Transcriptional signatures of ancient floraldevelopmental genetics in avocado(Persea americana; Lauraceae)Andre S. Chanderbalia,b,1, Victor A. Albertc, Jim Leebens-Mackd, Naomi S. Altmane, Douglas E. Soltisa,and Pamela S. Soltisb

aDepartment of Biology and bFlorida Museum of Natural History, University of Florida, Gainesville, FL 32611; cDepartment of Biological Sciences, Universityat Buffalo, State University of New York, Buffalo, NY 14260; dDepartment of Plant Biology, University of Georgia, Athens, GA 30602; and eDepartment ofStatistics, Pennsylvania State University, University Park, PA 16802

Edited by M. T. Clegg, University of California, Irvine, CA, and approved February 25, 2009 (received for review November 12, 2008)

The debate on the origin and evolution of flowers has recentlyentered the field of developmental genetics, with focus on thedesign of the ancestral floral regulatory program. Flowers candiffer dramatically among angiosperm lineages, but in general,male and female reproductive organs surrounded by a sterileperianth of sepals and petals constitute the basic floral structure.However, the basal angiosperm lineages exhibit spectacular diver-sity in the number, arrangement, and structure of floral organs,whereas the evolutionarily derived monocot and eudicot lineagesshare a far more uniform floral ground plan. Here we show thatbroadly overlapping transcriptional programs characterize the flo-ral transcriptome of the basal angiosperm Persea americana (avo-cado), whereas floral gene expression domains are considerablymore organ specific in the model eudicot Arabidopsis thaliana. Ourfindings therefore support the ‘‘fading borders’’ model for organidentity determination in basal angiosperm flowers and extend itfrom the action of regulatory genes to downstream transcriptionalprograms. Furthermore, the declining expression of components ofthe staminal transcriptome in central and peripheral regions ofPersea flowers concurs with elements of a previous hypothesis fordevelopmental regulation in a gymnosperm ‘‘floral progenitor.’’Accordingly, in contrast to the canalized organ-specific regulatoryapparatus of Arabidopsis, floral development may have beenoriginally regulated by overlapping transcriptional cascades withfading gradients of influence from focal to bordering organs.

ABC model � basal angiosperms � comparative transcriptome analysis �fading borders model � floral evolution

Recent insights into the genetic regulation of floral organidentity suggest that simple switches in the regulatory pro-

grams of nonflowering seed plants could have produced theoriginal f lowers (1–4) (Fig. 1). Although differing in details,these hypotheses involve transformation of a unisexual repro-ductive shoot into a bisexual structure with female organs(carpels) above male organs (stamens), through modifications toancient genetic programs for male and female organ identity.Condensation of this cone-like axis, followed by development ofan envelope of sterile perianth appendages surrounding thesexual organs, and perianth dimorphism into outer sepals (leaf-like) and inner petals (usually attractively colored), produced thetypical f lower (2). In the eudicot genetic model Arabidopsisthaliana, the development of such flowers is regulated by the‘‘ABC model,’’ in which A and C represent mutually antagonisticfunctions that combine with B function to specify floral organidentity: A specifies sepals, AB specify petals, BC specifystamens, and C specifies carpels (5). E function (D having beenassigned to ovule specification) is hypothesized to coordinatecombinatorial activity between AB and BC factors through theassembly of tetrameric protein complexes, or ‘‘f loral quartets’’(6, 7). In Arabidopsis, A function is provided by APETALA1(AP1) and APETALA2 (AP2), B function by APETALA3 (AP3)

and PISTILLATA (PI), C function by AGAMOUS (AG), and Efunction by multiple SEPALLATA gene products (SEP1–4)(5–7). All but AP2 belong to the large MADS domain family oftranscription factors (8).

Conserved expression patterns and successful complementa-tion experiments for homologues of AG and AP3/PI in diverseangiosperms and gymnosperms suggest that B and C functionsform the ancestral nucleus of the ABC(E) model, whereas A andE functions arose later in angiosperm evolution (2). Controversysurrounds A function, which may not be separable from floralmeristem specification (9), but E function seems to operate inrice (10), suggesting an origin before the separation of monocotsand eudicots. Genetic models have yet to be established amongbasal angiosperms, but the expression domains of ABC homo-logues, B homologues in particular, often extend beyond theirexpected boundaries (11, 12), supporting the ‘‘fading borders’’modification of the ABC model for organ identity in theirf lowers (13–15) (Fig. 1).

Here, we have drawn upon progress in the characterization ofglobal transcriptional patterns in Arabidopsis f lowers for com-parisons with the basal angiosperm Persea americana (avocado),to elucidate commonalities and features of the ancestral f loraldevelopmental program. Persea is a member of the magnoliidclade of basal angiosperms that, together with Chloranthales, liessister to the eudicot � monocot clade in recent phylogeneticreconstructions (16, 17). Persea f lowers are bisexual, with bothstamens and carpels, and bear an undifferentiated perianth ofpetaloid organs, termed tepals (Fig. 1). An undifferentiatedperianth is characteristic of basal angiosperms (18) and repre-sents an intermediate stage in the evolutionary scenario forflowers, before the differentiation of sepals and petals (2).

ResultsCustom microarrays targeting 6,068 genes collected from Perseaf loral buds by the Floral Genome Project (19, 20) identified4,797 significantly differentially expressed genes (P � 0.05; falsediscovery rate � 0.62%) among 8 sampled tissues: inflorescencebuds, premeiotic f loral buds, outer tepals, inner tepals, stamens(including staminodes), carpels, initiating fruit, and leaves (Fig.S1). For Arabidopsis, measures of gene expression in wild-type

Author contributions: A.S.C., V.A.A., J.L.-M., N.S.A., D.E.S., and P.S.S. designed research;A.S.C. performed research; A.S.C. contributed new reagents/analytic tools; A.S.C. and N.S.A.analyzed data; and A.S.C., V.A.A., J.L.-M., D.E.S., and P.S.S. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The experimental data reported in this paper have been deposited in theNCBI database (GEO accession no. GSE 13737).

Freely available online through the PNAS open access option.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: achander@botany.ufl.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811476106/DCSupplemental.

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vs. mutant flowers have identified large numbers of genes thatare not expressed in the absence of ABC gene activity and likelyact downstream of these transcription factors (21, 22). However,wild-type expression levels in floral organs, reported in theAtGenExpress dataset (23), are more comparable to our Perseadata. The AtGenExpress data also include measures of geneexpression in leaves, the vegetative organ we assayed to providea benchmark for assessing florally biased expression in Persea.Because Arabidopsis f loral organs are transformed into leaves inthe absence of ABC(E) gene activity, and vice versa (6, 7),elevated expression in flowers relative to leaves likely applies tocomponents of the transcriptome operating downstream of thesefloral regulators. Illustrating this relationship, positive log2 floral

organ/leaf expression ratios calculated from the AtGenExpressdataset generally match negative mutant/wild-type ratios pre-dicted by the ABC(E) model (Fig. S2). Notably, amidst thepredominant pairing of downregulation in mutants and elevatedf loral expression, the anticipated upregulation of petal-expressed genes in ag f lowers (22) is also evident. Furthermore,these ratios describe the established expression patterns of theABC genes (Table S1), supporting their use in standardizingcomparisons of transcriptional patterns in Persea and Arabidop-sis f lowers.

Unsupervised hierarchical clustering (24) of differentiallyexpressed Persea genes assembled log2 expression ratios intogroups, accommodating, and potentially regulated by, homo-

Fig. 1. On the origin and evolution of the floral developmental genetic program. Relationships between angiosperm and gymnosperm subjects of currentgenetic studies are indicated in accordance with current phylogenetic trees, and hypothetical ‘‘protoangiosperm’’ stages are inserted below angiosperms toillustrate hypothesized steps in floral origin from gymnosperm cones (1, 2). Color schemes on the right depict hypothesized (1, 2) and established gene expressionpatterns of ABC genes (5–7, 11, 12) in the evolution of the floral regulatory program. Reduction of B function in the distal region of male gymnosperm conesresults in female development (carpels) in a bisexual protoangiosperm I, and subsequent reduction of C function at the proximal region leads to the replacementof stamens by sterile perianth organs in protoangiosperm II. Expression data for basal angiosperms (11, 12) suggest that a ‘‘fading borders’’ mechanism of organidentity determination was active before the establishment of the strict expression domains of the ABC(E) model in Arabidopsis (13–15). For comparativepurposes, protoangiosperm ‘‘flowers’’ are depicted with condensed axes, although this step may have occurred between stages I and II (2); A function is includeddespite its controversial status. Persea americana and Arabidopsis thaliana flowers are enlarged to compare their morphologies: both have whorled phyllotaxy,but in Persea an undifferentiated perianth of tepals surrounds the stamens and carpels, whereas a dimorphic perianth with green sepals and white petalssurrounds the reproductive organs of Arabidopsis. Otp, outer tepals; Itp, inner tepals; Stm, stamens; Stmde, staminodes; Car, carpels; Sep, sepals; Pet, petals. Photocredits: S. Kim for Amborella; Yi Hu for Nuphar; A.S.C. for Persea; A. Morris for Illicium; and public domain for the remaining.

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logues of AG, AP3 and PI, AP1, and a fifth MADS domain gene,AGAMOUS-LIKE 6 (AGL6), respectively (Fig. 2). These genesare among the most highly expressed components of theirrespective clusters and exemplify the expression patterns ofgenes ostensibly downstream of them (Fig. 2). The broadest geneexpression pattern is shown by genes clustered with homologuesof AP3 and PI, the expression levels of which peak in stamens but

Fig. 2. Transcriptional patterns in Persea flowers. (A) Hierarchical clusteringof significantly differentially expressed genes assembled elevated floral ex-pression into 4 groups accommodating homologues of MADS domain geneswith prominent (AG, AP3/PI, AP1) and unclear (AGL6) roles in floral develop-mental genetics. The hierarchy of tissue relationships indicates transcriptionalsimilarities for inflorescences and floral buds, carpels and fruits, and tepals andstamens. (B) Homologues of AG, AP3, PI, AGL6, and AP1 (positions indicatedby black bars) rank among the most highly expressed members of theirrespective clusters sorted by expression levels in outer tepals (AGL6 cluster),stamens (AP3/PI and AG clusters), and inflorescence buds (AP1 cluster). Thescale bar of log2 expression ratios ranges from saturated yellow (1 andhigher � at least 2-fold upregulated) to saturated blue (�1 and lower � atleast 2-fold downregulated). Car, carpels; Flb, floral buds; Frt, fruits; IFb,inflorescence buds; Itp, inner tepals; Otp, outer tepals; Stm, stamens.

Fig. 3. The expression domains of genes with elevated floral expression arebroader in Persea than in Arabidopsis. (A) Positive log2 floral organ/leaf ratiosfor Arabidopsis from the AtGenExpresss dataset (23) and Persea are ranked byorgan of peak expression. (B) As above, but with the subset of genes withreciprocal homology sorted by Arabidopsis organs of peak expression. Thescale of log2 expression ratios ranges from saturated yellow (1 and higher �at least 2-fold upregulated) to saturated blue (�1 and lower � at least 2-folddownregulated). Otp, outer tepals; Itp, inner tepals; Stm, stamens; Car, car-pels; Sep, sepals; Pet, petals.

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remain high in tepals and are also detectable in carpels (TableS1; see also ref. 11). AG homologues are also broadly expressed,most strongly in stamens and carpels but still detectably in tepals(Table S1; see also ref. 11). In general, coexpressed genes areprimarily upregulated in stamens and carpels. Genes groupedwith homologues of AGL6 [which have uncertain function (25)]exhibit the opposite expression pattern [i.e., upregulation intepals (Table S1; see also ref. 11)], whereas in a departure fromperianth specification in Arabidopsis, the AP1 homologue issharply downregulated after strong initial expression in inflo-rescence buds (Fig. 2). Thus, expression patterns in Perseaf lowers correspond well with individual A, B, and C functions,while organ-specific biases are largely absent.

With these insights, we sought to determine how expressiondomains in Persea f lowers compare with those in Arabidopsis.First, in global assessments of their transcriptional landscapes,organ-wise partitioning of elevated floral expression ranked indescending order indicates that, in Arabidopsis, expression do-mains are predominantly tightly constrained to individual organcategories with little ‘‘leakage’’ into adjacent whorls, whereas inPersea, all but the most weakly expressed genes extend from sitesof peak expression into adjacent whorls and beyond (Fig. 3A).Almost all genes with peak expression in Arabidopsis carpelshave negative expression ratios in stamens, and vice versa. The

same is true for sepals and petals, and although genes with peakexpression in Arabidopsis stamens are often also upregulated inpetals, the reverse is not true. Curiously, this reciprocal rela-tionship exists for petals and carpels, organs that, following theABC model, do not share genetic developmental instructions. InPersea, however, genes expressed in tepals and carpels representa subset of those expressed in tepals and stamens. Second,similar analyses comparing the expression domains of homolo-gous genes (Fig. 3B) indicate that expression peaks restricted toindividual Arabidopsis organs universally correspond to broaderexpression domains in Persea. Third, plots of log2 floral organ/leaf expression ratios comparing the transcriptional profiles ofadjacent floral whorls generally assemble relative transcriptfrequencies close to the regression line in Persea, whereas theseare skewed toward the axes in Arabidopsis (Fig. 4 A–C). Evenlower correlations are obtained for Arabidopsis when only ho-mologues of Persea genes are considered (compare Fig. 4 B andC), making it unlikely that our particular sample of Persea genesartificially increases correlations between floral whorls. Takentogether, these results describe 2 distinct f loral genetic land-scapes: transcriptional programs in Persea f lowers are deployedin broad domains that overlap across adjacent whorls andbeyond, whereas those in Arabidopsis are more tightly con-strained to individual organ categories.

Fig. 4. Transcriptional profiles of floral organs are more strongly correlated in Persea than Arabidopsis. Scatterplots of log2 floral organ/leaf ratios in adjacentfloral organs of (A) Persea and (B and C) Arabidopsis. Pearson correlations of gene expression levels in the transcriptional profiles of adjacent floral organs aregiven. Plots and correlation values are calculated from genes in (A) the entire Persea dataset, (B) the entire AtGenExpress dataset (23) and (C) the subset ofArabidopsis genes with homologues in the Persea dataset. Pair-wise comparisons of correlations computed for Persea organs and the corresponding Arabidopsisorgans (across rows) are all significantly different (z � 2.56).

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DiscussionOur comparisons of the transcriptional patterns in Persea andArabidopsis f lowers provide insights into the ancestral f loraldevelopmental genetic program and its subsequent evolution.Persea f lowers bear 3 types of f loral organs [tepals, stamens(including staminodes), and carpels]; therefore, 3 distinguish-able transcriptional programs should be, and are, deployed—but not 1 per organ category. Instead of being organ specific,they are overlapping, with broad domain expansions from sitesof peak expression contributing to strong correlations betweenthe expression profiles of adjacent f loral whorls.

Earlier studies of ABC homologues in phylogenetically pivotalbasal angiosperms, including Persea (11, 12), have supported the‘‘fading borders’’ model of floral organ identity in basal angio-sperms (13–15) (Fig. 1). Originally developed to explain theintergrading morphologies of spirally arranged organs in Am-borella and other basal angiosperm flowers, ‘‘fading borders’’posits that gradually fading influence toward the periphery oforgan identity functions, of B function in particular, impartssome features of adjacent floral organs onto each other. A contin-uum of staminal features can be traced from stamens to tepals andfrom stamens to carpels in Persea flowers (26), and this seems to bewell reflected in organ-wise transcriptional profiles.

Our findings therefore extend the ‘‘fading borders’’ scenariofrom morphology and the key organ identity genes to the floraltranscriptome (i.e., those genes including and lying downstream

of the ABC genes). As such, fading activities of B and C classorgan identity genes in central and peripheral f loral regions,respectively, may be a vestige of the contraction of the maledevelopmental program (BC) in evolutionary scenarios forfloral origin (reviewed in ref. 1). Accordingly, carpel develop-ment proceeds where C function is the primary influence,whereas sterile perianth organs develop where B function isdominant (Fig. 5). The perianth identity program may be derivedfrom interrupted stamen identity functions (2), and several genesimplicated in AG repression participate in the Arabidopsis petalidentity program (27). AG homologues are, however, expressedin Persea tepals, but at lower levels than in stamens and carpels,suggesting that genetic mechanisms for their repression exist,albeit in rudimentary form, in this basal angiosperm.

Transcriptional programs remarkably similar to those in Per-sea are deployed across the intergrading floral organs of thewater lily Nuphar (Nymphaeales; M.-J. Yoo, A.S.C., N.S.A.,D.E.S., and P.S.S., unpublished data), representing the lineagethat likely lies sister to all extant flowering plants except Am-borella (16, 17). In Arabidopsis, in contrast, expression patternshighlighted here resemble the organ-specific transcriptionalcascades predicted by the standard ABC(E) model (Fig. 3). Onthe basis of our data, we hypothesize that broadly overlappingtranscriptional programs, promoted by B, C, and perhaps yetunknown A functions, act to orchestrate floral development inbasal angiosperms through shifting gradients of influence thatimpart intergrading morphologies on floral organs (Fig. 5). Assuch, the ancestral f lower may have been built by an incom-pletely partitioned regulatory mechanism that was subsequentlymodified into the highly canalized organ identity programs ofArabidopsis.

MethodsArray Design, Experimental Design, and Implementation. Custom Persea mi-croarrays, printed by Agilent Technologies, contain 10,187 randomly arrayedin situ–synthesized 60-mer oligonucleotide probes, designed as previouslydescribed (20), targeting 6,068 unique floral transcripts, collected and se-quenced by the Floral Genome Project (19). Except for phylogenetic analysesof MADS domain genes (11), homology between 3,790 of these Persea genesand 3,187 Arabidopsis genes was assigned through best reciprocal BLAST Escores �10�5. Expression profiles of inflorescence buds, premeiotic floral buds,inner and outer tepals, stamens, carpels, initiating fruit, and leaves wereassessed in an interwoven double-loop design (28) for 8 samples with 16 arrays(Fig. S1), which, compared with a reference design, minimizes the variance ofpair-wise comparisons and increases power to detect differential gene ex-pression (20). Sample materials were collected from 2 individuals (biologicreplicates) cultivated on the University of Florida’s Gainesville campus (Kim1135 and Chanderbali 618, respectively; vouchers deposited at the Universityof Florida Herbarium), and RNA was isolated twice for technical replicationusing a method described for basal angiosperms (11, 12). RNA transcripts werehybridized to the arrays in accordance with Fig. S1.

Data Acquisition and Analyses. Arrays were scanned with an Agilent DNAmicroarray scanner using Agilent’s Feature Extraction Software 9.1.3. Rawdata were read into Bioconductor’s Limma package (29) for normalization anddifferential expression analysis. Arrays passing graphic quality-control checkswere background corrected and loess normalized within, and A-quantilenormalized between, arrays (29, 30). A 1-way empirical Bayes ANOVA, usingsingle-channel analysis while accounting for correlation between channels(29, 30), identified significantly differentially expressed genes. Expressiondata for Arabidopsis (22, 23) were obtained from the Gene Expression Omnibus(accession no. GSE1275) and the Arabidopsis Information Resource (accession no.ME00319). Expression data were visualized using Java TreeView (31).

ACKNOWLEDGMENTS. We thank C. dePamphilis, J. Carlson, K. Wall, L. Muel-ler, D. Ilut, and W. Farmerie for EST sequencing and analysis; and M. Popp andthe staff of the Interdisciplinary Center for Biotechnology Research at Uni-versity of Florida for technical assistance with microarray processing. Oligo-nucleotide probes for the Persea arrays were designed by R. Gharaibeh and C.Gibbas of University of North Carolina-Charlotte. This study was supported byNational Science Foundation Grants PRG-0115684 to the Floral GenomeProject and PGR-0638595 the Ancestral Angiosperm Genome Project.

Fig. 5. A ‘‘fading borders’’ model for Persea floral organ identity. While itremains to be shown whether A function exists, the present data indicate thatfloral organs are all influenced by B and C functions, but in varying propor-tions. Accordingly, transcriptional cascades regulated by ‘‘Bc,’’ ‘‘BC,’’ and ‘‘bC’’activities, where lowercase font indicates lower functional influence, promotethe development of tepals, stamens, and carpels, respectively. These shiftingbalances in B and C influence are evident in the relative correlations betweentranscriptional patterns in Persea flowers and B (rB) and C (rC) expressiondomains (Bottom). Genes with similar rB and rC values (rB � rC � 0.0) areexpressed primarily in stamens, whereas those with greater rB and rC scoresare, respectively, primarily expressed in tepals and carpels. The scale of log2

expression ratios ranges from saturated yellow (1 and higher � at least 2-foldupregulated) to saturated blue (�1 and lower � at least 2-fold downregu-lated). The numeric scale measures the difference in rB and rC values (rB � rC �1.8 to �1.8). Otp, outer tepals; Itp, inner tepals; Stm, stamens, Car, carpels.

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