An Evaluation of A‐Function: Evidence from the APETALA1 and APETALA2 Gene Lineages

An Evaluation of A‐Function: Evidence from the APETALA1 and APETALA2 Gene LineagesAuthor(s): Amy LittSource: International Journal of Plant Sciences, Vol. 168, No. 1, SpecialIssue<break></break>Discerning Homology: Gene Expression, Development, andMorphology<break></break><italic>Edited by Elena Kramer</italic> (January 2007), pp. 73-91Published by: The University of Chicago PressStable URL: http://www.jstor.org/stable/10.1086/509662 .

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AN EVALUATION OF A-FUNCTION: EVIDENCE FROM THE APETALA1AND APETALA2 GENE LINEAGES

Amy Litt1

New York Botanical Garden, New York, New York 10458, U.S.A.

The ABC model for the specification of floral organ identity, published 15 years ago, has proven to be auseful framework for interpreting floral development data from a wide variety of species. In Arabidopsis, A-function (specification of sepal and petal identity) is attributed to two unrelated genes, APETALA1 (AP1) andAPETALA2 (AP2). An examination of the available information regarding orthologues and paralogues ofthese genes in other species shows that although some are required for sepal identity, none is required for bothsepal and petal identity. Combined with phylogenetic analyses that show gene duplication and loss specific toBrassicaceae, this suggests that the two-whorl phenotype attributed to loss of A-function in Arabidopsis maybe unique to Brassicaceae. Furthermore, all genes that are required for proper sepal identity, including AP1 andAP2, are also implicated in floral meristem identity, suggesting that these two functions may not be separable.Available data are all consistent with a previous Antirrhinum-based model for floral organ identity thatrequired only two gene functions. The loss of sepal identity seen in some AP1- and AP2-lineage mutants can beexplained as loss of floral meristem identity; the available evidence suggests that a discrete perianth identitygene function is not required.

Keywords: A-function, APETALA1, AP1/FUL gene lineage, APETALA2, AP2/ERF gene family, ABC model,flower development.

The ABC model of flower development was formulated byCoen and Meyerowitz (1991) based on research on two well-known model plant species, Antirrhinum majus and Arabi-dopsis thaliana (Bowman et al. 1991; Coen and Meyerowitz1991). In each species, phenotypically similar mutants hadbeen described in which the identity of the floral organs wasaffected in pairs of adjacent whorls: sepals and petals, petalsand stamens, and stamens and carpels. In most cases, theseorgans were homeotically transformed into other floral or-gans, so that the number and position of organs was correctbut the identity was altered. Three molecular functions—A,B, and C—were postulated in the model, with each functionactive in two whorls of the meristem: A-function in the outertwo whorls, C-function in the inner two whorls, and B-functionin the middle whorls (fig. 1). In addition, A- and C-functiongenes were predicted to negatively regulate each other’s ex-pression, confining A to the outer two whorls and C to theinner two. Each whorl therefore would have a unique combi-nation of gene functions that would act to specify the organtype in that whorl: A ¼ sepals, Aþ B ¼ petals, Bþ C ¼ stamens,C ¼ carpels. Thus, if one gene function were missing, the identityof the organs in two adjacent floral whorls would be affected,which is indeed what has been observed in Arabidopsis and Antir-rhinum floral organ identity mutants.

In Arabidopsis, A-function is encoded by APETALA1(AP1) (Irish and Sussex 1990; Mandel et al. 1992; Bowmanet al. 1993), a member of the MADS-box family of transcrip-

tion factors, and APETALA2 (AP2) (Komaki et al. 1988;Bowman et al. 1989, 1991; Kunst et al. 1989; Jofuku et al.1994), an AP2/ERF (or AP2/EREBP) transcription factor. Inmutants of both of these genes, the organs of the outer whorlof the flower—the sepals—are transformed into leaflike orbractlike organs (or develop carpelloid features), and theorgans of the second whorl—the petals—either are absent orare transformed into stamenlike structures. Thus, both ofthese genes appear to be required for correct specification ofthe identity of sepals and petals. In addition, AP2 negativelyregulates C-function gene activity (Drews et al. 1991); ectopicexpression of the Arabidopsis C-function gene AGAMOUS isresponsible for the carpelloid and stamenoid features of theouter-whorl organs in some ap2 mutant alleles (Mizukamiand Ma 1992). Loss-of-function ap1 mutants do not showectopic AG expression (Weigel and Meyerowitz 1993); thus,the A-function role of repressing C-class gene activity has notbeen attributed to AP1. Recently, however, it has been sug-gested that AP1 interacts with LEUNIG and SEUSS to re-press AG activity (Gregis et al. 2006; Sridhar et al. 2006) ina complex including the MADS-box genes AGL24 and SVP(Gregis et al. 2006).

In the years since its publication, the ABC model has be-come widely accepted and has been used as a general frame-work for interpreting flower development in a variety ofspecies, from maize (e.g., Ambrose et al. 2000) to basal angio-sperms (e.g., Kim et al. 2005b; Li et al. 2005) to tulip (e.g.,Kanno et al. 2003), and for interpreting reproductive struc-tures of gymnosperms (e.g., Sundstrom et al. 1999; Fukuiet al. 2001; Sundstrom and Engstrom 2002). Some elementsof the model have been shown to be consistent with

1 E-mail [email protected].

Manuscript received February 2006; revised manuscript received September

2006.

73

Int. J. Plant Sci. 168(1):73–91. 2007.

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observations of flower development in a wide variety of spe-cies, with most of the attention focused on the evolution andconservation of B-function (e.g., Kramer et al. 1998; Sund-strom et al. 1999; Ambrose et al. 2000; Tzeng and Yang2001; Becker et al. 2002; Lamb and Irish 2003; Tzeng et al.2004; Vandenbussche et al. 2004). In addition, since the pub-lication of the model, the SEPALLATA genes (SEP1–SEP4)have been shown in Arabidopsis to provide an E-functionthat is also required for flower formation and for properspecification of floral organ identity (Pelaz et al. 2000, 2001b;Honma and Goto 2001; Theissen and Saedler 2001; Vanden-bussche et al. 2003b; Ditta et al. 2004). However, little atten-tion has been paid to the role of the putative A-functiongenes, the AP1-like and AP2-like genes, in flower developmentof other plant species. In fact, it is often overlooked that at thetime of the publication of the ABC model, no A-function genehad been identified in Antirrhinum.

Recently a model of MADS-domain protein activity hasbeen proposed (Theissen 2001; Theissen and Saedler 2001;Kaufmann et al. 2005) based on reports of interactionsamong Antirrhinum SQUA, DEF, and GLO proteins (ortho-logues of AP1 and of the B-function AP3 and PI, respectively)(Egea-Cortines and Davies 2000) and among ArabidopsisAP1, AP3, PI, AG, and SEP proteins (Honma and Goto2001). The quartet model explains the requirement for A-,B-, C-, and E-function proteins in the specification of organidentity by postulating the formation of interacting quartetsof proteins in each whorl. In the sepal whorl, the quartet isAP1/AP1/SEP/SEP, and in the petal whorl it is AP1/SEP/AP3/PI (Theissen 2001; Theissen and Saedler 2001; Kaufmannet al. 2005). These quartets are consistent with the whorls inwhich mutant phenotypes are observed for each of the genesencoding these proteins, and the proteins have been shown toform these associations in yeast (Honma and Goto 2001).

These observations have lent support to the notion that theseare functional protein associations in Arabidopsis and otherspecies and have reinforced the notion that AP1 is requiredfor A-function. However, although the quartet model re-mains a useful hypothesis consistent with genetic and proteininteraction evidence, these quartets have not been shown toform in planta, and it has not yet been demonstrated thatthese MADS-box proteins function as heterotetramers inspecifying organ identity in plant systems. Furthermore, alter-native hypotheses, discussed below, explain the phenotypicand genetic data regarding protein function in the outer twowhorls equally as well as the quartet model.

Although A-function has been well documented in Arabi-dopsis, examination of the available information suggests thatthis function may be confined to Arabidopsis or Brassicaceae(e.g., Shepard and Purugganan 2002). Further, it can be arguedthat the phenotypic characters associated with loss of A-functionmay in fact be by-products of loss of floral meristem identity(Davies et al. 1999b). In addition, there is evidence to suggestthat the function of AP1-like genes may have changed duringthe course of angiosperm evolution, suggesting that func-tional data from Arabidopsis should not be extrapolated toall angiosperms (Litt and Irish 2003). In this article, availabledata regarding the evolution of the AP1 gene lineage and theevidence regarding A-function are considered, hypotheses re-garding the function of the AP1 and AP2 lineages are evalu-ated, and an alternative model for the genetic specification offloral organ identity, first published by Schwarz-Sommer et al.(Schwarz-Sommer et al. 1990), is shown to fully explain theavailable data.

Arabidopsis A-Function Genes: APETALA2

The AP2 gene has been demonstrated to play a role in avariety of developmental processes, including maintenance ofthe stem cell population (Wurschum et al. 2006), ovule andseed development (Jofuku et al. 1994, 2005; Leon-Kloosterzielet al. 1994; Modrusan et al. 1994; Western et al. 2001; Ohtoet al. 2005), specification of floral meristem identity (Bow-man et al. 1989; Irish and Sussex 1990; Schultz and Haughn1993; Shannon and Meeks-Wagner 1993; Okamuro et al.1997b), regulation of homeotic gene function (Drews et al.1991), and definition of floral organ identity, particularly inthe outer two whorls (Komaki et al. 1988; Bowman et al.1989, 1991; Kunst et al. 1989). The Arabidopsis apetala2 mu-tant has a variable and complex phenotype, but all alleles showsome disruption of organ identity in the outer two whorls. Thephenotype of the weak allele ap2-1 varies with temperature(Bowman et al. 1989): the outer whorl is transformed into leaf-like structures with increasing degrees of carpellody (reflectingectopic expression of the C-function AGAMOUS; Mizukamiand Ma 1992) as the temperature increases; the second whorlis transformed generally into leaflike structures at low tempera-tures, but at higher temperatures the organs are stamenlike orabsent. Other alleles show even more variability (Kunst et al.1989; Bowman et al. 1991; Jofuku et al. 1994); for instance,medial sepals may show a mix of sepal and carpel characters,while lateral sepals are wild-type, carpelloid, or absent. Second-whorl organs may vary from petal to stamen (ap2-5), may be

Fig. 1 Schematic representation of the ABC model as described by

Coen and Meyerowitz (1991), superimposed on a longitudinal section(A) and a cross section or bird’s-eye view of a floral meristem (B).

Three gene functions, A, B, and C, are active in two adjacent domains

of the meristem—A in the outer two (perianth whorls), B in the middle

two (petal and stamen), and C in the inner two (reproductive organs).The result is a unique combination of gene functions in each whorl

that serves to specify a particular floral organ.

74 INTERNATIONAL JOURNAL OF PLANT SCIENCES



absent in strong alleles (ap2-7), or may consist of sepal-carpelchimeras and carpelloid structures. In some alleles, under short-day conditions, secondary flowers form in this whorl (Komakiet al. 1988). In most ap2 mutant alleles, the reproductive organsare not transformed, although stamen number may be reducedand the carpels may remain unfused. In strong alleles such asap2-6, ovules may take on carpellike features, suggesting ectopicC-function (Kunst et al. 1989). The role of AP2 in restrictingAG activity is further supported by the suppression of this phe-notype in the ap2 ag double mutant (Bowman et al. 1991) andby the expansion of the AG expression domain in the ap2 mu-tant (Drews et al. 1991).

These data demonstrate that AP2 is required in Arabidop-sis for correct sepal and petal identity and for repressingC-function in the outer two whorls, the two attributes of anA-function gene. In addition, the transformation in manyap2 mutant alleles of outer-whorl organs into leaflike orbractlike structures, along with the formation of secondaryflowers in the second whorl in some alleles, indicates that inthe ap2 mutant, the meristem is not completely converted tofloral identity. The outer two whorls display evidence of in-florescence character—bracts and branches bearing addi-tional flowers—thus, in addition to being an A-functiongene, AP2 is also required for the correct transition to a flo-ral meristem. However, the evidence that AP2 is required forsepal and petal identity rests on the same phenotypic charac-teristics that indicate that AP2 is required for floral meristemidentity. Although AP2 is formally required for correct speci-fication of sepal and petal identity, the disruptions to peri-anth identity may be explained by an incomplete transitionto a floral meristem coupled with variable expansion of the do-main of C-function in the absence of the boundary-definingcadastral activity of AP2.

AP2 Gene Lineage Phylogeny

The AP2 gene is one of ca. 145 members of the AP2/ERF(ethylene response factor) family (also known as the AP2/EREBP [ethylene response element-binding protein] family)(Okamuro et al. 1997a; Riechmann and Meyerowitz 1998;Sakuma et al. 2002; Kim et al. 2006; Shigyo et al. 2006)identified in the Arabidopsis genome (fig. 2). These putativetranscription factors are characterized by a conserved DNA-binding domain (AP2 domain; Weigel 1995), of which twocopies are present in the AP2 subfamily. Phylogenetic analy-ses of this subfamily have identified a number of duplicationsin the history of this family, including a deep split betweenthe AP2 and ERF genes that appears to have occurred beforethe diversification of vascular plants (Kim et al. 2006; Shigyoet al. 2006) (fig. 2). Subsequently, within the AP2 subfamilyclade, a domain duplication occurred, producing the twoAP2 domains that are characteristic of AP2-like sequences.After this domain duplication, a second gene lineage duplica-tion occurred. This duplication, which preceded the origin ofthe seed plants (Shigyo and Ito 2004; Kim et al. 2006; Shigyoet al. 2006), produced two gene clades that contain the twobest-known members of this family, AP2 (euAP2 clade) andANT (AINTEGUMENTA; Elliott et al. 1996; Klucher et al.1996) (ANT clade) (fig. 2). The euAP2 clade, which includesgenes from both gymnosperms and angiosperms, is character-

ized by the acquisition of a microRNA (miRNA) binding site(Kim et al. 2006; Shigyo et al. 2006), and posttranscriptionalregulation of some members of this clade has been shownto be mediated by miRNA172 (Aukerman and Sakai 2003;Chen 2004; Lauter et al. 2005). AP2 activity, for instance,has been shown to be confined to the outer two whorls ofthe flower because of the accumulation of miRNA172 in theinner two whorls that represses AP2 activity through transla-tional inhibition (Chen 2004). It is likely that all membersof the euAP2 clade are regulated through the action ofmiRNA172, although the precise mechanism may vary (Auker-man and Sakai 2003; Kim et al. 2006; Shigyo et al. 2006).

Other Arabidopsis euAP2 Genes

Another factor that may contribute to the discrepancy be-tween the size of the domain of expression of AP2 and thesize of the domain of observed function is functional redun-dancy with other AP2-like genes. Within the Arabidopsisgenome, there are five other related euAP2 genes (Kim et al.2006), which are candidates for this redundancy (fig. 2).However, of the four that have been characterized, all appearto play a role in floral repression, a function that has notbeen described for AP2. SNZ and SMZ, which fall in theeuAP2 clade but have only one AP2-binding domain, areclosely related and redundant paralogues that were isolatedthrough activation tagging (Schmid et al. 2003). TOE1(TARGET OF EAT1) and TOE2 were isolated in a screenfor targets of EAT1 (EARLY ACTIVATION TAGGED1),which proved to be a precursor RNA for a member of themiRNA172 family (Aukerman and Sakai 2003). These genesare all implicated in phase transition, but none has beenshown to play a role in meristem identity, organ identity, orcadastral activity.

The presence of a total of six euAP2 genes and ca. 140 ad-ditional AP2/ERF genes in the Arabidopsis genome (Riech-mann and Meyerowitz 1998; Sakuma et al. 2002; Kim et al.2006) highlights the complexity of this gene family. Duplica-tions have been common and are represented by ancientsplits, such as the ERF/AP2 split, as well as by recent ones,

Fig. 2 Schematic phylogeny of the AP2/ERF gene family, after Kimet al. (2006). The relationships among Arabidopsis’s six euAP2 and 12

ANT (including eight euANT) genes are indicated.

75LITT—EVALUATION OF A-FUNCTION



such as the SNZ/SMZ split (fig. 2). These duplications pres-ent two challenges for studies of AP2-like genes. First, func-tional redundancy is common, as illustrated by SNZ/SMZ.It is likely that even in the case of the well-characterizedAP2 gene there are paralogues with redundant functions thathave not yet been discovered. For instance, Wurschum et al.(2006) recently determined that as-yet-unidentified factorsact redundantly with AP2 in stem cell maintenance. Second,orthology of genes from other species is difficult to deter-mine. Sampling is thorough from only Arabidopsis and rice,and it is almost certain that in other species many paralogueshave yet to be identified. This incomplete sampling makes itdifficult to pinpoint the phylogenetic position of duplicationevents. As a result, it is not possible to determine whethercharacterized genes from other species are paralogues or or-thologues of AP2, leaving functional comparisons on a some-what shaky foundation.

euAP2 Genes from Other Species

Two putative euAP2 orthologues from Antirrhinum, LIP-LESS1 and LIPLESS2 (LIP1/2; Keck et al. 2003), probablyarose through a recent duplication and appear to be com-pletely functionally redundant. The single lip1 and lip2 mu-tants show no abnormal phenotype, but the double lip1/2mutant suggests a role for these genes in flower organ devel-opment that shares features with that of AP2. In the outerwhorl, sepals are transformed into leaflike organs, as evidencedby their larger size and the presence of a glandular tip similarto that seen in leaves and bracts (Keck et al. 2003). Second-whorl organs have petal identity but are smaller, and the sizesof the lip and palate regions are reduced. Unlike AP2, LIP1/2 also strongly affect reproductive organ development: sta-men filaments are shorter than in the wild type, as is thestyle, whereas the ovary is larger, and ovules may have car-pelloid characteristics, as in some AP2 alleles. Secondaryflowers are not reported, nor is the Antirrhinum C-functiongene PLENA expressed ectopically in the absence of LIP1/2function, except perhaps in the ovules. Thus, lip1 lip2 flow-ers share with ap2 flowers the partial loss of sepal identi-ty—one of the characteristics of A-function mutants—butnot the control of petal identity or the regulation of C-func-tion activity. Keck et al. (2003) interpret the phenotype oflip1 lip2 mutants as showing that the organ identity and ca-dastral components of A-function can be separated. How-ever, another interpretation consistent with the data is thatLIP1/2 are required for the transition to a floral meristem, asevidenced by the transformation of sepals to leaflike organsin the double mutant and misregulation of aspects of growthcontrol, but that they do not have a distinct function in speci-fying organ identity or regulating C-function activity. Thus,similar to AP2, LIP1/2 do not appear to have organ identityactivity that can be separated from their role in specifyingmeristem identity.

No other putative orthologues of AP2 have been shown tohave an endogenous function in flower development. The pu-tative petunia orthologue of AP2 is PhAP2A, which canfunctionally substitute for AP2 in Arabidopsis; however, thePetunia mutant shows no floral phenotype (Maes et al.2001). Maes et al. suggest that the role of Arabidopsis AP2

in directing sepal identity rests largely in specifying the epi-dermal characteristics of sepals; in the absence of AP2 func-tion, leaf epidermal characteristics are retained. In petunia,the epidermis of the sepals and leaves is similar; therefore,PhAP2A does not share this role with AP2 (Maes et al.2001). Thus, PhAP2A does not appear to have A-function,but there may be redundant factors as yet unidentified.

Two AP2-like genes from maize, INDETERMINATESPIKELET 1 (IDS1; Chuck et al. 1998) and GLOSSY15(GL15; Moose and Sisco 1994, 1995), have also been char-acterized. GL15 plays a role in maintaining the juvenilestate; in loss-of-function mutants, the juvenile state is abbre-viated as determined by the epidermal features of the leaves(Moose and Sisco 1994, 1996). Moose and Sisco (1996) notethat GLOSSY15 plays a role in lateral organ identity similarto that of AP2, but in this case it is the identity of vegetativeorgans. An apparently unrelated role has been reported forIDS1, which is required for formation of a determinate spike-let meristem (Chuck et al. 1998). In the absence of IDS1 func-tion, extra florets are produced. IDS1 does not complementthe ap2 mutant in Arabidopsis, and the sequences are similarmainly in the conserved AP2-binding domain (Chuck et al.1998); thus, Chuck et al. (1998) suggest that IDS1 is not theorthologue of AP2, and the analysis of Kim et al. (2006) pla-ces it in a separate subclade of the euAP2 gene clade. Thus, todate, there are no data to suggest that either GL15 or IDS1plays a role in flower structure or floral organ identity.

Additional information is available regarding the mRNAexpression patterns of these and other putative AP2 ortho-logues, including some from gymnosperms (Vahala et al.2001; Shigyo and Ito 2004; Kim et al. 2006); however, wewould not expect these patterns to be informative if AP2-likegenes are regulated posttranscriptionally, as appears to be thecase. In Arabidopsis, for instance, AP2 is expressed in imma-ture flower buds, in all four developing floral organ types, inovules and seeds, and in stem and leaf tissue (Jofuku et al.1994; Okamuro et al. 1997a) (root tissue was not reported).However, the mutant phenotype is largely confined to theseed coat, the outer two whorls of the flower, and, in somealleles, the ovules. Thus, the expression domain is consider-ably larger than the functional domain—reflecting the modeof regulation as well as possible redundancy—and whetheror not an AP2-like gene in a different species shows thissame transcriptional expression pattern is likely not to be in-formative. At the very least, information on miRNA172 ex-pression, in conjunction with AP2-like gene expression, isneeded in order to estimate functional domains of AP2-likegenes.

The available functional data suggest a possible role forAP2 and its paralogues and orthologues in phase change toreproduction and do not support the hypotheses that euAP2genes are required for specifying perianth identity or restrict-ing C-function gene activity. These features are found to-gether only in Arabidopsis AP2, but even here the role insepal and petal identity is complicated by the additional rolein meristem identity. Furthermore, the ap2 ag double mutantforms petals and petaloid second-whorl organs (Bowmanet al. 1989, 1991); therefore, AP2 is not strictly required forspecification of petal identity. No other members of theeuAP2 clade have been shown to restrict C-function activity,




although few genes have been characterized, and even inthose cases, redundancy cannot be ruled out.

Arabidopsis A-Function Genes: ANT

The potential for functional redundancy, which may maskA-function in some AP2-like genes, is not limited to membersof the euAP2 clade. The sister ANT clade includes 12 genesthat must also be considered in this analysis (fig. 2). AINTEG-UMENTA, for which the ANT clade of AP2-like genes isnamed, plays a wide variety of roles in floral development,mainly through promotion of growth of floral organs. Mu-tants show decreased number and size of floral organs aswell as alterations in the structure of all floral organs (Kunstet al. 1989; Elliott et al. 1996; Klucher et al. 1996; Bakeret al. 1997; Sanders et al. 1999). ANT function is also re-quired for ovule integument initiation and megagametophyteformation (Elliott et al. 1996; Klucher et al. 1996) and forfusion of gynoecium margins (Liu et al. 2000). Flowers ofant mutants do not show homeotic organ transformation;however, the abaxial epidermis of second-whorl organs showssome loss of petal identity: cells in the center of the abaxialside resemble anther epidermis, and stomata are present (Krizeket al. 2000). In ap2 ant double mutants, an enhanced ap2second-whorl phenotype is seen, varying from leaflike inearly flowers to stamenlike in later flowers; a similar en-hancement of the ap1 phenotype is seen in ap1 ant doublemutants. The ap2 ant double mutant also shows increasedectopic AG expression over the ap2 mutant; however, loss ofANT function alone results in no change in AG expression.

The evidence suggests that ANT has roles in both organidentity and C-function gene regulation, but only in the sec-ond whorl (Krizek et al. 2000). Krizek et al. (2000) thus sug-gest ANT as a second-whorl A-function gene. ANT does playa role in these two components of A-function; however, therestriction of activity to the second whorl indicates that ANTis not a canonical A-function gene, since ‘‘ABC’’ genes are de-fined as acting in two adjacent whorls. Among the many rolesplayed by ANT in floral development, two are specificationof abaxial petal epidermis identity and second-whorl AG reg-ulation, but ANT cannot be considered a true A-function genewithout redefining the ABC model.

Other Arabidopsis ANT Genes

The Arabidopsis genome possesses 11 additional genesthat fall into the ANT clade (Kim et al. 2006) (fig. 2) andthat should be examined for possible redundancy that maymask A-function. Seven of these, referred to as AINTEGU-MENTA-like (AIL) genes (Nole-Wilson et al. 2005), belong,with ANT, to the euANT subclade of Kim et al. (2006). Sev-eral AIL genes have been characterized, and all have beenproposed to play a role in developmental processes involvingcell proliferation, similar to what has been proposed forANT (Krizek 1999; Mizukami and Fischer 2000). None todate has been identified as having a role in organ identity orAG regulation. PLETHORA1 and PLETHORA2 (PLT1 andPLT2), highly similar and redundant genes, are required inthe root apical meristem for specification and maintenance of

stem cell identity (Aida et al. 2004), similar to the role playedby AP2 in the shoot meristem (Wurschum et al. 2006). Over-expression of AINTEGUMENTA-like5 resulted in increasedfloral organ size as a result of increased cell proliferation(Nole-Wilson et al. 2005) similar to that seen with overex-pression of ANT (Krizek 1999). BABY BOOM (AtBBM) hasbeen suggested to play a role in embryo development basedon studies of the putative Brassica napus orthologue BBM(Boutilier et al. 2002). Overexpression of BBM in both B. napusand Arabidopsis caused the formation of ectopic embryosand cotyledon-like structures on seedlings. At later stages, trans-genic Arabidopsis showed a variety of pleiotropic effects, in-cluding dwarfing and alterations of leaf and flower structure(Boutilier et al. 2002). WRINKLED1, an Arabidopsis ANT-clade gene that falls outside of the euANT clade (Kim et al.2006) and is not included in the AIL genes (Nole-Wilsonet al. 2005) (fig. 2), shows a divergent function in regulatingseed oil content (Cernac and Benning 2004).

Nole-Wilson et al. (2005) analyzed expression of the sevenAIL genes in comparison with ANT, and all showed patternsconsistent with roles in developing plant tissues similar tothat of ANT. ANT is expressed broadly in vegetative and re-productive tissues, particularly in developing organ primor-dia (including floral organs and leaves) and strongly in ovules(especially the developing integuments), but also in floralmeristems, embryos, and roots (Elliott et al. 1996; Klucheret al. 1996). Quantitative RT-PCR studies show that allAIL genes (PLT1, PLT2, AtBBM, and AIL1, 5, 6, 7) are ex-pressed at higher levels in young developing tissues such asroots and siliques and at lower levels in mature tissues. Insitu mRNA hybridization of several AIL genes in inflores-cence tissue shows significant overlap of expression; combinedwith the absence of an observable phenotype in transgenicArabidopsis plants in which no AIL5 mRNA was detectable,these data reinforce the pervasiveness of redundancy in theAP2 gene family. To date, no data suggest a role for theseANT-like genes as A-function, petal identity, or cadastralgenes, but future teasing apart of the functional repertoiresof the euANT genes and other AP2-like genes may yet bringthese functions to light.

ANT Genes from Other Species

Relatively few data are available regarding ANT-clade genesfrom other species. Expression data are available for two genesthat appear to be orthologues of ANT (Kim et al. 2006),NtANTL from Nicotiana tabacum (Rieu et al. 2005) andPtANTL1 from Pinus thunbergii (Shigyo and Ito 2004). Bothgenes are expressed strongly in female reproductive structuresand in leaves, as is ANT. PtANTL1 is also weakly expressed inmale cones. The lack of additional data from these genes, fromother ANT-like genes in these species, and from other species,makes it difficult to formulate hypotheses regarding the role ofthese genes in species beyond Arabidopsis.

A-Function in the AP2/ERF Gene Family

The available data for genes belonging to either the euAP2or the ANT clade of the AP2/ERF gene family do not cur-rently suggest that these genes generally play a significant role




in either perianth identity or regulation of C-function geneactivity. In fact, those roles have been demonstrated only forthe two eponymous members of those clades, AP2 and ANT.In the case of AP2, interpretation of the perianth phenotypeis complicated by the role of the gene in meristem identity. Inthe case of ANT, the restriction of the functions to the secondwhorl rules this gene out as a canonical A-function gene.

The functional data for other euAP2 genes from Arabidop-sis, as well as from other species, show significant variability,which may reflect the fact that all paralogues are not repre-sented from other species. For instance, SNZ/SMZ from Arabi-dopsis function in floral repression (Schmid et al. 2003),whereas LIP1/2 from Antirrhinum function in meristem iden-tity and floral organ size (Keck et al. 2003). Whereas the func-tions of these euAP2 genes are very different, the two pairsof genes belong to different euAP2 subclades, and Antirrhinumorthologues of SNZ/SMZ have not been identified. Thus, al-though all are euAP2 genes, a comparison between SMZ/SNZ and LIP1/2 may be inappropriate. In fact, LIP1/2 be-long to the same euAP2 subclade as AP2 (Kim et al. 2006)and are functionally most similar to that Arabidopsis gene.Thus, characterization of additional Antirrhinum genes (andgenes from other species) may turn up genes that are ortholo-gous to and functionally similar to SMZ/SNZ and may lendmore order to the patterns observed. Furthermore, identifica-tion of redundant functions may also clarify the array offunctions represented by euAP2 genes.

Just as examination of data available on other euAP2genes does not turn up evidence for other A-function genesin Arabidopsis or other species, examination of other ANTgenes does not turn up such evidence either. In both geneclades there is ample evidence of redundancy, so the posses-sion of A-function by some of these genes cannot be ruledout at this point. All of the genes characterized in the ANTclade, including ANT, belong to the euANT subclade exceptone (WRINKLED1) (Kim et al. 2006), and it is plausiblethat these all play roles in cell proliferation and developmentof young organs (Boutilier et al. 2002; Aida et al. 2004;Nole-Wilson et al. 2005). None has been demonstrated tospecify organ identity or to regulate the expression of floralhomeotic genes, but much remains to be known about thesegenes. In particular, there are virtually no available data onthe expression or function of ANT-like genes in other species.

Arabidopsis A-Function Genes: APETALA1

Like most of the genes identified as performing A-, B-, orC-functions (all except AP2, in fact), AP1 is a member of theMADS-box family of transcription factors (Mandel et al.1992). These genes, which have diversified greatly in plants,play key roles in a variety of developmental processes in-cluding floral induction, embryo development, floral organspecification, fruit development, and others. MADS-domain-containing proteins all share a highly conserved MADSDNA-binding domain at or near the N terminus (Ma et al.1991; Riechmann et al. 1996b). A subset of plant proteins,including those identified so far as being important in devel-opmental processes, also share a structurally conserved Kdomain, implicated in dimerization (Riechmann et al. 1996a)

and separated from the MADS domain by a short, somewhatmore variable I (intervening) domain (Munster et al. 1997;Alvarez-Buylla et al. 2000). The C-terminal domain of theseproteins is highly variable in sequence and function and isstill poorly characterized. However, many of the ABC line-ages, including the AP1 lineage, have been shown to be char-acterized by short, highly conserved amino acid motifslocated at the C terminus of the protein (Kramer et al. 1998;Litt and Irish 2003; Zahn et al. 2005).

In the absence of AP1 function, the organs in the outerwhorls of the Arabidopsis flower are homeotically trans-formed into bractlike structures (occasionally with carpelloidfeatures at 30�C) (Irish and Sussex 1990; Bowman et al.1993). Petals are missing. Instead, in the second whorl thereare secondary flowers, which do not directly replace thepetals but are subtended by the first-whorl organs. The sec-ondary flowers may repeat the pattern, giving rise to tertiaryflowers with the same morphology. In the center of the pri-mary flower, the stamens and carpels are relatively normaland are fertile. Thus, AP1 provides the A-class function ofspecifying the identity of the sepals and petals; however, incontrast to what is observed in the ap2 mutant, the expres-sion domain of AG is not altered in the ap1 mutant, indicat-ing that AP1 does not have a role in regulating C-functiongene activity. In fact, AP1 was not included as an A-functiongene in early formulations of the ABC model (Bowman et al.1991; Coen and Meyerowitz 1991; Meyerowitz et al. 1991).It was only when AP1 expression was discovered to be re-stricted to the outer two whorls by the C-function locus AG(Gustafson-Brown et al. 1994) that it was included as anArabidopsis A-function gene along with AP2.

AP1 shares an additional function with AP2, that of deter-mining the identity of the floral meristem. The reduction inflowering and the increase in branching of the ap1 mutant in-dicate that AP1 function is required for the transition from abranched inflorescence to a determinate flower. This is sup-ported by the homeotic conversion of the sepals to bractlikeorgans that would characterize an inflorescence rather than aflower. Therefore, as with AP2, the same phenotype is usedboth to designate AP1 as an A-function gene and to ascribe toit a role in floral meristem determination. And as with AP2, itcan be argued that the role of AP1 as an organ identity genemay not be separable from its role as a meristem identitygene: loss of floral identity in the outer whorls leads to the for-mation of bracts in the outer whorl and branches in the sec-ond whorl. Furthermore, evidence that AP1 is not requiredfor the formation of petals comes from the ap1 ag double mu-tant (Bowman et al. 1993) and ap1 mutants in which SEP3 isconstitutively expressed (Castillejo et al. 2005), in whichnearly wild-type petals may be found in the second whorl.

The quartet model (Theissen 2001; Theissen and Saedler2001; Kaufmann et al. 2005) postulates that AP1 is one com-ponent of functional tetramers required for the specificationof sepals (AP1/AP1/SEP/SEP) and petals (AP1/SEP/AP3/PI).However, although we know that petal identity is not specifiedwithout these proteins, we have no direct evidence that theyare acting as a tetramer in Arabidopsis. It is equally consistentwith the data that AP1 and SEP have roles in meristem iden-tity, while AP3 and PI play a separate role in petal identityonce the meristem has been florally determined. In fact, the




observation that the second-whorl phenotype of the ap1 mu-tant (complete loss of petals) is very different from that of theap3 or pi mutant (petals converted to sepals) suggests that ei-ther these proteins are in fact not acting in a complex or AP1has other roles that, when absent, abolish petal formation. Sucha role could be the specification of floral meristem identity.

In the first whorl, the quartet model postulates that anAP1/AP1/SEP/SEP quartet is required for specification of se-pal identity. But the homeotic conversion of sepals to bractsis also consistent with a loss of floral meristem identity, afunction that has been attributed to AP1 and SEP3 singly aswell as to combinations of SEP genes (Honma and Goto2001; Pelaz et al. 2001a, 2001b; Ditta et al. 2004). Thus,there is no evidence that this quartet is explicitly specify-ing sepal, rather than meristem, identity; a quartet may beformed from AP1 and SEP proteins, but that quartet may beimplicated exclusively in meristem identity. AP1 and SEP di-mers functioning separately must also be ruled out; it re-mains to be demonstrated that the quartets specified in thequartet model exist as functional units in Arabidopsis.

The Role of AP1 in Repressing AGL24

The hypothesis that AP1 does not have a discrete role inorgan identity is borne out by the finding that a crucial func-tion of AP1 is the repression of AGL24, which has beenshown to play a key role in the maintenance of vegetativeand inflorescence meristem identity (Yu et al. 2004). Duringthe transition from an inflorescence to a floral meristem, AP1and LFY act to repress AGL24 expression in the corpus ofthe meristem and in the developing sepal and petal primordia(it is detected in stamen and carpel primordia, where AP1 isnot known to function). In the absence of AP1 function,AGL24 expression continues throughout the meristem and inthe developing organs, which in the outer two whorls developas inflorescence structures (bracts subtending secondarybranches) instead of floral structures. Loss of AGL24 functionsignificantly reduces the inflorescence characteristics of theap1 mutant; in the ap1 agl24 double mutant, meristems gener-ally develop as single flowers, as in the wild type, and perianthdefects such as the absence of petals are partly restored (Yuet al. 2004). This partial restoration of petal identity inthe ap1 agl24 mutant demonstrates further that AP1 functionis not strictly required for the formation or identity of theseorgans. Thus, the role of AP1 in determining the identity ofperianth organs, although formally recognizable by the mis-specification of these structures in the ap1 mutant, cannot beseparated from its role in determining the identity of the floralmeristem (Theissen et al. 2000), which it achieves by repres-sing the function of AGL24 (Yu et al. 2004).

Arabidopsis AP1 Paralogues: Redundancy

As is the case in the AP2 gene family, the AP1 lineage hasundergone numerous duplications in various angiospermclades (Litt and Irish 2003), but in this case we have a betterunderstanding of the phylogeny and the function of some ofthe paralogues. One duplication is probably confined to theBrassicaceae (Purugganan 1997; Lowman and Purugganan1999) (fig. 3) and produced the Arabidopsis paralogueCAULIFLOWER (CAL), which is nearly identical in se-

quence to AP1 and is redundant for specifying floral meri-stem identity (Bowman et al. 1993; Kempin et al. 1995). Thecal mutant has no abnormal phenotype, but the ap1 cal dou-ble mutant shows a further decrease in flowering and in-crease in meristem proliferation.

Fig. 3 Schematic phylogeny of the APETALA1 (AP1) gene lineage,after Litt and Irish (2003) and A. Litt (unpublished data). SEPALLATAand AGL6 genes were used as outgroups. The types of genes (AGL6,

SEPALLATA, FUL-like, euFUL, euAP1) found in each gene clade areindicated to the left of the vertical lines. The taxa from which those genes

were obtained are indicated to the right of the lines. The SEPALLATAgenes were obtained from across the angiosperms, and the AGL6 genes

were obtained from across both angiosperms and gymnosperms; each isshown as one clade here. Core eudicot AP1-lineage gene clades are

indicated by light gray triangles, non-core-eudicot clades by dark gray,

and the outgroups by black. Dotted lines indicate branches lacking high

bootstrap support. The position of the core eudicot FUL-like clade isuncertain; some analyses resolve this clade as sister to the euFUL clade,

whereas others resolve it as an earlier branch that precedes the euFUL/

euAP1 split.Within the three core eudicot clades, the relationshipsamongthe various Arabidopsis (AP1, CAL, FUL, T6J22.1) and Antirrhinumgenes (SQUA, AmFUL, DEFH28) are indicated. T6J22.1 has been

identified from genomic sequencing as being similar to AP1 lineage genes,

but a transcript has never been identified.




Recent analyses of phenotypes that resulted from constitu-tively expressing SEP3 in Arabidopsis uncovered a crypticrole for CAL in petal formation (Castillejo et al. 2005). In ap1mutants transformed with 35S::SEP3, petals were formed inthe second whorl. However, in ap1 cal double mutants simi-larly transformed, petals were not formed. This evidence sug-gests that in the absence of AP1 function, CAL may becapable of contributing to second-whorl floral organ identity,perhaps through a role in specifying floral meristem identity.

A third related and partially redundant gene, FUL (Mandeland Yanofsky 1995a; Ferrandiz et al. 2000a) (fig. 3), is theresult of a more ancient duplication that coincided with theorigin of the core eudicot clade that includes Arabidopsis andAntirrhinum. FUL shares with AP1 and CAL a function infloral meristem specification (Mandel and Yanofsky 1995a;Ferrandiz et al. 2000a): the triple mutant rarely forms flow-ers but instead branches prolifically. This meristem identityrole of FUL is manifest only in the absence of AP1 activity,which normally excludes FUL from the floral meristem. FULalso has a unique function in fruit development that is dis-tinct from those of AP1 and CAL (Gu et al. 1998; Ferrandizet al. 2000b; Liljegren et al. 2000, 2004; Ferrandiz 2002).No role is known for FUL in organ identity.

The presence in the Arabidopsis genome of three paralo-gous genes, each making varying contributions to floral meri-stem identity, complicates attempts to interpret the roles ofAP1-lineage genes in other species. The frequent occurrenceof gene duplications in this lineage means we must evaluatethe functions of AP1/CAL and FUL orthologues and para-logues in other species in order to understand the roles ofthese genes, how they have diversified, and the extent towhich the functions of the Arabidopsis representatives of thislineage are conserved in other species.

AP1 and FUL Orthologues in Antirrhinum

The Antirrhinum orthologue of AP1, SQUAMOSA (SQUA),does not appear to fill any of the functional roles of an A-classgene. The squa mutant shows decreased flowering and signif-icantly increased branching in the inflorescence, indicatingthat SQUA shares the role of AP1 in determining floral meri-stem identity (Huijser et al. 1992). But in the case of squa,when flowers are formed, the identity of the organs is cor-rect, although the flowers may be malformed. As with AP1,SQUA does not appear to regulate the function of PLENA,the Antirrhinum C-function gene.

Because the CAL duplication is confined to the Brassica-ceae, Antirrhinum has only one locus, SQUA, that is ortholo-gous to both AP1 and CAL. It may be that SQUA fills thefunction of both these genes combined; indeed, the severityof the loss of floral identity of squa is more similar to that inap1 cal than to that in ap1. It is notable, however, that whenap1 cal plants do form flowers, the structure of the flowers issimilar to that of ap1 flowers, in contrast to the flowersformed by squa. This suggests functional divergence betweenAP1þCAL, on the one hand, and SQUA, on the other, incontrol of the identity of the outer whorls, and it may reflectthe cryptic role of CAL in second-whorl identity.

Because the duplication that produced AP1 and FUL pre-dates the evolutionary separation of the angiosperm lineages

that produced Arabidopsis and Antirrhinum, Antirrhinumdoes have a FUL orthologue, AmFUL (Litt and Irish 2003).However, this gene has not been functionally characterized.The Antirrhinum genome also contains a second FUL-likegene that is highly similar to AmFUL (DEFH28) and showsan expression pattern similar to that of FUL (Muller et al.2001) but is in a paralogous clade (Litt and Irish 2003) (fig. 3).Expression of DEFH28 in Arabidopsis under the control ofthe constitutive CaMV 35S promoter causes early floweringand production of an abnormally constructed terminal flower(Muller et al. 2001) as well as indehiscent fruits, similar towhat has been described for constitutive expression of FUL(Ferrandiz et al. 2000b). Thus it is plausible that DEFH28plays a role in Antirrhinum similar to that of FUL in Arabi-dopsis, including a role in meristem identity. Arabidopsisdoes not appear to have an orthologue of DEFH28, althougha possible pseudogene with strong sequence similarity to FULhas been identified from genomic sequencing and may repre-sent a divergent member of that clade (Carlsbecker et al.2003; A. Litt, unpublished data) (fig. 3). On the basis of thesedata, a reasonable hypothesis is that in Antirrhinum, at leastboth SQUA and DEFH28 are involved in floral meristem de-velopment. Furthermore, the contributions of AmFUL and/orDEFH28 to flower development in Antirrhinum, and of CALin Arabidopsis, may be responsible for the phenotypic differ-ences between Antirrhinum squa and Arabidopsis ap1 mu-tants. None of these Antirrhinum genes have been implicatedin organ identity or regulation of C-function activity.

AP1-Lineage Genes from Other Species

The AP1-lineage duplication at the base of the core eudicotsproduced two distinct core eudicot gene clades, the euAP1clade, which includes AP1 (and CAL) and SQUA, and theeuFUL clade, which includes FUL and AmFUL (the positionof the DEFH28-containing clade is ambiguous; see fig. 3)(Litt and Irish 2003). Phylogenetic analyses show us that an-giosperm taxa that diverged before this duplication have onlyone type of gene. Notably, these genes show sequence simi-larity to euFUL genes rather than euAP1 genes (Litt and Irish2003) (fig. 4). This sequence similarity suggests that theseFUL-like genes will be functionally more similar to euFULgenes than to euAP1 genes. Nonetheless, a number of geneswere evaluated as AP1-like genes but were subsequently shownby phylogenetic analyses to be euFUL or FUL-like genes (seebelow). This has produced a confusing array of data and in-terpretations regarding the activity of genes of the AP1 line-age. In the section below, available functional and expressiondata are considered in the context of gene lineage phylogenyin order to assess whether they provide evidence that AP1-lineage genes play a role in specifying perianth identity orregulating C-function activity.

euAP1 Genes

Information from other members of the euAP1 clade,which includes AP1 and SQUA, does not provide additionalevidence for a role for these genes in sepal and petal identitybut suggests that these genes play a role in determining flo-ral meristem identity. The pim mutant (PROLIFERATING




INFLORESCENCE MERISTEM) from pea (Pisum sativum)is caused by a defect in the PEAM4 gene. Although pea ismore closely related to Arabidopsis than to Antirrhinum (An-giosperm Phylogeny Group 2003), the pim phenotype is simi-lar to that of squa (Berbel et al. 2001; Taylor et al. 2002),with an increase in branching and a decrease in flowering butno homeotic transformation of perianth organs. LeMADS_MC,an orthologue from tomato (Solanum lycopersicum, which ismore closely related to Antirrhinum; Angiosperm PhylogenyGroup 2003), also has a phenotype consistent with a role infloral meristem identity: the sepals are enlarged in the mutant,

and inflorescence determinacy is lost (Vrebalov et al. 2002). Innone of these mutants is there evidence that the euAP1 ortho-logue plays a role in petal identity.

Other functional data come from heterologous transforma-tion experiments. In general, these experiments show eitherpartial rescue of the Arabidopsis ap1 mutant phenotype, in-cluding perianth identity (e.g., Berbel et al. 2001; Shchenni-kova et al. 2004), or recapitulation of the Arabidopsis AP1overexpression phenotype of early flowering and formationof a compound terminal flower (e.g., Berbel et al. 2001;Shchennikova et al. 2003; Fernando and Zhang 2006). Eloet al. (2001) expressed the birch (Betula pendula) BpMADS3constitutively in tobacco, resulting in early flowering. Althoughthese experiments tell us what those genes will do in an Arabi-dopsis or tobacco genetic background, they do not necessarilypredict their endogenous roles. Furthermore, overexpressionexperiments do not give us information on a possible role forthese genes in perianth identity because constitutive expressionof AP1 produces no organ identity phenotype (Mandel and Ya-nofsky 1995b). As a result, these data are consistent with a rolefor euAP1 genes in floral meristem identity but are not infor-mative regarding a role in organ identity.

Comparison of expression patterns of euAP1 orthologues(as well as of euFUL and FUL-like genes) is limited by thedifferent techniques used and different sets of organs assayedby different researchers. Nonetheless, it can be said thateuAP1 genes are expressed in floral meristems and often inperianth parts (table 1). Both AP1 (Mandel et al. 1992) andPEAM4 (Berbel et al. 2001) are expressed in the floral meri-stem, developing perianth, and pedicel, while SQUA is addi-tionally expressed in bracts and carpels (Huijser et al. 1992).Expression of SLM4, the orthologue from Silene latifolia,conforms to the same pattern as SQUA, with expressionalso seen in the inflorescence meristem (Hardenack et al.1994); Hardenack et al. (1994) suggest that the expression ofSLM4 in both inflorescence and floral meristems reflects thepresence of a terminal flower in Silene. CDM111 from chry-santhemum (Dendranthema 3 grandiflorum) is expressed in in-florescence meristems and bracts as well as in petals, stamens,and carpels (the last only in disk flowers; Shchennikova et al.2003). In other cases, data from meristems are not reported;TOBM2, from tobacco (Nicotiana tabacum), is expressed

Fig. 4 Representative predicted amino acid sequences of AGL6-

like, SEPALLATA, FUL-like, euFUL, and euAP1 genes. Only theC-terminus is shown. Conserved motifs are in bold and boxed. The FUL-

like motif can be seen in the SEPALLATA sequences and the AGL6sequences, although it is somewhat less conserved in the latter. Notethe strictly conserved tryptophan (W). The FUL-like motif is absent

from euAP1 sequences, but two new motifs (transcription activation

and farnesylation) are seen. Question marks indicate missing data.

Table 1

Summary of Published Angiosperm Expression Patterns of Genes Belonging tothe AP1/FUL Gene Lineage and the AGL6 Gene Lineage

LeavesCauline leaves/

bractsInflorescence

meristemFloral

meristem Sepals Petals Stamens Carpels

euAP1 � 6 6 þþ þþ þþ � þeuFUL þþ þþ 6 6 þ þ 6 þþFUL-like þþ þ 6 6 þ þ þ þþAGL6-like � 6 6 þþ þþ þ þþ

Note. Blank cells indicate that insufficient data have been reported to assess expression in those or-

gans. � ¼ no expression reported; 6 ¼ expression in fewer than 50% of reports; þ ¼ expression in

50%–80% of reports; þþ ¼ expression in greater than 80% of reports. The assessments are approxi-mate because different studies have assayed different organs and structures using different techniques;

thus, in some cases, categories do not exactly match. In addition, relatively few AGL6-like genes, and

few FUL-like genes outside of the grasses, have been studied; thus, the patterns documented may not

hold when additional species are included.




in all four whorls of floral organs and particularly strongly incarpels (Wu et al. 2000), and LeMADS_MC is expressedin all floral organs except stamens (Vrebalov et al. 2002; Hile-man et al. 2006). A divergent pattern is found in the case ofGSQUA, from Gerbera hybrida–like chrysanthemum, a mem-ber of the Asteraceae with a capitulum inflorescence. GSQUAappears to be expressed in developing vascular bundles withinflowers and the capitulum receptacle (Yu et al. 1999). Thesedata in general are consistent with a role in meristem identityas well as a role in perianth identity (A-function), although ex-amples such as Gerbera suggest there has been divergence offunction in some taxa.

euFUL Genes

Within the euFUL gene clade there have been numerous du-plications within various core eudicot groups, and in generalneither expression nor functional data are available for allparalogues in any species. In fact, few FUL orthologues havebeen functionally characterized; thus, it is difficult to form acomprehensive picture of the activity of euFUL genes. In oneof the few studies to obtain direct functional data, cosuppres-sion of one of the several Petunia 3 hybrida FUL orthologues,PFG, was shown to result in plants that remain vegetative in-definitely (Immink et al. 1999).

Several heterologous-transformation experiments have beenperformed using euFUL genes, and in most cases these wereundertaken with comparisons with AP1 in mind (e.g., Kyozukaet al. 1997; Jang et al. 2002). In general these experimentsshow that the orthologues can either partially complementthe ap1-1 phenotype (e.g., Jang et al. 2002) or recapitulatethe 35S::AP1 phenotype in Arabidopsis (e.g., Kyozuka et al.1997). MdMADS2 from apple (Sung et al. 1999) andBpMADS4 and BpMADS5 from birch (Elo et al. 2001) pro-duced early flowering when constitutively expressed in tobacco.These results are interpreted as supporting the identificationof these genes as AP1-like (Mandel and Yanofsky 1995b), butthey are also consistent with their identification as FUL-like(Ferrandiz et al. 2000b). No fruit phenotypes are describedexcept for DEFH28 (Muller et al. 2001), possibly because thisis the only example where a direct comparison to FUL activitywas made. The results of these experiments are consistentwith a role for euFUL genes in meristem identity, but moreevidence is required.

The euFUL and FUL-like genes are most consistently ex-pressed in carpels and in meristems and vegetative tissues;however, broad expression patterns are common (table 1).FUL is expressed in cauline leaves, inflorescence meristem,and carpels (Mandel and Yanofsky 1995a; Gu et al. 1998). Itis worth noting that whereas AP1 and FUL are expressed inmutually exclusive reproductive domains (floral meristemand perianth vs. inflorescence meristem and carpels/fruit),SLM4 and SLM5, AP1 and FUL orthologues from Silene,are expressed in identical domains that encompass both theAP1 and FUL domains (inflorescence and floral meristem,bracts, perianth, and carpels; Hardenack et al. 1994), high-lighting the variability seen in these patterns.

Other members of the euFUL clade, cloned from a varietyof species of Solanaceae and Myrtaceae as well as apple,birch, and other core eudicot species, are generally expressed

in vegetative organs (e.g., Immink et al. 1999; Wu et al.2000; Hart and Hannapel 2002; Jang et al. 2002; van derLinden et al. 2002; Hileman et al. 2006), including bracts(Sung et al. 1999; Immink et al. 2003; Sreekantan et al.2004). Expression is also commonly seen in carpels and/orfruits (e.g., Immink et al. 1999, 2003; Wu et al. 2000; Sunget al. 2001; Jang et al. 2002; Busi et al. 2003; Hileman et al.2006). In some cases, expression has been documented in in-florescence (Sung et al. 1999; Hart and Hannapel 2002) andfloral meristems (Pnueli et al. 1991; Hart and Hannapel2002), in perianth organs (Kyozuka et al. 1997; Imminket al. 1999, 2003; Sung et al. 1999; Jang et al. 2002; Hile-man et al. 2006), and occasionally in stamens (Kyozukaet al. 1997; Sung et al. 1999; Wu et al. 2000; Hileman et al.2006). In euFUL genes isolated from tobacco (Wu et al.2000), tomato (Hileman et al. 2006), and apple (Sung et al.1999), expression is seen in all four floral whorls. Expressionhas even been recorded in roots for the petunia FBP29 (Im-mink et al. 2003). The broad and varied expression patterns,the absence of uniformity in data collection, and the lack ofdata for paralogues make it difficult to formulate hypothesesregarding the functions that might be represented by theseexpression patterns. However, relatively consistent expressionin vegetative organs is consistent with a role in phase change(table 1), as Sung et al. (1999) propose for MdMADS12from apple. There is less consistent evidence for a specificrole in meristem identity and fruit development, but expres-sion data from these tissues are still limited.

FUL-like Genes

FUL-like genes found in taxa outside of the core eudicotshave also undergone numerous duplications in different an-giosperm clades, and as with euFUL genes, data characteriz-ing all paralogues in any species are generally lacking. Theonly member of the AP1 lineage outside of the core eudicotsfor which we have a mutant phenotype is WAP1, fromwheat. WAP1 has no known role in flower development butis required for vernalization and phase transition (Danyluket al. 2003; Murai et al. 2003; Trevaskis et al. 2003; Yanet al. 2003). Other Poaceae genes have been assayed viaheterologous expression, and as with euFUL genes, these ex-periments have used Arabidopsis as a system to comparethe functional capabilities of FUL-like genes and AP1.OsMADS14 (Jang et al. 2002) and LtMADS15 (Gocal et al.2001), from rice (Oryza sativa) and Lolium temulentum, re-spectively, are able to partially complement strong ap1-1 mu-tants of Arabidopsis when constitutively expressed. However,we do not have evidence of their endogenous roles, nor dowe have a functional comparison with FUL.

As with euFUL genes, FUL-like genes tend to have broadand varied expression patterns, as documented from Poaceaeand other monocots as well as basal angiosperms (table 1).These genes are consistently expressed in leaves and vegeta-tive organs (Schmitz et al. 2000; Yu and Goh 2000; Gocalet al. 2001; Murai et al. 2003; Trevaskis et al. 2003; Petersenet al. 2004; Tsaftaris et al. 2004; Kim et al. 2005a, 2005b;Preston and Kellogg 2006). Expression is also seen in repro-ductive meristems (Schmitz et al. 2000; Yu and Goh 2000;Gocal et al. 2001; Pelucchi et al. 2002; Murai et al. 2003; Li




et al. 2005), although these tissues are not always reported.

Many of these genes are also expressed in floral organs—in

sterile organs (Mena et al. 1995; Kyozuka et al. 2000; Gocal

et al. 2001), in stamens and carpels (Jia et al. 2000; Yu and

Goh 2000; Kim et al. 2005a, 2005b; Li et al. 2005), or in all

whorls (Pelucchi et al. 2002; Tsaftaris et al. 2004; Kim et al.

2005a, 2005b; Preston and Kellogg 2006). Whereas lack of

data from other paralogues and additional species makes it

difficult to reach conclusions regarding the possible functional

implications of these expression patterns, the consistent ex-

pression in vegetative tissues (table 1) lends support to the hy-

pothesis that these genes function in the transition from the

vegetative to the reproductive phases, as is seen for WAP1. A

role in floral development is also supported by the data.

AP1 Gene Lineage Phylogeny: Summary and Caveats

Expression and functional data. When expression data areorganized according to phylogenetic relationships and wheneuFUL and FUL-like genes are distinguished from euAP1genes, some general patterns can be recognized (table 1; seealso Kim et al. 2005b). The euAP1 genes are rarely expressedin vegetative organs and are generally expressed in floral mer-istems, sometimes in inflorescence meristems, and often inperianth organs. In contrast, euFUL and FUL-like genes arealmost always expressed in vegetative organs and are oftenexpressed in carpels, although expression in other floral or-gans is variable. Expression in meristems is also common, al-though these are often not explicitly assayed for expression,as is also the case with fruits.

The functional significance of these patterns of expressionremains unclear, requiring additional data to evaluate theendogenous roles of AP1-lineage genes. To date, such func-tional data are available only for a half-dozen species, andonly in Arabidopsis are they available for all representativesof the AP1 lineage. Many of the heterologous expressiondata are based on overexpressing euFUL and FUL-like genesin Arabidopsis (or tobacco) and comparing the phenotypewith that from overexpressed AP1. These experiments gener-ally produce early flowering and an abnormal terminalflower, leading authors to conclude that the gene under studyplays a role similar to that of AP1. However, ectopic expres-sion of both FUL and AP1 results in this phenotype, whichreflects their shared role in meristem specification (Mandeland Yanofsky 1995b; Ferrandiz et al. 2000b). In a few cases,euFUL and FUL-like genes are used in complementation ex-periments, and in some cases, partial rescue of the ap1 organidentity phenotype is reported (e.g., Gocal et al. 2001; Janget al. 2002). This demonstrates that euFUL and FUL-likegenes share some biochemical functional similarities withAP1, but it does not give us information regarding their insitu function. Thus, we have virtually no information on theendogenous roles performed by euFUL and FUL-like genes.From expression data we can conjecture that genes of theAP1 lineage may play a conserved role in floral meristemidentity. FUL-like and euFUL genes show expression patternsconsistent with a role in phase change to reproduction aswell as possible roles in carpel and fruit development; thesehypotheses require testing. Expression patterns shown byeuAP1 genes are consistent with a role in sepal and petal de-

velopment. However, phenotypic data consistent with a con-tribution to petal identity have been observed only for AP1,and in this case, as noted, the data are equally congruentwith the role in meristem identity.

Orthology and Paralogy

It is clear that both core eudicot euFUL genes and non-core-eudicot FUL-like genes, for instance, WAP1, have beencompared with AP1 as well as named for this gene (Muraiet al. 2003). FUL-like genes from species outside the coreeudicots are equally orthologous to euAP1 and euFUL genes,but sequence analysis indicates that they are more similar toeuFUL (fig. 4). Based on this similarity, it would be informa-tive to include comparisons with euFUL and FUL-like genesin future characterizations of FUL-like genes.

Confusion exists in analyses of core eudicot genes as well;in the absence of a phylogenetic analysis of gene sequences,the only way to diagnose a sequence as euAP1 or euFUL isto examine the final ca. 20 amino acids. In fact, it has beenshown that at least one of the conserved motifs of euAP1proteins may have evolved by a frameshift possibly causedby the insertion of a single nucleotide (Litt and Irish 2003;Vandenbussche et al. 2003a), indicating that the nucleotidesequences are similar. These factors make determination oforthology, in the case of core eudicot genes, difficult to deter-mine without a phylogenetic analysis, and even BLASTsearches pull up a mix of euAP1 and euFUL genes. Thus,characterizations of core eudicot genes would benefit frominclusion of a rigorous phylogenetic analysis, to help deter-mine proper identification of gene orthology.

Sequence Diversification of the AP1 Lineage

Analysis of the predicted protein sequences of euAP1, eu-FUL, and FUL-like genes provides some evidence that euAP1genes may have functionally diverged from euFUL and FUL-like genes. All proteins of the AP1 lineage are highly similarthroughout the MADS, I, and K domains, and, like otherMADS-box proteins, are highly variable in the C-terminaldomain (Ma et al. 1991). But as has been observed in severalMADS-domain-containing protein groups, there are short,highly conserved motifs at the C termini of the predicted pro-tein sequences (Kramer et al. 1998; Litt and Irish 2003;Zahn et al. 2005) (fig. 4). The euFUL and FUL-like proteinsshare a motif comprising six hydrophobic amino acids; euAP1sequences lack this motif and instead have two completelydifferent C-terminal motifs. The FUL-like motif is also seenin the predicted amino acid sequences of the E-function SEPgenes and to some extent the AGL6-like genes (Litt and Irish2003; Zahn et al. 2005). These three groups together (AP1,SEP, AGL6) form one of the highly supported clades ofMADS-box genes that are consistently recovered in phylogeneticanalyses of MADS-box genes (Purugganan et al. 1995; Theis-sen et al. 1996, 2000; Hasebe and Banks 1997; Purugganan1997; Lawton-Rauh et al. 2000). In most reconstructions,the SEP and AGL6 clades are sister groups, and together theSEP/AGL6 clade is sister group to the AP1-like gene clade.The six-amino-acid FUL-like motif can be recognized in




essentially all members of this MADS-box clade, with the ex-ception of the euAP1 proteins (Litt and Irish 2003; Zahnet al. 2005). We currently have no data regarding the func-tion of the FUL-like motif, but we do have some data fromArabidopsis on the functions of the two C-terminal motifs ofeuAP1 proteins that reinforce the notion that the motifs mayconfer new biochemical capabilities on euAP1 proteins.

Functions of the euAP1 Amino Acid Motifs

The two conserved C-terminal motifs that are characteris-tic of euAP1 proteins, herein referred to collectively as theeuAP1 motifs, have both been functionally characterized inArabidopsis. They will therefore in the following discussionsbe referred to individually by their functions. The final fouramino acids are a farnesylation signal; just before this motifthere is a transcription activation domain. These two motifsare discussed below.

Farnesylation. The final four amino acids of most euAP1protein sequences form a canonical farnesylation motif(CaaX). In the process of farnesylation, a form of prenylation,the cysteine acts as an acceptor for a farnesyl molecule, whichgenerally causes the protein to be targeted to a membrane. Inthe case of transcription factors such as AP1, this can be aform of posttranslational regulation or can be required for theassembly of protein complexes (Yalovsky et al. 2000). AP1has been shown to be farnesylated in planta, but it has notbeen shown to be localized to a membrane (Yalovsky et al.2000). To determine whether farnesylation is necessary forproper AP1 function, Yalovsky et al. (2000) generated a mu-tant AP1 nucleotide sequence that would result in a serine be-ing substituted for the acceptor cysteine, and they expressed itin wild-type Arabidopsis under the control of the constitutiveCaMV 35S promoter. As with overexpression of wild-typeAP1, the mutant construct caused early flowering. However,it did not promote the formation of a compound terminalflower. This suggests only partial loss of function in the ab-sence of farnesylation; however, overexpression of a numberof MADS-box genes causes early flowering (Blazquez et al.2001), so this outcome may be nonspecfic. Furthermore, theoverexpression phenotype of AP1 does not allow evaluationof a potential role for farnesylation in organ identity.

The importance of farnesylation for the function of AP1 andorthologues has been variously interpreted as strong (Yalovskyet al. 2000) and weak (e.g., Berbel et al. 2001; Jang et al.2002). In fact, not all euAP1 proteins appear to have this motif.For instance, PEAM4 from pea apparently does not, but it isnonetheless capable of partially rescuing the strong ap1-1 mu-tant of Arabidopsis (Berbel et al. 2001). Thus, it may be thatprenylation is not strictly required or that the requirement hasbeen lost in certain core eudicot lineages (e.g., inflorescencearchitecture is very different in pea and Arabidopsis). Addi-tional data from Arabidopsis and other species may help toclarify whether this euAP1 motif biochemically distinguisheseuAP1 proteins from euFUL and FUL-like proteins.

Transcription activation. The other euAP1 motif that hasbeen characterized is an acidic transcription activation do-main, located just upstream of the C-terminal prenylation mo-tif. Cho et al. (1999) have demonstrated in yeast that thisdomain confers activation capability to AP1 as well as to the

AP1 orthologues of radish (Raphanus sativus) and tobacco(Nicotiana tabacum and Nicotiana sylvestris). This domain isabsent from euFUL and FUL-like proteins. Furthermore, FULand orthologues that have been used in yeast two-hybrid stud-ies have not shown this property (e.g., Immink et al. 2003).This again suggests that euAP1 proteins possess biochemicalfunctions that are absent from euFUL and FUL-like proteins.

Implications of Sequence Divergence in the euAP1 Clade

Early discussions of the ABC model focused on the evolu-tionary distance between Arabidopsis and Antirrhinum andsuggested that a model that described floral development inboth of those species was likely to be widely applicable acrossangiosperms (e.g., Coen and Meyerowitz 1991; Bowman1997). But although Arabidopsis is a member of the rosidsand Antirrhinum is a member of the asterids, these two cladesboth belong to the monophyletic core eudicots (AngiospermPhylogeny Group 1998, 2003). Thus, a model that works forboth of them may be robust across the core eudicots, but itdoes not necessarily follow that it will hold outside of thisclade. This uncertainty is enhanced by the duplication and se-quence divergence at the base of the core eudicots in the AP1lineage (Litt and Irish 2003). In fact, there were also duplica-tions in the B-function AP3 lineage (Kramer et al. 1998) andthe C-function AG lineage (Davies et al. 1999a; Kramer et al.2004) that coincide with the origin of the core eudicots. Fur-thermore, sequence divergence in the AP3 lineage is similar tothat seen in the AP1 lineage (Kramer et al. 1998), suggestingpossible changes in the regulation of floral development in thecore eudicots. AP1 is one of the A-function genes on which theABC model is based, yet we can show that AP1 and its euAP1orthologues are likely to possess functional capabilities that arenot found in the related proteins of non-core-eudicot species.Thus, it may be inappropriate to extrapolate from core eudicotsto the rest of the angiosperms regarding either the predictedroles of AP1 genes or the universality of A-function.

The Ancestral role of AP1-Lineage Genes

The hypothesis most consistent with available functionaland expression data would posit an ancestral role for theAP1 gene lineage in meristem specification (Theissen et al.1996, 2000) or phase change to reproduction. But we canalso look to sister gene clades for information with which toform a hypothesis of ancestral function. Of the AP1/SEP/AGL6 clade of MADS-box genes, to date only AGL6-likegenes have been found in gymnosperms; genes belonging tothe AP1 and SEP lineages have been isolated only from an-giosperms. Phylogenetic reconstructions suggest that all threegene lineages—AP1, SEP, and AGL6—were present in thecommon ancestor of angiosperms and gymnosperms and thatthe AP1 and SEP lineages were lost in gymnosperms. The rel-ative relationships of the three gene clades are not stronglysupported, and the complete absence of AP1- and SEP-lineagegenes from extant gymnosperms and non–seed plants makes itdifficult to reconcile this topology with hypotheses regardingthe roles of the AP1- and SEP-lineage genes during plant evo-lution. Additional data from gymnosperms and basal angio-sperm lineages may help to clarify the relationships.




It is intriguing to note that both AP1 and SEP genes are re-quired for the formation of a floral meristem (Irish and Sus-sex 1990; Mandel et al. 1992; Bowman et al. 1993; Mandeland Yanofsky 1995b; Pelaz et al. 2001a; Ditta et al. 2004).Other genes, such as LFY, that are required for floral meri-stem formation (Schultz and Haughn 1991; Weigel et al.1992) are found in gymnosperms (e.g., Mellerowicz et al.1998; Mouradov et al. 1998a; Frohlich and Parker 2000).Likewise, other MADS-box genes required for flower forma-tion, such as members of the B- and C-function clades, arealso found in gymnosperms (e.g., Tandre et al. 1995; Mun-ster et al. 1997; Mouradov et al. 1999; Shindo et al. 1999;Sundstrom et al. 1999; Becker et al. 2002, 2003; Carlsbeckeret al. 2003); this is logical, since these genes are required forthe formation of the reproductive organs and therefore arelikely to be conserved. Thus, of the identified genes that arecritical for flower formation, only the closely related AP1and SEP genes appear so far to be restricted to angiosperms.A tempting, although probably untestable, hypothesis is thatthe origin of these genes was either responsible for or a prereq-uisite for the origin of the flower. However, the contradictorytopology found in phylogenetic analyses and the paucity ofdata on the function of these genes, particularly from speciesoutside the core eudicots, where there is little evidence regard-ing the role of FUL-like genes, leave this hypothesis unsup-ported at this time.

The AGL6-like gene clade has not been well characterizedfunctionally, but it may hold the key to the ancestral role ofthese genes because it is the only sublineage of the AP1/SEP/AGL6 gene clade that unequivocally predates the origin ofthe flowering plants (fig. 3). It therefore also predates the or-igin of the floral meristem, the perianth, and the fruit—allfeatures that are characteristic of angiosperms and that ap-pear to require AP1/SEP genes for proper development.Some information is available from heterologous-transformationexperiments: in Arabidopsis and tobacco, expression of theorchid gene OMADS1 caused early flowering, loss of de-terminacy, and homeotic organ transformation that indicatedectopic C-function activity (Hsu and Yang 2002). Overexpres-sion of the spruce gene DAL1 in Arabidopsis produced a se-verely shortened juvenile phase (Carlsbecker et al. 2004). Bothphenotypes involve disrupted phase transition, consistentwith this hypothesized ancestral role for this gene family.

Expression data are available only for AGL6 and for asmall handful of monocots, basal angiosperms, and gymno-sperms (summarized in table 1). Unfortunately, there are fewdata on the expression of AGL6 and orthologues in meri-stems or vegetative organs, making it difficult to evaluate thehypothesis that the ancestral role of this gene clade is in meri-stem identity or phase transition. Arabidopsis AGL6 is ex-pressed in the inflorescence axis and in all floral organs(Mouradov et al. 1998b). OMADS1 is expressed in the transi-tional meristem (Hsu and Yang 2002), perianth, and carpel,and Mena et al. (1995) found expression of a maize AGL6 or-thologue also in perianth and carpels. Kim et al. (2005b) de-tected expression in the perianth in Magnolia and in all floralorgans in Amborella. In neither species was expression seenin leaves. Kim et al. (2005b) suggest, on the basis of theseexpression data, that AGL6 orthologues may be acting asA-function genes in basal angiosperms, but functional data

are clearly needed. The angiosperm expression patterns are re-markably similar to the expression seen in euAP1 and someeuFUL genes (table 1) and suggest roles in meristem identityand flower development, although more data from vegetativeorgans and meristems would help in evaluating this hypothe-sis. Gymnosperm AGL6 orthologues in general are expressedin both mega- and microsporangiate strobili, including sterileand reproductive components in different species (Tandreet al. 1995; Mouradov et al. 1998b; Winter et al. 1999; Beckeret al. 2003). DAL1 is also expressed vegetatively in a patternconsistent with a role in phase transition (Carlsbecker et al.2004), and PrMADS3 is expressed only vegetatively (Mouradovet al. 1998b). These patterns from gymnosperms suggest arole in phase transition and reproduction but are difficult tocompare with the patterns seen in angiosperms.

Is There an A-Function?

Several authors have raised the possibility that A-functionis confined to Arabidopsis or Brassicaceae or may not be uni-versal (e.g., Theissen et al. 1996, 2000; Maes et al. 2001;Shepard and Purugganan 2002; Smyth 2005). AP2 is theonly gene encoding both components of A-function (perianthidentity and C-function repression) (Bowman et al. 1989;Drews et al. 1991), and AP1 is the only other gene shown toplay a role in specifying the identity of both types of perianthorgans. Other members of these gene families have beenshown to play a role in meristem specification, and it is possi-ble that this is the primary function of these gene lineages. Infact, all AP1- and AP2-lineage genes shown to have a rolein perianth identity (AP2, LIP1/2, LeMADS_MC, and AP1;Bowman et al. 1989, 1991, 1993; Irish and Sussex 1990;Jofuku et al. 1994; Vrebalov et al. 2002; Keck et al. 2003)are also implicated in the specification of the floral meristem.This is one of the patterns that emerges from the literature: genesthat are required for proper sepal identity are all also requiredfor proper floral meristem identity. The evidence may be dra-matic, as in the secondary flowers of some AP1 and AP2 alleles.But it may also be manifest in the homeotic transformation ofthe outer-whorl organs from floral sepals to inflorescence bractsor vegetative leaves. The residual inflorescence or vegetativecharacter of those organs points to an incomplete transition tothe flowering state. Thus, one phenotype—transformation of se-pals toward bracts or leaves—is evidence of two functions: meri-stem identity and sepal identity.

An examination of the data, which to date are undeniablyscant, shows that it is essentially impossible to untangle thesetwo functions and identify a gene that determines sepal iden-tity without also playing a role in floral meristem identity(Theissen et al. 2000). In fact, the ABC model stipulates thatA- and C-functions negatively regulate each other so that inthe absence of A-function, carpels would be expected in theouter whorl (Coen and Meyerowitz 1991). AP1 and LIP1/2lack this cadastral function (Irish and Sussex 1990; Bowmanet al. 1993; Keck et al. 2003), but the ABC model fails to pre-dict what organs will be formed in the outer whorl under thosecircumstances. To date, AP2 is the only putative A-functiongene shown to restrict C-function to the inner two whorls andis the only example where outer-whorl organs are regularly




carpelloid. In other examples of putative loss of A-function(Irish and Sussex 1990; Bowman et al. 1993; Keck et al. 2003),the outer-whorl organs become leaflike or bractlike.

It is interesting to note that to date it is only in Arabidop-sis that we see mutant phenotypes that affect the identityof both the first- and second-whorl organs. For instance,LeMADS_MC (Vrebalov et al. 2002) and LIP1/2 (Keck et al.2003) show loss only of sepal identity. Currently there are noother described examples where loss of function of an AP1or AP2 orthologue results in loss of identity of both sepalsand petals, suggesting that this is a Brassicaceae-specific phe-nomenon. This implies that there is something distinct aboutfloral development in Arabidopsis that affects the outer twowhorls and that this is not characteristic of floral develop-ment in other species. This difference is perhaps related tosome of the unique features of the Arabidopsis (or Brassica-ceae) genome, such as the presence of a second euAP1 gene(CAL), which plays a role in meristem identity and may beparticularly important in the second whorl (Bowman et al.1993; Kempin et al. 1995; Castillejo et al. 2005), and theloss of a DEFH28 orthologue.

Resurrecting a Two-Gene-Function Model

In fact, the data to date are all consistent with a model,published by Schwarz-Sommer et al. (1990) before the ABCmodel, based on Antirrhinum phenotypes (fig. 5). This modelpostulated only two gene functions, called A and B but fillingthe roles of what are now referred to as B- and C-functiongenes, and it is sufficient to account for all observed pheno-types and interactions. A general characteristic of shoot meri-stems is to produce lateral organs; if this is a ground-statefunction encoded in the pathways that direct a group of cellsto be shoot meristem, additional instructions to promote for-mation of lateral organs are not required (see also Schwarz-Sommer et al. 1990; Motte et al. 1998; Theissen et al. 2000).The identity of the lateral organs varies from leaves producedby vegetative meristems to bracts produced by inflorescencemeristems to sepals produced by floral meristems. This isconsistent with data that demonstrate a molecular connec-tion between meristems and lateral organs, for instance, the

expression of AGL24 in both vegetative meristems and leavesand in inflorescence meristems and cauline leaves (Yu et al.2002). Thus, when the ‘‘floral meristem identity’’ program isactivated in a group of cells by the activity of LFY, AP1, andother genes (Motte et al. 1998), the lateral organs that areproduced become sepals. In the second whorl, the activity ofthe B-function genes is added to this ground state, and the or-gans produced are petals (fig. 5). In the third whorl, C-functionis added, and stamens are produced. In the fourth whorl, onlyC-function is present, and carpels are produced. This ‘‘BC’’model is consistent with all available data regarding the specifi-cation of organ identity in all species studied.

How does this model explain the ap1 phenotype? AP1 isexpressed in the floral meristem, and as the flower developsit becomes confined to the outer two whorls (Mandel et al.1992). In the absence of AP1 function, the outer two whorlsdo not receive sufficient signal to become florally determined,even though other meristem identity genes such as LFY andSEP3 are active. In that case, in these two whorls, one wouldpredict finding inflorescence, rather than flower, structures.Indeed, that is what we find: ‘‘bracts’’ in the outer whorl sub-tending branches bearing secondary flowers (fig. 5). In thesesecondary flowers the same phenomenon occurs, and the outerwhorl is specified as bracts and the second as flower-bearingbranches. Variations in functional domains and redundant fac-tors can account for differences in phenotypes among ap1,squa, pim, and lemads_mc, but in no case is a separate genefunction needed to specify sepal and petal identity.

The phenotype of the other Arabidopsis floral organ iden-tity mutants can also be explained by the BC model. AG re-stricts the expression of AP1 to the outer two whorls; thus,in the ag mutant, AP1 is ectopically expressed throughoutthe developing flower (Gustafson-Brown et al. 1994). In thethird whorl, organs develop as petals, and in the fourthwhorl, a new flower is initiated that reiterates the sepal-petal-petal pattern (Yanofsky et al. 1990). Thus, there is ahomeotic transformation as well as loss of floral determinacy.According to the ABC model, A- and B-function will be ac-tive in the third whorl, producing petals, and A-functionalone will be active in the inner whorl, producing sepals(Coen and Meyerowitz 1991). However, an alternative ex-planation based on the hypothesis that AP1 has no direct

Fig. 5 Comparison of the ABC model (Coen and Meyerowitz 1991) and a two-gene-function model (Schwarz-Sommer et al. 1990) for thespecification of floral organ identity. Gene activity and resultant organ formed are indicated in domains of a floral meristem shown in longitudinal

section. A, ABC model, in which three gene functions are each active in two adjacent domains of the meristem. B, BC, or two-gene-function,

model (published as A and B gene functions; Schwarz-Sommer et al. 1990), in which no floral organ identity gene function is required in the sepal

whorl. C, Interpretation of the ap1 mutant, in which the outer two whorls remain specified as inflorescence and the inner two whorls are specifiedas flower, with development of corresponding organs in each pair of whorls.




role in organ identity is that the B-function genes in whorlthree are sufficient to specify petals and that the lack ofany organ-identity gene function in the fourth whorl leavesthe meristem in the floral ground state, in which sepals areproduced.

Synthesis

Although the phenotypes of ap1 and ap2 mutants of Arabi-dopsis can both be interpreted as consistent with A-function,to date there are no other examples of floral mutants in anyspecies in which both of the outer whorls are affected.In addition, AP2 is the only identified gene that fills bothA-function roles of specifying organ identity and restrictingC-function activity from the outer two whorls. Taken to-gether, this information suggests, at best, that A-function asdefined in the ABC model may be restricted to Brassicaceae.Even here, however, the existence of a discrete gene functionrequired for sepal and petal identity is cast into doubt by theroles played by AP1 and AP2 in specifying meristem identity;the phenotypic features attributed to loss of this functioninclude the phenotypic features attributed to A-function,namely, homeotic conversion of sepals to bracts and presenceof secondary flowers in the second whorl. As noted by Theis-sen et al. (2000), these two functions do not appear separa-ble. Thus, it may be that A-function does not exist as adiscrete gene function.

Evidence that the role played by AP1 in Arabidopsis maybe divergent from its role in other species comes from an un-derstanding of the phylogeny of this gene lineage. Not onlydo we find a second euAP1 paralogue (CAL) in Brassicaceae,but we also do not find a DEFH28 paralogue. This suggests

that even within the core eudicots there has been diversifica-tion in this gene lineage. In species outside of the core eudi-cots there is even greater difference, with only FUL-likegenes present. Given the divergence in amino acid sequencemotifs, it is plausible that the euAP1 proteins of core eudicotshave different functional capabilities than FUL-like and eu-FUL proteins, adding to the circumstantial evidence that mo-lecular processes involving AP1 lineage genes may havechanged during the course of angiosperm evolution.

Thus, there is little evidence to date to support the notionof a universal or even a core-eudicot-wide A-function. How-ever, this function is not needed to completely and uniquelyspecify the four types of floral organs, as noted by Schwarz-Sommer et al. (1990). Sepal development seems intimatelylinked with floral meristem identity such that a gene functionmay not be needed to specify the identity of these organsonce the identity of the meristem has been established. In thisscenario, only two additional gene functions are required tospecify petal, stamen, and carpel, and this is completely con-sistent with all available data (fig. 5). It is plausible that newmutants and new gene functions will be discovered that willdemonstrate the existence of a class of genes responsible forspecifying sepal and petal identity; however, the two-gene-function model (originally A- and B-functions; Schwarz-Sommeret al. 1990) is sufficient to explain the current state of ourknowledge.

Acknowledgments

I would like to thank two anonymous reviewers for theirsuggestions and E. Kramer and V. Irish for comments on themanuscript and valuable discussions of the ideas.

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An Evaluation of A‐Function: Evidence from the APETALA1 and APETALA2 Gene Lineages