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Genome-Scale Transcriptomic Insights into Early-Stage Fruit
Development in Woodland Strawberry Fragaria vesca
CW
Chunying Kang,a Omar Darwish,b Aviva Geretz,a Rachel Shahan,a Nadim Alkharouf,b and Zhongchi Liua,1
a Department
b Department
of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742
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 fruit
development. The strawberry fruit is unique in that the edible flesh is actually enlarged receptacle tissue. The true fruit are the
numerous dry achenes dotting the receptacle’s surface. Auxin produced from the achene is essential for the receptacle fruit
set, a paradigm for studying crosstalk between hormone signaling and development. To investigate the molecular mechanism
underlying strawberry fruit set, next-generation sequencing was employed to profile early-stage fruit development with five
fruit tissue types and five developmental stages from floral anthesis to enlarged fruits. This two-dimensional data set provides
a systems-level view of molecular events with precise spatial and temporal resolution. The data suggest that the endosperm
and seed coat may play a more prominent role than the embryo in auxin and gibberellin biosynthesis for fruit set. A model is
proposed to illustrate how hormonal signals produced in the endosperm and seed coat coordinate seed, ovary wall, and
receptacle fruit development. The comprehensive fruit transcriptome data set provides a wealth of genomic resources for
the strawberry and Rosaceae 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 model
for the cultivated octoploid strawberry as well as the Rosaceae
family 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 investigate signal coordination and communication between different organs due to strawberry’s unique fruit structure. Its fleshy
fruit is actually the stem tip, the receptacle, while the true fruit is
the achene, a dried up ovary. More than two hundred achenes
dot the surface of the receptacle, each consisting of a single
fused ovary with one seed inside. Complex signaling over space
and developmental time between the seed-bearing achene and
the supporting receptacle ensures proper reproductive success
and seed dispersal.
In most plants, early fruit development consists of three phases
(Gillaspy et al., 1993). The earliest phase, the decision to abort or
to proceed with fruit development, is referred to as fruit set. The
second phase involves cell division and fruit growth, and the third
phase involves cell expansion. Fruit set is always regulated by
positive signals generated during fertilization, although fleshy
fruits can develop from a variety of floral parts, such as ovary in
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions 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 and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.111732
tomato (Solanum lycopersicum), receptacle in strawberry, or
fused sepals in apple (Malus domestica) (Nitsch, 1950; Gillaspy
et al., 1993; Yao et al., 2001; Fuentes and Vivian-Smith, 2009).
Fertilization-dependent fruit set ensures that the maternally derived structure, the fruit, only forms when fertilization is successful. In 1950, Nitsch showed that the removal of fertilized
ovaries (achenes) from a strawberry receptacle prevented receptacle fruit enlargement. However, exogenous auxin application acted as a substitute for achenes and stimulated receptacle
fruit growth. Thus, auxin is essential to strawberry receptacle enlargement, and the achenes are the source of auxin
(Nitsch, 1950; Dreher and Poovaiah, 1982; Archbold and
Dennis, 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, which
was extracted from receptacle and achenes together, were
shown 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 and
subsequent fall in auxin level correlates with auxin’s stimulating
effect on receptacle growth and its inhibitory effect on coloration
and ripening, respectively (Symons et al., 2012). The level of
GA1, an active form of gibberellin (GA), followed a similar trend,
except that GA1’s rise and fall lags behind that of auxin (Symons
et al., 2012). This finding is consistent with a similar effect of GA
on fruit development; GA alone or in combination with auxin
stimulated strawberry fruit development in the absence of pollination (Thompson, 1969). Studies in Arabidopsis thaliana and
tomato indicated that auxin acts upstream of GA by stimulating
GA biosynthesis during fruit set (Serrani et al., 2008; Dorcey
et al., 2009; Fuentes et al., 2012).
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Understanding the hormonal regulation of fruit set is of considerable agronomic value. As illustrated above, exogenous hormone application can bypass the requirement of pollination and
fertilization for fruit set. This so-called “fertilization-independent
fruit set” or “parthenocarpy” could ensure high yields even under
conditions unfavorable for fertilization, such as too high or low
temperatures or a lack of pollinators. In addition to the induction of
parthenocarpic fruit by exogenous applications of auxin and GA in
a variety of plant species (Ozga and Reinecke, 1999; Vivian-Smith
and Koltunow, 1999), transgenic approaches that increase the
expression of auxin biosynthesis genes in ovules led to parthenocarpic eggplant (Solanum melongena), tomato, strawberry, and
raspberry (Rubus idaeus) (Rotino et al., 1997; Ficcadenti et al.,
1999; Mezzetti et al., 2004). Further, mutations affecting auxin
transport (Sl-PIN4) and signaling (At-ARF8, Sl-ARF7, and Sl-IAA9)
or GA signaling (Sl-DELLA) also induced parthenocarpic fruit in
Arabidopsis 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 and
signaling in fruit crops will pinpoint key genes and pathways as
targets of genetic manipulation for inducing parthenocarpic fruit
development.
A recent report suggests that auxin biosynthesis in Arabidopsis
involves only a two-step pathway, in which TAA1 (for TRYPTOPHAN AMIONOTRANSFERASE OF ARABIDOPSIS1) and its homologs TAR1-4 (for TRYPTOPHAN AMIONOTRANSFERASE
RELATED1 to 4) convert Trp to IPA (for INDOLE-3-PROPIONIC
ACID). The YUCCA (YUC) family of flavin monooxygenases then
catalyzes the conversion of IPA to auxin (indole-3-acetic acid
[IAA]) (Won et al., 2011). Auxin transport is vital to auxin function
and is based on the chemiosmotic model (Rubery and Sheldrake,
1973; Feraru and Friml, 2008), in which auxin enters cells by
AUXIN RESISTANT1/LIKE AUXIN RESISTANT (AUX/LAX) auxin
influx carriers and exits cells directionally through the PINFORMED (PIN) family of efflux carriers. The asymmetric localization of PIN on the plasma membrane directs the polarity of the
transport. Two types of auxin receptors have been reported (reviewed in Hayashi, 2012). The TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) family auxin
receptors are the F-box subunits of the SKP-CULLIN-F-BOX
(SCF) ubiquitin ligase. They facilitate ubiquitination and subsequent protein degradation of Aux/IAA repressors that normally
bind and inhibit AUXIN RESPONSE FACTORS (ARFs). The released ARFs can act upon downstream target genes (Mockaitis
and Estelle, 2008). AUXIN BINDING PROTEIN1 (ABP1) represents
a second type of auxin receptors implicated in rapid auxin responses on the plasma membrane (Dahlke et al., 2010).
In the GA biosynthesis pathway, geranylgeranyl diphosphate
is converted to GA12 through successive steps catalyzed by
ent-copalyl diphosphate synthase, ent-kaurene synthase, entkaurene oxidase, and ent-kaurenoic acid oxidase. GA12 is then
converted to bioactive GA1 and GA4 through a series of oxidation
steps catalyzed by GA 20-oxidases (GA20ox) and GA3-oxidases
(GA3ox) (reviewed in Sun, 2011). By contrast, GA2ox converts
active GA1 and GA4 to an inactive form, playing a critical role in
maintaining GA homeostasis (Sun, 2011). GA signaling causes
the protein degradation of DELLA transcriptional repressors, which
are encoded by five genes in Arabidopsis: REPRESSOR OF GA1-3
(RGA), GA-INSENSITIVE (GAI), RGA-LIKE1 (RGL1), RGL2, and
RGL3. GA binding to its receptor GIBBERELLIN INSENSITIVE
DWARF1 (GID1) increases the affinity of GID1 for DELLA proteins;
the stable GID1-DELLA complex enables efficient recognition of
DELLA by SCFSLY1 and subsequent degradation of DELLA proteins by the 26S proteasome (Sun, 2011).
Despite significant progress in illuminating the molecular basis
of auxin, GA, and other phytohormone biosynthesis and signaling
pathways in model species, how these hormonal pathway genes
operate in the context of fruit set and fruit development is not well
understood. Furthermore, almost all past molecular studies of
strawberry fruit were focused on the ripening of the fruit (Aharoni
and O’Connell, 2002; Garcia-Gago et al., 2009), reflecting the
agronomic interests and the value of strawberry as a crop. In
comparison, much less is known about the temporal and spatial
regulation of fruit set and fruit growth, although these aspects are
of equally critical importance to agriculture. In addition, almost all
prior strawberry studies focused on the commercial cultivar F. x
ananassa, an octoploid lacking a sequenced genome. Finally,
most molecular genetic studies of fruit development have been
on Arabidopsis and tomato, which develop true fruit from ovary
walls. Molecular genetic studies of strawberry, an accessory fruit,
will greatly broaden our understanding of fruit development in
general. To date, there is still a need for an overall molecular
framework that can be used to address fundamental questions of
fruit development. For example, where precisely are the phytohormones produced inside a seed? What are the relative contributions of auxin and GA to fruit set and growth? How are the
hormonal signals transported to the receptacle and via what
types of transport mechanisms? How does the receptacle, a tissue quite different from the true fruit (ovary), respond to auxin and
GA, and how have different plant species evolved to develop
fleshy 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 the
molecular characterization of fruit development. Taking advantage of powerful second-generation sequencing technology, we
profiled early stage fruit transcriptomes of F. vesca, producing
high-resolution digital profiles of global gene expression in five
different fruit tissues at five developmental stages. Since samples
were collected from well-characterized stages and tissues, the
transcriptome data is highly conducive to cross-lab or crossspecies comparisons. Here, we report initial analysis of this wealth
of molecular information, which yields unprecedented molecular
insight 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 to
green fruit was divided into five stages that encompass complex developmental, morphological, physiological, and hormonal
changes (Hollender et al., 2012) (Figure 1). Stage 1 is the prefertilization stage when flowers have just opened. Stage 2 is 2 to
4 d postanthesis, when fertilization has just occurred and signs of
Transcriptome of Strawberry Fruit Set
senescence begin to show, including complete loss of petals,
pink styles, and enlargement of the ovary. A globular stage embryo is inside the stage 2 seed (Hollender et al., 2012). Stage 3 is
typified by red and dry styles, complete loss of anthers, and
a 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 the
entire seed. The cotyledons turn from transparent to white, indicative of embryo maturation. Hence, stage 5 marks the maturation of the embryos and achenes.
Size-wise, achenes undergo progressive enlargement from
stage 1 to stage 3 but remain relatively constant from stages 3 to
5 (Figure 1A). Conversely, the receptacle remains relatively constant in size between stages 1 and 2 but increases progressively
from stages 2 to 5, exposing more and more receptacle tissue
between achenes. The receptacle size, which is proportional to
the number of successfully fertilized achenes, is a poor measurement of developmental stages due to varying degrees of
successful fertilization. Instead, the morphology of the developing
embryo inside each developing seed serves as a more reliable
temporal marker (Hollender et al., 2012). Based on the timing of
fruit coloration in Ruegen, a variety of F. vesca with red berries,
stages 1 to 5 described here for F. vesca correspond to early
stage fruit development from prefertilization to “big green” in
cultivated strawberry (see Supplemental Figure 1 online).
Global Analysis of Fruit Transcriptome
To investigate the underlying molecular changes that accompany the morphological changes described above, we used
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RNA-seq to generate transcriptome profiles for each of the five
stages. For each stage, we hand-dissected individual achenes
into three tissues: (1) ovary wall (wall, for short), (2) ghost, which
refers to the entire seed with its embryo removed (and thus
consists predominantly of endosperm and seed coat tissues),
and (3) embryo (Figures 1A and 1B). Furthermore, we separated
receptacle tissues into (4) cortex, the fleshy tissue immediately
underneath achenes, and (5) pith, the interior tissue of the receptacle (Figure 1C). Cortex and pith are separated by a ring of
vascular bundles, most of which were included in the pith tissue
collection. For simplicity, we use “fruit tissues” to indicate all
tissues described above (embryo, ghost, wall, pith, and cortex),
“achene” to refer to embryo, ghost, and ovary wall, “seed” to
refer to embryo and ghost, and “receptacle” to indicate both pith
and 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 has
ovule tissue instead. Stage 2 has only “seed” tissue since the
globular embryo is too small to be dissected out of the seed.
Additionally, we sampled two vegetative tissues, seedling and
leaf, as controls. Two biological replicates were harvested for
each sample. For convenience, tissue name plus stage number is
used as sample name hereafter. After removing low-quality reads,
12 to 40 million reads per sample were mapped against the
F. 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 the
genome (see Supplemental Table 1 online). Only reads mapped
against 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 shows
individual achenes. Third row shows a single seed dissected out of each individual achene. “Ovary” and “Ovule” correspond to the prefertilization
structure of achene and seed, respectively. The fourth (bottom) row indicates individual embryos dissected from individual seeds. The inset is an
enlarged 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.
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The correlation dendrogram illustrates the global relative relationships among the 25 tissues (Figure 2A). All paired biological replicates, with the exception of stage 4 pith, clustered
together. For all fruit tissue types, stage 1 to 2 samples clustered
together and stage 3 to 5 tissues clustered together (Figure 2A).
This may suggest a delayed global response to fertilization due
to the time needed to transmit fertilization-induced signals across
layers of cells and tissues as well as signal amplifications needed
to effect the entire fruit development. For example, the stage 3-5
ovary wall tissues were more distant from stage 1-2 ovary walls,
which grouped together with stages 1 and 2 ovule/seed, suggesting significant transcriptomic change at stage 3 ovary wall
during its transition from a female floral organ into the true fruit.
Another example is the pith and cortex; they clustered together at
stage 1-2 but become distant at stage 3-5, suggesting that one
hallmark of fruit set is the differentiation between pith and cortex.
Seeds from stage 3 to 5 fruit were dissected to give rise to
embryo and the remaining tissue, the ghost. Transcriptomes of
embryo 3-5 were most distinct; they were clustered distant from
all 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 were
clustered between the maternal reproductive tissues and the
ghost/embryo tissues (Figure 2A).
Genes with normalized reads lower than 0.3 “Reads Per Kilobase per Million”. (RPKM) were considered “too lowly expressed” and removed from analysis. Between 17,935 (embryo
5) and 22,171 (seed 2) genes out of 34,809 predicted F. vesca
genes remained in each transcriptome, 80% of which were in
the 1 to 100 RPKM range (Figure 2B). These genes from all five
stages of the same tissue were combined, and a Venn diagram
was used to reveal unique or commonly expressed genes
among the fruit tissues (Figure 2C; see Supplemental Data Set 1
online). In total, 19,236 genes were common among all five fruit
tissues. While ghost (combining ovule 1, seed 2, and ghosts 3,
4, and 5) had 990 tissue-specific genes, cortex and pith had only
76 and 130 specific genes, respectively. This correlates with
lower tissue complexity in cortex and pith. Transcription factors
(TFs) unique to each tissue are also shown (Figure 2C; see
Supplemental 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 the
degree of variance (see Methods). In all figures to follow, samples are named as “Tissue_stage_replicate” or Tissue_stage.” Embryo 3.1 means
Embryo_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 in
parentheses.
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
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Venn diagram (see Supplemental Data Set 1 online). In total, 3434
fruit-specific and 518 vegetative-specific genes were identified;
22,611 genes were expressed in both tissues (see Supplemental
Data Set 1 online).
Identification of Temporal or Spatial Expression Trends
across F. vesca Fruit Transcriptome
To identify different gene expression profiles across all five
stages, we first filtered out genes with constant or low expression levels (see Supplemental Figure 2 online). After filtering,
15,754 genes remained. Next, relative RPKM value was calculated
for each gene (RPKMX-tissue/RPKMaverage). The relative RPKM
value allows for identification of genes with low absolute RPKM
values (such as TFs) while still showing significant expression
changes in specific tissues or stages. Thirty-six out of 50 k-means
clusters representing 5432 genes exhibited specific temporal or
spatial expression patterns (Figure 3A). Clusters with similar expression trends were further combined into 16 superclusters, each
with a unique gene expression profile across stages and tissues
(Figure 3A; see Supplemental Data Set 2 and Supplemental Table
2 online).
In temporally regulated clusters, supercluster 1 showed highest
expression at stage 1 (prefertilization) and gradual downregulation
from stage 2 to 5 in all fruit tissues. By contrast, supercluster 3
showed gradual upregulation from stage 1 to 5 in all tissues except embryos (Figure 3A; see Supplemental Table 2 online).
Some superclusters were highly expressed only in one tissue
such as embryo (supercluster 5), ghost (supercluster 6), and wall
(supercluster 7). Other superclusters showed high levels of expression in several tissues, such as supercluster 8 (achene: embryo, ghost, and wall), supercluster 9 (receptacle: cortex and pith),
and supercluster 10 (wall, cortex, and pith). Enriched Gene Ontology (GO) categories could be identified for some of the superclusters with reasonable confidence (false discovery rate
<0.05; see Supplemental Data Set 2 online), while other superclusters did not have enriched GO terms due to a smaller gene
number in the supercluster.
Among the 5432 genes that made up the 16 superclusters, 472
were TFs belonging to 41 families (Figure 3B; see Supplemental
Data Set 2 online). These TFs showed distinct stage or tissuespecific expression patterns mirroring each of the 16 clusters
from which they came (Figure 3B; see Supplemental Table 2
online). Dynamic expression changes associated with these TFs
may reveal key functions they may play. For example, five APETALA2 (AP2)-like genes, ANT (AINTEGUMENTA gene02623),
AIL5 AINTEGUMENTA-LIKE 5 (gene16919), AIL6 (gene20828),
BBM1 (BABY BOOM gene21524), and PLT2 (PLETHORA 2
gene20607), were specifically upregulated only in embryos supercluster 5), especially at stages 3 and 4 (Figure 3B; see
Supplemental Table 2 online). This suggests conserved functions in embryo development and organ primordial formation
Figure 3. Sixteen Superclusters of Genes with Unique Stage or Tissue
Expression Profiles.
(A) Thirty-six K-means clusters of 5432 genes showing distinct stage and
tissue-specific expression patterns. The scale: averaged log2 “relative
RPKM value” of all genes in each cluster. Clusters with similar expression trends are combined to form 16 superclusters.
(B) The heat map showing log2 “relative RPKM values” of individual 472
TFs.
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similar to the Arabidopsis homologs (Okamuro et al., 1997; Aida
et al., 2004; Krizek, 2009). In addition, WOX3 (WUSCHEL RELATED HOMEOBOX 3 gene14025) and WUSCHEL (gene14621)
homologs were upregulated in receptacles and may regulate
stem cell proliferation in the stem tip.
Global Analysis of Auxin and GA Pathway Genes
We identified F. vesca auxin and GA pathway gene families involved in synthesis, transport, conjugation, and signaling by
BLAST searches in the F. vesca genome browser using Arabidopsis protein sequences as queries. Phylogenetic analysis
using protein sequences guided assignment of gene names
(see Supplemental Figures 3 and 4 online).
Differentially expressed auxin and GA pathway genes were
identified by pairwise comparisons between successive stages
(Figure 4; see Supplemental Data Set 3 online). The data suggest
an induction of a large number of auxin pathway genes in both
embryo and ghost. However, induced GA pathway genes mainly
occurred in the ghost and ovary wall, but not in embryos. Temporally, the number of upregulated auxin and GA genes showed
moderate increase at stage 2, peaked at stage 3, tapered off at
stage 4, and reached almost zero at stage 5 in all fruit tissues
(Figures 4A and 4C). However, downregulated auxin and GA genes
increased dramatically in achenes at stage 5 (Figure 4B), which
coincides with the completion of embryo development (Figure 1)
We examined in more detail the major auxin and GA gene
families with RPKM values of 10 or higher in any of the 23 fruit
transcriptomes (see Supplemental Data Set 4 online). Log2
RPKM values for these auxin and GA genes were subject to
hierarchical clustering analysis (Figure 5). Auxin biosynthesis
genes YUC5, YUC11, and TAR1 and GA biosynthesis genes
GA20ox3, GA3ox3, 4, 5, and 6 were predominantly expressed in
the achene (embryo, ghost, and wall) and largely absent from the
receptacle (pith and cortex) (Figure 5A). They were nearly all
highly expressed in the ghost and less highly in the embryo and
wall. YUC4 and TAR2 represented a second group that was
more abundant in the embryo (Figure 5A) and may play an important role in embryo development. A third group represented
by YUC10, GA20ox1, GA20ox2, and GA3ox1 was expressed in
all fruit tissues but showed low or absent expression in embryos.
Most of these biosynthesis genes showed gradual induction of
expression from low at stage 1 to high at stage 4 or 5; this likely
reflects the effect of fertilization. By contrast, GA2ox2, GA2ox4,
and GA2ox5, which are involved in GA catabolism, were expressed at a low level in all fruit tissues and at all stages (Figure
5A). Taken together, auxin and GA biosynthesis genes were
more highly expressed in the achene, particularly in the ghost.
The data strongly indicate that achenes are the site of fertilization-induced auxin synthesis. Furthermore, the data pinpoint the
ghost, containing predominantly the endosperm and seed coat,
as the main tissue for auxin and GA biosynthesis.
Auxin efflux and influx transporters did not exhibit obvious
tissue or stage specificity and were more broadly expressed
(Figure 5B). The exceptions were PIN10 (gene12312), which was
highly 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 expressed
both in embryo and in pith. Quantitative RT-PCR validated the
RNA-seq data for several auxin transport and biosynthesis
genes (see Supplemental Figure 5 online).
Auxin receptor genes TIR1, AFB2, and AFB5 were expressed
in all fruit tissues but exhibited downward regulation from high in
stages 1-2 to low in stages 3-5 (Figure 5C). ABP1, an endoplasmic reticulum–localized auxin receptor (Dahlke et al., 2010),
was mainly expressed in the embryo and ghost. Additionally, the
GA receptor homolog GID1a was expressed 20-fold more highly
in the receptacle than in the achene, which was indicative of active
GA signaling in the receptacle. Furthermore, GAI and RGA1, both
DELLA repressors of GA signaling, were more abundantly expressed in the receptacle. Together, the data suggest that the
receptacle expresses an abundance of receptors for auxin and GA.
The increased GAI and RGA1 transcripts may be caused by rapid
turnover of these two DELLA proteins or feedback regulation
during 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 expression is auxin inducible, forming a negative feedback loop to
control auxin homoeostasis. Multiple members of the GH3
family, such as GH3.1, GH3.5, GH3.9, and GH3.17, were highly
expressed in seed 2 and ghost 3-5 (Figure 5D). Therefore, IAA
conjugation 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 majority
of the AUX/IAA and ARF genes were highly expressed in pith
and cortex, less so in the ovary wall, and extremely lowly expressed in the embryos and ghosts (Figure 5E). This indicates
active auxin signaling in the receptacles and, to a lesser degree,
in the ovary walls. Each AUX/IAA protein is known to inhibit
a partner ARF, but specific partnership between AUX/IAA and
ARF is difficult to discern. Hierarchical clustering (Figure 5E) revealed potential functional pairs based on coexpression, such as
IAA8c 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 and
ghost, the signaling components of GA and auxin were more
specifically and highly expressed in cortex and pith. Ovary walls
expressed biosynthesis as well as signaling genes. The spatial
separation of hormone biosynthesis and signaling observed
here is consistent with earlier observations implicating the
achene as the source of auxin and the receptacle as the responding tissue. Our data also indicate achenes as the source
of GA.
Fertilization-Independent F. vesca Fruit Development
Induced by Auxin and GA
We tested the effect of exogenous auxin and GA application on
induction of fertilization-independent fruit set in F. vesca. Previous experiments were done in the cultivated strawberry (F. x
ananassa), but the hormonal effect on F. vesca can be better
correlated with molecular events revealed by the F. vesca
transcriptomes. Synthetic auxin (1-naphthaleneacetic acid [NAA])
and GA (GA3) were applied to emasculated F. vesca flowers, with
Transcriptome of Strawberry Fruit Set
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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 (fold
change >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 earlier
stage in the same tissue. Number of total selected auxin genes was 78 and GA genes was 39 (see Supplemental Data Set 3 online).
mock treatment and hand pollination serving as negative and
positive controls, respectively. NAA or GA3 single hormone application induced fruit set and enlargement in both the achene
and the receptacle, but the fruit size was smaller than the handpollinated control (Figures 6A to 6F). When GA3 and NAA were
applied together to the same emasculated flower, both the
achene fruit and the receptacle fruit were larger in size than with
either single hormone treatment and were similar in size to the
pollinated fruit (Figures 6A to 6F). This indicates an additive effect
of GA and auxin on fruit size. Additionally, the ovary wall hardened just as in normal fertilization. The unfertilized seed inside the
NAA- or GA-treated achene was enlarged as well (Figure 6C).
When the auxin efflux transport inhibitor N-1-naphthylphthalamic
acid (NPA) was applied, fertilization-independent fruit enlargement
was 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 fruit
was reported for tomato and Arabidopsis (Serrani et al., 2008;
Dorcey et al., 2009). This was interpreted to be a result of pooling
IAA in the ovule due to a block of IAA transport by NPA, leading
to an artificially elevated level of auxin even in the absence of
fertilization.
Analysis of Transcriptomic Changes Accompanying
Fertilization
Since fertilization is the key event required for fruit set, we identified differentially expressed genes between stage 2 (immediately
postfertilization) and stage 1 (prefertilization) in each tissue. The
seed 2 versus ovule 1 comparison had the most differentially
expressed genes, with 1176 up and 1302 down (Figures 7A and
7C; see Supplemental Data Set 5 online), suggesting that the
impact of fertilization is largely restricted to the seed at this early
stage or/and that the newly fertilized seeds have the highest
transcriptome complexity. Surprisingly, only 14 and 6 genes were
simultaneously up- or downregulated, respectively, in all four fruit
tissues, 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 enriched
among the differentially expressed genes identified above (Figures 7B and 7D; see Supplemental Data Set 5 online). The ovary
wall and seed showed similarly enriched MapMan or GO categories, such as secondary metabolism, cell wall synthesis, and
organization (Figure 7B), indicating coupling of certain gene
expression networks between seeds and ovary walls. However,
the MapMan categories of pith 2 and cortex 2 were independent
from each other (Figures 7B and 7D; see Supplemental Data Set
5 online), despite their close physical proximity and similar
overall gene expression shown in Figure 2A.
Among the differentially expressed genes, TFs with increased
expression in seed 2 when compared with ovule 1 included eight
MADS box genes (two AGAMOUS-LIKE (AGL)80, three AGL62,
one each of AGL15, AGL92, and AGL8), most of which were
upregulated 8- to 30-fold (see Supplemental Data Set 5 online).
AGL80 and AGL62 were shown in Arabidopsis to regulate endosperm and central cell development (Portereiko et al., 2006;
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The Plant Cell
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.
Kang et al., 2008). Two AP2 TFs with similarity to BBM and PLT2
were also highly induced in seed 2 (see Supplemental Data Set 5
online). In Arabidopsis, BBM and PLT2 are key regulators of
embryo development (Smith and Long, 2010). The induced expression of AGL62/80 and BBM/PLT2 in fertilized seed 2 is indicative of active endosperm and embryo development in seed 2.
We investigated all six major phytohormone genes and their
expression in the stage 2 versus stage 1 comparison (Table 1).
Consistent with previous analysis (Figure 5), auxin biosynthesis
enzymes YUC10, TAA1, and TAR1 were induced 11-, 153-, and
10-fold, respectively. The auxin efflux carrier PIN10 was induced
22-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 auxin
biosynthesis genes were upregulated in the pith or cortex (Table
1). The GA biosynthesis gene GA3ox1 was induced in seed and
wall by 85- and 43-fold, respectively (Table 1). By contrast, almost
all of the ethylene biosynthesis genes were downregulated (Table
1). Interestingly, the ABA biosynthesis, catabolism, and signaling
genes were mostly downregulated in the achene and upregulated
in the pith, but none showed any change in the cortex.
Seed Anatomy and Subregion Transcriptional Activity
As products of double fertilization, embryo and endosperm
are both enclosed within the ghost, yet they possess distinct
developmental programs. We observed their development
Transcriptome of Strawberry Fruit Set
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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. Mock
and hand-pollination serve as the negative and positive controls, respectively. Photos were taken at day 12 after the first hormone application. Bar = 5
mm.
(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 mocktreated 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.
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The Plant Cell
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 downregulated (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 downregulated genes in each tissue with available MapMan bins is indicated next to the color code of tissues. Percentage of total annotated genes in the
genome belonging to each MapMan bin serves as a control and is coded gray.
through microscopy (Figure 8). The F. vesca female gametophyte at the time of anthesis consisted of a long and narrow
embryo sac with a highly visible central cell (Figure 8A). Surrounded by integument layers, the embryo sac positioned the
egg cell at the top (toward the style) and the chalaza end at
the base. The ovule, which will become the seed upon fertilization, was connected to the ovary wall with visible vascular
bundles (Figure 8B). After fertilization, confocal images of
propidium iodide–stained seeds indicated an eight-cell octant
embryo zygote encased in a sac of micropylar endosperm nuclei
(Figure 8C).
The complex tissue and anatomy of seeds raise an important
question: Are auxin and GA produced uniformly throughout the
seed? Our transcriptome data indicate that the ghost plays
a more prominent role in auxin and GA biosynthesis (Figure 5).
We mined an Arabidopsis transcriptome database (Belmonte
et al. 2013), which provides a higher tissue resolution of gene
expression 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 expression (see
Supplemental Figure 6 online). Similarly, Arabidopsis orthologous
genes in GA biosynthesis appeared to be expressed in either the
chalazal endosperm (At-GA3ox3, 4; At-GA20ox4, 5), the chalazal
seed coat (At-GA20ox2), or in both chalazal seed coat and chalazal endosperm (At-GA20ox1) (see Supplemental Figure 6 online).
The chalazal seed coat– and endosperm-specific localization of
hormonal gene expression may directly affect maternal fruit tissue
development.
To directly test region-specific expression of F. vesca genes
within the strawberry seed, we fused FvPIN1 and FvPIN5 promoters to the b-glucuronidase (GUS) reporter and transformed
the reporter genes into F. vesca (YW5AF7) plants. In stage 1
ovules, pFvPIN1:GUS and pFvPIN5:GUS similarly showed chalazal domain–specific expression (Figures 8D and 8G). At stage 2,
both genes were induced in the seed integuments, the precursors
to seed coat (Figures 8E and 8H). At stage 3, when heart stage
embryos became visible, pFvPIN1:GUS was highly expressed in
Transcriptome of Strawberry Fruit Set
the embryo (Figure 8F), while pFvPIN5:GUS was only weakly expressed in the embryo (Figure 8I). Both genes remained highly
expressed in the chalazal end of the seed coat throughout seed
development (Figures 8F, 8I, and 8J). These distinct expression
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patterns were consistent with the RNA-seq data (Figure 5B).
Furthermore, the persistent chalazal seed coat expression of
both auxin efflux carriers highlights the importance of chalazal
seed coat in auxin transport.
Table 1. Differentially Expressed Genes in Hormonal Pathways Identified by Comparing Stage 2 (Postfertilization)
with Stage 1 (Prefertilization) Transcriptomes
Hormone
Auxin
Tissue
Gene ID
Gene Name
Putative Function
Fold Change
Seed
gene27796
gene03586
gene31791
gene24005
gene23026
gene04070
gene01267
gene12312
gene16571
gene25723
gene27891
gene22779
gene23280
gene30882
gene07619
gene27796
gene23026
gene08336
gene27891
gene22779
gene23280
gene22838
gene03265
gene07619
gene22838
gene08194
gene27792
gene22838
gene18907
gene27740
gene01376
gene08194
gene08492
gene12917
gene27792
gene31790
gene30702
gene27891
gene05990
gene19699
gene13360
gene06004
gene01058
gene27756
gene06004
gene07935
gene06947
None
gene06004
gene27756
YUC10
TAA1
TAR1
GH3.2
GH3.17
PIN4
PIN8
PIN10
IAA16
IAA26b
IAA12
IAA31
ARF16c
YUC2
LAX3
YUC10
GH3.17
IAA11
IAA12
IAA31
ARF16c
GH3.1
GH3.5
LAX3
GH3.1
IAA14b
ARF1b
GH3.1
GH3.11
GH3.12
AFB2
IAA14b
ARF5
ARF19a
ARF1b
TAR2
GH3.6
IAA12
IAA19
KS1
GA20ox1
GA3ox1
GA3ox3
GID1b
GA3ox1
GA2ox4
RGL3
Biosynthesis
Biosynthesis
Biosynthesis
Conjugation
Conjugation
Transport
Transport
Transport
Signaling
Signaling
Signaling
Signaling
Signaling
Biosynthesis
Transport
Biosynthesis
Conjugation
Signaling
Signaling
Signaling
Signaling
Conjugation
Conjugation
Transport
Conjugation
Signaling
Signaling
Conjugation
Conjugation
Conjugation
Receptor
Signaling
Signaling
Signaling
Signaling
Biosynthesis
Conjugation
Signaling
Signaling
Biosynthesis
Biosynthesis
Biosynthesis
Biosynthesis
Signaling
Biosynthesis
Deactivation
Signaling
11.8
153.1a
10.7
12.3
3.3
2
4.2
22.6
2
2
2.6
6.5
3.1
0.014
0.38
2.2
3.4
2.2
2.2
2.4
4.2
0.35
0.18
0.41
2.9
3.1
2.9
2.7
2.5
3.5
2.9
2.1
4.6
2.2
2.2
0.15
0.34
0.48
0.21
2.9
7.7
84.9
Inf (0‒48.8)b
0.46
43.3
2.2
0.084
GA3ox1
GID1b
Biosynthesis
Signaling
3.7
3.2
Wall
Cortex
Pith
GA
Seed
Wall
Cortex
Pith
(Continued)
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The Plant Cell
Table 1. (continued).
Hormone
Cytokinin
Tissue
Gene ID
Gene Name
Putative Function
Fold Change
Seed
gene10304
gene14452
gene12648
gene13325
gene09744
gene30448
gene30654
gene15204
gene30477
gene04136
gene09501
gene21885
gene08409
gene27842
gene15382
gene08409
gene27842
gene02714
gene30477
gene14452
gene11196
gene09501
gene20962
gene20962
None
gene20962
gene30974
gene01195
gene29525
gene19023
gene19733
gene11421
gene19733
gene11421
gene01202
gene31045
gene11421
gene23460
gene01202
gene00379
gene11442
gene11421
gene31335
gene11291
gene26316
gene02502
gene10931
gene11969
None
gene09100
gene21044
gene29674
gene31902
gene24096
gene10769
gene16244
gene30616
CYP735A1
CKX1
CKX5
ARR2b
LOG3
LOG7
CKX2
CKX8
LOG9
AHK4
ARR5
ARR9a
ARR24a
IPT5
CKX6
ARR24a
IPT5
LOG6
LOG9
CKX1
ARR9c
ARR5
CPD
CPD
Biosynthesis
Degradation
Degradation
Signaling
Biosynthesis
Biosynthesis
Degradation
Degradation
Biosynthesis
Signaling
Signaling
Signaling
Signaling
Biosynthesis
Degradation
Signaling
Biosynthesis
Biosynthesis
Biosynthesis
Degradation
Signaling
Signaling
Biosynthesis
Biosynthesis
4.5
2.3
5.1
46.4
0.3
0.12
0.088
0.26
2.2
2.7
2.7
2.5
2.5
12.7
3.6
2.9
15.4
3.3
3.8
5.5
0.37
0.38
0.41
4.6
CPD
CYP72B1
BRL1
BZR2
ACS2
ACO2
ACO4
ACO2
ACO4
ACO1
EBF2
ACO4
EIN5c
ACO1
EIN3b
ERF1
ACO4
NCED3
PP2C5
PP2C7
CHLH/ABAR
CYP707A1/3
SnRK2.10
Biosynthesis
Deactivation
Signaling
Signaling
Biosynthesis
Biosynthesis
Biosynthesis
Biosynthesis
Biosynthesis
Biosynthesis
Signaling
Biosynthesis
Signaling
Biosynthesis
Signaling
Signaling
Biosynthesis
Biosynthesis
Signaling
Signaling
Signaling
Catabolism
Signaling
18.3
3.1
2.1
2.2
0.081
0.12
0.16
0.21
0.12
6.6
2.4
0.043
0.39
5.8
2.4
Inf (0‒14.5)b
0.044
0.26
0.43
0.49
2
0.47
0.49
CYP707A4a
PYL5
PYL6
OST1
SnRK2.3
SnRK2.4
SnRK2.6
NCED5
Catabolism
Signaling
Signaling
Signaling
Signaling
Signaling
Signaling
Biosynthesis
5.6
4.9
3.7
4.5
18.6
2.2
2.9
0.26
Wall
Cortex
Pith
BR
Ethylene
Seed
Wall
Cortex
Pith
Seed
Wall
Cortex
Pith
ABA
Seed
Wall
Cortex
Pith
Bold font highlights downregulated genes. ABA, abscisic acid; BR, brassinosteroid.
TAA1 was not identified in the global analysis (Figure 5), which focuses only on genes with a RPKM >10.
b
Inf, an expression difference between 0 (stage 1) and a specific RPKM (stage 2).
a
Transcriptome of Strawberry Fruit Set
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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 the
ovary 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 pole
endosperm 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).
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 seed
2, which contained both embryo and ghost (Figure 9A). Embryo
3 had a higher number of downregulated genes due to its distinct tissue composition from seed 2. A Venn diagram revealed
345 coordinately upregulated and 630 coordinately downregulated genes in embryo 3 and ghost 3 (Figure 9A; see
Supplemental Data Set 6 online). In total, 262 and 97 genes
exhibited opposite expression trends, up in ghost 3 and down in
embryo 3 or vice versa. The coordinated and opposite expression trends are summarized in drawings below the Venn diagram
(Figure 9A). Comparisons were also made between ghost 3
and embryo 3 to identify genes preferentially expressed in either tissue (Figure 9B; see Supplemental Data Set 7 online).
Embryo 3–abundant genes were enriched for GO terms in cell
division and ribosome biogenesis as well as developmental
regulation (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 auxin
biosynthesis.
DISCUSSION
Despite significant discoveries made in strawberry fruit research
in the 1950s (Nitsch 1950, 1955), progress has been limited in
elucidating the molecular mechanisms by which auxin drives
fruit development. Our work reported here provides crucial
molecular insights into this important developmental process.
The comprehensive two-dimensional tissue and stage collection
and in-depth RNA-seq data set enable genome-scale analyses
at a high resolution. The genome-scale approach allows us to
examine the expression profiles of all members of gene families
in an unbiased manner and to simultaneously analyze multiple
hormonal pathways. Previously, Csukasi et al. (2011) examined
a single Fa-GA3ox gene (which corresponds to Fv-GA3ox1;
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The Plant Cell
in all fruit tissues while other family members, Fv-GA3ox4, 5,
and 6, are expressed many fold higher than Fv-GA3ox1 in the
ghost. This reveals a prominent role of ghost in GA biosynthesis,
a conclusion contrary to that of Csukasi et al. (2011). This illustrates the power of genome-wide studies in that they allow
simultaneous examination of all family members.
Taking advantage of the accessibility of achenes on the outside surface of the receptacle, we were able to dissect the seed
out of the achene and the embryo out of the seed. This provided
us an unprecedented opportunity to resolve gene expression
between embryo and the remaining seed (ghost) and enabled us
to discover that ghosts likely play a more important role than the
embryos in the synthesis of auxin and GA for fruit set. Such fine
dissection and separation of embryos from remaining seeds
would prove highly challenging in other fruit types, such as tomato, the seeds of which are embedded and mixed with the
internal tissues.
Our study provides a wealth of genomic information on the
earliest stages of fruit development, which is an understudied
but critically important area of fruit research. Past molecular
studies have largely focused on the ripening of the strawberry
fruit (Aharoni and O’Connell, 2002; Garcia-Gago et al., 2009).
The availability of our RNA-seq data to the entire research
community (via Sequence Read Archive at the National Center
for Biotechnology Information (NCBI) or via http://bioinformatics.
towson.edu/strawberry/Default.aspx) paves the way for future
functional dissection of genes and networks that regulate fruit
set and fruit growth.
Figure 9. Differential Gene Expression of Embryo and Ghost.
(A) A Venn diagram reveals overlapping sets of genes between upregulated and downregulated genes in embryo 3 and ghost 3 when each is
compared with seed 2. Beneath the Venn diagram are graphic representations of stage 3 seeds showing different sets of genes with similar
or opposite expression trends in embryo 3 and ghost 3. Pink indicates upregulation; green indicates downregulation; white indicates no change. Number
of genes in each expression category is written beneath the seed diagram.
(B) Top 10 overrepresented GO terms of embryo 3–abundant and ghost
3–abundant genes.
gene 06004 in this study) during cultivated strawberry fruit development and reported that Fa-GA3ox was expressed at a level
40-fold higher in the receptacle than in the achene. They concluded that the receptacle was the main source of GA biosynthesis. We show that Fv-GA3ox1 is expressed at low levels
Figure 10. A Model of Strawberry Fruit Development.
A diagram illustrating auxin and GA biosynthesis and transport in the
achene soon after fertilization. Fertilization-induced auxin and GA accumulation in the ghost are transported to the ovary wall (route 1) and the
receptacle (route 2), shown by the two red arrows. The arrow from auxin
to GA within the ghost indicates the positive effect of auxin on GA biosynthesis. In route 1 to the ovary wall, PIN-dependent transport of auxin
may be necessary to promote local auxin and GA biosynthesis in the
ovary wall. In route 2 from ghost to receptacle, GA and auxin may be
transported via a PIN-independent mechanism (or both PIN-dependent
and independent mechanisms). Upon arriving at the receptacle, auxin
and GA exert their effects by stimulating downstream signaling events for
receptacle growth.
Transcriptome of Strawberry Fruit Set
Insights into Auxin Biosynthesis and Signaling during
Fruit Development
Previous studies using DR5 reporters indicated a surge of auxin
in the ovule/seed postfertilization and a lack of auxin in the ovary
wall/pericarp, suggesting that ovary wall does not play a major
role in auxin biosynthesis (Hocher et al., 1992; Pattison and
Catala, 2012). Our data (Figure 5A) also show lower expression
levels of auxin biosynthesis genes in the ovary wall when compared with the ghost. Furthermore, our data show that ghosts
have the highest expression levels of auxin biosynthesis genes
among the achene tissues (embryo, ghost, and ovary wall) and
may be the main site of auxin biosynthesis.
The strong expression of auxin receptors and signaling components in the receptacle (Figures 5C and 5E) indicates that auxin
is likely transported to the receptacle and exerts a direct role in
stimulating receptacle growth. This differs from earlier suggestions of an indirect role of auxin in ovary wall fruit set through
auxin-mediated promotion of GA synthesis (Dorcey et al., 2009).
An indirect role of auxin in strawberry receptacle fruit set could
also explain an absence of free IAA in the receptacle (Nitsch,
1950; Symons et al., 2012). However, the IAA measurement
method in 1950 was less sensitive than modern methods. Additionally, Symons et al. (2012) measured free IAA from the white
stage receptacle, a late stage by which the IAA level may have
significantly declined. A recent immunohistochemical study detected strong signals of IAA in the phloem of the receptacles (Hou
and Huang, 2004), but the immunohistological staining may not
be able to distinguish free IAA from conjugated forms. Therefore,
it remains unresolved as to whether free IAA exists in the receptacle at the early stages of fruit development.
Based on our data, we propose that a majority of the auxin
made in the ghost may be conjugated and then transported to
the 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 expressed auxin conjugating GH3 genes in ghost and ovary wall
(Figure 5D, Table 1). Furthermore, IAA amide conjugates (Archbold
and 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 of
GA in fruit set and suggested that auxin acts upstream of GA in
a linear pathway to stimulate fruit development (Serrani et al.,
2008; Dorcey et al., 2009; Fuentes et al., 2012). Here, we demonstrated that single GA3 application to emasculated F. vesca
flowers caused fertilization-independent fruit enlargement in both
fruit types (achene and receptacle) (Figure 6) and combined application of auxin (NAA) and GA3 stimulated both fruit types to
grow to wild-type size in the absence of fertilization. This indicates that GA and auxin may have common as well as unique
roles 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 export. Interestingly, the degree of receptacle enlargement appeared to be greater than that of the ovary wall. This suggests
15 of 19
that auxin polar transport (via PIN proteins) is required for ovary
wall enlargement and is consistent with the chalazal localization
of FvPIN1:GUS and FvPIN5:GUS in prefertilization ovules. In
Arabidopsis, NPA-treated siliques were more slender than GA3treated siliques (Dorcey et al., 2009), indicating a similarly PINdependent transport of auxin from the seed to the ovary wall.
We propose that NPA treatment resulted in an artificially elevated auxin level inside the ovule. This increased IAA could not
be transported to the ovary wall due to nonfunctional PIN proteins. However, the increased IAA in ovules could trigger the
synthesis of secondary signals, such as GA in ovules. A second
and nonexclusive scenario is that the elevated IAA could be
conjugated in the ovule. Both GA and conjugated IAA could be
transported to the receptacle (and less so to the ovary wall) via
PIN-independent transport routes.
Parthenocarpic fruit is of significant agronomic value because it
can ensure high fruit yields even under unfavorable growing
conditions. In Arabidopsis and tomato, a mutant form of At-ARF8,
a knockdown of Sl-ARF7 by RNA interference, or a knockdown of
Sl-IAA9 by RNA interference were shown to induce parthenocarpy (Wang et al., 2005; Goetz et al., 2006; Goetz et al., 2007; de
Jong et al., 2009). It was proposed that the IAA9/ARF7/ARF8
repressor complex inhibits fruit set in the absence of fertilization,
perhaps by inhibiting GA synthesis (Wang et al., 2009). Our study
provides the molecular basis for the selection of certain strawberry auxin and GA pathway genes, such as the receptaclespecific Fv-IAA16 or Fv-GAI, as targets for genetic manipulation
to induce parthenocarpic strawberry fruit.
A Model of Strawberry Receptacle Fruit Development: A
Spatial Consideration
We propose that upon fertilization, auxin is synthesized in the
products of double fertilization: the embryo and the endosperm
(as well as seed coat). In our model, auxin made in the embryo is
involved in embryo development and has less to do with fruit
development; auxin synthesized in the ghost is responsible for
fruit set (Figure 10). PIN-dependent transport of auxin is necessary for botanical fruit (ovary wall) development. Once transported
to the ovary wall, auxin induces the secondary biosynthesis of GA
and auxin in the ovary wall. Blocking the initial transport of auxin
to the ovary wall by NPA would limit secondary GA and IAA
biosynthesis and thus result in slower ovary wall growth. Auxin
also stimulates GA biosynthesis inside the seed, mainly in the
chalazal end. Both auxin and GA in the seed are transported to
the receptacle, perhaps via a PIN-independent mechanism (or both
PIN-dependent and independent mechanisms), to promote downstream 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 Fragaria
vesca, Yellow Wonder 5AF7 (YW5AF7, Slovin et al., 2009). Plants were
grown in a growth chamber with 12 h light at 25°C followed by 12 h dark at
20°C. All tissues were hand-dissected under a stereomicroscope and
frozen immediately in liquid nitrogen. The tissues from at least three fruits
were combined to form one biological replicate and there were two
16 of 19
The Plant Cell
biological replicates for each tissue. Around 1000 heart stage embryos
were dissected and pooled into two biological replicates.
RNA was extracted using the RNeasy plant mini kit (Qiagen); oncolumn DNase digestion with the RNase-Free DNase set (Qiagen) was
performed to remove contaminating DNA. A total of 0.5 to 2 µg RNA per
sample was sent to the Genomics Resources Core Facility at Weill Cornell
Medical College for library preparation with Illumina TruSeq RNA sample
preparation kit and sequencing with Illumina HiSeq2000. In most cases,
six libraries were bar-coded and sequenced in one lane. About 12 to 40
million 51-bp single-end reads were generated for each sample (see
Supplemental Table 1 online).
Initial Mapping of Reads
The raw reads were filtered with FASTQ_Quality_Filter tool from the
FASTX-toolkit. Reads with more than 90% of their bases having a quality
score higher than 28 were kept. The filtered reads were aligned to the
F. vesca genome (v1.1) and CDS (v1.0) (Shulaev et. al., 2011) using Bowtie
1.0 with default settings, allowing two mismatches. The F. vesca reference
genome and CDS files were downloaded from the Genome Data for
Rosaceae website at www.rosaceae.org/species/fragaria/fragaria_vesca.
Alignment output files from Bowtie were parsed using scripts written in
Perl to calculate the number of reads for each gene. The script is included
in the Supplemental Methods 1 online. Reads mapped against CDS were
used in all subsequent analyses.
Identification of F. vesca TFs and Hormonal Pathway Genes
Arabidopsis thaliana TF protein sequences were downloaded from
PlantTFDB (http://planttfdb.cbi.edu.cn) (Zhang et al., 2011), which were
used to blast against the F. vesca GeneMark hybrid proteins with annotations (downloaded from www.rosaceae.org) by BLASTP using blast2.2.26+ from NCBI on local computers. Stringent criteria (e-value < 10220;
bit score > 100; pident > 30%) were applied to select the set of F. vesca
TFs, which were then manually checked against the TF families in
Plaza2.5 (http://bioinformatics.psb.ugent.be/plaza/) to yield the final set
of F. vesca TFs listed in Supplemental Data Set 8 online.
Hormonal genes were identified by BLAST against Fragaria vesca
Gene 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 database. All the F. vesca hormonal genes are shown in Supplemental Data
Set 4 online.
Gene Expression Analysis
The dendrogram in Figure 2A was made by the function cor () in R with
default settings. The y axis is computed as 1 minus cor (correlation) to
reflect the degree of variance. Log2-transformed mapped read counts
without normalization were used in the computation. The bar graph in
Figure 2B and the Venn diagram in Figure 2C are based on RPKM. The
Venn diagram in Figure 2C was made by function venn () in R using gene
list for each tissue type, which is derived by combining genes from all
stages of the same fruit tissue (see Supplemental Data Set 1 online). A
similar comparison was conducted between vegetative tissues (seedling
and leaf) and fruit tissues (all five fruit tissues) (see Supplemental Data Set
1 online).
For Figure 3, only 17,108 differentially expressed genes were selected
based on the selection scheme described in Supplemental Figure 2
online. DESeq in R (Anders and Huber, 2010) was used to select for
genes with differential gene expression between stages and tissues
(padj < 0.0001). After removing low abundance genes or genes highly
expressed 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). Log2transformed relative RPKM value (RPKMgeneX in each tissue divided by
average RPKMgeneX across all fruit tissues) was imported into MeV4.8.
The optimal number of clusters was determined to be 50 based on figure
of merit analysis within MeV4.8 (Yeung et al., 2001). Only 36 clusters with
distinct tissue- or stage-specific expression profiles are shown in Figure
3A. TFs were extracted from the 16 superclusters (Figure 3A; see
Supplemental Data Set 2 online) based on the F. vesca TF table (see
Supplemental Data Set 8 online); the log 2 -transformed relative RPKM
value of each TF was used to yield Figure 3B via Mev4.8.
The differentially expressed genes in auxin and GA pathways
shown in Figure 4 were extracted from the differentially expressed
gene lists in pairwise comparisons with successive stages conducted
with DESeq in R. Other differentially expressed genes between stages
2 and 1 or between embryo 3 and ghost 3 (Figures 7 and 9) were
similarly identified using DESeq in R following its vignette. In all DESeq
analyses, mapped read counts against CDS without normalization
were used as input. In pairwise comparisons with DESeq in R, the
functions were set at newCountDataSet, estimateSizeFactors, estimateDispersions, and nbinomTest; P value was adjusted using the
Benjamini-Hochberg method; cutoff was set at fold change > 2 and
padj < 0.01. In the multifactor design that tests all fruit tissues, stage
and tissue were used as the two factors; the functions include
newCountDataSet, estimateSizeFactors, estimateDispersions, fitNbinomGLMs, and nbinomGLMTest; P value was adjusted using the
Benjamini-Hochberg method; differentially expressed genes with
padj # 0.0001 were kept.
The hierarchical clustering shown in Figure 5 uses average linkage
clustering and Pearson correlation within MeV4.8 and RPKM as input. The
heat map in Supplemental Figure 6 online was made in MeV4.8 based on
the globally normalized microarray data downloaded from the Gene
Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) (Belmonte et al.
2013). Venn diagrams in Figures 7 and 9 were made by R package
VennDiagram.
Assignment of MapMan Bins and GO Terms
MapMan bins of F. vesca genome were assigned by the Mercator pipeline
for 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 to
BLAST against the nonredundant database in NCBI by BLASTX (e-value
1023). Subsequent “GO-annotation” and “InterPro annotation” functions
within Blast2GO were used to yield the GO annotation file for ;20,003
F. vesca genes (see Supplemental Data Set 9 online). GO enrichment
was derived with Fisher’s exact test and a cutoff of false discovery rate <
0.05; the genome annotation file described above was used as the
reference. Only Biological Process GO terms are shown in tables or
figures.
Phylogenetic Analysis
The unrooted phylogenetic tree shown in Supplemental Figures 3 and 4
online 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
Hormonal Treatment of Flowers
About 50 mL hormone solution was pipetted onto the receptacle of each
emasculated flower. Stock solutions of 50 mM NAA (Sigma-Aldrich),
50 mM NPA (Sigma-Aldrich), and 100 mM GA3 (Sigma-Aldrich) were made
in ethanol and were diluted with two drops of Tween 20 and water before
application. The final treatment concentrations were 500 µM for NAA and
GA3 and 100 µM for NPA. The solutions were applied every 2 d until day 12
when photos were taken. Fruit size was measured with ImageJ of photographed 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 into
pMDC162 binary vector, and transformed into YW5AF7 according to
a published protocol (Slovin et al., 2009; Chatterjee et al., 2011). A more
detailed 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 anthesis
were stained with Safranin-O/Fast Green and photographed under
a Nikon LABOPHOT-2 microscope equipped with an AxioCam digital
camera as described previously (Hollender et al., 2012). For confocal
images, seeds were dissected out of the ovary and stained with propidium iodide following a published protocol (Running et al., 1995). The
photo was taken with a Leica SP5 X confocal microscope by threedimensional projection with eight Z-stacks of the same seed. GUS
staining and photography are described in the Supplemental Methods 1
online.
17 of 19
Supplemental Data Set 2. Genes and Transcription Factors in the 16
Superclusters in Figure 3.
Supplemental Data Set 3. Up- or Downregulated Hormonal Genes
Shown in Figure 4.
Supplemental Data Set 4. Plant Hormone Pathway Gene Expression
across Fruit Tissues and Stages, Correlating with Figure 5.
Supplemental Data Set 5. Up- or Downregulated Genes in the Stage
2 to Stage 1 Comparison Shown in Figure 7.
Supplemental Data Set 6. Differentially Expressed Genes in Embryo 3
and Ghost 3 Shown in Figure 9A.
Supplemental Data Set 7. Embryo 3– and Ghost 3–Abundant Genes
Shown in Figure 9B.
Supplemental Data Set 8. Summary of Transcription Factors in the
F. vesca Genome.
Supplemental Data Set 9. GO Annotation File for F. vesca.
Supplemental Data Set 10. Sequence Alignment File Used to
Generate Phylogenetic Trees Shown in Supplemental Figures 3 and 4.
ACKNOWLEDGMENTS
We thank Hector Bravo for advice on data analysis, Kevin Folta for
training on F. vesca transformation, Charles Hawkins for drawing Figure
10, Julie Caruana, Courtney Hollender, and Jing Wang for helpful comments, and Jenny Xiang at Weill Cornell Medical College for sequencing.
This work was supported by National Science Foundation Grant
MCB0923913 to Z.L. and N.A. and by the Maryland MAES Hatch Project
(MD-CBMG-0525).
Accession Numbers
Illumina reads of all 50 samples have been submitted to the Sequence
Read Archive at NCBI (http://www.ncbi.nlm.nih.gov/sra). The submission
code is SRA065786.
AUTHOR CONTRIBUTIONS
C.K. and Z.L. designed the experiments. C.K., A.G., and R.S. performed
the experiments. C.K., O.D., and N.A. analyzed the data. C.K. and Z.L.
wrote the article.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. F. vesca Ruegen Late-Stage Fruit Development.
Received March 20, 2013; revised May 19, 2013; accepted June 7, 2013;
published June 28, 2013.
Supplemental Figure 2. Filtering Strategy for Differentially Expressed
Genes Shown in Figure 3.
Supplemental Figure 3. Phylogenetic Trees of Major Auxin Pathway
Genes.
Supplemental Figure 4. Phylogenetic Trees of Major GA Pathway
Genes.
Supplemental Figure 5. qRT-PCR Verification of Selective Genes in
the Auxin Pathway.
Supplemental Figure 6. Expression of Auxin and GA Genes in
Arabidopsis Seeds.
Supplemental Table 1. Summary of RNA-Seq Read Statistics
Supplemental Table 2. Summary of 16 Superclusters Shown in
Figure 3
Supplemental Table 3. List of qPCR and Cloning Primers Used in
This Study.
Supplemental Methods 1. Detailed Description of Methods.
Supplemental Data Set 1. Fruit and Vegetative Tissue-Specific Genes
and Transcription Factors Shown in Figure 2C.
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Genome-Scale Transcriptomic Insights into Early-Stage Fruit Development in Woodland
Strawberry Fragaria vesca
Chunying Kang, Omar Darwish, Aviva Geretz, Rachel Shahan, Nadim Alkharouf and Zhongchi Liu
Plant Cell; originally published online June 28, 2013;
DOI 10.1105/tpc.113.111732
This information is current as of June 16, 2017
Supplemental Data
/content/suppl/2013/06/28/tpc.113.111732.DC1.html
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