stages and in both stamen (yellow arrowhead) and carpel (red asterisks) primordia at later stages of flower development. (C) Longitudinal section of an L. schismatica flower hybridized with the Ls-STK probe. Ls-STK mRNA is absent in stamen primordia and is strongly expressed in carpel primordia. (D) Hybridization with a sense Ls-AG probe. (E) Superposition of antisense Ls-AG signal (in red) shown in (A) over a bright-field view. (F) and (G) Bright-field images of (B) and (C), respectively, for structural reference. c, carpel; s, stamen. Bars = 100 μm. [See online article for color version of this figure.]

Figure 4.

L. schismatica B Genes Are Able to Specify Petal and Stamen Cell Identity in Arabidopsis Transgenic Lines. (A) The Ls-PI overexpression line, 35S:LsPI, in a wild-type background. (B) The Ls-AP3 overexpression line, 35S:LsAP3, in a wild-type background. (C) to (E) Flowers of the F2 descendants of the double overexpressor line 35S:LsAP3 35S:LsPI. The flower in (C) is similar to the single Ls-PI overexpression line in (A). Carpels do not show conversion to stamens (D). However, aberrant filamentous carpels occasionally are observed (E). (F) pi-1 homozygous flower. (G) 35S:LsPI in the pi-1 mutant background, showing the ability of Ls-PI overexpression to partially rescue petals and stamens of the pi-1 mutant flower. (H) to (K) Scanning electron microscopy images of the line in (G) showing immature stamens (H) and mature stamens with viable pollen (I). Flower with a chimeric stamen/carpel organ in the third whorl (J). Second whorl petal-like cells from (G) are shown in (K). (L) An ap3-3 homozygous flower. (M) to (P) Examples of progeny from 35S:LsAP3 lines crossed to ap3-3 plants. (N) Scanning electron microscopy image of the dissected perianth organs of a flower, showing at least four stamens. (O) and (P) Scanning electron microscopy images of an opened anther. (P) Enlargement of viable pollen. Bars = 1 mm. (Q) Summary of floral organ phenotypes observed in overexpression lines harboring L. schismatica B genes in Arabidopsis wild-type (Wt) backgrounds and B mutant plants compared with the corresponding phenotypes in lines harboring Arabidopsis B genes (Jack et al., 1994; Krizek and Meyerowitz, 1996). Asterisk indicates the phenotype reported for the pi mutant line complemented with the Arabidopsis cDNA corresponds to data using the pi-4 allele (Piwarzyk et al., 2007). CA, carpels; PE, petals; SE, sepals; ST, stamens. [See online article for color version of this figure.]

Figure 5.

Modified ABC Model for L. schismatica Based on Expression Data Reported Here and Compared with the ABC Model of Arabidopsis. (A) The combinatorial ABC model of flower organ identity conserved among eudicots, with B function restricted to the petal and stamen primordia (Coen and Meyerowitz, 1991). (B) Proposed molecular genetics hypothesis to explain the inside-out flower of L. schismatica: AP3-PI dimer function is restricted to the flower center, where stamens initiate and develop. A (green), B (AP3 dark blue and PI light blue), and C (orange) function. CA, carpels; PE, petals; SE, sepals; ST, stamens; TE, tepals. Bar = 500 μm. (C) Changes in the in situ expression patterns of B gene mRNA along flower developmental series comparing Arabidopsis (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996) and L. schismatica (this article). The differences between these two species represent two instances of the evolutionary shifts of such patterns during angiosperm evolution. Schematic representation of the developmental stages of Arabidopsis and L. schismatica flowers, starting at stage 3 of Arabidopsis flower development when sepal primordia are already visible. Sepal primordia continue to grow until they enclose the flower meristem, from stage 4 to 6. Meanwhile, at stage 5, petal and stamen primordia start to appear, and the gynoecium starts to form at stage 6. Similar stages of L. schismatica flower development were selected based on morphological traits. [See online article for color version of this figure.]

Figure 6.

The Evolution of ABC Genes in Angiosperms. Simplified angiosperm phylogeny (based on the Angiosperm Phylogeny Group III, 2009). Variations in the ABC model of floral organ formation for selected taxa, as well as the site where the BC combinatory function defines the development of stamens, are mapped. Representative angiosperm groups are shown in the branches of the tree with their taxonomic family in parentheses, except for Orchidaceae and Solanaceae (ª), for which the ABC model depicted stands for several species that have been experimentally analyzed (for Orchidaceae, Phalaenopsis spp [Tsai et al., 2004] and D. crumenatum [Xu et al., 2006]; for Solanaceae, Petunia hybrida [Vandenbussche et al., 2004] and tomato [Solanum lycopersicum] de Martino et al., 2006). The A function for all lineages is included for all taxa to enable comparison with Arabidopsis, although a question mark was added to emphasize its dubious role in other flowering plants, where it has not been shown to be necessary for perianth formation; rather, it is fundamental for floral meristem determination (Soltis et al., 2007). Along the phylogeny branches, the moments when the variants of the ABC genes could have evolved are indicated with orange rectangles (synapomorphies; characters shared by several lineages derived from a common ancestor). Purple rectangles indicate the appearance of traits that favored homeosis in the Triuridaceae lineage (an autapomorphy; a derived character unique to a single lineage/taxon). This autapomorphy pertains to L. schismatica AP3 expression and stamen development, which are constrained to the center of the flower during early and late developmental stages, in contrast with the early expression of AP3 orthologs in other hermaphroditic flowering plants, which can encompass several whorls, although the combined B (joint expression of AP3 and PI orthologs) and C functions are restricted to the third whorl, where stamens usually develop. ca, carpels; lo, lodicules; pe, petals; pl, petal-like tepals; ps, palea/lemma; sd, staminodes; se, sepals; sl, sepal-like tepals; st, stamens; te, petaloid tepals; sp, sepaloid petals; W1-W4, whorls within the flower primordia numbered from the outermost position to the center; asterisk, while in numerous angiosperms, W1 would stand for the sepal whorl, W2 for the petals, W3 for stamens, and W4 for the carpels, several angiosperm lineages differ from this organization either because they have a spiral arrangement (Amborella and Nimphaea), by having very modified perianth organs (Oryza), or by lacking a defined sepal/petal identity (tepals; Amborella, Lacandonia, and orchids). ABC model modified from Figure 25 of Álvarez-Buylla et al. (2010b) (data summarized from Xiao et al., 2003; Irish and Litt, 2005; Kim et al., 2005; Soltis et al., 2007; Theissen and Melzer, 2007; Whipple et al., 2007; Mondragón-Palomino and Theissen, 2009). [See online article for color version of this figure.]