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Expression of Paralogous SEP-, FUL-, AG- And STK-like MADS-box Genes in Wild-Type and Peloric Phalaenopsis Flowers

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Expression of Paralogous SEP-, FUL-, AG- And STK-like MADS-box Genes in Wild-Type and Peloric Phalaenopsis Flowers

Roberta Acri-Nunes-Miranda et al. Front Plant Sci.

Abstract

The diverse flowers of Orchidaceae are the result of several major morphological transitions, among them the most studied is the differentiation of the inner median tepal into the labellum, a perianth organ key in pollinator attraction. Type A peloria lacking stamens and with ectopic labella in place of inner lateral tepals are useful for testing models on the genes specifying these organs by comparing their patterns of expression between wild-type and peloric flowers. Previous studies focused on DEFICIENS- and GLOBOSA-like MADS-box genes because of their conserved role in perianth and stamen development. The "orchid code" model summarizes this work and shows in Orchidaceae there are four paralogous lineages of DEFICIENS/AP3-like genes differentially expressed in each floral whorl. Experimental tests of this model showed the conserved, higher expression of genes from two specific DEF-like gene lineages is associated with labellum development. The present study tests whether eight MADS-box candidate SEP-, FUL-, AG-, and STK-like genes have been specifically duplicated in the Orchidaceae and are also differentially expressed in association with the distinct flower organs of Phalaenopsis hyb. "Athens." The gene trees indicate orchid-specific duplications. In a way analogous to what is observed in labellum-specific DEF-like genes, a two-fold increase in the expression of SEP3-like gene PhaMADS7 was measured in the labellum-like inner lateral tepals of peloric flowers. The overlap between SEP3-like and DEF-like genes suggests both are associated with labellum specification and similar positional cues determine their domains of expression. In contrast, the uniform messenger levels of FUL-like genes suggest they are involved in the development of all organs and their expression in the ovary suggests cell differentiation starts before pollination. As previously reported AG-like and STK-like genes are exclusively expressed in gynostemium and ovary, however no evidence for transcriptional divergence was found in the stage investigated. Gene expression suggests a developmental regulatory system based on the combined activity of duplicate MADS-box genes. We discuss its feasibility based on documented protein interactions and patterns of expression.

Keywords: Orchidaceae; evo-devo; flower evolution; gene duplication; labellum; qPCR.

Figures

Figure 1
Figure 1
Phalaenopsis flower structure, distinctive floral features of wild-type and peloric hybrid “Athens.” Phylogenetic relationships the sequences studied. (A) Flower organs of wild-type and peloric mutant represented in the analysis. The stamen is located under the anther cap, a white laminar structure on top of the wild-type gynostemium (indicated with an arrow). The labella developing in place of inner lateral tepals as well as the organs missing in the gynostemium of peloric flowers are indicated with arrows. (B) Systematic relationships of the plant families represented in the phylogenetic analyses of MADS-box genes from monocots (based on Angiosperm phylogeny website version 12, www.mobot.org). The number sequences from every group in the dataset is indicated between brackets. (C) Systematic relationships of Orchidaceae subfamilies. The stars mark the points where the Orchidaceae subfamily composition of the gene trees suggests duplications might have occurred in SEP3-like genes (pale and dark blue), AG-like genes (red) and FUL-like II (green). The colors of the stars correspond to those employed to indicate these duplications in Figures 2–4.
Figure 2
Figure 2
Maximum Likelihood phylogeny of monocot FUL-like genes. The two major monocot clades previously identified are indicated with a black frame (Litt and Irish, 2003). The clades containing sequences from Orchidaceae are highlighted if gene expression was characterized in Phalaenopsis hyb. “Athens.” Otherwise the non-isolated ortholog is marked with an asterisk. The numbers on every node indicate branch support above 0.80. The bars indicate clades of FUL-like genes from the plant families represented in the dataset as well as the outgroup. Black arrows point at the sequences employed as templates for qPCR primer design. Stars indicate gene duplications.
Figure 3
Figure 3
Maximum Likelihood phylogeny of monocot SEP-like genes. The two major monocot clades previously identified are indicated with a black frame (Zahn et al., 2005a). Orchidaceae-specific clades and other annotations are as described in Figure 2.
Figure 4
Figure 4
Maximum Likelihood phylogeny of monocot AG- and STK-like genes. Major monocot clades previously identified are indicated with a black frame (Kramer et al., 2004). Orchidaceae-specific clades and other annotations are as described in Figure 2.
Figure 5
Figure 5
Normalized expression of orchid FUL-like genes. Expression of PhaMADS1 and PhaMADS2 in six flower organs of wild-type and peloric of Phalaenopsis hybrid “Athens.” OLT, outer lateral tepals; OMT, outer median tepals; ILT, inner lateral tepals; L, labellum; G, gynostemium; O, ovary. Expression of the target genes was normalized to the geometric average expression of three internal control genes: Actin, EF1α and Ubiquitin. Each column represents the expression obtained from six samples (three replicates from each of two different cDNA pools). The error bars represent the standard errors of the replicates. The y-axis is in arbitrary fluorescence units. The qPCR products from each sample series are presented below their corresponding columns in the graph.
Figure 6
Figure 6
Normalized expression of orchid SEPALLATA-like genes. Expression of PhaMADS4 and PhaMADS5 and PhaMADS7 in six flower organs of wild-type and peloric of Phalaenopsis hybrid “Athens.” Replicates, normalization, graphics and abbreviations are as in Figure 5.
Figure 7
Figure 7
Normalized expression of orchid AGAMOUS- and SEEDSTICK-like genes. Expression of PhaMADS8, PhaMADS9 and PhaMADS10 in six flower organs of wild-type and peloric Phalaenopsis hybrid “Athens.” Replicates, normalization, graphics and abbreviations are as described in Figure 5.
Figure 8
Figure 8
Normalized expression of orchid FUL-, SEP-, AG-, and STK-like MADS-box genes in developing ovary. (A) Normalized expression of MADS-box genes PhaMADS1, PhaMADS2 (FUL-like), PhaMADS4, PhaMADS5, PhaMADS7 (SEP-like), PhaMADS8, PhaMADS10 (AG-like) and PhaMADS9 (STK-like) in developing ovary of Phalaenopsis hybrid “Athens.” (B) Expression of three internal control genes (Actin, EF1α and Ubiquitin) in the developing ovaries. Replicates, normalization, graphics and abbreviations are as described in Figure 5.
Figure 9
Figure 9
A transcriptional model for Phalaenopsis flower development. Summary of expression patterns of orchid paralogous FUL-, DEF-, GLO-, AG-, STK-, and SEP-like genes in Phalaenopsis perianth and reproductive organs. The combined differential activity and levels of expression of genes from different clades in each organ are represented with blocks of different colors and mapped on their domains of expression in the perianth, column and ovary. Genes corresponding to a clade with an asterisk were isolated and measured in other studies and their pattern of expression is included here for completion.

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