Gene Duplication and Transference of Function in the paleoAP3 Lineage of Floral Organ Identity Genes

Abstract

The floral organ identity gene APETALA3 (AP3) is a MADS-box transcription factor involved in stamen and petal identity that belongs to the B-class of the ABC model of flower development. Thalictrum (Ranunculaceae), an emerging model in the non-core eudicots, has AP3 homologs derived from both ancient and recent gene duplications. Prior work has shown that petals have been lost repeatedly and independently in Ranunculaceae in correlation with the loss of a specific AP3 paralog, and Thalictrum represents one of these instances. The main goal of this study was to conduct a functional analysis of the three AP3 orthologs present in Thalictrum thalictroides, representing the paleoAP3 gene lineage, to determine the degree of redundancy versus divergence after gene duplication. Because Thalictrum lacks petals, and has lost the petal-specific AP3, we also asked whether heterotopic expression of the remaining AP3 genes contributes to the partial transference of petal function to the first whorl found in insect-pollinated species. To address these questions, we undertook functional characterization by virus-induced gene silencing (VIGS), protein–protein interaction and binding site analyses. Our results illustrate partial redundancy among Thalictrum AP3s, with deep conservation of B-class function in stamen identity and a novel role in ectopic petaloidy of sepals. Certain aspects of petal function of the lost AP3 locus have apparently been transferred to the other paralogs. A novel result is that the protein products interact not only with each other, but also as homodimers. Evidence presented here also suggests that expression of the different ThtAP3 paralogs is tightly integrated, with an apparent disruption of B function homeostasis upon silencing of one of the paralogs that codes for a truncated protein. To explain this result, we propose two testable alternative scenarios: that the truncated protein is a dominant negative mutant or that there is a compensational response as part of a back-up circuit. The evidence for promiscuous protein–protein interactions via yeast two-hybrid combined with the detection of AP3 specific binding motifs in all B-class gene promoters provide partial support for these hypotheses.

Spontaneous homeotic mutants and genetic control of floral organ identity in a ranunculid

“…We analyzed coding regions of B‐, C‐, and E‐class genes in search of candidate mutations (Table 1). PPI motifs in their protein products were identified to check for the potential functional significance of amino acid substitutions (Figure 5), as PPI had been previously demonstrated to occur in vitro amongst the products of B‐, C‐, and E‐class Thalictrum genes (Galimba & Di Stilio, 2015; Galimba et al, 2018). Overall, ThtAP3‐1 and ThtSEP1 had the most significant nonsynonymous (NS) substitutions, synonymous substitutions were also found (Figures S2–S5).…”

“…Our goals include (a) investigate the extent to which homeotic changes in flower organ identity in the mutants correspond to changes in B‐, C‐, and E‐class gene expression, and generate modified BCE models of flower development; (b) determine whether gene duplicates have diverged in function; and (c) determine whether changes to transcription factor binding sites and/or protein–protein interaction (PPI) motifs underlie the phenotypic changes. The forward approach undertaken here complements prior expression and functional work by targeted gene silencing of B‐, C‐, and E‐class genes in T. thalictroides (Galimba & Di Stilio, 2015; Galimba et al, 2018; LaRue et al, 2013; Soza et al, 2016). Studies in Ranunculaceae contribute to a more comprehensive depiction of the genetics underlying flower development across eudicots.…”

Genomic and transcriptomic resources for candidate gene discovery in the Ranunculids

“…Thalictrum is a temperate genus of approximately 200 species with floral diversity that encompasses unisexual and wind‐pollinated flowers, in association with polyploidy (Soza et al, 2012, 2013). Certain species, mainly T. thalictroides (L.) A. J. Eames & B. Boivin, are amenable to virus‐induced gene silencing (Di Stilio et al, 2010), which has enabled functional studies of ABC model floral MADS‐box genes (Galimba et al, 2012, 2018; Di Stilio et al, 2013; Galimba and Di Stilio, 2015; Soza et al, 2016). Two chloroplast genomes have been assembled to date for this genus, for T. coreanum H. Lév.…”

Facilitating candidate gene discovery in an emerging model plant lineage: Transcriptomic and genomic resources forThalictrum(Ranunculaceae)

“…Thalictrum is a temperate genus of circa 200 species with floral diversity that encompasses unisexual and wind-pollinated flowers, in association with polyploidy (Soza et al, 2012(Soza et al, , 2013. Certain species, mainly T. thalictroides, are amenable to virus-induced gene silencing (Di Stilio et al, 2010), which has enabled functional studies of ABC model floral MADS-box genes (Di Galimba and Di Stilio, 2015;Galimba et al, 2012Galimba et al, , 2018Soza et al, 2016). Two chloroplast genomes have been assembled to date for the genus, for T. coreanum (Park et al, 2015) and T. thalictroides (from this dataset, Morales Briones et al, 2019).…”