Publications

In most dioecious angiosperm species, flowers are initially perfect but abort either stamens or carpels during their development, indicating that sex determination occurs after floral organ identity has been established. Dioecious members of the genus Thalictrum (meadow-rue), however, produce flowers that lack aborted organs. Examination of early flower development of T. dioicum confirms that flowers are male or female from inception, raising the possibility that genetic mechanisms working at or above the level of organ identity promote sex determination through a homeotic-like mechanism. In order to investigate this possibility, we identified homologs of the organ identity genes PISTILLATA (PI), APETALA3 (AP3) and AGAMOUS (AG) from T. dioicum and the hermaphroditic species T. thalictroides. A combination of early and late duplication events was uncovered in these gene lineages and expression analyses indicate that these events are generally associated with divergence in gene regulation. In light of these findings, we discuss the potential of T. dioicum as a new model for the study of sex determination in the basal eudicots.

Of the many innovations associated with the radiation of the angiosperms, the evolution of a petal identity program is among the best understood from a genetic standpoint. Although the existing data do indicate that similar genetic mechanisms control petal development across diverse taxa, there is also considerable evidence for variability in petal identity programs, likely due to a number of factors. These points are illustrated through a review of our current knowledge on the subject, integrating phylogenetic, morphological, and genetic studies. Comparative studies of petal identity highlight the complex nature of homology in plants and stand as a cautionary tale for the interpretation of gene expression data.

A major component of evolutionary developmental (evo-devo) genetics is the analysis of gene expression patterns in nonmodel species. This comparative approach can take many forms, including reverse-transcriptase polymerase chain reaction, Northern blot hybridization, and in situ hybridization. The choice of technique depends on several issues such as the availability of fresh tissue, as well as the expected expression level and pattern of the candidate gene in question. Although the protocols for these procedures are fairly standard, optimization is often required because of the specific characteristics of the species under analysis. This chapter describes several methods commonly used to determine gene expression patterns in angiosperms, particularly in floral tissues. Suggestions for adapting basic protocols for diverse taxa and troubleshooting are also extensively discussed.

Several lines of evidence suggest that sterile floral organs, collectively known as the perianth, have evolved multiple times during the evolution of the angiosperms. In the family Aristolochiaceae, the perianth is formed by two whorls of organs in the genus Saruma but by only one whorl in the remaining genera, including Aristolochia. Although the morphology of Saruma is similar in appearance to the core eudicot perianth, with leaf-like sepals and showy colored petals, the unipartite perianth of Aristolochia combines morphological aspects of both calyx and corolla. To investigate the organ identity program functioning in the novel perianth of Aristolochia, we identified homologs of the B-class genes APETALA3 (AP3) and PISTILLATA (PI) in both Saruma and Aristolochia. The expression patterns of these genes in Saruma indicate they are functioning in the development of the second whorl petaloid organs and third whorl stamens. In Aristolochia, however, the expression of AP3 and PI homologs in the perianth does not suggest a role in organ identity but, rather, in promoting late aspects of cell differentiation. The implications of these findings for the evolution of both petaloidy and B gene function are discussed.

The B class genes, including homologs of the Arabidopsis loci APETALA3 (AP3) and PISTILLATA (PI ), appear to play a conserved role in the determination of petal and stamen identity across core eudicot angiosperms. Understanding how and when these functions evolved is a critical component of elucidating the evolution of flowers, particularly the appearance of petaloid perianth organs. Before comparisons of gene expression patterns or functions can be made, however, it is necessary to establish the orthology of AP3 and PI homologs from basal angiosperms. Here, we report the identification and analysis of 29 new representatives of the B gene lineage from basal ANITA and magnoliid dicot angiosperms. These studies indicate that gene duplications have occurred at every phylogenetic level, both before and after the duplication that produced the separate AP3 and PI lineages. Comparison of genomic structure among PI homologs indicates that a 12-nucleotide deletion that had been considered synapomorphic for the whole PI lineage actually arose within the ANITA grade, after the split of the Nymphaeales but before the separation of the Austrobaileyales. Evidence for alternative splicing of the Nymphaea AP3 homolog is also presented. The implications of these findings for angiosperm systematics, the conservation of AP3 and PI gene function, and the evolution of the ABC program are discussed.

Members of the AGAMOUS (AG) subfamily of MIKC-type MADS-box genes appear to control the development of reproductive organs in both gymnosperms and angiosperms. To understand the evolution of this subfamily in the flowering plants, we have identified 26 new AG-like genes from 15 diverse angiosperm species. Phylogenetic analyses of these genes within a large data set of AG-like sequences show that ancient gene duplications were critical in shaping the evolution of the subfamily. Before the radiation of extant angiosperms, one event produced the ovule-specific D lineage and the well-characterized C lineage, whose members typically promote stamen and carpel identity as well as floral meristem determinacy. Subsequent duplications in the C lineage resulted in independent instances of paralog subfunctionalization and maintained functional redundancy. Most notably, the functional homologs AG from Arabidopsis and PLENA (PLE) from Antirrhinum are shown to be representatives of separate paralogous lineages rather than simple genetic orthologs. The multiple subfunctionalization events that have occurred in this subfamily highlight the potential for gene duplication to lead to dissociation among genetic modules, thereby allowing an increase in morphological diversity.

Shifts in flower symmetry have occurred frequently during the diversification of angiosperms, and it is thought that such shifts play important roles in plant-pollinator interactions. In the model developmental system Antirrhinum majus (snapdragon), the closely related genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) are needed for the development of zygomorphic flowers and the determination of adaxial (dorsal) identity of floral organs, including adaxial stamen abortion and asymmetry of adaxial petals. However, it is not known whether these genes played a role in the divergence of species differing in flower morphology and pollination mode. We compared A. majus with a close relative, Mohavea confertiflora (desert ghost flower), which differs from Antirrhinum in corolla (petal) symmetry and pollination mode. In addition, Mohavea has undergone a homeotic-like transformation in stamen number relative to Antirrhinum, aborting the lateral and adaxial stamens during flower development. Here we show that the patterns of expression of CYC and DICH orthologs have shifted in concert with changes in floral morphology. Specifically, lateral stamen abortion in Mohavea is correlated with an expansion of CYC and DICH expression, and internal symmetry of Mohavea adaxial petals is correlated with a reduction in DICH expression during petal differentiation. We propose that changes in the pattern of CYC and DICH expression have contributed to the derived flower morphology of Mohavea and may reflect adaptations to a pollination strategy resulting from a mimetic relationship, linking the genetic basis for morphological evolution to the ecological context in which the morphology arose.

Molecular genetic studies in Arabidopsis thaliana and other higher-eudicot flowering plants have led to the development of the 'ABC' model of the determination of organ identity in flowers, in which three classes of gene, A, B and C, are thought to work together to determine organ identity. According to this model, the B-class genes APETALA3 (AP3) and PISTILLATA (PI) act to specify petal and stamen identity. Here we test whether the roles of these genes are conserved throughout the angiosperms by analysing the expression of AP3 and PI orthologues in the lower eudicot subclass Ranunculidae. We show that, although expression of these orthologues in the stamens is conserved, the expression patterns in the petals differ from those found in the higher eudicots. The differences between these expression patterns suggest that the function of AP3 and PI homologues as B-class organ-identity genes is not rigidly conserved among all angiosperms. These observations have important implications for understanding the evolution of both angiosperm petals and the genetic mechanisms that control the identities of floral organs.

We show by molecular analysis of behavioral and physiological mutants that the Drosophila Dmca1A calcium-channel alpha1 subunit is encoded by the cacophony (cac) gene and that nightblind-A and lethal(1)L13 mutations are allelic to cac with respect to an expanded array of behavioral and physiological phenotypes associated with this gene. The cacS mutant, which exhibits defects in the patterning of courtship lovesong and a newly revealed but subtle abnormality in visual physiology, is mutated such that a highly conserved phenylalanine (in one of the quasi-homologous intrapolypeptide regions called IIIS6) is replaced by isoleucine. The cacH18 mutant exhibits defects in visual physiology (including complete unresponsiveness to light in certain genetic combinations) and visually mediated behaviors; this mutant (originally nbAH18) has a stop codon in an alternative exon (within the cac ORF), which is differentially expressed in the eye. Analysis of the various courtship and visual phenotypes associated with this array of cac mutants demonstrates that Dmca1A calcium channels mediate multiple, separable biological functions; these correlate in part with transcript diversity generated via alternative splicing.

The specification of floral organ identity in the higher dicots depends on the function of a limited set of homeotic genes, many of them members of the MADS-box gene family. Two such genes, APETALA3 (AP3) and PISTILLATA (PI), are required for petal and stamen identity in Arabidopsis; their orthologs in Antirrhinum exhibit similar functions. To understand how changes in these genes may have influenced the morphological evolution of petals and stamens, we have cloned twenty-six homologs of the AP3 and PI genes from two higher eudicot and eleven lower eudicot and magnolid dicot species. The sequences of these genes reveal the presence of characteristic PI- and AP3-specific motifs. While the PI-specific motif is found in all of the PI genes characterized to date, the lower eudicot and magnolid dicot AP3 homologs contain distinctly different motifs from those seen in the higher eudicots. An analysis of all the available AP3 and PI sequences uncovers multiple duplication events within each of the two gene lineages. A major duplication event in the AP3 lineage coincides with the base of the higher eudicot radiation and may reflect the evolution of a petal-specific AP3 function in the higher eudicot lineage.