The role of APETALA1 in petal number robustness
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 .
The flowers of A . thaliana and other Brassicaceae species are readily distinguished by their four petals . This phenotype is robust to natural genetic variation; for example , flowers from genetically diverse A . thaliana accessions consistently produce four petals ( Figure 1a ) . C . hirsuta lacks this robustness and shows variation in petal number . For example , we found similar frequencies of each petal number between zero and four in flowers from 39 C . hirsuta accessions sampled from across the species range ( Figure 1b ) ( Hay et al . , 2014 ) . Therefore , petal number varies within C . hirsuta and is a species-level trait that distinguishes C . hirsuta from A . thaliana . Petals initiate in the second whorl of A . thaliana and C . hirsuta flowers , each positioned between two outer sepals , with the inner whorls being occupied by male and female reproductive organs ( stamens and carpels ) ( McKim et al . , 2017; Smyth et al . , 1990 ) . Small petal primordia are readily observed in the second whorl of A . thaliana floral buds , located between first-whorl sepals and third-whorl stamens ( Figure 1c , d ) . In contrast to this , petal primordia were often missing in C . hirsuta flowers at similar developmental stages ( Figure 1e ) . Instead , we observed a flat surface in the second whorl with no indication of outgrowths ( Figure 1f ) . However , when we found petal primordia in C . hirsuta flowers , their development appeared indistinguishable from those in A . thaliana ( Figure 1—figure supplement 1 ) . These petals occupied any of the four positions available in the second whorl , with a slightly higher frequency in abaxial positions ( Figure 1—figure supplement 2 ) . Therefore , the number of petals in a C . hirsuta flower is determined at early stages of petal initiation and outgrowth . To study the earliest stages of petal initiation , we tracked floral meristem development using time-lapse confocal laser scanning microscopy and analysed growth in these 4-dimensional image stacks ( Barbier de Reuille et al . , 2015 ) . We followed the formation of auxin activity maxima during petal initiation in A . thaliana and C . hirsuta using the DR5::VENUS and DR5v2::VENUS auxin activity sensors ( Barkoulas et al . , 2008; Heisler et al . , 2005; Liao et al . , 2015 ) . At sites of petal initiation in A . thaliana , auxin activity maxima formed in 2 – 3 epidermal cells on the floral meristem flank prior to growth of these cells ( Figure 2a , b; 83% DR5::VENUS observation rate , Figure 2—figure supplement 1 ) . However , in C . hirsuta , auxin activity maxima often failed to form on the floral meristem , and instead were either absent or aberrantly located in the first whorl between sepals ( Figure 2c , d; 36% DR5v2::VENUS observation rate , Figure 2—figure supplement 1 ) . Therefore , four sites of petal initiation are usually marked by auxin activity maxima in the second whorl of the floral meristem in A . thaliana , but not C . hirsuta . The ptl mutant in A . thaliana mimics the variable petal number found in C . hirsuta and shows a similar distribution of auxin activity during petal initiation as wild-type C . hirsuta ( Figure 2d ) ( Griffith et al . , 1999; Lampugnani et al . , 2013 ) . Therefore , we tested whether differences in PTL function could explain why petal number is robust in A . thaliana but variable in C . hirsuta . A functional fusion protein of A . thaliana PTL ( AtPTL::AtPTL:YFP ) was sufficient to restore four petals in the ptl mutant , but did not alter petal number when expressed in C . hirsuta ( Figure 2e; Figure 2—figure supplement 2 ) . Given that AtPTL::AtPTL:YFP expressed correctly in the regions between sepals in C . hirsuta and A . thaliana flowers ( Figure 2f , g ) , similar to the endogenous PTL transcripts in C . hirsuta and A . thaliana ( Figure 2—figure supplement 3 ) ( Brewer et al . , 2004; Lampugnani et al . , 2012; Lampugnani et al . , 2013 ) , these results indicate that differences in PTL function are unlikely to account for the variation in petal number between C . hirsuta and A . thaliana . Petals are defined by both their identity and position within a flower . To test whether the variable number of petals in C . hirsuta is dependent on their identity or on the location where they arise in the second whorl , we used the homeotic mutant agamous ( ag ) , to alter floral organ identity . In C . hirsuta ag mutants , four petals replaced the four stamens normally found in the third whorl of wild-type flowers , while petal number remained variable and lower than four in the second whorl ( Figure 2h , i ) . This means that floral organs with petal identity show no variation in number if they arise outside the second whorl . As predicted by the ABC model ( Coen and Meyerowitz , 1991 ) , we found AP1 ectopically expressed in third whorl petals of ag mutants in C . hirsuta ( Figure 2j ) . Therefore , ectopic expression of AP1 is associated with an invariant number of petals , whereas endogenous AP1 expression in whorl two of the C . hirsuta floral meristem is associated with variable petal number . We reasoned that AP1 might be a good candidate to contribute to petal number variation in C . hirsuta , particularly given that ap1 mutants in both C . hirsuta and A . thaliana show variable petal loss ( Bowman et al . , 1993; Monniaux et al . , 2017 ) . To test this possibility , we used a genomic construct of A . thaliana AP1 ( AtAP1::AtAP1:GFP ( Urbanus et al . , 2009 ) ) , which was sufficient to restore four petals in the ap1 mutant ( Mann-Whitney U test , p=1 . 07e-06 ) and eliminate the ectopic flowers that characterize the partial loss of floral meristem identity of ap1 mutants ( Mann-Whitney U test , p=2 . 92e-06; Figure 3a–c ) . We found that this transgene was sufficient to convert C . hirsuta petal number from variable to robust , elevating petal number towards the A . thaliana value of four petals ( pairwise Mann-Whitney U test with Bonferroni correction , p=2 . 4e-08; Figure 3a , d , e ) . In contrast to this , the distribution of petal number remained variable in C . hirsuta lines expressing the endogenous AP1 locus ( ChAP1::ChAP1:GFP; pairwise Mann-Whitney U test with Bonferroni correction , p=0 . 07: Figure 3a ) . This suggests that the function of the endogenous AP1 locus to confer four petals is attenuated in C . hirsuta . Therefore , divergence in AP1 function likely contributed to the variation in petal number between A . thaliana and C . hirsuta . This raises the question whether petal number variation both between and within species may be caused by similar genetic changes . If AP1 divergence contributed to petal number variation between species , do AP1 polymorphisms contribute to this phenotype within C . hirsuta ? To address this question , we inspected the locations of petal number QTL previously identified in five mapping populations derived from bi-parental crosses of different C . hirsuta accessions ( Pieper et al . , 2016 ) , and an additional population constructed in this study ( Figure 3f ) . We found that none of the QTL mapped to the AP1 locus , which was represented by a specific genetic marker on chromosome 2 ( Figure 3f , Table 1 ) ( Pieper et al . , 2016 ) . Therefore , allelic variation at AP1 does not contribute to the quantitative variation in petal number mapped in C . hirsuta . However , an alternative possibility is that AP1 divergence indirectly caused petal number to vary within C . hirsuta by altering the robustness of this phenotype to genetic variation . Given that petal number is a canalized trait in A . thaliana and robust to genetic variation ( Figure 1a ) , we hypothesized that AP1 divergence may have decanalized petal number in C . hirsuta , giving phenotypic expression to formerly cryptic variation ( Figure 1b ) ( Félix , 2007; Paaby and Rockman , 2014 ) . A key prediction of this hypothesis is that A . thaliana AP1 should canalize petal number in C . hirsuta via epistatic interactions with petal number QTL . We tested this genetic prediction in an F2 population created by crossing C . hirsuta Ox containing the A . thaliana AP1 genomic locus ( AtAP1::AtAP1:GFP ) , with the Nz accession ( Figure 3f ) . We detected nine petal number QTL in addition to the A . thaliana AP1 transgene locus ( Table 1 , Figure 3f ) . Strikingly , the allelic effects of these 9 QTL were undetectable in the presence of the A . thaliana AP1 genomic locus ( Table 1 ) . This epistasis was readily observed in the distribution of petal number between plants homozygous for the A . thaliana AP1 transgene , which had four petals , and plants that lacked the transgene , which had variable petal number ( Figure 3g ) . Thus , A . thaliana AP1 canalized C . hirsuta petal number by masking the phenotypic effects of at least 9 QTL . Our findings suggest that the AP1 genes from A . thaliana and C . hirsuta may have a differential ability to confer four petals . To test whether or not C . hirsuta AP1 could fully complement the function of A . thaliana AP1 , we introduced a ChAP1::ChAP1:GFP transgene into an ap1 mutant background in A . thaliana . Rather than restoring four petals like ap1-12; AtAP1::AtAP1:GFP ( Figure 3a ) , we found that the distribution of petal number remained variable in ap1-12; ChAP1::ChAP1:GFP flowers ( homogeneity of variance accepted by Levene’s test , p=0 . 282; Figure 4a , b ) , mimicking the variable petal number found in C . hirsuta . Petal number varied between zero and four , and the average petal number was significantly lower in ap1 plants expressing the AP1 genomic clone from C . hirsuta rather than A . thaliana ( Figures 3a and 4a , b , Mann-Whitney U test p=2 . 08e-07 ) . In contrast , ChAP1::ChAP1:GFP expression was sufficient to reduce the ectopic flowers in ap1 mutants ( Figure 4b ) , indicating that AP1 divergence between C . hirsuta and A . thaliana affected petal number independently of floral meristem identity . Therefore , the results of these gene swaps indicate that C . hirsuta AP1 has a reduced ability to promote four petals when compared to A . thaliana AP1 . Next , we considered the relative contributions of regulatory and coding sequences to this species-specific difference in AP1 function . To address this question , we expressed endogenous and chimeric versions of A . thaliana and C . hirsuta AP1 , swapping the promoter and coding sequences , in an ap1 mutant background in A . thaliana . We found that petal number was significantly elevated by the A . thaliana AP1 promoter , compared to the C . hirsuta AP1 promoter , irrespective of the AP1 coding sequence driven by these promoters ( Figure 4c ) . Whereas all constructs functioned equivalently to rescue the ectopic flowers found in ap1 mutants ( Figure 4—figure supplement 1 ) . Therefore , functional differences in AP1 that are responsible for petal number variation between A . thaliana and C . hirsuta , are more likely to reside in regulatory regions of the gene rather than coding sequences . Since upstream regulatory regions contributed to AP1 divergence , we investigated whether expression differed between C . hirsuta and A . thaliana AP1 . We reasoned that the reduced function of C . hirsuta AP1 to promote four petals may reflect reduced levels of gene expression . To test this prediction , we compared AP1 expression between C . hirsuta lines with matched copy numbers of either AtAP1::AtAP1:GFP or ChAP1::ChAP1:GFP transgenes , and found that expression levels were significantly lower in floral tissues of ChAP1::ChAP1:GFP than AtAP1::AtAP1:GFP lines ( Figure 4d ) . To visualize the spatiotemporal dynamics of expression , we localized AP1::AP1:GFP fusion proteins from each species in the four-dimensional context of the growing C . hirsuta flower ( Figure 4—figure supplement 2 ) . In stage four floral buds , we observed A . thaliana AP1::AP1:GFP nuclear signal in the sepal whorl and on the flanks of the floral meristem , in the small regions where petals initiate in whorl two ( Figure 4e ) . In contrast to this , C . hirsuta AP1::AP1:GFP was essentially restricted to the sepal whorl throughout stages 4 and 5 ( Figure 4f , Figure 4—figure supplement 2 ) . Using top-view , two-dimensional snapshots of these curved surface images , we measured how far the AP1::AP1:GFP signal extended into whorl two in each transgenic line ( Figure 4—figure supplements 3–5 ) . We found that the expression of A . thaliana AP1::AP1:GFP extended significantly further than C . hirsuta AP1::AP1:GFP ( approximately 5 µm , Figure 4g ) . Moreover , we found no significant change in size or geometry between flowers expressing either the A . thaliana or C . hirsuta AP1 genomic constructs ( Figure 4g , Figure 4—figure supplement 6 ) . This contrasts with the changes in growth and maturation of floral buds that are associated with the regulation of C . hirsuta petal number by seasonal changes in temperature ( McKim et al . , 2017 ) . Therefore , the expression domain of ChAP1::ChAP1:GFP is reduced compared to AtAP1::AtAP1:GFP , comprising fewer cells in the petal whorl on the flanks of the floral meristem in C . hirsuta . By co-localizing the expression of both AtAP1::AtAP1:GFP and ChAP1::ChAP1:RFP in stage 4 flowers of C . hirsuta and A . thaliana , we also found that C . hirsuta AP1::AP1:RFP expression is enriched in the regions between sepals ( Figure 4—figure supplement 7 ) . Since peaks of auxin activity are displaced away from the petal whorl to the region between sepals in C . hirsuta flowers ( Figure 2d ) , and since distortions of this region have been shown to influence petal initiation in A . thaliana ( Baker et al . , 2005; Lampugnani et al . , 2012; Lampugnani et al . , 2013; Laufs et al . , 2004; Mallory et al . , 2004 ) , the enrichment of C . hirsuta AP1 in this domain might be relevant for petal number variation in C . hirsuta . Our findings suggest that petal number is a robust phenotype in A . thaliana that became decanalized in C . hirsuta , such that AP1 divergence allowed previously cryptic loci to quantitatively affect petal number . Allelic variation at these QTL maintains the distribution of petal numbers found among natural accessions of C . hirsuta ( Pieper et al . , 2016 ) . A striking feature of this distribution is the absence of natural accessions with four petals ( Figure 5a ) . Moreover , few accessions have an average petal number approaching four ( Figure 5a ) . To explore the genetic basis of this phenotypic distribution , we constructed experimental populations from founder accessions with phenotypes close to the mean ( arrowheads , Figure 5a ) . These recombinant inbred populations contained a high frequency of lines with high average petal numbers , including four petals ( Figure 5b , and other examples in ( Pieper et al . , 2016 ) ) . This shows that there is sufficient standing genetic variation to produce phenotypes through recombination that are not observed in natural accessions . To explore this finding , we reasoned that producing four-petalled flowers could incur an energetic cost . However , in standard growth conditions , we could not detect any difference in seed output between C . hirsuta wild-type and AtAP1::AtAP1:GFP plants that differed in petal number ( Figure 5—figure supplement 1 ) . Alternatively , variable petal number could be the cause or consequence of variation in other trait ( s ) that contribute to maintaining petal number within its variable range . Given that petals are responsible for the opening of most flowers ( van Doorn and Van Meeteren , 2003 ) , thus allowing cross-pollination , we tested whether petal number was associated with outcrossing rate in C . hirsuta . Using field conditions to grow two genotypes that differed significantly in petal number ( 2 . 65 ± 0 . 08 vs 3 . 45 ± 0 . 05 , Mann-Whitney U test p<0 . 001; Figure 5c , Figure 5—figure supplement 2 ) , we paternity-tested the progeny of these plants and found a significantly higher outcrossing rate in the genotype with higher petal number ( Chi-square test p<0 . 05 , Monte-Carlo permutation test p<0 . 001 , Figure 5d , Figure 5—figure supplement 2 ) . Therefore , outcrossing frequency is associated with petal number in C . hirsuta .
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 .
The wild-type genotype in C . hirsuta is the reference Oxford ( Ox ) accession , herbarium specimen voucher Hay 1 ( OXF ) ( Hay and Tsiantis , 2006 ) , and in A . thaliana , the reference Col-0 accession . DR5::VENUS transgenic lines in A . thaliana ( Heisler et al . , 2005 ) and C . hirsuta ( Barkoulas et al . , 2008 ) have been described previously . NASC accession numbers for A . thaliana mutants: ap1-12 ( N6232 ) , ptl-1 ( N276 ) . Additional A . thaliana and C . hirsuta accessions have been described previously ( 1001 Genomes Consortium . Electronic address: magnus . nordborg@gmi . oeaw . ac . at and 1001 Genomes Consortium , 2016; Hay et al . , 2014 ) . C . hirsuta genome assembly gene identifiers: ChAP1 ( CARHR062020 ) , ChPTL ( CARHR209620 ) , ChAG ( CARHR225900 ) , Clathrin/AP2M ( CARHR174880 ) ( Gan et al . , 2016 ) . All plants were grown in long day conditions unless otherwise stated . Greenhouse: 16 hr light ( 22°C ) , 8 hr dark ( 20°C ) ; controlled environment room long days: 16 hr light ( 21°C ) , 8 hr dark ( 20°C ) and short days: 10 hr light ( 21°C ) , 14 hr dark ( 21°C ) ; growth cabinet short days: 8 hr light ( 22°C ) , 16 hr dark ( 20°C ) . Petal number was generally scored on 10–15 plants from each genotype , except when scoring the T1 generation of transgenic lines . Flowers were scored every second day by removing them from the plant with tweezers , and observing them with a head band magnifier or stereo microscope . For the C . hirsuta ag mutant , flowers from four plants were removed every second day and scored with a binocular loop . Whorl one was removed to allow scoring of whorl two organs , which were then removed to score whorl three organs . C . hirsuta Ox seeds were mutagenized by agitation with ethyl methanesulfonate ( EMS , Sigma ) , sown on soil , harvested as pools of five M1 plants , and M2 plants were screened for floral phenotypes as described previously ( Pieper et al . , 2016 ) . The four petals 2 ( fp2 ) ( Pieper et al . , 2016 ) and agamous ( ag-1 ) mutants were isolated and backcrossed twice to Ox . The ag-1 sequence bears a C to T single nucleotide change predicted to convert a Gln residue to a stop codon and produce a truncated 33 AA protein . Expressing an AtAG:GFP translational fusion ( gift from G . Angenent ( Urbanus et al . , 2009 ) ) complemented the C . hirsuta ag-1 mutant phenotype . Five and ten flowers from one plant of C . hirsuta wild-type and ag-1 , respectively , were pooled to generate one biological replicate for RNA extraction . Three biological replicates were generated per genotype . For quantification of AP1 expression levels in C . hirsuta AtAP1::AtAP1:GFP and ChAP1::ChAP1:GFP lines , a secondary inflorescence from 29 and 24 plants from five independent lines , respectively , was used for RNA extraction , together with three wild-type biological replicates . For these plants , transgene copy number was determined from genomic DNA by a Taqman qPCR assay using the Hygromycin resistance gene ( IDna Genetics , Norwich , UK ) . RNA was extracted using Spectrum Plant Total RNA kit ( Sigma ) and DNA was digested by on-column DNase I digestion ( Sigma ) . Reverse Transcription was performed with SuperScript III Reverse Transcriptase ( ThermoFisher Scientific ) using 1 µg of RNA template . Quantitative PCR was performed with the Power SYBR Green Master Mix ( ThermoFisher Scientific ) with the following primers: AP1-qPCR-F ( 5’- CCAGAGGCATTATCTTGGGGAAGACTTG ) and AP1-qPCR-R ( 5’- GCTCATTGATGGACTCGTACATAAGTTGGT ) to amplify either ChAP1 or AtAP1 , and Clathrin-qPCR-F ( 5’- TCGATTGCTTGGTTTGGAAGATAAGA ) and Clathrin-qPCR-R ( 5’- TTCTCTCCCATTGTTGAGATCAACTC ) to amplify the reference gene Clathrin/AP2M . Expression was calculated with the ΔΔCt method ( Pfaffl , 2001 ) , normalized against the reference gene , and expressed relative to wild-type levels . For the AtPTL::AtPTL:YFP construct , a 2 . 8 kb PTL promoter up to the second exon , driving functional PTL expression ( Lampugnani et al . , 2012 ) , was amplified with primers pPTL-F ( 5’-ATATATTGAGAAGAGATTAAAAACTTAG ) and pPTL-R ( 5’-GTATCCATGTTCCTCGGACA ) from Col-0 genomic DNA and cloned into the multiSite Gateway donor vector pDONR-P4-P1R . The full 2 . 9 kb genomic sequence of PTL was amplified with primers gPTL-F ( 5’-ATGGATCAAGATCAGCATC ) and gPTL-R ( 5’-CTGATTCTCTTCTTTACTGAGC ) from Col-0 genomic DNA and cloned into the multiSite Gateway donor vector pDONR-221 . The YFP coding sequence was cloned into the multiSite Gateway donor vector pDONR-P2R-P3 . The AtPTL::AtPTL:YFP construct was created by recombining together the three previous vectors into the pGII-0229 destination vector , as described in the MultiSite Gateway manual ( Thermo Fisher Scientific ) . Eight and nineteen independent lines of AtPTL::AtPTL:YFP were generated in C . hirsuta wild type and A . thaliana ptl-1 respectively . Petal number was scored on all independent lines in the T1 generation , together with C . hirsuta wild type and A . thaliana ptl-1 . Twenty independent lines of AtAP1::AtAP1:GFP ( gift from G . Angenent ( Urbanus et al . , 2009 ) ) were generated in C . hirsuta wild-type and A . thaliana ap1-12 backgrounds . This translational fusion contains a 6 . 6 kb genomic fragment of A . thaliana AP1 including 3 kb of regulatory sequence upstream of the translational start . ChAP1::ChAP1:GFP was previously described ( Monniaux et al . , 2017 ) and contains a comparable 6 . 6 kb genomic fragment of C . hirsuta AP1 . Ten independent lines of ChAP1::ChAP1:GFP were generated in C . hirsuta wild type and A . thaliana ap1-12 . For all the AP1-related lines , petal number was scored on 2 to 5 single-insertion homozygous T3 lines , together with wild-type C . hirsuta and A . thaliana ap1-12 . ChAP1::ChAP1:RFP was constructed in the modified destination vector pB7RWG2 ( gift from M . Kater ( Gregis et al . , 2009 ) ) by recombining the same C . hirsuta AP1 genomic fragment used above . Six independent lines were generated in A . thaliana AtAP1::AtAP1:GFP for co-localisation studies . Four independent lines were generated in C . hirsuta and selected for strong expression in the third generation . Homozygous plants were crossed to an AtAP1::AtAP1:GFP strong expressing line for co-localisation studies . The pAtAP1::AtAP1 , pAtAP1::ChAP1 , pChAP1::AtAP1 and pChAP1::ChAP1 constructs were generated by three-fragment multi-site Gateway in the pGII-0227 destination vector . The AtAP1 and ChAP1 promoters contain 2 . 9 kb upstream of the start codon and were amplified with primers pAtAP1-F ( 5’- CGAACGTGGTGGTTAGAAGA ) and pAtAP1-R ( 5’-TTTTGATCCTTTTTTAAGAAACTT ) , and primers pChAP1-F ( 5’-CATATAGCTTGGATCATGCTC ) and pChAP1-R ( 5’-TTTGATCCTATTTTGAGAAACTTCTT ) respectively . Ten independent lines , with five plants per line , were scored for petal and ectopic flower number together with A . thaliana wild type and ap1-12 . Lines with a clear ap1-12 phenotype were considered not to be complemented by the transgene and were removed from the analysis . The DR5-v2::3xVENUS plasmid was a gift from Dolf Weijers ( Liao et al . , 2015 ) . Eleven independent insertion lines were generated in C . hirsuta wild type . All lines were checked for expression in the T1 generation and two representative lines were selected to image by time-lapse CLSM in the T2 generation . All binary vectors were transformed into C . hirsuta or A . thaliana by Agrobacterium tumefaciens ( strains GV3101 or C58 ) -mediated floral dip . Shoot apices were induced to flower by a shift from short to long day conditions and fixed in FAA , post-fixed in osmium tetraoxide , dehydrated , critical point dried and dissected before coating with gold/palladium for viewing in a JSM-5510 microscope ( JEOL ) . Floral primordia were staged according to ( Smyth et al . , 1990 ) . Time-lapse imaging was performed using 4–5 week old plants grown on soil in long day conditions . The inflorescence was cut and flowers were dissected off to uncover young floral primordia at the shoot apex . Dissected shoots ( around 0 . 5 cm long sections ) were transferred to ½ MS medium supplemented with 1 . 5% plant agar , 1% sucrose and 0 . 1% Plant Preservative Mixture ( Plant Cell Technology ) . To outline cells , samples were stained with 0 . 1% propidium iodide ( PI , Sigma ) for 2 – 5 min before each observation . Floral primordia were immersed in water and imaged from the top at 24 hr intervals . Confocal imaging was performed using a Leica SP8 up-right confocal microscope equipped with a long working-distance water immersion objective ( L 40x/0 . 8 W ) ( Leica ) and HyD hybrid detectors ( Leica ) . Excitation was achieved using an argon laser with 514 nm for VENUS and PI . Images were collected at 526 – 545 nm for VENUS , and 600 – 660 nm for PI . Between imaging , samples were transferred to a growth cabinet and cultured in vitro in standard long day conditions at 20°C . Confocal image stacks of time-lapse series were analyzed using MorphoGraphX software ( Barbier de Reuille et al . , 2015; Kierzkowski et al . , 2012 ) . The outer 10 Full datasets of A . thaliana and C . hirsuta time-lapse series used to track growth and DR5::VENUS and DR5v2::VENUS expression shown in Figure 2—figure supplement 1 . To measure physical boundary size and size of AP1-GFP expression , the epidermal ( 2 to 5 µm ) GFP signal was projected on the surface of the sample with MorphoGraphX . Top-view snapshots of the flower meristem with GFP-projected signal were acquired and subsequently analyzed with FiJi ( Schindelin et al . , 2012 ) as described in Figure 4—figure supplement 3 . Shoot apices were induced to flower by a shift from short to long day conditions . Digoxigenin-labeled antisense RNA probes to C . hirsuta ChPTL were generated by mixing together three synthetic probes covering the whole ChPTL cDNA ( GenScript HK Limited , USA ) . 8 – 10 µm inflorescence cross-sections were fixed , embedded in paraffin and hybridized with the ChPTL probe as previously described ( Vlad et al . , 2014 ) . The signal was observed and images were acquired with a Zeiss AxioImager . M2 light microscope equipped with an Axiocam HR color camera . To cover the entire hybridization pattern in depth , images of two consecutive sections were registered and minimal projections generated using the image processing package Fiji ( Schindelin et al . , 2012 ) . Cropping , gamma and colour correction were done using Photoshop CS5 and performed on entire images only . For semi-thin sections , apices were fixed in 2 . 5% glutaraldehyde in phosphate buffer , dehydrated , step-wise infiltrated with and embedded in TAAB Low Viscosity resin ( TAAB ) and 1 . 5 μm sections were stained with 0 . 05% toluidine blue . QTL analysis of petal number was performed on a C . hirsuta F2 population derived from a cross between an AtAP1::AtAP1:GFP transgenic line in the Ox accession and the Nz accession . Petal number was quantified in 312 individuals that were pre-screened by PCR amplification of the GFP sequence such that approximately 1/4 of the plants were wild-type . The first 25 flowers on each plant were removed on the day they opened and petal number was counted using a dissecting microscope . The 312 F2 plants were genotyped with 155 Sequenom markers ( Welcome Trust Center for Human Genetics , High Throughput Genomics , Oxford , UK ) designed to cover the whole genome according to an early version of the C . hirsuta genome assembly ( Gan et al . , 2016 ) , and a dCAPS marker was generated for the C . hirsuta AP1 locus using primers AP1cisF1 ( 5’-TCCCTAAAACCGCTCTTAGC ) and AP1cisR1 ( 5’-AGAGAGATAAAGAAGAGTTCAGGC ) and the restriction enzyme AluI . The genetic map was made using JoinMap 4 ( Van Ooijen , 2006 ) , including the genotype for AtAP1::AtAP1:GFP as a dominant marker to determine the location of the transgene , and had a total length of 910 centiMorgans in eight linkage groups . QTL analyses were performed with Genstat 13th Edition ( VSN International , Hemel Hempstead , UK ) using all 312 F2 plants . Genetic predictors were calculated with a maximum distance of 2 cM between them from the molecular marker data and the genetic map . Average petal number per F2 plant was used as a phenotype for QTL analysis . Simple interval mapping and composite interval mapping were performed . The latter procedure was repeated several times while adding and/or removing cofactors until no further improvement could be made . The resulting set of 10 cofactors was used in a final QTL model to estimate QTL effects . A model with nine cofactors , when excluding the AtAP1::AtAP1:GFP locus , was fitted to data from F2 plants that were either homozygous for the AtAP1::AtAP1:GFP transgene or wild-type . Seeds from Ox and fp2 ( Pieper et al . , 2016 ) were stratified for 1 week at 4°C and sown on 15 . 03 . 2016 on hydrated Jiffy plugs . Seedlings were first grown in a greenhouse without temperature or light control , and later transferred to the field on 13 . 04 . 2016 . More details on experimental design can be found in Figure 5 and Figure 5—figure supplement 2 . Genomic DNA was extracted from parent and progeny plants with Edwards Buffer and isopropanol precipitation and amplified with primers m458 ( 5’-GCCTAATCTTGCACAACACGAAATCT ) and m459 ( 5’-GATTCTAAAGTTCTGTCAAAAGGAGAAACCTGA ) , designed with dCAPS Finder ( http://helix . wustl . edu/dcaps/dcaps . html ) , to genotype SNP:2:2905982 by dCAPS . PCR was performed with Mango Taq polymerase ( Bioline ) under the following conditions: initial denaturation of 5 min at 95°C , 40 cycles of 30 s at 95°C , 30 s at 56°C and 30 s at 72°C , final extension of 10 min at 72°C . 1/5th volume of the reaction was digested with 2 . 5 units of DdeI ( New England Biolabs ) for 2 hr at 37°C and migrated on a 3% agarose gel to resolve the uncut 141 bp amplicon for fp2 , and the two cut fragments of 116 bp and 25 bp for Ox . 40 plants of C . hirsuta wild type and a homozygous T4 line of AtAP1::AtAP1:GFP were grown in a greenhouse with standard conditions ( 20°C , long days ) . 20 of these plants were scored for petal number , and the other 20 were bagged carefully to recover all seeds . A fraction of the seeds was counted and weighed with the seed analyser MARVIN ( GTA Sensorik GmbH ) and total seed number was estimated by proportionality with the total seed weight .
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 .
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 .