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Title: The role of APETALA1 in petal number robustness}
Article:
Abstract:
Invariant floral forms are important for reproductive success and robust to natural perturbations. Petal number, for example, is invariant in Arabidopsis thaliana flowers. However, petal number varies in the closely related species Cardamine hirsuta, and the genetic basis for this difference between species is unknown. Here we show that divergence in the pleiotropic floral regulator APETALA1( AP1) can account for the species-specific difference in petal number robustness. This large effect of AP1 is explained by epistatic interactions: A. thaliana AP1 confers robustness by masking the phenotypic expression of quantitative trait loci controlling petal number in C. hirsuta. We show that C. hirsuta AP1 fails to complement this function of A. thaliana AP1, conferring variable petal number, and that upstream regulatory regions of AP1 contribute to this divergence. Moreover, variable petal number is maintained in C. hirsuta despite sufficient standing genetic variation in natural accessions to produce plants with four-petalled flowers.
Introduction:
Determining the genetic basis of developmental traits, and how these evolve to generate novelties, is a major goal of evolutionary developmental biology. Developmental systems are remarkably robust to natural perturbations, such that individuals tend to develop normally despite variation in the environment or their genetic make-up( Wagner, 2005). Therefore, a particular challenge is to understand the developmental transitions between the robust morphology of individuals within a species, and the variation in form between species. Petal number is a robust trait in flowering plants and usually invariant within species and even higher taxonomic orders. For example, three petals are commonly found in monocots while five petals are characteristic of many core eudicots( Endress, 2011; Specht and Bartlett, 2009). On the other hand, petal number is much more labile in basal angiosperms( Endress, 2001; Endress, 2011; Specht and Bartlett, 2009), suggesting that this trait was canalized during angiosperm evolution to produce a stable phenotype in the face of genetic and environmental perturbation. Petals are usually required to open the flower and to help attract pollinators( Fenster et al., 2004; van Doorn and Van Meeteren, 2003), therefore a stable number of petals could ensure a reliable display of the reproductive organs and a reproducible cue for pollinators. The model plant Arabidopsis thaliana belongs to the Brassicaceae family, which are commonly called crucifers after the cross-shaped arrangement of four petals in their flowers( Endress, 1992). Petals acquire their identity via the combined activity of A- and B-class floral organ identity genes, according to the ABC model of flower development( Coen and Meyerowitz, 1991). However, less is known about the genetic control of petal number. In A. thaliana flowers, initiation of four petals depends on the size of the floral meristem, the establishment of boundaries that demarcate the position of petal primordia on the floral meristem, the transient formation of auxin activity maxima in these positions, and the general mechanisms of lateral organ outgrowth( Huang and Irish, 2015; Huang and Irish, 2016; Irish, 2008). Consistent with this complexity, few A. thaliana mutants specifically affect petal number; among them is petal loss( ptl), which displays a variable loss of petals caused by mutation of the PTL trihelix transcription factor( Brewer et al., 2004; Griffith et al., 1999). Other mutants pleiotropically affect petal number, such as mutations in the MADS-box transcription factor APETALA1( AP1), which shows floral meristem identity defects in addition to variable petal loss( Irish and Sussex, 1990; Mandel et al., 1992). Cardamine hirsuta is a close relative of A. thaliana that lacks a robust phenotype of four petals( Hay et al., 2014). Instead, C. hirsuta flowers display a variable number of petals, between zero and four, on a single plant( Monniaux et al., 2016; Pieper et al., 2016). This phenotype varies in response to both environmental and genetic perturbation. C. hirsuta flowers show seasonal variation in petal number, with spring-flowering plants producing more petals than summer-flowering plants( McKim et al., 2017). Seasonal cues, such as day length, winter cold, and particularly ambient temperature, all influence the number of petals produced in C. hirsuta flowers( McKim et al., 2017). Furthermore, petal number is strongly influenced by natural genetic variation in C. hirsuta. A polygenic architecture of small to moderate effect quantitative trait loci( QTL), that shift the trait in both directions, contribute to petal number variation in C. hirsuta( Pieper et al., 2016). Alleles of large effect and low pleiotropy were identified in genetic screens for four-petalled mutants in C. hirsuta, but were not detected by QTL analysis in natural accessions( Pieper et al., 2016). Thus, the distribution of allelic effects found in natural populations of C. hirsuta is more likely to maintain variation, rather than robustness, in petal number. Petal number varies both within and between species, evolving from a robust state of four petals, typified by A. thaliana, to a variable state in C. hirsuta. Phenotypic divergence between species is necessarily derived from variation within species, but identifying these evolutionary transitions is not a straight-forward task. This is because similar phenotypes that vary within and between species may or may not be caused by similar genetic mechanisms. For example, the same light-pigmentation alleles that are fixed in a yellow-bodied Drosophila species, segregate in a closely related brown-bodied species and contribute to clinal variation in its body colour( Wittkopp et al., 2009). However, in another example, genes responsible for leaf shape differences between A. thaliana and C. hirsuta were not detected as leaf shape QTL in C. hirsuta( Cartolano et al., 2015). Therefore, to understand how petal number variation is produced and how it evolved, it is important to investigate both the genetic basis of variation within species and divergence between species. For example, to address questions such as: How many genes contribute to trait divergence between species? How large are their effects? Do they have pleiotropic functions? How do they interact with genes causing variation in natural populations? A simple prediction about robust phenotypes, such as petal number in A. thaliana, is that they are invariant because genetic variation is reduced by stabilizing selection on the phenotype. On the other hand, a developmental pathway might be robust because certain alleles prevent the phenotypic effects of new mutations. This would effectively buffer the phenotype and hide underlying genetic variation. Previous studies of vulva development in Caenorhabditis( Félix, 2007), and eye development in cavefish( Rohner et al., 2013), support the latter view, showing that there is extensive, selectable genetic variation affecting robust phenotypes, which can be exposed by genetic or environmental perturbation. Moreover, studies that use gene expression as a trait, have mapped QTL that influence variance rather than mean expression level( Hulse and Cai, 2013), and identified selection acting on expression noise rather than mean level( Metzger et al., 2015). However, there are few examples( Rohner et al., 2013) where the genetic basis of morphological differences between species can be traced to the release of cryptic variation. In this study, we investigate the evolutionary transition from a robust phenotype of four petals, typified by A. thaliana, to a variable petal number in C. hirsuta. We show that divergence in the pleiotropic floral regulator AP1 can account for the difference in petal number robustness between species. This large effect of AP1 is explained by epistatic interactions: A. thaliana AP1 masks the phenotypic expression of all petal number QTL in C. hirsuta and, in this way, confers robustness. We show that C. hirsuta AP1 fails to complement this function of A. thaliana AP1, conferring variable petal number, and that upstream regulatory regions of AP1 contribute to this divergence.
Discussion:
In this study, we identified AP1 as a gene of major effect in the evolutionary transition from a robust phenotype of four petals, typified by A. thaliana, to a variable petal number in C. hirsuta. Despite this large effect, AP1 polymorphisms do not contribute directly to within-species variation in C. hirsuta petal number. Instead, the decanalization of petal number in C. hirsuta involved regulatory changes in AP1 that relaxed its epistasis over alleles that cause petal number to vary. Therefore, our results suggest that AP1 divergence likely exposed cryptic genetic variation in C. hirsuta that contributes directly to maintaining variable petal number within natural accessions of C. hirsuta. AP1 is an important regulator of A. thaliana flower development, acting early to initiate flowers and later to specify floral organs( Bowman et al., 1993; Irish and Sussex, 1990; Mandel et al., 1992). It functions predominantly as a transcriptional repressor during floral initiation and later as an activator during sepal and petal initiation, and has been described as a ‘true hub’ in the gene regulatory network that initiates flower development( Kaufmann et al., 2010). We found that AP1 genes from A. thaliana and C. hirsuta diverged in their ability to specify an invariant number of four petals. This difference mapped broadly to upstream regulatory rather than coding regions of AP1, and did not alter the function of AP1 in flower initiation or sepal specification; suggesting that regulatory sequence divergence can disable specific linkages in gene regulatory networks while maintaining pleiotropic functions in other tissues( Rebeiz and Tsiantis, 2017). We showed that differences in AP1 expression are associated with the functional divergence in AP1 between A. thaliana and C. hirsuta. Specifically, C. hirsuta AP1 transcript levels are reduced in floral tissues and protein abundance is reduced in the small regions of stage four flowers where petals initiate. Robustness to variation in developmental systems is often a consequence of nonlinear dose-response curves between gene activity and phenotype( Félix and Barkoulas, 2015). For example, vulva development in Caenorhabditis elegans is robust to genetic variation in lin-3 expression, such that 15–50 lin-3 mRNA molecules defines a robust range that allows wild-type cell fate patterning, bounded by two thresholds, beyond which the phenotype varies( Barkoulas et al., 2013). We propose that the expression of A. thaliana AP1 defines a robust range where petal number is buffered against perturbations( Figure 6a). C. hirsuta AP1 is expressed below this threshold, such that petal number varies in response to genetic, environmental, and stochastic perturbations( Figure 6a)( McKim et al., 2017; Monniaux et al., 2016; Pieper et al., 2016). Waddington’s model of canalization invokes a similar concept of phenotypic buffering against natural variation. In his classical metaphor of marbles rolling down canals, he depicted the surface of the landscape being pulled down by guy ropes and fastened to anchors that represented genes( Waddington, 1957). Like this, the genetic underpinnings of the landscape may vary, but produce a consistent phenotype( e. g. A. thaliana petal number, Figure 6b)( Paaby and Rockman, 2014). Petal number is invariant among natural accessions of A. thaliana, reflecting its robustness to genetic variation. However, we could change A. thaliana petal number to variable by complementing ap1 mutants with the AP1 genomic locus from C. hirsuta. These flowers mimicked the variable petal number found in C. hirsuta, suggesting that there may be variability in the gene regulatory network controlling petal number in A. thaliana that is hidden beneath the uniformity of wild-type development. In C. hirsuta, petal number is decanalized, such that natural genetic variation deforms Waddington’s landscape, producing a variable phenotype( Figure 6b). We mapped this natural variation to specific QTL in the C. hirsuta genome and showed that introducing the A. thaliana AP1 locus masked the effects of all QTL. Therefore, petal number was effectively canalized in C. hirsuta via epistasis of A. thaliana AP1 over existing QTL. Based on these results, we propose that AP1 divergence perturbed the genetic equilibrium in C. hirsuta that confers petal number robustness. Given the large effect of the A. thaliana AP1 locus on petal number, it is interesting that endogenous AP1 polymorphisms do not contribute directly to petal number variation in C. hirsuta. Rather, it is the ability of AP1 to exert epistasis over other loci that diverged between A. thaliana and C. hirsuta( Figure 6b). We propose that this change in genetic interactions was the likely mechanism by which cryptic variation was exposed in C. hirsuta, contributing to the evolution of variable petal number.
Many plants produce flowers that attract insects to land on them . Different insects are attracted to flowers of different shapes and colors . Therefore , it is generally advantageous for plants of the same species to produce flowers that look very similar . For example , a small weed known as Arabidopsis – which is often used in research studies – produces little white flowers that all have four petals . Thus , the number of petals in Arabidopsis flowers is said to be a ‘robust’ trait . However , a closely-related plant called hairy bittercress produces flowers with any number of petals between zero and four . Studying the genetic differences between Arabidopsis and hairy bittercress can help to reveal why the numbers of petals on hairy bittercress flowers vary . A gene called APETALA1 helps to control how petals form . Monniaux , Pieper et al . found that Arabidopsis and hairy bittercress have different versions of this gene that determine whether the number of petals may vary between individual flowers . Inserting the Arabidopsis version of APETALA1 into hairy bittercress plants caused the plants to produce flowers that had more similar numbers of petals to each other , that is , the petal number became more robust . Monniaux , Pieper et al . then used a statistical method called quantitative trait locus analysis to identify the precise location of regions in the hairy bittercress genome that control petal number . This showed that the Arabidopsis version of APETALA1 , but not the hairy bittercress version , conceals the action of these genes that could alter petal number . These findings reveal that evolutionary change in a single gene of hairy bittercress unmasked the action of other genes that caused petal number to vary . A next step will be to identify some of these genes and understand how they control petal number .