LARGE-SCALE BIOLOGY ARTICLE

Genome-Scale Transcriptomic Insights into Early-Stage FruitDevelopment in Woodland Strawberry Fragaria vescaC W

Chunying Kang,a Omar Darwish,b Aviva Geretz,a Rachel Shahan,a Nadim Alkharouf,b and Zhongchi Liua,1

a Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742bDepartment of Computer and Information Sciences, Towson University, Towson, Maryland 21252

ORCID IDs: 0000-0001-9969-9381 (Z.L.); 0000-0002-6902-740X (C.K.).

Fragaria vesca, a diploid woodland strawberry with a small and sequenced genome, is an excellent model for studying fruitdevelopment. The strawberry fruit is unique in that the edible flesh is actually enlarged receptacle tissue. The true fruit are thenumerous dry achenes dotting the receptacle’s surface. Auxin produced from the achene is essential for the receptacle fruitset, a paradigm for studying crosstalk between hormone signaling and development. To investigate the molecular mechanismunderlying strawberry fruit set, next-generation sequencing was employed to profile early-stage fruit development with fivefruit tissue types and five developmental stages from floral anthesis to enlarged fruits. This two-dimensional data set providesa systems-level view of molecular events with precise spatial and temporal resolution. The data suggest that the endospermand seed coat may play a more prominent role than the embryo in auxin and gibberellin biosynthesis for fruit set. A model isproposed to illustrate how hormonal signals produced in the endosperm and seed coat coordinate seed, ovary wall, and receptaclefruit development. The comprehensive fruit transcriptome data set provides a wealth of genomic resources for the strawberry andRosaceae communities as well as unprecedented molecular insight into fruit set and early stage fruit development.

INTRODUCTION

Fragaria vesca, the woodland strawberry, is emerging as a modelfor the cultivated octoploid strawberry as well as the Rosaceaefamily due to its small and sequenced genome, diploidy (2n = 14,240 Mb genome), small stature, ease of growth, short life cycle,and facile transformation (Shulaev et al., 2011). Furthermore,F. vesca fruit development offers an unusual opportunity to in-vestigate signal coordination and communication between dif-ferent organs due to strawberry’s unique fruit structure. Its fleshyfruit is actually the stem tip, the receptacle, while the true fruit isthe achene, a dried up ovary. More than two hundred achenesdot the surface of the receptacle, each consisting of a singlefused ovary with one seed inside. Complex signaling over spaceand developmental time between the seed-bearing achene andthe supporting receptacle ensures proper reproductive successand seed dispersal.

In most plants, early fruit development consists of three phases(Gillaspy et al., 1993). The earliest phase, the decision to abort orto proceed with fruit development, is referred to as fruit set. Thesecond phase involves cell division and fruit growth, and the thirdphase involves cell expansion. Fruit set is always regulated bypositive signals generated during fertilization, although fleshyfruits can develop from a variety of floral parts, such as ovary in

tomato (Solanum lycopersicum), receptacle in strawberry, orfused sepals in apple (Malus domestica) (Nitsch, 1950; Gillaspyet al., 1993; Yao et al., 2001; Fuentes and Vivian-Smith, 2009).Fertilization-dependent fruit set ensures that the maternally de-rived structure, the fruit, only forms when fertilization is suc-cessful. In 1950, Nitsch showed that the removal of fertilizedovaries (achenes) from a strawberry receptacle prevented re-ceptacle fruit enlargement. However, exogenous auxin applica-tion acted as a substitute for achenes and stimulated receptaclefruit growth. Thus, auxin is essential to strawberry recep-tacle enlargement, and the achenes are the source of auxin(Nitsch, 1950; Dreher and Poovaiah, 1982; Archbold andDennis, 1984).In the cultivated strawberry F. x ananassa, fruit development

from fruit set to ripened fruit has been divided into seven stages:flower/anthesis, small green, medium green, large green, white,turning, and red (Fait et al., 2008). The levels of free auxin, whichwas extracted from receptacle and achenes together, wereshown to surge post fertilization, peak at the small green stage,and then decline during fruit maturation (Nitsch, 1950, 1955;Dreher and Poovaiah, 1982; Symons et al., 2012). This rise andsubsequent fall in auxin level correlates with auxin’s stimulatingeffect on receptacle growth and its inhibitory effect on colorationand ripening, respectively (Symons et al., 2012). The level ofGA1, an active form of gibberellin (GA), followed a similar trend,except that GA1’s rise and fall lags behind that of auxin (Symonset al., 2012). This finding is consistent with a similar effect of GAon fruit development; GA alone or in combination with auxinstimulated strawberry fruit development in the absence of pol-lination (Thompson, 1969). Studies in Arabidopsis thaliana andtomato indicated that auxin acts upstream of GA by stimulatingGA biosynthesis during fruit set (Serrani et al., 2008; Dorceyet al., 2009; Fuentes et al., 2012).

1 Address correspondence to zliu@umd.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Zhongchi Liu (zliu@umd.edu).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.113.111732

The Plant Cell, Vol. 25: 1960–1978, June 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

Understanding the hormonal regulation of fruit set is of con-siderable agronomic value. As illustrated above, exogenous hor-mone application can bypass the requirement of pollination andfertilization for fruit set. This so-called “fertilization-independentfruit set” or “parthenocarpy” could ensure high yields even underconditions unfavorable for fertilization, such as too high or lowtemperatures or a lack of pollinators. In addition to the induction ofparthenocarpic fruit by exogenous applications of auxin and GA ina variety of plant species (Ozga and Reinecke, 1999; Vivian-Smithand Koltunow, 1999), transgenic approaches that increase theexpression of auxin biosynthesis genes in ovules led to parthe-nocarpic eggplant (Solanum melongena), tomato, strawberry, andraspberry (Rubus idaeus) (Rotino et al., 1997; Ficcadenti et al.,1999; Mezzetti et al., 2004). Further, mutations affecting auxintransport (Sl-PIN4) and signaling (At-ARF8, Sl-ARF7, and Sl-IAA9)or GA signaling (Sl-DELLA) also induced parthenocarpic fruit inArabidopsis and tomato (Wang et al., 2005; Goetz et al., 2007;Marti et al., 2007; de Jong et al., 2009; Mounet et al., 2012).Therefore, better understanding of hormonal biosynthesis andsignaling in fruit crops will pinpoint key genes and pathways astargets of genetic manipulation for inducing parthenocarpic fruitdevelopment.

A recent report suggests that auxin biosynthesis in Arabidopsisinvolves only a two-step pathway, in which TAA1 (for TRYPTO-PHAN AMIONOTRANSFERASE OF ARABIDOPSIS1) and its ho-mologs TAR1-4 (for TRYPTOPHAN AMIONOTRANSFERASERELATED1 to 4) convert Trp to IPA (for INDOLE-3-PROPIONICACID). The YUCCA (YUC) family of flavin monooxygenases thencatalyzes the conversion of IPA to auxin (indole-3-acetic acid[IAA]) (Won et al., 2011). Auxin transport is vital to auxin functionand is based on the chemiosmotic model (Rubery and Sheldrake,1973; Feraru and Friml, 2008), in which auxin enters cells byAUXIN RESISTANT1/LIKE AUXIN RESISTANT (AUX/LAX) auxininflux carriers and exits cells directionally through the PIN-FORMED (PIN) family of efflux carriers. The asymmetric locali-zation of PIN on the plasma membrane directs the polarity of thetransport. Two types of auxin receptors have been reported (re-viewed in Hayashi, 2012). The TRANSPORT INHIBITOR RE-SPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) family auxinreceptors are the F-box subunits of the SKP-CULLIN-F-BOX(SCF) ubiquitin ligase. They facilitate ubiquitination and sub-sequent protein degradation of Aux/IAA repressors that normallybind and inhibit AUXIN RESPONSE FACTORS (ARFs). The re-leased ARFs can act upon downstream target genes (Mockaitisand Estelle, 2008). AUXIN BINDING PROTEIN1 (ABP1) representsa second type of auxin receptors implicated in rapid auxin re-sponses on the plasma membrane (Dahlke et al., 2010).

In the GA biosynthesis pathway, geranylgeranyl diphosphateis converted to GA12 through successive steps catalyzed byent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, and ent-kaurenoic acid oxidase. GA12 is thenconverted to bioactive GA1 and GA4 through a series of oxidationsteps catalyzed by GA 20-oxidases (GA20ox) and GA3-oxidases(GA3ox) (reviewed in Sun, 2011). By contrast, GA2ox convertsactive GA1 and GA4 to an inactive form, playing a critical role inmaintaining GA homeostasis (Sun, 2011). GA signaling causesthe protein degradation of DELLA transcriptional repressors, whichare encoded by five genes in Arabidopsis: REPRESSOR OF GA1-3

(RGA), GA-INSENSITIVE (GAI), RGA-LIKE1 (RGL1), RGL2, andRGL3. GA binding to its receptor GIBBERELLIN INSENSITIVEDWARF1 (GID1) increases the affinity of GID1 for DELLA proteins;the stable GID1-DELLA complex enables efficient recognition ofDELLA by SCFSLY1 and subsequent degradation of DELLA pro-teins by the 26S proteasome (Sun, 2011).Despite significant progress in illuminating the molecular basis

of auxin, GA, and other phytohormone biosynthesis and signalingpathways in model species, how these hormonal pathway genesoperate in the context of fruit set and fruit development is not wellunderstood. Furthermore, almost all past molecular studies ofstrawberry fruit were focused on the ripening of the fruit (Aharoniand O’Connell, 2002; Garcia-Gago et al., 2009), reflecting theagronomic interests and the value of strawberry as a crop. Incomparison, much less is known about the temporal and spatialregulation of fruit set and fruit growth, although these aspects areof equally critical importance to agriculture. In addition, almost allprior strawberry studies focused on the commercial cultivar F. xananassa, an octoploid lacking a sequenced genome. Finally,most molecular genetic studies of fruit development have beenon Arabidopsis and tomato, which develop true fruit from ovarywalls. Molecular genetic studies of strawberry, an accessory fruit,will greatly broaden our understanding of fruit development ingeneral. To date, there is still a need for an overall molecularframework that can be used to address fundamental questions offruit development. For example, where precisely are the phyto-hormones produced inside a seed? What are the relative con-tributions of auxin and GA to fruit set and growth? How are thehormonal signals transported to the receptacle and via whattypes of transport mechanisms? How does the receptacle, a tis-sue quite different from the true fruit (ovary), respond to auxin andGA, and how have different plant species evolved to developfleshy or dry fruit from different maternal structures?Previously, we conducted a detailed morphological description

of flower development and early fruit development in F. vesca(Hollender et al., 2012), placing the necessary groundwork for themolecular characterization of fruit development. Taking advan-tage of powerful second-generation sequencing technology, weprofiled early stage fruit transcriptomes of F. vesca, producinghigh-resolution digital profiles of global gene expression in fivedifferent fruit tissues at five developmental stages. Since sampleswere collected from well-characterized stages and tissues, thetranscriptome data is highly conducive to cross-lab or cross-species comparisons. Here, we report initial analysis of this wealthof molecular information, which yields unprecedented molecularinsight into early stage fruit development.

RESULTS

In-Depth Description of Early Fruit Development in F. vesca

Early F. vesca fruit development from anthesis to fertilization togreen fruit was divided into five stages that encompass com-plex developmental, morphological, physiological, and hormonalchanges (Hollender et al., 2012) (Figure 1). Stage 1 is the pre-fertilization stage when flowers have just opened. Stage 2 is 2 to4 d postanthesis, when fertilization has just occurred and signs of

Transcriptome of Strawberry Fruit Set 1961

senescence begin to show, including complete loss of petals,pink styles, and enlargement of the ovary. A globular stage em-bryo is inside the stage 2 seed (Hollender et al., 2012). Stage 3 istypified by red and dry styles, complete loss of anthers, anda heart stage embryo inside each seed (Figure 1A). At stage 4,embryos adopt torpedo or walking stick morphology. At stage 5,the two cotyledons of the embryo stay upright and fill up theentire seed. The cotyledons turn from transparent to white, in-dicative of embryo maturation. Hence, stage 5 marks the matu-ration of the embryos and achenes.

Size-wise, achenes undergo progressive enlargement fromstage 1 to stage 3 but remain relatively constant from stages 3 to5 (Figure 1A). Conversely, the receptacle remains relatively con-stant in size between stages 1 and 2 but increases progressivelyfrom stages 2 to 5, exposing more and more receptacle tissuebetween achenes. The receptacle size, which is proportional tothe number of successfully fertilized achenes, is a poor mea-surement of developmental stages due to varying degrees ofsuccessful fertilization. Instead, the morphology of the developingembryo inside each developing seed serves as a more reliabletemporal marker (Hollender et al., 2012). Based on the timing offruit coloration in Ruegen, a variety of F. vesca with red berries,stages 1 to 5 described here for F. vesca correspond to earlystage fruit development from prefertilization to “big green” incultivated strawberry (see Supplemental Figure 1 online).

Global Analysis of Fruit Transcriptome

To investigate the underlying molecular changes that accom-pany the morphological changes described above, we used

RNA-seq to generate transcriptome profiles for each of the fivestages. For each stage, we hand-dissected individual achenesinto three tissues: (1) ovary wall (wall, for short), (2) ghost, whichrefers to the entire seed with its embryo removed (and thusconsists predominantly of endosperm and seed coat tissues),and (3) embryo (Figures 1A and 1B). Furthermore, we separatedreceptacle tissues into (4) cortex, the fleshy tissue immediatelyunderneath achenes, and (5) pith, the interior tissue of the re-ceptacle (Figure 1C). Cortex and pith are separated by a ring ofvascular bundles, most of which were included in the pith tissuecollection. For simplicity, we use “fruit tissues” to indicate alltissues described above (embryo, ghost, wall, pith, and cortex),“achene” to refer to embryo, ghost, and ovary wall, “seed” torefer to embryo and ghost, and “receptacle” to indicate both pithand cortex.Sampling these five developmental stages and the five different

tissues resulted in 23 different reproductive transcriptomes.Stage 1, a prefertilization stage, lacks embryo and seed but hasovule tissue instead. Stage 2 has only “seed” tissue since theglobular embryo is too small to be dissected out of the seed.Additionally, we sampled two vegetative tissues, seedling andleaf, as controls. Two biological replicates were harvested foreach sample. For convenience, tissue name plus stage number isused as sample name hereafter. After removing low-quality reads,12 to 40 million reads per sample were mapped against theF. vesca reference genome (see Methods). An average of 62.6%of filtered reads was mapped to coding DNA sequence (CDS),and an average of 79.6% of filtered reads was mapped to thegenome (see Supplemental Table 1 online). Only reads mappedagainst CDS were used in subsequent analyses.

Figure 1. Detailed Staging of F. vesca Fruit Development.

(A) Five stages of F. vesca early fruit development. Top row shows each receptacle fruit dotted with spirally arranged achenes. Second row showsindividual achenes. Third row shows a single seed dissected out of each individual achene. “Ovary” and “Ovule” correspond to the prefertilizationstructure of achene and seed, respectively. The fourth (bottom) row indicates individual embryos dissected from individual seeds. The inset is anenlarged heart stage embryo. Bars = 2 mm in the top row and 0.2 mm in rows 2 to 4.(B) A dissected achene showing ovary wall and the seed inside.(C) A receptacle cross section showing pith (center) and cortex (flanking) tissues.

1962 The Plant Cell

The correlation dendrogram illustrates the global relative re-lationships among the 25 tissues (Figure 2A). All paired bi-ological replicates, with the exception of stage 4 pith, clusteredtogether. For all fruit tissue types, stage 1 to 2 samples clusteredtogether and stage 3 to 5 tissues clustered together (Figure 2A).This may suggest a delayed global response to fertilization dueto the time needed to transmit fertilization-induced signals acrosslayers of cells and tissues as well as signal amplifications neededto effect the entire fruit development. For example, the stage 3-5ovary wall tissues were more distant from stage 1-2 ovary walls,which grouped together with stages 1 and 2 ovule/seed, sug-gesting significant transcriptomic change at stage 3 ovary wallduring its transition from a female floral organ into the true fruit.Another example is the pith and cortex; they clustered together atstage 1-2 but become distant at stage 3-5, suggesting that onehallmark of fruit set is the differentiation between pith and cortex.

Seeds from stage 3 to 5 fruit were dissected to give rise toembryo and the remaining tissue, the ghost. Transcriptomes ofembryo 3-5 were most distinct; they were clustered distant fromall maternal tissues: ovary wall, cortex, and pith (Figure 2A).Likewise, ghosts were clustered next to embryos and distant

from the maternal tissues (Figure 2A). The two vegetative tissues(seedlings and leaves) were more related to each other and wereclustered between the maternal reproductive tissues and theghost/embryo tissues (Figure 2A).Genes with normalized reads lower than 0.3 “Reads Per Ki-

lobase per Million”. (RPKM) were considered “too lowly ex-pressed” and removed from analysis. Between 17,935 (embryo5) and 22,171 (seed 2) genes out of 34,809 predicted F. vescagenes remained in each transcriptome, 80% of which were inthe 1 to 100 RPKM range (Figure 2B). These genes from all fivestages of the same tissue were combined, and a Venn diagramwas used to reveal unique or commonly expressed genesamong the fruit tissues (Figure 2C; see Supplemental Data Set 1online). In total, 19,236 genes were common among all five fruittissues. While ghost (combining ovule 1, seed 2, and ghosts 3,4, and 5) had 990 tissue-specific genes, cortex and pith had only76 and 130 specific genes, respectively. This correlates withlower tissue complexity in cortex and pith. Transcription factors(TFs) unique to each tissue are also shown (Figure 2C; seeSupplemental Data Set 1 online). The vegetative transcriptome(seedling and leaf) was compared with fruit transcriptomes by

Figure 2. Analysis of Global Gene Expression among Fruit Tissues.

(A) Cluster dendrogram showing global relationships between biological replicates and among different stages and tissues. The y axis measures thedegree of variance (see Methods). In all figures to follow, samples are named as “Tissue_stage_replicate” or Tissue_stage.” Embryo 3.1 meansEmbryo_Stage 3_Replicate 1.(B) Number of genes expressed in each tissue with an average RPKM higher than 0.3.(C) A Venn diagram showing the number of commonly and uniquely expressed genes among the fruit tissues. The number of TFs is shown inparentheses.Expressed genes (RPKM > 0.3) in each tissue were combined for the analysis.[See online article for color version of this figure.]

Transcriptome of Strawberry Fruit Set 1963

Venn diagram (see Supplemental Data Set 1 online). In total, 3434fruit-specific and 518 vegetative-specific genes were identified;22,611 genes were expressed in both tissues (see SupplementalData Set 1 online).

Identification of Temporal or Spatial Expression Trendsacross F. vesca Fruit Transcriptome

To identify different gene expression profiles across all fivestages, we first filtered out genes with constant or low expres-sion levels (see Supplemental Figure 2 online). After filtering,15,754 genes remained. Next, relative RPKM value was calculatedfor each gene (RPKMX-tissue/RPKMaverage). The relative RPKMvalue allows for identification of genes with low absolute RPKMvalues (such as TFs) while still showing significant expressionchanges in specific tissues or stages. Thirty-six out of 50 k-meansclusters representing 5432 genes exhibited specific temporal orspatial expression patterns (Figure 3A). Clusters with similar ex-pression trends were further combined into 16 superclusters, eachwith a unique gene expression profile across stages and tissues(Figure 3A; see Supplemental Data Set 2 and Supplemental Table2 online).In temporally regulated clusters, supercluster 1 showed highest

expression at stage 1 (prefertilization) and gradual downregulationfrom stage 2 to 5 in all fruit tissues. By contrast, supercluster 3showed gradual upregulation from stage 1 to 5 in all tissues ex-cept embryos (Figure 3A; see Supplemental Table 2 online).Some superclusters were highly expressed only in one tissuesuch as embryo (supercluster 5), ghost (supercluster 6), and wall(supercluster 7). Other superclusters showed high levels of ex-pression in several tissues, such as supercluster 8 (achene: em-bryo, ghost, and wall), supercluster 9 (receptacle: cortex and pith),and supercluster 10 (wall, cortex, and pith). Enriched Gene On-tology (GO) categories could be identified for some of the su-perclusters with reasonable confidence (false discovery rate<0.05; see Supplemental Data Set 2 online), while other super-clusters did not have enriched GO terms due to a smaller genenumber in the supercluster.Among the 5432 genes that made up the 16 superclusters, 472

were TFs belonging to 41 families (Figure 3B; see SupplementalData Set 2 online). These TFs showed distinct stage or tissue-specific expression patterns mirroring each of the 16 clustersfrom which they came (Figure 3B; see Supplemental Table 2online). Dynamic expression changes associated with these TFsmay reveal key functions they may play. For example, five APE-TALA2 (AP2)-like genes, ANT (AINTEGUMENTA gene02623),AIL5 AINTEGUMENTA-LIKE 5 (gene16919), AIL6 (gene20828),BBM1 (BABY BOOM gene21524), and PLT2 (PLETHORA 2gene20607), were specifically upregulated only in embryos su-percluster 5), especially at stages 3 and 4 (Figure 3B; seeSupplemental Table 2 online). This suggests conserved func-tions in embryo development and organ primordial formation

Figure 3. Sixteen Superclusters of Genes with Unique Stage or TissueExpression Profiles.

(A) Thirty-six K-means clusters of 5432 genes showing distinct stage andtissue-specific expression patterns. The scale: averaged log2 “relative

RPKM value” of all genes in each cluster. Clusters with similar expres-sion trends are combined to form 16 superclusters.(B) The heat map showing log2 “relative RPKM values” of individual 472TFs.

1964 The Plant Cell

similar to the Arabidopsis homologs (Okamuro et al., 1997; Aidaet al., 2004; Krizek, 2009). In addition, WOX3 (WUSCHEL RE-LATED HOMEOBOX 3 gene14025) and WUSCHEL (gene14621)homologs were upregulated in receptacles and may regulatestem cell proliferation in the stem tip.

Global Analysis of Auxin and GA Pathway Genes

We identified F. vesca auxin and GA pathway gene families in-volved in synthesis, transport, conjugation, and signaling byBLAST searches in the F. vesca genome browser using Arabi-dopsis protein sequences as queries. Phylogenetic analysisusing protein sequences guided assignment of gene names(see Supplemental Figures 3 and 4 online).

Differentially expressed auxin and GA pathway genes wereidentified by pairwise comparisons between successive stages(Figure 4; see Supplemental Data Set 3 online). The data suggestan induction of a large number of auxin pathway genes in bothembryo and ghost. However, induced GA pathway genes mainlyoccurred in the ghost and ovary wall, but not in embryos. Tem-porally, the number of upregulated auxin and GA genes showedmoderate increase at stage 2, peaked at stage 3, tapered off atstage 4, and reached almost zero at stage 5 in all fruit tissues(Figures 4A and 4C). However, downregulated auxin and GA genesincreased dramatically in achenes at stage 5 (Figure 4B), whichcoincides with the completion of embryo development (Figure 1)

We examined in more detail the major auxin and GA genefamilies with RPKM values of 10 or higher in any of the 23 fruittranscriptomes (see Supplemental Data Set 4 online). Log2

RPKM values for these auxin and GA genes were subject tohierarchical clustering analysis (Figure 5). Auxin biosynthesisgenes YUC5, YUC11, and TAR1 and GA biosynthesis genesGA20ox3, GA3ox3, 4, 5, and 6 were predominantly expressed inthe achene (embryo, ghost, and wall) and largely absent from thereceptacle (pith and cortex) (Figure 5A). They were nearly allhighly expressed in the ghost and less highly in the embryo andwall. YUC4 and TAR2 represented a second group that wasmore abundant in the embryo (Figure 5A) and may play an im-portant role in embryo development. A third group representedby YUC10, GA20ox1, GA20ox2, and GA3ox1 was expressed inall fruit tissues but showed low or absent expression in embryos.Most of these biosynthesis genes showed gradual induction ofexpression from low at stage 1 to high at stage 4 or 5; this likelyreflects the effect of fertilization. By contrast, GA2ox2, GA2ox4,and GA2ox5, which are involved in GA catabolism, were ex-pressed at a low level in all fruit tissues and at all stages (Figure5A). Taken together, auxin and GA biosynthesis genes weremore highly expressed in the achene, particularly in the ghost.The data strongly indicate that achenes are the site of fertiliza-tion-induced auxin synthesis. Furthermore, the data pinpoint theghost, containing predominantly the endosperm and seed coat,as the main tissue for auxin and GA biosynthesis.

Auxin efflux and influx transporters did not exhibit obvioustissue or stage specificity and were more broadly expressed(Figure 5B). The exceptions were PIN10 (gene12312), which washighly induced in ghost immediately after fertilization, PIN5(gene16792), which was highly expressed in pith, and LAX1(gene20938), which was highly expressed in ghosts and

embryos (Figure 5B). In addition, PIN1 was highly expressedboth in embryo and in pith. Quantitative RT-PCR validated theRNA-seq data for several auxin transport and biosynthesisgenes (see Supplemental Figure 5 online).Auxin receptor genes TIR1, AFB2, and AFB5 were expressed

in all fruit tissues but exhibited downward regulation from high instages 1-2 to low in stages 3-5 (Figure 5C). ABP1, an endo-plasmic reticulum–localized auxin receptor (Dahlke et al., 2010),was mainly expressed in the embryo and ghost. Additionally, theGA receptor homolog GID1a was expressed 20-fold more highlyin the receptacle than in the achene, which was indicative of activeGA signaling in the receptacle. Furthermore, GAI and RGA1, bothDELLA repressors of GA signaling, were more abundantly ex-pressed in the receptacle. Together, the data suggest that thereceptacle expresses an abundance of receptors for auxin and GA.The increased GAI and RGA1 transcripts may be caused by rapidturnover of these two DELLA proteins or feedback regulationduring active GA signaling in the receptacle.GH3 proteins catalyze the conjugation of IAA to amino acids,

thereby reducing free IAA (Staswick et al., 2005). GH3 expres-sion is auxin inducible, forming a negative feedback loop tocontrol auxin homoeostasis. Multiple members of the GH3family, such as GH3.1, GH3.5, GH3.9, and GH3.17, were highlyexpressed in seed 2 and ghost 3-5 (Figure 5D). Therefore, IAAconjugation may be induced as IAA is being synthesized.AUX/IAAs are known to be transcriptionally induced by auxin

(Paponov et al., 2008). Strikingly, our data show that the majorityof the AUX/IAA and ARF genes were highly expressed in pithand cortex, less so in the ovary wall, and extremely lowly ex-pressed in the embryos and ghosts (Figure 5E). This indicatesactive auxin signaling in the receptacles and, to a lesser degree,in the ovary walls. Each AUX/IAA protein is known to inhibita partner ARF, but specific partnership between AUX/IAA andARF is difficult to discern. Hierarchical clustering (Figure 5E) re-vealed potential functional pairs based on coexpression, such asIAA8c and ARF2, IAA4a/16 and ARF6a/8, and IAA8a and ARF9.In general, IAAs were expressed at higher levels than ARFs.To summarize, while the biosynthesis genes for auxin and GA

were more highly and specifically expressed in the embryo andghost, the signaling components of GA and auxin were morespecifically and highly expressed in cortex and pith. Ovary wallsexpressed biosynthesis as well as signaling genes. The spatialseparation of hormone biosynthesis and signaling observedhere is consistent with earlier observations implicating theachene as the source of auxin and the receptacle as the re-sponding tissue. Our data also indicate achenes as the sourceof GA.

Fertilization-Independent F. vesca Fruit DevelopmentInduced by Auxin and GA

We tested the effect of exogenous auxin and GA application oninduction of fertilization-independent fruit set in F. vesca. Pre-vious experiments were done in the cultivated strawberry (F. xananassa), but the hormonal effect on F. vesca can be bettercorrelated with molecular events revealed by the F. vescatranscriptomes. Synthetic auxin (1-naphthaleneacetic acid [NAA])and GA (GA3) were applied to emasculated F. vesca flowers, with

Transcriptome of Strawberry Fruit Set 1965

mock treatment and hand pollination serving as negative andpositive controls, respectively. NAA or GA3 single hormone ap-plication induced fruit set and enlargement in both the acheneand the receptacle, but the fruit size was smaller than the hand-pollinated control (Figures 6A to 6F). When GA3 and NAA wereapplied together to the same emasculated flower, both theachene fruit and the receptacle fruit were larger in size than witheither single hormone treatment and were similar in size to thepollinated fruit (Figures 6A to 6F). This indicates an additive effectof GA and auxin on fruit size. Additionally, the ovary wall hard-ened just as in normal fertilization. The unfertilized seed inside theNAA- or GA-treated achene was enlarged as well (Figure 6C).

When the auxin efflux transport inhibitor N-1-naphthylphthalamicacid (NPA) was applied, fertilization-independent fruit enlargementwas observed in the receptacle (Figures 6A and 6D). However,the achene fruit enlargement was not as obvious (Figures 6B,6C, 6E, and 6F). A similar effect of NPA on parthenocarpic fruitwas reported for tomato and Arabidopsis (Serrani et al., 2008;Dorcey et al., 2009). This was interpreted to be a result of poolingIAA in the ovule due to a block of IAA transport by NPA, leadingto an artificially elevated level of auxin even in the absence offertilization.

Analysis of Transcriptomic Changes AccompanyingFertilization

Since fertilization is the key event required for fruit set, we iden-tified differentially expressed genes between stage 2 (immediately

postfertilization) and stage 1 (prefertilization) in each tissue. Theseed 2 versus ovule 1 comparison had the most differentiallyexpressed genes, with 1176 up and 1302 down (Figures 7A and7C; see Supplemental Data Set 5 online), suggesting that theimpact of fertilization is largely restricted to the seed at this earlystage or/and that the newly fertilized seeds have the highesttranscriptome complexity. Surprisingly, only 14 and 6 genes weresimultaneously up- or downregulated, respectively, in all four fruittissues, as revealed by the Venn diagrams (Figures 7A and 7C).Both MapMan (Thimm et al., 2004) and GO terms were used

to identify specific categories of genes or pathways enrichedamong the differentially expressed genes identified above (Fig-ures 7B and 7D; see Supplemental Data Set 5 online). The ovarywall and seed showed similarly enriched MapMan or GO cate-gories, such as secondary metabolism, cell wall synthesis, andorganization (Figure 7B), indicating coupling of certain geneexpression networks between seeds and ovary walls. However,the MapMan categories of pith 2 and cortex 2 were independentfrom each other (Figures 7B and 7D; see Supplemental Data Set5 online), despite their close physical proximity and similaroverall gene expression shown in Figure 2A.Among the differentially expressed genes, TFs with increased

expression in seed 2 when compared with ovule 1 included eightMADS box genes (two AGAMOUS-LIKE (AGL)80, three AGL62,one each of AGL15, AGL92, and AGL8), most of which wereupregulated 8- to 30-fold (see Supplemental Data Set 5 online).AGL80 and AGL62 were shown in Arabidopsis to regulate en-dosperm and central cell development (Portereiko et al., 2006;

Figure 4. Global Views of Differentially Expressed Auxin or GA Pathway Genes through Pairwise Comparisons between Successive Stages.

Percentage of auxin or GA pathway genes showing upregulation or downregulation through successive stages. Differentially expressed genes (foldchange >2, padj < 0.01) for stage 3 embryo was obtained by comparing embryo 3 with seed 2. All other tissues compare a later stage with an earlierstage in the same tissue. Number of total selected auxin genes was 78 and GA genes was 39 (see Supplemental Data Set 3 online).

1966 The Plant Cell

Kang et al., 2008). Two AP2 TFs with similarity to BBM andPLT2 were also highly induced in seed 2 (see Supplemental DataSet 5 online). In Arabidopsis, BBM and PLT2 are key regulatorsof embryo development (Smith and Long, 2010). The inducedexpression of AGL62/80 and BBM/PLT2 in fertilized seed 2 isindicative of active endosperm and embryo development inseed 2.

We investigated all six major phytohormone genes and theirexpression in the stage 2 versus stage 1 comparison (Table 1).Consistent with previous analysis (Figure 5), auxin biosynthesisenzymes YUC10, TAA1, and TAR1 were induced 11-, 153-, and10-fold, respectively. The auxin efflux carrier PIN10 was induced22-fold and the auxin conjugating enzymes GH3.2 and GH3.17

were induced 12.3- and 3.3-fold in seed 2. None of the auxinbiosynthesis genes were upregulated in the pith or cortex (Table1). The GA biosynthesis gene GA3ox1 was induced in seed andwall by 85- and 43-fold, respectively (Table 1). By contrast, almostall of the ethylene biosynthesis genes were downregulated (Table1). Interestingly, the ABA biosynthesis, catabolism, and signalinggenes were mostly downregulated in the achene and upregulatedin the pith, but none showed any change in the cortex.

Seed Anatomy and Subregion Transcriptional Activity

As products of double fertilization, embryo and endospermare both enclosed within the ghost, yet they possess distinct

Figure 5. Expression of Major Auxin and GA Pathway Genes across Tissues and Stages.

Heat maps depicting log2 RPKM value for major auxin and GA genes that had an RPKM >10 in at least one fruit tissue.(A) Biosynthetic genes.(B) Auxin efflux transporters (PIN) and influx transporters (AUX/LAX).(C) Auxin receptors, GA receptors, and DELLA repressors of GA signaling.(D) GH3 auxin conjugating enzymes.(E) Auxin signaling components, ARF and IAA.

Transcriptome of Strawberry Fruit Set 1967

Figure 6. Parthenocarpic Fruit Development Induced by Auxin and GA.

(A) Fertilization-independent fruit enlargement induced by exogenous applications of GA3, NPA, NAA, or GA3 plus NAA to emasculated flowers. Mockand hand-pollination serve as the negative and positive controls, respectively. Photos were taken at day 12 after the first hormone application. Bar = 5mm.(B) Photos showing achenes of the corresponding treatments in (A). Bar = 0.5 mm.(C) Achenes dissected to show a single white seed (white arrow) inside. Bar = 0.2 mm.(D) Quantitation of receptacle fruit length (mm) in the respective treatments. n = 10 to 14 fruits for each treatment.(E) and (F) Relative achene length (E) and width (F), derived by dividing experimentally treated achene size with mock-treated achene size. The mock-treated achene size is designated as 100%. n = ;15 achenes for each treatment.Asterisks denote statistically significant differences at *P < 0.05 and **P < 0.01, respectively, in the indicated comparisons by t test.

1968 The Plant Cell

developmental programs. We observed their developmentthrough microscopy (Figure 8). The F. vesca female gameto-phyte at the time of anthesis consisted of a long and narrowembryo sac with a highly visible central cell (Figure 8A). Sur-rounded by integument layers, the embryo sac positioned theegg cell at the top (toward the style) and the chalaza end atthe base. The ovule, which will become the seed upon ferti-lization, was connected to the ovary wall with visible vascularbundles (Figure 8B). After fertilization, confocal images ofpropidium iodide–stained seeds indicated an eight-cell octantembryo zygote encased in a sac of micropylar endosperm nuclei(Figure 8C).

The complex tissue and anatomy of seeds raise an importantquestion: Are auxin and GA produced uniformly throughout theseed? Our transcriptome data indicate that the ghost playsa more prominent role in auxin and GA biosynthesis (Figure 5).We mined an Arabidopsis transcriptome database (Belmonteet al. 2013), which provides a higher tissue resolution of geneexpression within the seed. Arabidopsis homologs of F. vesca

auxin-related genes At-YUC10 and At-TAR1 showed chalazal-endosperm-specific expression, whereas At-YUC6, At-TAR2,and At-PIN3 exhibited chalazal-seed coat-specific expres-sion (see Supplemental Figure 6 online). Similarly, Arabi-dopsis orthologous genes in GA biosynthesis appeared tobe expressed in either the chalazal endosperm (At-GA3ox3,4; At-GA20ox4, 5), the chalazal seed coat (At-GA20ox2), orin both chalazal seed coat and chalazal endosperm (At-GA20ox1) (see Supplemental Figure 6 online). The chalazalseed coat– and endosperm-specific localization of hormonalgene expression may directly affect maternal fruit tissuedevelopment.To directly test region-specific expression of F. vesca genes

within the strawberry seed, we fused FvPIN1 and FvPIN5 pro-moters to the b-glucuronidase (GUS) reporter and transformedthe reporter genes into F. vesca (YW5AF7) plants. In stage 1ovules, pFvPIN1:GUS and pFvPIN5:GUS similarly showed chala-zal domain–specific expression (Figures 8D and 8G). At stage 2,both genes were induced in the seed integuments, the precursors

Figure 7. Gene Expression Comparisons between Stage 1 and Stage 2 Fruit Tissues.

(A) and (C) Differentially expressed genes between stage 2 and stage 1 in each of the fruit tissues. Overlapping sets of upregulated (A) or down-regulated (C) genes between fruit tissues are shown in the Venn diagram.(B) and (D) Percentage of upregulated (B) or downregulated (D) genes belonging to specific MapMan bin categories. The number of up- or down-regulated genes in each tissue with available MapMan bins is indicated next to the color code of tissues. Percentage of total annotated genes in thegenome belonging to each MapMan bin serves as a control and is coded gray.

Transcriptome of Strawberry Fruit Set 1969

to seed coat (Figures 8E and 8H). At stage 3, when heart stageembryos became visible, pFvPIN1:GUS was highly expressed inthe embryo (Figure 8F), while pFvPIN5:GUS was only weakly ex-pressed in the embryo (Figure 8I). Both genes remained highlyexpressed in the chalazal end of the seed coat throughout seed

development (Figures 8F, 8I, and 8J). These distinct expressionpatterns were consistent with the RNA-seq data (Figure 5B).Furthermore, the persistent chalazal seed coat expression ofboth auxin efflux carriers highlights the importance of chalazalseed coat in auxin transport.

Table 1. Differentially Expressed Genes in Hormonal Pathways Identified by Comparing Stage 2 (Postfertilization)with Stage 1 (Prefertilization) Transcriptomes

Hormone Tissue Gene ID Gene Name Putative Function Fold Change

Auxin Seed gene27796 YUC10 Biosynthesis 11.8gene03586 TAA1 Biosynthesis 153.1a

gene31791 TAR1 Biosynthesis 10.7gene24005 GH3.2 Conjugation 12.3gene23026 GH3.17 Conjugation 3.3gene04070 PIN4 Transport 2gene01267 PIN8 Transport 4.2gene12312 PIN10 Transport 22.6gene16571 IAA16 Signaling 2gene25723 IAA26b Signaling 2gene27891 IAA12 Signaling 2.6gene22779 IAA31 Signaling 6.5gene23280 ARF16c Signaling 3.1gene30882 YUC2 Biosynthesis 0.014gene07619 LAX3 Transport 0.38

Wall gene27796 YUC10 Biosynthesis 2.2gene23026 GH3.17 Conjugation 3.4gene08336 IAA11 Signaling 2.2gene27891 IAA12 Signaling 2.2gene22779 IAA31 Signaling 2.4gene23280 ARF16c Signaling 4.2gene22838 GH3.1 Conjugation 0.35gene03265 GH3.5 Conjugation 0.18gene07619 LAX3 Transport 0.41

Cortex gene22838 GH3.1 Conjugation 2.9gene08194 IAA14b Signaling 3.1gene27792 ARF1b Signaling 2.9

Pith gene22838 GH3.1 Conjugation 2.7gene18907 GH3.11 Conjugation 2.5gene27740 GH3.12 Conjugation 3.5gene01376 AFB2 Receptor 2.9gene08194 IAA14b Signaling 2.1gene08492 ARF5 Signaling 4.6gene12917 ARF19a Signaling 2.2gene27792 ARF1b Signaling 2.2gene31790 TAR2 Biosynthesis 0.15gene30702 GH3.6 Conjugation 0.34gene27891 IAA12 Signaling 0.48gene05990 IAA19 Signaling 0.21

GA Seed gene19699 KS1 Biosynthesis 2.9gene13360 GA20ox1 Biosynthesis 7.7gene06004 GA3ox1 Biosynthesis 84.9gene01058 GA3ox3 Biosynthesis Inf (0‒48.8)b

gene27756 GID1b Signaling 0.46Wall gene06004 GA3ox1 Biosynthesis 43.3

gene07935 GA2ox4 Deactivation 2.2gene06947 RGL3 Signaling 0.084

Cortex NonePith gene06004 GA3ox1 Biosynthesis 3.7

gene27756 GID1b Signaling 3.2

(Continued)

1970 The Plant Cell

Table 1. (continued).

Hormone Tissue Gene ID Gene Name Putative Function Fold Change

Cytokinin Seed gene10304 CYP735A1 Biosynthesis 4.5gene14452 CKX1 Degradation 2.3gene12648 CKX5 Degradation 5.1gene13325 ARR2b Signaling 46.4gene09744 LOG3 Biosynthesis 0.3gene30448 LOG7 Biosynthesis 0.12gene30654 CKX2 Degradation 0.088gene15204 CKX8 Degradation 0.26

Wall gene30477 LOG9 Biosynthesis 2.2gene04136 AHK4 Signaling 2.7gene09501 ARR5 Signaling 2.7gene21885 ARR9a Signaling 2.5gene08409 ARR24a Signaling 2.5

Cortex gene27842 IPT5 Biosynthesis 12.7gene15382 CKX6 Degradation 3.6gene08409 ARR24a Signaling 2.9

Pith gene27842 IPT5 Biosynthesis 15.4gene02714 LOG6 Biosynthesis 3.3gene30477 LOG9 Biosynthesis 3.8gene14452 CKX1 Degradation 5.5gene11196 ARR9c Signaling 0.37gene09501 ARR5 Signaling 0.38

BR Seed gene20962 CPD Biosynthesis 0.41Wall gene20962 CPD Biosynthesis 4.6Cortex NonePith gene20962 CPD Biosynthesis 18.3

gene30974 CYP72B1 Deactivation 3.1gene01195 BRL1 Signaling 2.1gene29525 BZR2 Signaling 2.2

Ethylene Seed gene19023 ACS2 Biosynthesis 0.081gene19733 ACO2 Biosynthesis 0.12gene11421 ACO4 Biosynthesis 0.16

Wall gene19733 ACO2 Biosynthesis 0.21gene11421 ACO4 Biosynthesis 0.12

Cortex gene01202 ACO1 Biosynthesis 6.6gene31045 EBF2 Signaling 2.4gene11421 ACO4 Biosynthesis 0.043gene23460 EIN5c Signaling 0.39

Pith gene01202 ACO1 Biosynthesis 5.8gene00379 EIN3b Signaling 2.4gene11442 ERF1 Signaling Inf (0‒14.5)b

gene11421 ACO4 Biosynthesis 0.044ABA Seed gene31335 NCED3 Biosynthesis 0.26

gene11291 PP2C5 Signaling 0.43gene26316 PP2C7 Signaling 0.49

Wall gene02502 CHLH/ABAR Signaling 2gene10931 CYP707A1/3 Catabolism 0.47gene11969 SnRK2.10 Signaling 0.49

Cortex NonePith gene09100 CYP707A4a Catabolism 5.6

gene21044 PYL5 Signaling 4.9gene29674 PYL6 Signaling 3.7gene31902 OST1 Signaling 4.5gene24096 SnRK2.3 Signaling 18.6gene10769 SnRK2.4 Signaling 2.2gene16244 SnRK2.6 Signaling 2.9gene30616 NCED5 Biosynthesis 0.26

Bold font highlights downregulated genes. ABA, abscisic acid; BR, brassinosteroid.aTAA1 was not identified in the global analysis (Figure 5), which focuses only on genes with a RPKM >10.bInf, an expression difference between 0 (stage 1) and a specific RPKM (stage 2).

Transcriptome of Strawberry Fruit Set 1971

Comparing Embryo and Ghost Transcriptomes

To compare the contributions of embryo and ghost to fruit set,we compared stage 3 embryo and ghost, respectively, to seed2, which contained both embryo and ghost (Figure 9A). Embryo3 had a higher number of downregulated genes due to its dis-tinct tissue composition from seed 2. A Venn diagram revealed345 coordinately upregulated and 630 coordinately down-regulated genes in embryo 3 and ghost 3 (Figure 9A; seeSupplemental Data Set 6 online). In total, 262 and 97 genesexhibited opposite expression trends, up in ghost 3 and down inembryo 3 or vice versa. The coordinated and opposite expres-sion trends are summarized in drawings below the Venn diagram(Figure 9A). Comparisons were also made between ghost 3and embryo 3 to identify genes preferentially expressed in ei-ther tissue (Figure 9B; see Supplemental Data Set 7 online).Embryo 3–abundant genes were enriched for GO terms in celldivision and ribosome biogenesis as well as developmentalregulation (Figure 9B). By contrast, apoptosis, auxin biosynthetic

process, and signal transduction were overrepresented in ghost 3(Figure 9B), again supporting a prominent role of ghost in auxinbiosynthesis.

DISCUSSION

Despite significant discoveries made in strawberry fruit researchin the 1950s (Nitsch 1950, 1955), progress has been limited inelucidating the molecular mechanisms by which auxin drivesfruit development. Our work reported here provides crucialmolecular insights into this important developmental process.The comprehensive two-dimensional tissue and stage collectionand in-depth RNA-seq data set enable genome-scale analysesat a high resolution. The genome-scale approach allows us toexamine the expression profiles of all members of gene familiesin an unbiased manner and to simultaneously analyze multiplehormonal pathways. Previously, Csukasi et al. (2011) examineda single Fa-GA3ox gene (which corresponds to Fv-GA3ox1;

Figure 8. Domain-Specific Transcription of FvPIN1 and FvPIN5 within a F. vesca Seed.

(A) A longitudinal section of an ovary in an open flower before fertilization. Ovary wall (w), the central cell (arrowhead), the Chalazal (ch) end of the ovule,and the style (st) are indicated.(B) Nomarski image of a prefertilization ovary showing the vascular (v; false colored blue) connection between the ovule (false colored beige) and theovary wall (w).(C) A confocal three-dimensional projection stack of a fertilized seed (stage 2). The octant stage embryo (white arrow) is surrounded by micropylar poleendosperm nuclei (white arrowhead).(D) to (F) Images of pFvPIN1:GUS expression.(G) to (J) Images of pFvPIN5:GUS expression.(D) and (G) Stage 1 prefertilized ovule.(E) and (H) Stage 2 newly fertilized seed.(F) and (I) Stage 3 seed.(J) Dorsal view of a stage 3 seed showing the chalazal staining.Bars = 100 µm in (A), (B), (D), and (G), 25 µm in (C), and 200 µm in (E), (F), (H), (I), and (J).

1972 The Plant Cell

gene 06004 in this study) during cultivated strawberry fruit de-velopment and reported that Fa-GA3ox was expressed at a level40-fold higher in the receptacle than in the achene. They con-cluded that the receptacle was the main source of GA bio-synthesis. We show that Fv-GA3ox1 is expressed at low levels

in all fruit tissues while other family members, Fv-GA3ox4, 5,and 6, are expressed many fold higher than Fv-GA3ox1 in theghost. This reveals a prominent role of ghost in GA biosynthesis,a conclusion contrary to that of Csukasi et al. (2011). This il-lustrates the power of genome-wide studies in that they allowsimultaneous examination of all family members.Taking advantage of the accessibility of achenes on the out-

side surface of the receptacle, we were able to dissect the seedout of the achene and the embryo out of the seed. This providedus an unprecedented opportunity to resolve gene expressionbetween embryo and the remaining seed (ghost) and enabled usto discover that ghosts likely play a more important role than theembryos in the synthesis of auxin and GA for fruit set. Such finedissection and separation of embryos from remaining seedswould prove highly challenging in other fruit types, such as to-mato, the seeds of which are embedded and mixed with theinternal tissues.Our study provides a wealth of genomic information on the

earliest stages of fruit development, which is an understudiedbut critically important area of fruit research. Past molecularstudies have largely focused on the ripening of the strawberryfruit (Aharoni and O’Connell, 2002; Garcia-Gago et al., 2009).The availability of our RNA-seq data to the entire researchcommunity (via Sequence Read Archive at the National Centerfor Biotechnology Information (NCBI) or via http://bioinformatics.towson.edu/strawberry/Default.aspx) paves the way for futurefunctional dissection of genes and networks that regulate fruitset and fruit growth.

Figure 9. Differential Gene Expression of Embryo and Ghost.

(A) A Venn diagram reveals overlapping sets of genes between upre-gulated and downregulated genes in embryo 3 and ghost 3 when each iscompared with seed 2. Beneath the Venn diagram are graphic repre-sentations of stage 3 seeds showing different sets of genes with similaror opposite expression trends in embryo 3 and ghost 3. Pink indicates upre-gulation; green indicates downregulation; white indicates no change. Numberof genes in each expression category is written beneath the seed diagram.(B) Top 10 overrepresented GO terms of embryo 3–abundant and ghost3–abundant genes.

Figure 10. A Model of Strawberry Fruit Development.

A diagram illustrating auxin and GA biosynthesis and transport in theachene soon after fertilization. Fertilization-induced auxin and GA ac-cumulation in the ghost are transported to the ovary wall (route 1) and thereceptacle (route 2), shown by the two red arrows. The arrow from auxinto GA within the ghost indicates the positive effect of auxin on GA bio-synthesis. In route 1 to the ovary wall, PIN-dependent transport of auxinmay be necessary to promote local auxin and GA biosynthesis in theovary wall. In route 2 from ghost to receptacle, GA and auxin may betransported via a PIN-independent mechanism (or both PIN-dependentand independent mechanisms). Upon arriving at the receptacle, auxinand GA exert their effects by stimulating downstream signaling events forreceptacle growth.

Transcriptome of Strawberry Fruit Set 1973

Insights into Auxin Biosynthesis and Signaling duringFruit Development

Previous studies using DR5 reporters indicated a surge of auxinin the ovule/seed postfertilization and a lack of auxin in the ovarywall/pericarp, suggesting that ovary wall does not play a majorrole in auxin biosynthesis (Hocher et al., 1992; Pattison andCatala, 2012). Our data (Figure 5A) also show lower expressionlevels of auxin biosynthesis genes in the ovary wall when com-pared with the ghost. Furthermore, our data show that ghostshave the highest expression levels of auxin biosynthesis genesamong the achene tissues (embryo, ghost, and ovary wall) andmay be the main site of auxin biosynthesis.

The strong expression of auxin receptors and signaling com-ponents in the receptacle (Figures 5C and 5E) indicates that auxinis likely transported to the receptacle and exerts a direct role instimulating receptacle growth. This differs from earlier sugges-tions of an indirect role of auxin in ovary wall fruit set throughauxin-mediated promotion of GA synthesis (Dorcey et al., 2009).An indirect role of auxin in strawberry receptacle fruit set couldalso explain an absence of free IAA in the receptacle (Nitsch,1950; Symons et al., 2012). However, the IAA measurementmethod in 1950 was less sensitive than modern methods. Addi-tionally, Symons et al. (2012) measured free IAA from the whitestage receptacle, a late stage by which the IAA level may havesignificantly declined. A recent immunohistochemical study de-tected strong signals of IAA in the phloem of the receptacles (Houand Huang, 2004), but the immunohistological staining may notbe able to distinguish free IAA from conjugated forms. Therefore,it remains unresolved as to whether free IAA exists in the re-ceptacle at the early stages of fruit development.

Based on our data, we propose that a majority of the auxinmade in the ghost may be conjugated and then transported tothe receptacle as a conjugated form. In the receptacle, free IAA,which could be released to activate downstream signaling,would then be quickly degraded. Indeed, we observed highly ex-pressed auxin conjugating GH3 genes in ghost and ovary wall(Figure 5D, Table 1). Furthermore, IAA amide conjugates (Archboldand Dennis, 1984) and a highly abundant IAA-protein conjugate(Park et al., 2006) were reported in strawberry receptacles.

GA Signaling and Parthenocarpic Fruit Development

Previous studies in other plant species indicated a crucial role ofGA in fruit set and suggested that auxin acts upstream of GA ina linear pathway to stimulate fruit development (Serrani et al.,2008; Dorcey et al., 2009; Fuentes et al., 2012). Here, we dem-onstrated that single GA3 application to emasculated F. vescaflowers caused fertilization-independent fruit enlargement in bothfruit types (achene and receptacle) (Figure 6) and combined ap-plication of auxin (NAA) and GA3 stimulated both fruit types togrow to wild-type size in the absence of fertilization. This in-dicates that GA and auxin may have common as well as uniqueroles in fruit set and growth.

Application of NPA, an inhibitor of polar auxin transporters,also resulted in parthenocarpic strawberry fruit (Figure 6),probably by pooling IAA in the ovule due to a block of IAA ex-port. Interestingly, the degree of receptacle enlargement ap-peared to be greater than that of the ovary wall. This suggests

that auxin polar transport (via PIN proteins) is required for ovarywall enlargement and is consistent with the chalazal localizationof FvPIN1:GUS and FvPIN5:GUS in prefertilization ovules. InArabidopsis, NPA-treated siliques were more slender than GA3-treated siliques (Dorcey et al., 2009), indicating a similarly PIN-dependent transport of auxin from the seed to the ovary wall.We propose that NPA treatment resulted in an artificially ele-vated auxin level inside the ovule. This increased IAA could notbe transported to the ovary wall due to nonfunctional PIN pro-teins. However, the increased IAA in ovules could trigger thesynthesis of secondary signals, such as GA in ovules. A secondand nonexclusive scenario is that the elevated IAA could beconjugated in the ovule. Both GA and conjugated IAA could betransported to the receptacle (and less so to the ovary wall) viaPIN-independent transport routes.Parthenocarpic fruit is of significant agronomic value because it

can ensure high fruit yields even under unfavorable growingconditions. In Arabidopsis and tomato, a mutant form of At-ARF8,a knockdown of Sl-ARF7 by RNA interference, or a knockdown ofSl-IAA9 by RNA interference were shown to induce partheno-carpy (Wang et al., 2005; Goetz et al., 2006; Goetz et al., 2007; deJong et al., 2009). It was proposed that the IAA9/ARF7/ARF8repressor complex inhibits fruit set in the absence of fertilization,perhaps by inhibiting GA synthesis (Wang et al., 2009). Our studyprovides the molecular basis for the selection of certain straw-berry auxin and GA pathway genes, such as the receptacle-specific Fv-IAA16 or Fv-GAI, as targets for genetic manipulationto induce parthenocarpic strawberry fruit.

A Model of Strawberry Receptacle Fruit Development: ASpatial Consideration

We propose that upon fertilization, auxin is synthesized in theproducts of double fertilization: the embryo and the endosperm(as well as seed coat). In our model, auxin made in the embryo isinvolved in embryo development and has less to do with fruitdevelopment; auxin synthesized in the ghost is responsible forfruit set (Figure 10). PIN-dependent transport of auxin is neces-sary for botanical fruit (ovary wall) development. Once transportedto the ovary wall, auxin induces the secondary biosynthesis of GAand auxin in the ovary wall. Blocking the initial transport of auxinto the ovary wall by NPA would limit secondary GA and IAAbiosynthesis and thus result in slower ovary wall growth. Auxinalso stimulates GA biosynthesis inside the seed, mainly in thechalazal end. Both auxin and GA in the seed are transported tothe receptacle, perhaps via a PIN-independent mechanism (or bothPIN-dependent and independent mechanisms), to promote down-stream signaling events in receptacle fruit growth (Figure 10).

METHODS

Tissue Isolation and RNA Extraction

All fruit tissues were collected from a 7th generation inbred line of Fragariavesca, Yellow Wonder 5AF7 (YW5AF7, Slovin et al., 2009). Plants weregrown in a growth chamber with 12 h light at 25°C followed by 12 h dark at20°C. All tissues were hand-dissected under a stereomicroscope andfrozen immediately in liquid nitrogen. The tissues from at least three fruitswere combined to form one biological replicate and there were two

1974 The Plant Cell

biological replicates for each tissue. Around 1000 heart stage embryoswere dissected and pooled into two biological replicates.

RNA was extracted using the RNeasy plant mini kit (Qiagen); on-column DNase digestion with the RNase-Free DNase set (Qiagen) wasperformed to remove contaminating DNA. A total of 0.5 to 2 µg RNA persample was sent to the Genomics Resources Core Facility at Weill CornellMedical College for library preparation with Illumina TruSeq RNA samplepreparation kit and sequencing with Illumina HiSeq2000. In most cases,six libraries were bar-coded and sequenced in one lane. About 12 to 40million 51-bp single-end reads were generated for each sample (seeSupplemental Table 1 online).

Initial Mapping of Reads

The raw reads were filtered with FASTQ_Quality_Filter tool from theFASTX-toolkit. Reads with more than 90% of their bases having a qualityscore higher than 28 were kept. The filtered reads were aligned to theF. vesca genome (v1.1) and CDS (v1.0) (Shulaev et. al., 2011) using Bowtie1.0 with default settings, allowing twomismatches. The F. vesca referencegenome and CDS files were downloaded from the Genome Data forRosaceae website at www.rosaceae.org/species/fragaria/fragaria_vesca.Alignment output files from Bowtie were parsed using scripts written inPerl to calculate the number of reads for each gene. The script is includedin the Supplemental Methods 1 online. Reads mapped against CDS wereused in all subsequent analyses.

Identification of F. vesca TFs and Hormonal Pathway Genes

Arabidopsis thaliana TF protein sequences were downloaded fromPlantTFDB (http://planttfdb.cbi.edu.cn) (Zhang et al., 2011), which wereused to blast against the F. vesca GeneMark hybrid proteins with an-notations (downloaded from www.rosaceae.org) by BLASTP using blast-2.2.26+ from NCBI on local computers. Stringent criteria (e-value < 10220;bit score > 100; pident > 30%) were applied to select the set of F. vescaTFs, which were then manually checked against the TF families inPlaza2.5 (http://bioinformatics.psb.ugent.be/plaza/) to yield the final setof F. vesca TFs listed in Supplemental Data Set 8 online.

Hormonal genes were identified by BLAST against Fragaria vescaGene Models (Hybrid V2) using Arabidopsis protein sequences as query(https://strawberry.plantandfood.co.nz/cgi-bin/nph-blast.cgi?Jform=0).Top hits were confirmed by BLAST against Arabidopsis protein data-base. All the F. vesca hormonal genes are shown in Supplemental DataSet 4 online.

Gene Expression Analysis

The dendrogram in Figure 2A was made by the function cor () in R withdefault settings. The y axis is computed as 1 minus cor (correlation) toreflect the degree of variance. Log2-transformed mapped read countswithout normalization were used in the computation. The bar graph inFigure 2B and the Venn diagram in Figure 2C are based on RPKM. TheVenn diagram in Figure 2C was made by function venn () in R using genelist for each tissue type, which is derived by combining genes from allstages of the same fruit tissue (see Supplemental Data Set 1 online). Asimilar comparison was conducted between vegetative tissues (seedlingand leaf) and fruit tissues (all five fruit tissues) (see Supplemental Data Set1 online).

For Figure 3, only 17,108 differentially expressed genes were selectedbased on the selection scheme described in Supplemental Figure 2online. DESeq in R (Anders and Huber, 2010) was used to select forgenes with differential gene expression between stages and tissues(padj < 0.0001). After removing low abundance genes or genes highlyexpressed in nonfruit tissues, 15,754 remaining genes were subject to the

K-means clustering using the MultiExperiment Viewer 4.8 (MeV4.8; http://www.tm4.org/mev/) with Euclidean distance (Saeed et al., 2006). Log2-transformed relative RPKM value (RPKMgeneX in each tissue divided byaverage RPKMgeneX across all fruit tissues) was imported into MeV4.8.The optimal number of clusters was determined to be 50 based on figureof merit analysis within MeV4.8 (Yeung et al., 2001). Only 36 clusters withdistinct tissue- or stage-specific expression profiles are shown in Figure3A. TFs were extracted from the 16 superclusters (Figure 3A; seeSupplemental Data Set 2 online) based on the F. vesca TF table (seeSupplemental Data Set 8 online); the log2-transformed relative RPKMvalue of each TF was used to yield Figure 3B via Mev4.8.

The differentially expressed genes in auxin and GA pathwaysshown in Figure 4 were extracted from the differentially expressedgene lists in pairwise comparisons with successive stages conductedwith DESeq in R. Other differentially expressed genes between stages2 and 1 or between embryo 3 and ghost 3 (Figures 7 and 9) weresimilarly identified using DESeq in R following its vignette. In all DESeqanalyses, mapped read counts against CDS without normalizationwere used as input. In pairwise comparisons with DESeq in R, thefunctions were set at newCountDataSet, estimateSizeFactors, esti-mateDispersions, and nbinomTest; P value was adjusted using theBenjamini-Hochberg method; cutoff was set at fold change > 2 andpadj < 0.01. In the multifactor design that tests all fruit tissues, stageand tissue were used as the two factors; the functions includenewCountDataSet, estimateSizeFactors, estimateDispersions, fitN-binomGLMs, and nbinomGLMTest; P value was adjusted using theBenjamini-Hochberg method; differentially expressed genes withpadj # 0.0001 were kept.

The hierarchical clustering shown in Figure 5 uses average linkageclustering and Pearson correlation within MeV4.8 and RPKM as input. Theheat map in Supplemental Figure 6 online was made in MeV4.8 based onthe globally normalized microarray data downloaded from the GeneExpression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) (Belmonte et al.2013). Venn diagrams in Figures 7 and 9 were made by R packageVennDiagram.

Assignment of MapMan Bins and GO Terms

MapMan bins of F. vesca genome were assigned by the Mercator pipelinefor automated sequence annotation (http://mapman.gabipd.org/web/guest/app/mercator) (Thimm et al., 2004); 100 was used as the BLAST_-CUTOFF.

GO ontologies were assigned using Blast2GO (Conesa et al., 2005).Within the Blast2GO, CDS sequences of F. vesva genome at http://www.rosaceae.org/species/fragaria/fragaria_vesca/genome_v1.0 were used toBLAST against the nonredundant database in NCBI by BLASTX (e-value1023). Subsequent “GO-annotation” and “InterPro annotation” functionswithin Blast2GO were used to yield the GO annotation file for ;20,003F. vesca genes (see Supplemental Data Set 9 online). GO enrichmentwas derived with Fisher’s exact test and a cutoff of false discovery rate <0.05; the genome annotation file described above was used as thereference. Only Biological Process GO terms are shown in tables orfigures.

Phylogenetic Analysis

The unrooted phylogenetic tree shown in Supplemental Figures 3 and 4online was constructed using MEGA 5.05 (http://www.megasoftware.net/)with the neighbor-joining statistical method and bootstrap analysis(1000 replicates). Protein sequences were downloaded from Plaza2.5(http://bioinformatics.psb.ugent.be/plaza/); the sequence alignment(see Supplemental Data Set 10 online) was made using Clustal Omega(http://www.ebi.ac.uk/Tools/msa/clustalo).

Transcriptome of Strawberry Fruit Set 1975

Hormonal Treatment of Flowers

About 50 mL hormone solution was pipetted onto the receptacle of eachemasculated flower. Stock solutions of 50 mM NAA (Sigma-Aldrich),50mMNPA (Sigma-Aldrich), and 100mMGA3 (Sigma-Aldrich) weremadein ethanol and were diluted with two drops of Tween 20 and water beforeapplication. The final treatment concentrations were 500 µM for NAA andGA3 and 100 µM for NPA. The solutions were applied every 2 d until day 12when photos were taken. Fruit size was measured with ImageJ of pho-tographed fruits.

Promoter:GUS Construct and Strawberry Transformation

The Fv-PIN1 (gene09384; 2148 bp) and Fv-PIN5 (gene16792; 2298 bp)promoters were PCR amplified from YW5AF7 genomic DNA, cloned intopMDC162 binary vector, and transformed into YW5AF7 according toa published protocol (Slovin et al., 2009; Chatterjee et al., 2011). A moredetailed description is included in the Supplemental Methods 1 online.The primers are listed in Supplemental Table 3 online.

Microscopy Analysis

For light microscopy, histological sections of F. vesca flowers at anthesiswere stained with Safranin-O/Fast Green and photographed undera Nikon LABOPHOT-2 microscope equipped with an AxioCam digitalcamera as described previously (Hollender et al., 2012). For confocalimages, seeds were dissected out of the ovary and stained with pro-pidium iodide following a published protocol (Running et al., 1995). Thephoto was taken with a Leica SP5 X confocal microscope by three-dimensional projection with eight Z-stacks of the same seed. GUSstaining and photography are described in the Supplemental Methods 1online.

Accession Numbers

Illumina reads of all 50 samples have been submitted to the SequenceRead Archive at NCBI (http://www.ncbi.nlm.nih.gov/sra). The submissioncode is SRA065786.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. F. vesca Ruegen Late-Stage Fruit Development.

Supplemental Figure 2. Filtering Strategy for Differentially ExpressedGenes Shown in Figure 3.

Supplemental Figure 3. Phylogenetic Trees of Major Auxin PathwayGenes.

Supplemental Figure 4. Phylogenetic Trees of Major GA PathwayGenes.

Supplemental Figure 5. qRT-PCR Verification of Selective Genes inthe Auxin Pathway.

Supplemental Figure 6. Expression of Auxin and GA Genes inArabidopsis Seeds.

Supplemental Table 1. Summary of RNA-Seq Read Statistics

Supplemental Table 2. Summary of 16 Superclusters Shown inFigure 3

Supplemental Table 3. List of qPCR and Cloning Primers Used inThis Study.

Supplemental Methods 1. Detailed Description of Methods.

Supplemental Data Set 1. Fruit and Vegetative Tissue-Specific Genesand Transcription Factors Shown in Figure 2C.

Supplemental Data Set 2. Genes and Transcription Factors in the 16Superclusters in Figure 3.

Supplemental Data Set 3. Up- or Downregulated Hormonal GenesShown in Figure 4.

Supplemental Data Set 4. Plant Hormone Pathway Gene Expressionacross Fruit Tissues and Stages, Correlating with Figure 5.

Supplemental Data Set 5. Up- or Downregulated Genes in the Stage2 to Stage 1 Comparison Shown in Figure 7.

Supplemental Data Set 6. Differentially Expressed Genes in Embryo 3and Ghost 3 Shown in Figure 9A.

Supplemental Data Set 7. Embryo 3– and Ghost 3–Abundant GenesShown in Figure 9B.

Supplemental Data Set 8. Summary of Transcription Factors in theF. vesca Genome.

Supplemental Data Set 9. GO Annotation File for F. vesca.

Supplemental Data Set 10. Sequence Alignment File Used toGenerate Phylogenetic Trees Shown in Supplemental Figures 3 and 4.

ACKNOWLEDGMENTS

We thank Hector Bravo for advice on data analysis, Kevin Folta fortraining on F. vesca transformation, Charles Hawkins for drawing Figure10, Julie Caruana, Courtney Hollender, and Jing Wang for helpful com-ments, and Jenny Xiang at Weill Cornell Medical College for sequencing.This work was supported by National Science Foundation GrantMCB0923913 to Z.L. and N.A. and by the Maryland MAES Hatch Project(MD-CBMG-0525).

AUTHOR CONTRIBUTIONS

C.K. and Z.L. designed the experiments. C.K., A.G., and R.S. performedthe experiments. C.K., O.D., and N.A. analyzed the data. C.K. and Z.L.wrote the article.

Received March 20, 2013; revised May 19, 2013; accepted June 7, 2013;published June 28, 2013.

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1978 The Plant Cell

DOI 10.1105/tpc.113.111732; originally published online June 28, 2013; 2013;25;1960-1978Plant Cell

Chunying Kang, Omar Darwish, Aviva Geretz, Rachel Shahan, Nadim Alkharouf and Zhongchi LiuFragaria vescaStrawberry

Genome-Scale Transcriptomic Insights into Early-Stage Fruit Development in Woodland

 This information is current as of August 28, 2020

 

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