Petal‐specific subfunctionalization of an APETALA3 paralog in the Ranunculales and its implications for petal evolution

Abstract

Summary• The petals of the lower eudicot family Ranunculaceae are thought to have been derived many times independently from stamens. However, investigation of the genetic basis of their identity has suggested an alternative hypothesis: that they share a commonly inherited petal identity program. This theory is based on the fact that an ancient paralogous lineage of APETALA3 (AP3) in the Ranunculaceae appears to have a conserved, petal-specific expression pattern.• Here, we have used a combination of approaches, including RNAi, comparative gene expression and molecular evolutionary studies, to understand the function of this petal-specific AP3 lineage.• Functional analysis of the Aquilegia locus AqAP3-3 has demonstrated that the paralog is required for petal identity with little contribution to the identity of the other floral organs. Expanded expression studies and analyses of molecular evolutionary patterns provide further evidence that orthologs of AqAP3-3 are primarily expressed in petals and are under higher purifying selection across the family than the other AP3 paralogs.• Taken together, these findings suggest that the AqAP3-3 lineage underwent progressive subfunctionalization within the order Ranunculales, ultimately yielding a specific role in petal identity that has probably been conserved, in stark contrast with the multiple independent origins predicted by botanical theories.

Disruption of the petal identity gene APETALA3-3 is highly correlated with loss of petals within the buttercup family (Ranunculaceae)

“…Up to now, it is still unclear whether AP3-3 evolved its current functions through changes in coding, regulatory, or both regions, yet the available data suggest that (i) it is a key regulator of petal development and (ii) it does not have pleiotropic roles. Being a key regulator of petal development implies that each time when having petals is no longer advantageous it will become useless and accumulate destructive mutations; otherwise, it evolves under stringent functional constraints, as is confirmed by a recent molecular evolutionary study (24). Meanwhile, because it is not pleiotropic, and because of the existence of two functionally redundant (at least in stamens) paralogs (AP3-1 and AP3-2), down-regulation, silencing, or loss of it will only affect the expression of a few downstream genes and, consequently, is unlikely to cause obvious phenotypic alterations outside petals.…”

“…In Clematis, the AP3-3 orthologs have become pseudogenes, although the underlying mechanism for the possible pseudogenization or reduced expression is still unclear. This, together with the fact that knockdown of the AP3-3 gene in Aquilegia can result in the petal-to-sepal transformation (24), strongly suggests that AP3-3 is a petal identity gene, and that the specific role of AP3-3 in petal identity is broadly conserved across the Ranunculaceae. More importantly, because Aquilegia and Nigella are two deeply divergent members of the family, these results provide the most compelling argument yet that the AP3-3-based petal identity program did not evolve many times independently but rather was lost in numerous instances.…”

“…Unlike AP3-1 and AP3-2, whose expression patterns are rather broad and nonspecific, the AP3-3 orthologs are usually specifically expressed in petals, and the expression levels are generally exceptionally high (7,35). Silencing of AP3-3 in Aquilegia and Nigella only resulted in homeotic transformations of petals into sepals, whereas all other floral organs remained largely unchanged (24). Up to now, it is still unclear whether AP3-3 evolved its current functions through changes in coding, regulatory, or both regions, yet the available data suggest that (i) it is a key regulator of petal development and (ii) it does not have pleiotropic roles.…”

“…In the species lacking petals, however, AP3-3 expression could not be detected, despite considerable efforts. Based on these observations, it has been postulated that the independent petal losses in different Ranunculaceous lineages were caused by parallel inactivation of the AP3-3 orthologs (7,24). However, because only a few species have been investigated extensively, it is still unclear whether petal losses are indeed correlated with AP3-3 expression, how AP3-3 orthologs could have been silenced or down-regulated, and whether silencing/downregulation of AP3-3 was the only cause for parallel petal losses.…”

“…However, recent studies indicated that absences of petals in this and related families are correlated with lack of the expression of a floral organ identity gene, AP3-3 (7,23,24). AP3-3 is an AP3-like gene, which, along with its two closest paralogs, AP3-1 and AP3-2, was generated through two successive gene duplication events before the divergence of the Ranunculaceae from its allies (7,24).…”