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Funct Integr Genomics

DOI 10.1007/s10142-016-0510-3

ORIGINAL ARTICLE

Extensive transcriptomic studies on the roles played by abscisic
acid and auxins in the development and ripening of strawberry
fruits
Laura Medina-Puche 1 & Rosario Blanco-Portales 1 & Francisco Javier Molina-Hidalgo 1 &
Guadalupe Cumplido-Laso 1 & Nicolás García-Caparrós 1 & Enriqueta Moyano-Cañete 1 &
José Luis Caballero-Repullo 1 & Juan Muñoz-Blanco 1 & Antonio Rodríguez-Franco 1

Received: 17 February 2016 / Revised: 17 July 2016 / Accepted: 20 July 2016
# Springer-Verlag Berlin Heidelberg 2016

Abstract Strawberry is an ideal model for studying the molecular biology of the development and ripening of nonclimacteric fruits. Hormonal regulation of gene expression
along all these processes in strawberries is still to be fully
elucidated. Although auxins and ABA have been pointed
out as the major regulatory hormones, few high-throughput
analyses have been carried out to date. The role for ethylene
and gibberellins as regulatory hormones during the development and ripening of the strawberry fruit remain still elusive.
By using a custom-made and high-quality oligo microarray
platform done with over 32,000 probes including all of the
genes actually described in the strawberry genome, we have
analysed the expression of genes during the development and
ripening in the receptacles of these fruits. We classify these

genes into two major groups depending upon their temporal
and developmental expression. First group are genes induced
during the initial development stages. The second group encompasses genes induced during the final maturation and ripening processes. Each of these two groups has been also divided into four sub-groups according their pattern of hormonal regulation. By analyzing gene expression, we clearly show
that auxins and ABA are the main and key hormones that
combined or independently are responsible of the development and ripening process. Auxins are responsible for the
receptacle fruit development and, at the same time¸ prevent
ripening by repressing crucial genes. ABA regulates the expression of the vast majority of genes involved in the ripening.
The main genes expressed under the control of these hor-

Laura Medina-Puche and Rosario Blanco-Portales contributed equally to
this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s10142-016-0510-3) contains supplementary material,
which is available to authorized users.
* Juan Muñoz-Blanco
bb1mublj@uco.es

Enriqueta Moyano-Cañete
bb2mocae@uco.es

Laura Medina-Puche
b22mepul@uco.es

José Luis Caballero-Repullo
bb1carej@uco.es

Rosario Blanco-Portales
bb2blpor@uco.es

Antonio Rodríguez-Franco
arfranco@uco.es

Francisco Javier Molina-Hidalgo
b52mohif@uco.es
Guadalupe Cumplido-Laso
lupelaso@gmail.com
Nicolás García-Caparrós
bb2gacan@uco.es

1

Departamento de Bioquímica y Biología Molecular. Edificio Severo
Ochoa, Campus Universitario de Rabanales y Campus de Excelencia
Internacional Agroalimentario CEIA3, Universidad de Córdoba,
14071, Córdoba, Spain

Funct Integr Genomics

mones are presented and their physiological rule discussed.
We also conclude that ethylene and gibberellins do not seem
to play a prominent role during these processes.
Keywords Fragaria × ananassa . Microarray .
Transcriptome . ABA . Auxins . Fruit ripening

Abbreviations
ABA
Abscisic acid
Ct
Threshold cycle
EST
Expressed sequence tag
G1
Green1 development stage
G3
Green3 development stage
GAs
Gibberellins
GO
Gene ontology
IAA
Indole-3-acetic acid
MEP
2-C-methyl-D-erithritol 4-phosphate pathway
MVA
Mevalonate pathway
NCED
9-cis-Epoxycarotenoid dioxygenase
NDGA
Nordihydroguaiaretic acid
qRTQuantitative real-time-polymerase chain reaction
PCR
R
Red ripe ripening stage
TFs
Transcription factors
Tm
Melting temperature
u.a.i
Arbitrary units of intensity

Introduction
Fruit developing and ripening process is determinated by numerous factors including light, hormones, temperature and
genotype. Fruits can be classified as climacteric or non-climacteric, depending upon the presence or not of a burst in
respiration taking place at the onset of ripening. In climacteric
fruits, such as tomato, it is well known that the plant hormone
ethylene triggers the ripening process (Giovannoni 2001;
Giovannoni 2004; Giovannoni 2007; Klee and Giovannoni
2011). Grapes and strawberries, however, are two model nonclimacteric fruits where the role of ethylene and other plant
hormones in fruit development and ripening remains still uncertain (Chervin et al. 2004; Symons et al. 2006; Symons et al.
2012; Böttcher et al. 2013; Kuhn et al. 2014; Fortes et al. 2015).
Like many other fleshy fruits, the strawberry undergoes
four sequential stages. The first stage includes organogenesis
that involves fertilization and ovary development (fruit set).
The second stage is an active process of cell division accompanied by seed and early embryo formation (Wechter et al.
2008). In the third phase, fruit growth is produced by cell
expansion until large vacuolated cells are formed that make
up the flesh of the fruits. Cell expansion includes changes in

the cell wall structure and continuous accumulation in vacuoles of carbohydrate, organics acids and different compounds
that help to retain the osmotic pressure and the flow of water
into expanding cells (Wechter et al. 2008). The fourth is
the ripening phase that takes place after seed maturation
and is characterized by a rise in the content of soluble
solids in receptacles, a conversion of starch to sugars,
the production of natural aroma and flavour compounds,
alterations to fruit shape, size, texture and pigmentation
and a partial degradation of cell walls that lead to an
increased susceptibility to post-harvest pathogens
(Wechter et al. 2008). Most of these structural, biochemical and physiological events occurring in the fruit
receptacle are putatively regulated by hormones.
The hormonal control of the ripening of strawberry
(Fragaria × ananassa) is starting to be revealed. It has been
proposed that both ABA and auxins are heavily involved in
the development of this fruit (Perkins-Veazie 1995; MedinaPuche et al. 2014). Auxins, synthesized by the immature
achenes, seem to promote receptacle cell division and expansion and at the same time prevent ripening, so the removal of
achenes in immature fruits or the application of auxins synthesis inhibitors leads to dwarf fruits and to the premature
expression of many ripening-related genes (Medina-Escobar
et al. 1997; Trainotti et al. 1999; Redondo-Nevado et al. 2001;
Benítez-Burraco et al. 2003; Cumplido-Laso et al. 2012;
Daminato et al. 2013; Molina-Hidalgo et al. 2013, 2015;
Medina-Puche et al. 2014, 2015). ABA seems to play a major
role once the fruits have already developed in size. The endogenous content of ABA increases substantially at the final
stages of fruit ripening, and this was accompanied by the
induction of many ripening-related genes including those involved in anthocyanin production (Koyama et al. 2010; Chai
et al. 2011; Chen et al. 2011; Jia et al. 2011; Daminato et al.
2013; Molina-Hidalgo et al. 2013; Medina-Puche et al. 2014).
Therefore, the addition of ABA synthesis inhibitors such as
NDGA fully blocks the ripening process, whereas water deprivation, which is known to increase endogenous ABA biosynthesis, promotes premature ripening (Medina-Puche et al.
2014). However, other ABA-independent pathways could also
mediate some aspects of the receptacle ripening process (Jia
et al. 2013).
Some other plant hormones as ethylene or gibberellins seem
also to play minor roles in the ripening of strawberry. Along
the ripening, a very low level of ethylene compared with standard climacteric fruits is produced (Perkins-Veazie 1995;
Leshem and Pinchasov 2000). This low concentration of ethylene is present in green fruits, decay in white fruits and eventually increase at the red stage of ripening (Perkins-Veazie et al.
1996; Iannetta et al. 2006). Although the application of ethylene to strawberry fruits does not have important effects on
ripening process, it affects the expression of a subset of
ripening-related genes as β-galactosidase, pectin methyl

Funct Integr Genomics

esterase and β-xylosidase (Trainotti et al. 2001; Castillejo et al.
2004; Bustamante et al. 2009; Lee et al. 2010).
The enlargement of receptacle cells by cell expansion during strawberry fruit development is the critical factor determining fruit size. This process could be regulated by gibberellins (GA) (Csukasi et al. 2011). The endogenous content of
three different gibberellins (GA1, GA3 and GA4) was monitored during fruit development and ripening. GA4 content was
extremely high in receptacles with a maximum in the white
stage of ripening (Csukasi et al. 2011). Additionally, in this
study, genes with high homology to genes encoding GA pathway components, such as receptors and those involved in the
biosynthesis and catabolism of GA, were identified and their
expression in different tissues and developmental stages of
strawberry fruit was analysed. The expression of all of these
genes showed a stage-specific pattern during fruit development being highest in the receptacle, suggesting that GA4
could be biosynthesized in this tissue (Csukasi et al. 2011).
However, a relationship between GAs and ripening was not
demonstrated.
The aim of this study is to present and discuss an extensive
list of genes that are regulated during the development and
ripening of strawberry receptacles with a special emphasis in
clarifying how they are controlled by the hormones, auxins
and ABA. To do so, we have carried out a high-throughput
microarray analysis of the transcriptomic changes under different hormonal and ripening conditions. This approach has
been successfully applied in other similar studies (Seki et al.
2002; Waters et al. 2005). Clearly, our results support that
auxins are the key hormones that regulate receptacle fruit
growth and development, and that ABA regulates the expression of the vast majority of genes involved in the ripening.
Auxins and ABA-independent pathways were observed.

Results and discussion
The molecular characterization of the genes involved in the
development and ripening of strawberry receptacles as well as
the knowledge of their hormonal regulation is of biotechnological interest. To investigate this, we have compared
transcriptomes using microarrays with RNA isolated under 4
different contrast conditions (Fig. 1): (A) green immature versus red ripe receptacles (Table S3), (B) red ripe receptacles
versus those where the endogenous biosynthesis of ABA was
inhibited by NDGA (Table S4), (C) green immature versus
green de-achened immature receptacles and (D) red ripe receptacles versus vegetative tissues (Table S5). The correlation
coefficients (r value) of the signals obtained from the biological replicates are shown in supplementary Table S6.
After analyzing the data, we ended with 1805 differentially
regulated expressed genes (log2 fold change > ±2 and adjusted
p ≤ 0.05) putatively related with the strawberry fruit receptacle

development and ripening that could be classified in eight
different clusters (Table 1). On those, a total of 1281 and
524 genes were up- (R-up) and down-regulated (R-down) in
red in comparison with green receptacles, respectively
(Table 1).
The 1281 up-regulated genes were classified as follows:
Cluster UP(ABA + AUX-): contains 369 genes whose expression were activated by ABA and repressed by auxins in
the receptacle; Cluster UP(ABA + AUX0): contains 116
genes whose expression were activated by ABA, but were
not influenced by auxins in the receptacle; Cluster
UP(ABA0AUX-): contains a total of 356 genes whose expression were repressed by auxins and not affected by ABA; and
Cluster UP(ABA0AUX0): includes 440 ripening-related
genes whose expression was not influenced neither by ABA
nor for auxins (Fig. 2a; Table 1). Figure 3 depicts a tree map
showing the expression of these up-regulated clusters which
allow a semiquantitative comparison done by eye. From
Table 1 and Fig. 3, it can be deduced that approximately
29 % of all of the up-regulated and ripening-related genes were
activated by both ABA and auxins, 9 % activated only by
ABA, 28 % activated only by auxins and approximately
35 % were not affected by any of the two hormones, suggesting that either other hormones (e.g. ethylene or gibberellins)
and/or signals (e.g. oxidative stress) are involved in these processes of fruit formation (Martínez et al. 1996; Aharoni et al.
2002; Trainotti et al. 2001; Villarreal et al. 2010; Csukasi et al.
2011; Symons et al. 2012; Opazo et al. 2013).
The 524 down-regulated genes were classified in the following clusters: Cluster DOWN(ABA0AUX+): includes
338 genes whose expression were induced by auxins in green
G1 fruits and not influenced by ABA; Cluster DOWN(ABAAUX+): includes 6 genes whose expression was induced by
auxins in the G1 stage receptacles, but were repressed by
ABA in red ripe receptacles; Cluster DOWN(ABAAUX0): includes 133 genes that were induced in G1 stage
receptacles, were not affected by auxins but were repressed
by ABA in ripe receptacles; Cluster DOWN(ABA0AUX0):
includes 47 genes that were up-regulated in G1 stage receptacles and were not affected neither by ABA nor by auxins
(Fig. 2b; Table 1).
As in the case of the up-regulated genes, our results show
clearly that auxins play an important role at the earliest stages
of the receptacle growth. The expression of most up-regulated
genes in young immature receptacles declined in G1 receptacles when auxins were depleted after removing the achenes
(Medina-Escobar et al. 1997; Trainotti et al. 1999; RedondoNevado et al. 2001; Benítez-Burraco et al. 2003; CumplidoLaso et al. 2012; Daminato et al. 2013; Molina-Hidalgo et al.
2013, 2015; Medina-Puche et al. 2014, 2015). Auxins seem to
activate the expression of genes involved in the cell proliferation and flesh growth while repressing genes related to the
ripening. However, there were also significant number of

Funct Integr Genomics

Fig. 1 Transcriptomic analysis performed between different strawberry tissues in several experimental situations

genes whose expression was not affected by auxins, suggesting that other hormones or regulatory signals (e.g. ABA,
brassinosteroids and/or gibberellins) could be involved in this
process. In fact, a recent transcriptomic study performed in
strawberry has identified 41 unigenes whose expression was
regulated only by ABA (Chen et al. 2015).

Table 1
genes

Summary of Fragaria × ananassa differentially expressed

Ripening regulation

Cluster

Number of genes

R-up

UP(ABA+AUX-)
UP(ABA+AUX0)
UP(ABA0AUX-)

369
116
356

UP(ABA0AUX0)
Total R-up
R-down
DOWN(ABA0AUX+)
DOWN(ABA-AUX+)
DOWN(ABA-AUX0)
DOWN(ABA0AUX0)
Total R-down
Total differentially expressed 1805

440
1281
338
6
133
47
524

Qualitative and quantitative validation of the microarrays
data
We validated the differential expression of several genes by
real-time PCR (qRT-PCR) (Table S7). The oligonucleotide
sequences used are summarized in Table S1.
A Pearson’s correlation coefficient of 0.868 (p value < 0.01)
was calculated when we compared the microarray fold changes
with those obtained through qRT-PCR. These results indicate that our microarrays detect both low and high fold
changes with high accuracy under different experimental
conditions. However, fold changes obtained by qRT-PCR
were higher than those obtained by the microarray analysis. These discrepancies are higher in highly expressed
genes, possibly due to a saturation in the microarray hybridization (Wurmbach 2009). Furthermore, qRT-PCR
analysis presents a broad range of linearity than microarrays, resulting in more accurate measurements.
Functional annotation and enrichment analysis
of the microarray
Gene annotation with the 1805 differentially expressed genes
was done using Blast2Go using BLASTX (1.0E-10 cut-off

Funct Integr Genomics
Fig. 2 Venn diagrams showing
genes regulated by ABA and
auxins. Panel a shows ripeningrelated genes (R-up) regulated by
ABA and/or auxins. Panel b deploys development-related genes
(R-down) regulated by ABA and/
or auxins

value). GO terms were assigned to a total of 1335 sequences (aprox. 74 %) (Tables S7–S14), whereas 470
remained non-annotated possibly due to either a lack of
homologous sequences or the presence of non-coding
RNA in the population of analysed genes.
GO terms distribution at level 2 is summarized in pie charts
(Fig. S1). An enrichment for GO terms related to aroma, flavonols, biotic stress and ABA/ethylene hormones was found
during the fruit ripening process (R-up) whereas photosynthetic apparatus and auxin-related GO terms were present
mainly during the fruit development process (R-down). We
detected a similar enrichment in cell wall-related GO terms

Fig. 3 Treemap comparing fold
changes expression of sequences
corresponding to the four
different up-regulated clusters.
Each gene is represented by a
square cell placed in the same
place in each of the four graphics,
so a comparison of the level of
expression can be accomplished
by eye. A range of 1 to 30-fold
change level of expression is represented through colour intensity.
Any expression over 30-fold
change is considered saturated
and is represented by the same
dark colour. Grey cells represent
genes whose expression did not
pass a cutoff value of 2-fold
change. ABA+: genes induced by
ABA. ABA0, genes not influenced by ABA. AUX- genes repressed by auxins. AUX0 genes
not influenced by auxins

between ripening-related and development-related genes
(Tables S15–S28).

Criteria for the analysis and discussion of gene expression
We analyze and discuss the expression of relevant genes taking into account: (1) their putative physiological function, (2)
the levels of their expression in ripe fruit receptacles, (3) their
hormonal regulation by ABA (clusters UP(ABA + AUX-) and
UP(ABA + AUX0)) and (4) their levels of transcripts within
the red ripe receptacle transcriptome.

Funct Integr Genomics

Analysis of organoleptic and cell wall-related genes
In this functional group, the vast majority of the genes of
Cluster UP(ABA + AUX-) coding proteins related with fruit
organoleptic features are included. The physiological function
of many of them has been previously characterized as:
(i) Genes encoding enzymes responsible for the biogenesis
of aroma in fruit receptacles as alcohol acyltransferases
(gene34009, gene23453 and gene07931), pyruvate decarboxylase (gene13476), or quinone oxidoreductase
(gene28407), an enzyme involved in the biosynthesis of
furaneol (Table 2; Table S16).
(ii) Genes encoding cell wall hydrolases as pectate lyase B
(gene17555), polygalacturonases (gene21638 and
gene19241), endo-beta-1,4-glucanases (FaEG1)
(gene34867 and gene06191), rhamnogalacturonate lyase
(gene31030) as well as an expansin (gene21343). These
genes contribute, through cell wall degradation processes, to the fruit softening along the ripening process
(Table 3; Table S17).
(iii) Genes encoding enzymes of the flavonoids/
phenylpropanoids metabolic pathway including several glucosyl transferases (gene26265, gene20181,
gene26249, gene24225, gene26352 and gene12591),
an UDP-rhamnose:rhamnosyltransferase (gene14947),
a phenylalanine ammonia lyase (gene23261), a flavonol 3 hydroxylase (FaFHT) (gene14611), a flavonoid
3-O-glucosyltransferase (FaUFGT) (gene12591), a
cinnamoyl CoA reductase (FaCCR) (gene32333), a
cinnamyl alcohol dehydrogenase (FaCAD1)
(gene20700) and an isoflavone reductase (eugenol synthase 2; FaEGS2) (gene25260) (Table 4; Table S18).

Recently, Chen et al. (2015) have also described the induction of some of these genes, such as pectate lyase and

Table 2 Annotation of differentially regulated aroma-related genes
sorted into the four up-regulated clusters. Magnitudes of relative induction to ripe receptacles, p value (≤0.05) and arbitrary units of
intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.) indicates
AROMA-related genes
GENES

Putave funcon

cinnamyl alcohol dehydrogenase, after the application of
ABA in harvested strawberries.
We have found in this group novel genes, still not functionally characterized, coding enzymes potentially related to the
biosynthesis of aromas, such as alcohol acyltransferases
(gene27724, gene22294, gene34010 and gene19765), alcohol
dehydrogenases (gene06565, gene32435 and gene01341)
(Table 2; Table S16) and those that putatively are coding proteins involved in cell wall alterations, such as α-expansins
(gene28118), two xyloglucan xyloglucosyltransferase
(gene26403 and gene13718), a mannan endo-1,4-betamannosidase (gene15177), a beta-1,4-mannosyl-glycoprotein
(gene31094), a pectin methylesterase (gene05463) and a potential polygalacturonase (gene16186) (Table 3; Table S17).
In Cluster UP(ABA + AUX-), we have also identified new
genes probably related to the flavonoid/phenylpropanoid metabolism such as two anthranilate synthases (gene20939 and
gene04128), a 4-coumarate CoA ligase (gene09603), a
chalcone flavonone isomerase (CHI) (gene21346), two novel
cinnamoyl CoA reductases (CCR) (gene29482 and
gene29483), a cinnamoyl-CoA hydratase-dehydrogenase
(CHD) (gene30926), a cinnamyl alcohol dehydrogenase
(gene08569), a malonyl-CoA:isoflavone 7-O-glucoside-6'O-malonyltransferase (gene04262) and two glucosyl transferases (gene25577 and gene26353) (Table 4; Table S18). The
physiological function of these genes has yet to be clarified.
In Cluster UP(ABA + AUX0), our studies revealed the induction of a gene coding for cinnamyl alcohol dehydrogenase
(CAD) (gene24967), an enzyme potentially participating in
vascular lignifications and also in eugenol biosynthesis
(Aharoni et al. 2002; Blanco-Portales et al. 2002) and two
isoflavone reductases (IFR) (gene25258 and gene25257)
(Table 4; Table S18). IFR participates in the last steps of the
isoflavonoid biosynthetic pathway and is related to the regulation of lignin modifications (Hua et al. 2013) whose induction
has been previously described along the ripening process
(Chen et al. 2015). Additionally, the expression of

the total signal intensity for each feature on the microarray platform.
Gene ID in purple indicates specifity of expression in ripe receptacle
against to vegetative tissues

Ripe fruit receptacles
Fold

p-value

Up regulated
a.u.i.

CLOSER homologs in other plant species e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
gene27724
gene22294
gene34010
gene34009
gene23453
gene07931
gene01341
gene19765
gene13476
gene28407
gene06565
gene32435

ALCOHOL ACYL TRANSFERASE
ALCOHOL ACYL TRANSFERASE
ALCOHOL ACYL TRANSFERASE
ALCOHOL ACYL TRANSFERASE
ALCOHOL ACYL TRANSFERASE
ALCOHOL ACYL TRANSFERASE
ALCOHOL DEHYDROGENASE
LONG-CHAIN-ALCOHOL O-FATTY-ACYLTRANSFERASE
PYRUVATE DECARBOXYLASE
QUINONE OXIDOREDUCTASE
SHORT CHAIN DEHYDROGENASE
SHORT CHAIN DEHYDROGENASE/REDUCTASE

gene03054
gene15359

ALCOHOL DEHYDROGENASE
KETONE/ZINGERONE SYNTHASE

4.106
12.668
80.556
112.727
83.547
30.671
9.465
34.804
3.044
99.228
3.613
36.45

7.74E-04
7.74E-04
4.18E-04
4.94E-04
3.88E-04
3.84E-04
1.29E-03
4.58E-04
8.06E-04
6.91E-04
1.14E-03
4.01E-04

4.444
2.565

1.14E-03
7.05E-04

611.736
1822.214
13706.894
262737.325
41393.53
79123.951
102324.416
40077.082
21108.132
71563.74
29043.423
29440.949

Pyrus communis
Malus x domesca
Fragaria vesca
Fragaria x ananassa
Fragaria chiloensis
Fragaria x ananassa
Prunus armeniaca
Arabidopsis thaliana
Fragaria x ananassa
Fragaria x ananassa
Solanum tuberosum
Nandina domesca

7E-34
9E-177
0.0
1E-25
3E-52
5E-98
6E-142
3E-67
0.0
7E-102
3E-87
3E-98

AY534530
AY517491
AF193790
AF193789
FJ548610
JN089766
EU395433
NM_124916
AF193791
AY048861
AB192882
FJ789568

0.0
0

HM240512
JN166691

CLUSTER UP(ABA0AUX0)
34817.814 Prunus dulcis x Prunus persica
3645.113 Rubus idaeus

Aharoni, A. 2000
González, M. 2009
Cumplido-Laso, G. 2012

Aharoni, A. 2000
Raab, T. 2006

Funct Integr Genomics
intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.) indicates the total
signal intensity for each feature on the microarray platform

Table 3 Annotation of differentially regulated cell wall-related genes
sorted into the four up-regulated clusters. Magnitudes of relative
induction to ripe receptacles, p value (≤0.05) and arbitrary units of
CELL WALL-related genes
GENES

Putave funcon

Ripe fruit receptacles
Fold

p-value

Up regulated
a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
gene26030
gene14343
gene31094
gene34867
gene06191
gene28118
gene21343
gene06193
gene15177
gene17555
gene05463
gene16186
gene21638
gene19241
gene31030
gene26403
gene13718

ALPHA-EXPANSIN 3
ANNEXIN
BETA-1,4-MANNOSYL-GLYCOPROTEIN
ENDO-BETA-1,4-GLUCANASE
ENDO-1,4-BETA-GLUCANASE
EXPANSIN
EXPANSIN 2
INVERTASE/PECTIN METHYLESTERASE INHIBITOR
MANNAN ENDO-1,4-BETA-MANNOSIDASE
PECTATE LYASE B (FAPLB)
PECTIN METHYLESTERASE
POLYGALACTURONASE
POLYGALACTURONASE 1 (FAPG1)
POLYGALACTURONASE 2 (FAPG2)
RHAMNOGALACTURONATE LYASE (FARGLYASE)
XYLOGLUCAN GLYCOSYLTRANSFERASE
XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASE

gene04127
gene16326

HYDROXYPROLINE-RICH GLYCOPROTEIN
XYLOGLUCAN GALACTOSYLTRANSFERASE KATAMARI1

7.218
14.816
22.037
444.724
12.972
2.541
3.514
7.621
4.934
4.059
5.545
5.382
14.459
3.205
25.988
4.841
10.015

5.49E-04
4.16E-04
6.42E-04
3.82E-04
4.47E-04
2.96E-03
7.05E-04
1.44E-03
6.80E-04
1.11E-03
8.30E-04
1.07E-03
4.09E-04
1.45E-03
9.80E-04
1.93E-03
3.89E-04

2.847
6.428

9.78E-04
2.37E-03

1135.409
31932.04
5872.412
117102.147
101516.363
5686.578
27077.312
1576.893
44606.809
79298.451
27998.146
752.122
1905.208
15925.282
1799.52
2613.867
14733.559

Populus tremula x Populus tremuloides
Fragaria x ananassa
Arabidopsis thaliana
Fragaria x ananassa
Fragaria x ananassa
Rosa hybrid culvar
Fragaria x ananassa
Pyrus x bretschneideri
Prunus persica
Fragaria x ananassa
Arabidopsis thaliana
Vis vinifera
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa
Glycine max
Arabidopsis thaliana

3E-125
9E-180
3E-71
4E-45
0.0
2E-135
0.0
6E-76
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2E-132

AY435101
AF188832
NM_101170
AJ414709
AF074923
AB370116
AF159563
XP_009351520
EF568778
AF339024
U25649
XM_002274102
DQ458990
AY280662
AY282613
XM_003543785
NM_102950

9E-141
1E-78

XM_002311511
XM_003617783

Wang, GL. 2001
Spolaore, S. 2003
Harpster, MH. 1998
Civello, PM. 1999

Benítez-Burraco, A. 2003

Quesada, MA. 2009
Salenjn, EMJ. 2003
Molina-Hidalgo, FJ. 2013

CLUSTER UP(ABA+AUX0)
8638.53 Populus trichocarpa
1835.538 Medicago truncatula

CLUSTER UP(ABA0AUX-)
gene05164

BETA XYLOSIDASE

gene04618
gene03446
gene22434
gene10629
gene00522
gene11447
gene31031

BETA-1,3-GLUCANASE
BETA-1,3-GLUCANASE
PECTINESTERASE
PECTINESTERASE
POLYGALACTURONASE-INHIBITING PROTEIN
PROLINE-RICH CELL WALL PROTEIN
RHAMNOGALACTURONATE LYASE PROTEIN

gene12291
gene00663

XYLOGLUCAN ENDOTRANSGLYCOSYLASE
XYLOGLUCAN ENDOTRANSGLYCOSYLASE HYDROLASE

4.05

6.44E-04

5.65
25.36
5.32
3.89
13.34
8.58
54.25

2.51E-04
3.81E-04
1.64E-03
3.07E-03
4.10E-04
6.94E-04
5.73E-04

6.00
3.78

6.12E-04
8.07E-04

7362.412 Fragaria x ananassa
1114.343
24180.842
1499.436
1948.185
1114.74
30581.594
1094.262

Fragaria x ananassa
Ziziphus jujuba
Arabidopsis thaliana
Arabidopsis thaliana
Rubus idaeus
Vis vinifera
Arabidopsis thaliana

1574.01 Pyrus x bretschneideri
944.695 Apium graveolens

0.0

AY486104

0.0
0.0
7E-161
0.0
0.0
3E-04
0.0

AY170375
DQ093571
NM_115763
NM_116466
CAF04462
AJ237982
NM_127827

2E-165
3E-35

AGR44475
DQ204724

Marnez, G.A. 2004
Khan, A.A. 2003

CLUSTER UP(ABA0AUX0)
gene01893

ALPHA/BETA-HYDROLASE DOMAIN-CONTAINING PROTEIN

gene05671

BETA-1,3-GLUCANASE

gene12025

EXOPOLYGALACTURONASE

8.462

1.44E-04

13.436

5.73E-04

1038.505 Vis riparia

4E-118

EU676805

2.772

4.61E-03

2530.228 Populus trichocarpa

9E-116

XM_002301453

gene04127 (hydroxyproline-rich glycoprotein) and
gene16326 (xyloglucan galactosyltransferase) that belongs to Cluster UP(ABA + AUX0) was also up-regulated
(Table 3; Table S17). These genes encode cell wall proteins
involved in many aspects of plant growth and development
(Showalter et al. 2010) and have important roles in the
formation and function of the walls surrounding growing
plant cells (Kong et al. 2015).

21532.142 Arabidopsis thaliana

0.0

NM_105142

decarboxylase (gene19498) and several different
acyltransferases (gene14837, gene14839, gene15086 and
gene19766) (Table 5; Table S19). Their expression profiles
match with that of the strawberry alcohol acyltransferases involved in the biosynthesis of volatile ester (Aharoni et al.
2000; Cumplido-Laso et al. 2012).

Signaling and regulatory proteins
Fatty acid-derived and other lipophilic flavour
compounds
Alcohols and acyl-CoA formed during β-oxidation are used
for flavour ester production. In plants, the majority of volatiles
originate from the degradation of saturated and unsaturated
chain fatty acids through α- and β-oxidation processes
(Schwab et al. 2008). The de novo synthesis and hydrolysis
of fatty acids from acyl carrier proteins (acyl-ACP) also provide volatile acids (Schwab et al. 2008). In fruits, the hydrogenation of aldehydes produces alcohols through the action of
flavour-related alcohol dehydrogenases (ADHs) (Manríquez
et al. 2006). Several genes putatively encoding genes of the
fatty acid metabolism related with aroma production in ripe
strawberry fruits are included into Cluster UP(ABA + AUX-).
In this cluster, the vast majority of aroma-related genes are
also included (Fig. 3). The expression of many of these genes
was fruit specific and highly expressed in red ripe receptacle.
We detected a stearoyl ACP desaturase (gene21537), an acyl
carrier protein (gene16428), two different acyl-CoA
thioesterases (gene03716 and gene03720), a malonyl-CoA

Fleshy fruit development and ripening is a high complex process that includes the coordination of many metabolic pathways involved in colour, taste, firmness, flavour and turgor
required to produce a normal ripe fruit. All these process underlying fruit receptacle development and ripening must be
carefully regulated. Our microarray analysis has identified
several transcription factors (TFs) and regulatory proteins
whose expression pattern allowed its inclusion into each
ripening-related cluster (Fig. 3).
Up-regulated transcription factors
The microarray expression studies showed that 17 genes coding transcription factors belonging to Cluster UP(ABA +
AUX-) and 7 genes to Cluster UP(ABA + AUX0) (Fig. 3).
Six TFs present high level of transcripts in red ripe receptacles
(Table 6; Table S20). Genes gene31413, gene28435,
gene09407 and gene24494 code three R2R3 MYB TFs and
a MADS-box TF (FaSHP; SHATTERPROOF-like gene) respectively. FaMYB10 (gene 31413) has been described as a

Funct Integr Genomics
Table 4 Annotation of differentially regulated flavonol/phenylpropanoid
metabolism-related genes sorted into the four up-regulated clusters.
Magnitudes of relative induction to ripe receptacles, p value (≤0.05) and
arbitrary units of intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.)

indicates the total signal intensity for each feature on the microarray
platform. Gene ID in purple indicates specifity of expression in ripe
receptacle against to vegetative tissues

FLAVONOLS/PHENYLPROPANOID METABOLISM-related genes
GENES

Putave funcon

Ripe fruit receptacles Up regulated
Fold

p-value

a.u.i.

loser homologs in other plant species e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
gene09603
gene20939
gene04128
gene21346
gene32333
gene29482
gene29483
gene30926
gene20700
gene08569
gene14611
gene12591
gene07065
gene25577
gene25260
gene04262
gene23261
gene26353
gene18829
gene26265
gene20181
gene26249
gene24225
gene26352
gene12591
gene14947

4-COUMARATE-COA LIGASE
ANTHRANILATE SYNTHASE ALPHA SUBUNIT
ANTHRANILATE SYNTHASE BETA SUBUNIT
CHALCONE FLAVONONE ISOMERASE (CHI)
CINNAMOYL COA REDUCTASE (CCR)
CINNAMOYL COA REDUCTASE
CINNAMOYL COA REDUCTASE
CINNAMOYL-COA HYDRATASE-DEHYDROGENASE (CHD)
CINNAMYL ALCOHOL DEHYDROGENASE (CAD)
CINNAMYL ALCOHOL DEHYDROGENASE
FLAVANONE 3-HYDROXYLASE (FHT)
FLAVONOID 3-O-GLUCOSYLTRANSFERASE (FAUFGT)
FRA A 2 ALLERGEN (FRAA2)
GLUCOSYLTRANSFERASE
ISOFLAVONE REDUCTASE (EUGENOL SYNTHASE; FAEGS2)
MALONYL-COA:ISOFLAVONE 7-O-GLUCOSIDE-6''-O-MALONYLTRANSFERASE
PHENYLALANINE AMMONIA LYASE (PAL)
PHENYLPROPANOID:GLUCOSYLTRANSFERASE
POLYPHENOL OXIDASE 2 (PPO2)
UDP-GLUCOSE GLUCOSYLTRANSFERASE
UDP-GLUCOSE GLUCOSYLTRANSFERASE
UDP-GLUCOSE GLUCOSYLTRANSFERASE
UDP-GLUCOSE GLUCOSYLTRANSFERASE
UDP-GLUCOSE GLUCOSYLTRANSFERASE
UDP-GLUCOSE:CINNAMATE GLUCOSYLTRANSFERASE
UDP-RHAMNOSE:RHAMNOSYLTRANSFERASE

gene28093
gene24967
gene25258
gene25257

CINNAMATE-4-HYDROXYLASE
CINNAMYL ALCOHOL DEHYDROGENASE
ISOFLAVONE REDUCTASE
ISOFLAVONE REDUCTASE

gene15845

CINNAMOYL COA REDUCTASE

gene03747
gene30778

CINNAMOYL-COA REDUCTASE
FLAVONOL SYNTHASE/FLAVANONE 3-HYDROXYLASE

16.273
16.707
17.342
2.86
5.86
13.863
14.761
4.256
348.576
35.373
2.697
190.971
7.103
3.886
945.604
16.965
3.042
5.679
14.979
11.401
31.879
2.408
41.256
2.561
240.118
21.319

4.86E-04
4.22E-04
3.87E-04
1.03E-03
6.19E-04
4.57E-04
5.35E-04
1.42E-03
3.79E-04
3.90E-04
1.25E-03
8.27E-04
1.02E-03
5.82E-04
1.14E-03
1.03E-03
1.71E-03
5.08E-04
4.93E-04
4.08E-04
3.72E-04
3.06E-03
4.01E-04
1.46E-03
3.80E-04
3.77E-04

71314.032
2198.718
13939.922
219991.684
4490.154
5311.66
6119.27
40490.529
152638.488
15980.983
255100.62
34629.998
84071.637
755.83
10732.21
1048.548
57410.815
15288.679
2805.2
99900.157
1602.396
2467.037
6709.78
844.524
25063.16
3726.085

Arabidopsis thaliana
Arabidopsis thaliana
Populus trichocarpa
Prunus persica
Fragaria x ananassa
Populus trichocarpa
Populus trichocarpa
Petunia x hybrida
Fragaria x ananassa
Camellia sinensis
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa
Vis vinifera
Fragaria x ananassa
Glycine max
Fragaria x ananassa
Nicoana tabacum
Fragaria pentaphylla
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa
Fragaria x ananassa

0.0
0.0
2E-122
9E-116
3E-106
1E-132
1E-158
0.0
0.0
4E-176
0.0
0.0
1E-102
3E-27
0.0
2E-92
0.0
1E-163
0.0
0.0
0.0
0.0
0.0
3E-165
0.0
2.00E-179

NM_116755
M92353
XM_002314725
HM543569
AY285922
XM_002314017
XM_002314016
JX142126
U63534
GQ438848
AY691918
AY695815
GQ148818
JN164680
AGV02008.1
NM_001249890
HM641823
AF346431
HM063946
AY663785
AY663786
DQ289586
DQ289587
DQ289588
AY663784
AY663787

Salenjn, EMJ. 2003

Blanco-Portales, R. 2002
Almeida, JR. 2007
Almeida, JR. 2007
Muñoz, C. 2010
Aragüez, I. 2013
Pombo, MA. 2011

Lunkenbein, S. 2006
Lunkenbein, S. 2006
Griesser, M. 2008
Griesser, M. 2008
Griesser, M. 2008
Lunkenbein, S. 2006
Lunkenbein, S. 2006

CLUSTER UP(ABA+AUX0)
5.436
2.271
5.539
8.14

2.56E-04
1.36E-03
5.25E-04
9.10E-04

15892.832
11025.849
17012.885
2265.382

Rubus occidentalis
Sinopodophyllum hexandrum
Glycine max
Pyrus communis

0.0
0
1E-170
4E-109

FJ554629
HQ268590
AF202184
AF071477

CLUSTER UP(ABA0AUX-)
5.45

1.22E-03

13.45
3.058

4.06E-04
2.52E-03

40597.858 Fragaria x ananassa
823.034 Jatropha curcas
4135.94 Medicago truncatula

gene16550

HYDROXYCINNAMOYL-COENZYME A SHIKIMATE/QUINATE HYDROXYCINNAMOYLTRANSFERASE

12.37

4.73E-04

gene17831
gene04258
gene09318
gene26345

ISOFLAVONE 3'-HYDROXYLASE
ISOFLAVONE 7-O-GLUCOSIDE-6''-O-MALONYLTRANSFERASE
NAD(P)H-DEPENDENT 6'-DEOXYCHALCONE SYNTHASE
PHENYLPROPANOID :GLUCOSYLTRANSFERASE

52.37
8.62
2.348
2.406

3.80E-04
6.39E-04
1.47E-03
1.80E-03

2126.824
2844.052
759.62
714.89

Medicago truncatula
Glycine max
Medicago truncatula
Nicoana tabacum

gene30434
gene00494
gene26344
gene24224
gene06602

POLYPHENOL OXIDASE
PROBABLE CINNAMYL ALCOHOL DEHYDROGENASE
UDP-GLUCOSE GLUCOSYLTRANSFERASE
UDP-GLUCOSE GLUCOSYLTRANSFERASE

59.21
20.49
2.821
451.72
3.86

9.86E-04
1.19E-03
1.75E-03
4.86E-04
7.39E-04

2318.431
2041.434
881.11
2664.134
9323.865

Potenlla frucosa
Vis vinifera
Fragaria x ananassa
Fragaria x ananassa
Malus x domesca

gene03689
gene27021
gene01708

ISOFLAVONE REDUCTASE HOMOLOG
UDP-GLUCURONOSYLTRANSFERASE
UDP-GLYCOSYLTRANSFERASE

UDP-GLUCOSE:FLAVONOID 7-O-GLUCOSYLTRANSFERASE

866.869 Vis vinifera

0.0

AY285922

5E-162
4E-126

GQ149700
XM_003609420

3E-120

XP_002273086

1E-108
3.00E-73
2E-117
2E-177

AY278228
AB291059
XM_003607060
AF346431

0.0
6E-98
0.0
0.0
0.0

DQ851214
XP_002277375
DQ289588
AY663786
AY786997

3E-165
8E-174
0

XM_003533408
XM_002530352
XM_002267629

Salenjn, E.M.J. 2003

Griesser, M. 2008
Lunkenbein, S. 2006

CLUSTER UP(ABA0AUX0)
2.569
2.154
2.153

1.11E-03
1.16E-03
8.39E-04

ripening-related master regulatory gene of the structural
flavonoid/phenylpropanoid metabolic pathway genes
(Medina-Puche et al. 2014) whereas FaEOBII (gene28435)
is a positive regulator of two flavonoids/phenylpropanoids
volatile-related genes, FaCAD1 and FaEGS2, controlling eugenol production in ripe strawberry receptacles (MedinaPuche et al. 2015). FcMYB1 (gene09407) was functionally
characterized as regulator of the branching point of the
anthocyanins/proanthocyanidins biosynthesis (Salvatierra
et al. 2013). FaSHP (gene24494) codes a C-type MADSbox gene. Transient repression of this FaSHP gene in strawberry receptacle lead to a slightly shorter delay in the time
required to reach the pink stage of ripening (Daminato et al.
2013). In addition, expression of several ripening-related
genes as well as the content of different metabolites was altered in these transiently modified fruits (Daminato et al.
2013). A secondary role as modulator of ripening was proposed for the FaSHP gene (Daminato et al. 2013). Recently,

963.847 Glycine max
1690.034 Ricinus communis
2188.141 Vis vinifera

the role played by FaSCL8 along strawberry fruit ripening
was proposed (Pillet et al. 2015). This TF could modulate
the regulation of the expression of genes related to the
flavonoid/anthocyanin biosynthesis probably through their influence on FaMYB10 gene expression (Pillet et al. 2015). We
have identified three different WRKY (gene28720,
gene19478 and gene01340), two AP2/ERF (gene07057 and
gene32084), two R2R3 MYB (gene25060 and gene19715,),
two regulatory basic helix-loop-helix (bHLH) (gene30478
and gene23830), a NAC (gene29766), a DOF (gene32526),
a CH2H2L zinc finger (gene25547), a ZF-HD (gene26009)
and a SCL (gene13212) transcription factors that belong to
Cluster UP(ABA + AUX-) and UP(ABA + AUX0). The expression of FaMYB10 (gene31413), FaSHP (gene24494),
WRKY (gene28720), R2R3 MYB (gene25060), AP2/ERF
(gene32084), SCL (gene13212), DOF (gene32526) and
NAC (gene29766) were stronger in fruit receptacles compared to that of vegetative tissues (Table 6; Table S20).

Funct Integr Genomics
Table 5 Annotation of differentially regulated lipid metabolism-related
genes sorted into the four up-regulated clusters. Magnitudes of relative
induction to ripe receptacles, p value (≤0.05) and arbitrary units of
intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.) indicates the total
LIPID METABOLISM-related genes
GENES

Putave funcon

Fold

p-value

a.u.i.

signal intensity for each feature on the microarray platform. Gene ID in
purple indicates specifity of expression in ripe receptacle against to
vegetative tissues
Ripe fruit receptacles
Up regulated
CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
gene15086
gene16428
gene32451
gene19766
gene18789
gene10626
gene14921
gene22011
gene19498
gene30534
gene14839
gene14837
gene30403
gene21537
gene03720
gene03716

1-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE
ACYL CARRIER PROTEIN
ACYL:COA LIGASE
ACYL-COA--STEROL O-ACYLTRANSFERASE
ALPHA-CARBOXYLTRANSFERASE
EPOXIDE HYDROLASE
EPOXIDE HYDROLASE
GDSL ESTERASE/LIPASE
MALONYL-COA DECARBOXYLASE FAMILY PROTEIN
NON-SPECIFIC LIPID-TRANSFER PROTEIN 2
O-ACYLTRANSFERASE WSD1
O-ACYLTRANSFERASE WSD1
SPHINGOLIPID DELTA-4 DESATURASE
STEAROYL-ACP DESATURASE
THIOESTERASE
THIOESTERASE

14.735
9.246
4.454
27.514
8.458
6.538
6.244
12.902
6.193
48.826
621.993
106.07
2.825
9.78
53.345
65.231

7.70E-04
3.89E-04
7.71E-04
5.11E-04
8.70E-04
5.23E-04
5.25E-04
9.78E-04
7.85E-04
4.45E-04
4.33E-04
2.79E-02
1.05E-03
6.32E-04
5.48E-04
3.84E-04

gene12921
gene31042

BETA-KETOACYL-ACP SYNTHASE
PHOSPHOLIPASE D

2.01
36.321

4.06E-02
4.08E-04

gene25083
gene25061
gene16355
gene08561
gene05435
gene05818
gene08996
gene13454

12-OXOPHYTODIENOATE REDUCTASE
3-OXOACYL-[ACYL-CARRIER-PROTEIN] REDUCTASE
ACYL-COENZYME A OXIDASE
GDSL ESTERASE/LIPASE
GDSL ESTERASE/LIPASE
LONG CHAIN ACYL-COA SYNTHETASE
MONOGLYCERIDE LIPASE
OMEGA-3 DESATURASE

3.111
2.028
8.64
10.38
4.28
5.24
4.59
11.66

7.21E-04
2.11E-03
4.96E-04
5.26E-04
2.06E-03
1.32E-03
6.39E-04
4.57E-04

gene06158
gene00708
gene06841
gene13295
gene15770
gene11707

ACYL CARRIER PROTEIN
ACYL-COA DEHYDROGENASE
FATTY ACID BETA-OXIDATION MULTIFUNCTIONAL PROTEIN
PHOSPHOLIPASE A2 HOMOLOG
PHOSPHOLIPASE D ZETA
PROSTAGLANDIN E SYNTHASE

4.019
5.119
2.002
2.334
2.334
2.319

4.62E-03
1.67E-03
5.01E-04
2.90E-03
9.57E-04
1.41E-03

1533.064
250709.656
12468.596
35119.281
4229.298
4240.341
1359.326
2357.557
7642.606
16848.37
1423.823
951.027
4293.257
21938.505
1117.897
1332.166

Lotus japonicus
Fragaria vesca
Populus trichocarpa
Glycine max
Jatropha curcas
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis lyrata subsp. lyrata
Pyrus x bretschneideri
Vis vinifera
Populus trichocarpa
Arabidopsis thaliana
Vernicia montana
Vis vinifera
Arabidopsis thaliana

0.0
1E-94
0.0
2E-99
0.0
1E-155
1E-78
8E-103
2E-162
1E-25
3E-79
2E-37
0.0
0.0
3E-88
7E-64

AP004969
AJ001446
XM_002322437
XM_003534777
GQ845013
NM_116467
NM_114960
NM_102626
XM_002872641
XP_009357786
XM_002274486
XM_002328929
NM_116731
EU072353
AM447422
NM_105497

CLUSTER UP(ABA+AUX0)
715.004 Arachis hypogaea
2984.85 Cucumis savus

0.0
0.0

EU823327
EF363796

CLUSTER UP(ABA0AUX-)
2484.61
10889.97
1933.611
1459.027
3628.274
2526.885
10229.516
3907.154

Theobroma cacao
Glycine max
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Vis vinifera
Cicer arienum
Prunus persica

3E-89
3E-125
0.0
6.00E-118
8E-103
0.0
0.0

EOY17386
XM_003518929
NM_125910.5
NM_202420
NM_104270
XP_002277936
XP_004501143
AF517831

5E-32
0.0
0.0
5E-41
0.0
1E-135

FJ768733
XM_002314327
NM_111566
XM_004157866
XM_002516928
XM_003619775

CLUSTER UP(ABA0AUX0)
5411.52
704.991
1749.846
3023.973
1189.399
3941.997

These results clearly suggest that these TFs could regulate
the expression of genes involved in receptacle fruit ripening, as previously show for FaMYB10, FaSHP, FaEOBII
or FaSCL8 (Daminato et al. 2013; Medina-Puche et al. 2014;
Medina-Puche et al. 2015; Pillet et al. 2015).

Other regulatory proteins
Cluster UP(ABA + AUX-) also included two genes putatively
encoding two small proteins containing a C2 domain
(gene19112 and gene26051), as well as a gene corresponding
to a multiple C2 and transmembrane domain-containing protein (gene32454) (Table 7; Table S21). The C2 domains are
found in a large variety of proteins combined with a wide range
of other domains or modules encoding different enzymatic
activities involved in intracellular signal transduction and
membrane trafficking (Zhang and Aravind 2010). Recently, it
has been shown that a family of small proteins containing a
lipid binding C2 domain (CAR proteins) interacts with the
ABA-receptor PYR/PYLs and positively regulate ABA sensitivity (Rodríguez et al. 2014). Of these genes coding C2
domain-containing proteins, only the expression of gene19112

Arachis hypogaea
Populus trichocarpa
Arabidopsis thaliana
Cucumis savus
Ricinus communis
Medicago truncatula

was receptacle specific. Gene19112 code a small protein that
contains a C2 domain located into the N-terminal end of the
protein, suggesting that this C2 protein could be a CAR protein
involved in the positive regulation of ABA signaling along
fruit receptacle ripening process. In Cluster UP(ABA +
AUX0), there are two genes coding a similar N-terminal C2
domain-containing protein (gene09729 and gene19113).
Gene19113 is specifically and highly induced in ripe receptacles (Table 7; Table S21). The putative regulatory function of
these proteins remains to be elucidated.
Gene21905 encodes a putative BOP/NPR1/NIM-like regulatory protein (Table 7; Table S21). This type of protein is
named BTB-ankyrins protein because they contain two conserved protein–protein interactions motifs: a BOP/BOZ domain at the N-terminus and four ankyrins motifs near the Cterminus (Khan et al. 2014). These domains were also present
in the FaBOP bioinformatically deduced protein.
Functionally, these BTB-anyrin proteins are considered as
plant-specific transcriptional co-activators (Khan et al.
2014). This gene is expressed at very high levels and only in
ripe receptacles suggesting that this protein play an important
regulatory role as co-activator of the expression of many
ripening-related genes.

Funct Integr Genomics
Table 6 Annotation of differentially regulated transcription factors
sorted into the four up-regulated clusters. Magnitudes of relative
induction to ripe receptacles, p value (≤0.05) and arbitrary units of
intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.) indicates the total
Transcripon factors
GENES

Putave funcon

signal intensity for each feature on the microarray platform. Gene ID in
purple indicates specifity of expression in ripe receptacle against to
vegetative tissues

Ripe fruit receptacles
Fold

p-value

a.u.i.

Up regulated
CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
gene24494
gene07057
gene32084
gene30478
gene20865
gene25547
gene32526
gene13212
gene12519
gene29766
gene28435
gene31413
gene25060
gene28720

AGAMOUS (FASHP)
AP2 DOMAIN CLASS TRANSCRIPTION FACTOR
AP2/ERF DOMAIN-CONTAINING TRANSCRIPTION FACTOR
BASIC HELIX-LOOP-HELIX
BEL1 HOMEODOMAIN PROTEIN
C2H2L DOMAIN CLASS TRANSCRIPTION FACTOR
DOF ZINC FINGER PROTEIN
GRAS FAMILY TRANSCRIPTION FACTOR
LURP-ONE-RELATED PROTEIN (TUBBY C 2 DOMAIN CONTAINING PROTEIN)
NAC DOMAIN PROTEIN
R2R3 MYB TRANSCRIPTION FACTOR (FAEOBII)
R2R3 MYB TRANSCRIPTION FACTOR (FAMYB10)
R2R3 MYB TRANSCRIPTION FACTOR (TT2)
WRKY TRANSCRIPTION FACTOR

5.938
3.891
4.481
6.539
5.277
10.435
35.854
5.087
328.446
23.533
25.21
417.84
129.981
120.181

8.16E-04
2.31E-03
1.32E-02
3.66E-03
1.13E-03
1.44E-03
2.62E-03
5.05E-04
1.43E-03
5.20E-04
1.57E-03
3.79E-04
3.85E-04
4.48E-04

gene19478
gene01340
gene26009
gene21047
gene23830
gene02417
gene13301
gene19715
gene09407
gene30204

5907.602
4262.951
851.933
791.585
751.109
1333.306
2268.309
19090.26
924.15
3718.698
3198.538
25737.348
1398.983
11400.684

Fragaria x ananassa
Malus x domesca
Populus trichocarpa
Arabidopsis thaliana
Vis vinifera
Malus x domesca
Medicago truncatula
Fragaria x ananassa
Vis vinifera
Populus trichocarpa
Fragaria x ananassa
Fragaria x ananassa
Rosa rugosa
Medicago truncatula

WRKY TRANSCRIPTION FACTOR

10.244

2.36E-03

1327.876 (Populus tomentosa x P. bolleana) x P. tomentosa

WRKY TRANSCRIPTION FACTOR
ZF-HD HOMEOBOX PROTEIN

28.846
2.109

4.11E-04
7.51E-03

2564.835 Solanum tuberosum / Malus x domesca
2083.591 Populus trichocarpa

AP2 DOMAIN CLASS TRANSCRIPTION FACTOR
BHLH TRANSCRIPTION FACTOR
GENERAL TRANSCRIPTION FACTOR IIH SUBUNIT
HSF DOMAIN CLASS TRANSCRIPTION FACTOR
R2R3 MYB TRANSCRIPTION FACTOR
R2R3 MYB TRANSCRIPTION FACTOR
TRANSCRIPTION FACTOR PCL1

4.972
19.922
3.01
2.184
58.178
2.212
19.494

8E-177
3E-74
6E-63
4E-20
2E-80
3E-38
3E-49
2E-156
2E-77
6E-117
8E-82
1E-41
6E-46
5E-62

AGU92563.1
GU732439
XM_002304518
NM_179381
XM_002277567
HM122497
XM_003602109
XM_002305878
XM_002278173
XM_002305738
KM099230
EU155162
FR828543
XM_003617375

2E-82

GQ377448

Daminato, M. 2013

Pillet, 2015

Medina-Puche, L. 2015
Medina-Puche, L. 2014

1.00E-47 AB061245 / HM122717
1.00E-30
XP_002306048.1

CLUSTER UP(ABA+AUX0)
4.83E-04
1.04E-04
1.29E-03
2.47E-03
5.58E-04
1.76E-03
4.73E-04

1269.657
6757.179
2023.13
3060.134
1472.388
3987.974
729.478

Malus x domesca
Medicago truncatula
Medicago truncatula
Malus x domesca
Medicago truncatula
Fragaria chiloensis
Prunus persica

1E-45
2E-39
6E-97
1E-57
4E-52
8E-116
2E-122

GU732462
XM_003611682
XM_003601023
HM122592
XM_003601151
GQ867222
XP_007205582

Malus x domesca
Populus trichocarpa
Glycine max
Malus x domesca
Malus domesca
Rosa rugosa
Glycine max
Rosa rugosa
Humulus lupulus
Malus x domesca
Malus domesca
Malus x domesca
Malus domesca
Medicago truncatula
Arabidopsis thaliana
Medicago truncatula
Fragaria x ananassa
Solanum tuberosum
Nicoana tabacum
Medicago truncatula

4E-43
7.00E-37
1E-32
7E-179
1.00E-50
1E-118
2.00E-58
8E-153
2E-70
7E-107
1E-152
7.00E-100
5.00E-159
8E-46
7.00E-43
1E-149
3E-101
0.0
4.00E-81
1E-72

GU732450
XM_002327017
XM_003516732
HM122505
ADL36628
AFP55588
DQ822914
GU967443
FR751555
HM122664
ADL36805
HM122660
ACI13682
XM_003608713
NM_102343
XM_003590588
EU727547
AK329902
AB041520
XM_003604615

Humulus lupulus
Malus domesca
Medicago truncatula
Malus x domesca
Glycine max
Arabidopsis thaliana

7E-34
3.00E-16
1E-74
3E-54
1E-69
2E-53

FN395065
XP_008367213
XM_003590008
HM122489
XM_003556167
NM_119321

Salvaerra, A. 2013

CLUSTER UP(ABA0AUX-)
gene07304
gene04916
gene28601
gene08699
gene28452
gene12765
gene11419
gene03712
gene03865
gene07251
gene30439
gene20686
gene04424
gene32251
gene03713
gene20299
gene07210
gene03411
gene09147
gene13452

AP2 DOMAIN CLASS TRANSCRIPTION FACTOR
AP2/ERF DOMAIN-CONTAINING TRANSCRIPTION FACTOR
B3 DOMAIN-CONTAINING PROTEIN
C2H2L DOMAIN CLASS TRANSCRIPTION FACTOR
C2H2L DOMAIN CLASS TRANSCRIPTION FACTOR
HOMEOBOX LEUCINE ZIPPER
MYB TRANSCRIPTION FACTOR
MYB TRANSCRIPTION FACTOR
MYB TRANSCRIPTION FACTOR
NAC DOMAIN CLASS TRANSCRIPTION FACTOR
NAC DOMAIN CLASS TRANSCRIPTION FACTOR
NAC DOMAIN CLASS TRANSCRIPTION FACTOR
NAC DOMAIN CLASS TRANSCRIPTION FACTOR
YABBY
BHLH TRANSCRIPTION FACTOR
WD REPEAT-CONTAINING PROTEIN
WRKY TRANSCRIPTION FACTOR
WRKY TRANSCRIPTION FACTOR
WRKY TRANSCRIPTION FACTOR
ZINC FINGER AND SCAN DOMAIN-CONTAINING PROTEIN

gene08033
gene11645
gene32427
gene17037
gene23226
gene01153

BASIC-LEUCINE ZIPPER
FAR1 DNA-BINDING DOMAIN CONTAINING PROTEIN
GENERAL TRANSCRIPTION FACTOR IIE SUBUNIT2
MADS-BOX TRANSCRIPTION FACTOR 22-LIKE
LOB DOMAIN-CONTAINING PROTEIN
ZINC FINGER CCCH DOMAIN-CONTAINING PROTEIN 3

7.65
48.27
3.748
2.785
5.99
11.57
8.97
4.44
3.054
2.483
5.16
19.69
5.61
25.52
74.05
2.607
11.46
417.18
6.25
7.26

5.99E-04
6.43E-04
1.38E-03
9.23E-04
5.62E-04
3.72E-04
1.72E-03
4.60E-04
1.99E-03
1.57E-03
5.59E-04
3.79E-04
8.07E-04
3.80E-04
1.74E-04
1.21E-03
4.94E-04
1.34E-03
6.02E-04
4.97E-04

1724.632
3416.356
1146.75
4424.90
1153.625
1171.102
7626.273
4321
4524.99
12917.73
1275.898
1754.341
1212.353
2950.067
1343.654
5690.84
814.522
891.421
1959.55
2184.342

Encinas-Villarejo, S. 2009

CLUSTER UP(ABA0AUX0)
3.567
3.33
2.177
3.377
3.143
2.193

3.46E-03
3.87E-02
3.39E-03
2.52E-03
1.14E-03
1.65E-03

Gene30997 encodes a Ran protein that belongs to the small
GTP-binding proteins family and contains three ZnF-RB domains (Table 7; Table S21). Ran are small GTPases essential for
nuclear transport and nuclear assembly and are primarily located inside the nucleus. These proteins are involved in the plant
response to hormone or environmental signaling such as salt
stress or cold tolerance (Zang et al. 2010; Xu and Cai 2014).
Gene05293 and gene24830 were also up-regulated in ripe
receptacles and encoding two different putative leucine-rich
repeat (LRR) receptors (Table 7; Table S21). The protein deduced from the gene05293 sequence contains five leucinerich repeats constituting, at the N-terminal end of the protein, a typical domain of the LRR receptors. Additionally,
in the C-carboxyl end, a protein kinase domain is present.
The deduced protein corresponding to gene24830 include
four different leucine-rich repeat motifs organized as a
leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)like subfamily domains that participate in protein–protein
interactions. Both genes are included into Cluster
UP(ABA + AUX-), and their elevated expression in red
ripe receptacles compared to green receptacles and vegetative tissues is worth to highlight. This is not in agreement

3544.80
1749.296
2775.236
2257.018
4925.558
2161.806

with data obtained by Chen et al (2015), who described
two putative leucine-rich repeat receptor-like protein kinase genes (LRR-RLKs) whose expression was repressed
in the presence of IAA and ABA. Nevertheless, the LRRRLKs family is very broad and may include genes with
different expression along the ripening process. In fact,
the LRR receptors encoded by gene05293 and gene24830
have not still functionally characterized in plants and the
role played along the strawberry fruit ripening process
must be still determined.
SCF complexes (E3 ubiquitin ligases) are composed by
different proteins as RING finger proteins, the scaffold culin
1 protein and the substrate adaptor proteins, SKP1, Fbox
(FBX), BTB, VHL, SOCs or DWD (WD40). These components associate in a combinatorial fashion rendering diverse
protein adaptors able to specifically recruit appropriate substrates for selective ubiquitination by a wide range of different
SCF ligases. These ligases bind, through the RING protein,
the E2 ubiquitin ligases conjugating enzyme and bring together both the E2 and specific substrate targets. The catalytic core
of these complexes is constituted by the interaction between
culin and RING finger proteins (Hua and Vierstra 2011;

Funct Integr Genomics
(a.u.i.) indicates the total signal intensity for each feature on the
microarray platform. Gene ID in purple indicates specifity of expression
in ripe receptacle against to vegetative tissues

Table 7 Annotation of differentially regulated signaling and regulatory
proteins-related genes sorted into the four up-regulated clusters.
Magnitudes of relative induction to ripe receptacles, p value (≤0.05)
and arbitrary units of intensity (a.u.i.) are given. Intensity in blue scale
Signalling and Regulatory Proteins
GENES

Putave funcon

Ripe fruit receptacles
Fold

p-value

Up regulated

a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

CLUSTER UP(ABA+AUX-)
gene21905
gene26483
gene19112
gene26501
gene05089
gene32454
gene10659
gene02110
gene31093

BOP/NPR1/NIM1 REGULATORY PROTEIN
DVL
C2 DOMAIN CONTAINING PROTEIN ELICITOR RESPONSIVE GENE
C2 DOMAIN CONTAINING PROTEIN ELICITOR RESPONSIVE GENE
HYPERSENSITIVE REACTION ASSOCIATED CA2+-BINDING PROTEIN
MULTIPLE C2 AND TRANSMEMBRANE DOMAIN-CONTAINING PROTEIN
PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASE 5
RAS-RELATED PROTEIN RHN1-LIKE ISOFORM X1
WD40 DOMAIN CONTAINING PROTEIN

gene21168

WD-REPEAT PROTEIN

28.876
4.486
447.572
9.483
2.055
5.232
12.939
4.381

6.32E-04
1.03E-03
1.10E-03
6.50E-04
2.57E-03
7.42E-04
5.40E-04
5.53E-04

5.865

6.79E-04

12.423

1.98E-03

1952.712
11234.476
22676.928
840.288
19185.487
26361.513
1087.751
7385.48

Populus trichocarpa
Arabidopsis thaliana
Oryza sava
Medicago truncatula
Phaseolus vulgaris
Vis vinifera
Glycine max
Malus domesca

0.0
1E-20
1E-25
1E-54
3E-50
0.0
4E-94
4E-127

XM_002308869
BK001754
AF090698
XM_003616359
AF145386
XM_002271554
XM_003517865
XP_008337431

0.0
0.0

XP_006358293
XP_008376375

Vis vinifera
Medicago truncatula
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Medicago truncatula
Arabidopsis thaliana
Vis vinifera
Glycine max
Arabidopsis thaliana
Medicago truncatula

0.0
1E-160
0.0
2E-106
0.0
0.0
0.0
1E-148
0.0
3E-117
4E-63

XM_002272301
XM_003601211
NM_111649
NM_128240
NM_105452
XM_003608235
NM_180212
XM_002272976
XM_003549582
NM_113431.5
XM_003609219

Glycine max
Vis vinifera
Vis vinifera
Arabidopsis thaliana

3E-126
5E-69
1E-146
0.0

FJ014728
XM_003633734
XM_003633996
AF084035

5423.75 Solanum tuberosum
1747.34 Malus domesca

KINASES
gene23258
gene06226
gene15532
gene31007
gene22182
gene30640
gene13564
gene20321
gene32170
gene17422
gene30997

ADENOSINE KINASE
PANTOTHENATE KINASE
PROTEIN KINASE DOMAIN-CONTAINING PROTEIN
PROTEIN KINASE-LIKE PROTEIN
SENSOR KINASE (CSK)
SERINE/THREONINE PROTEIN KINASE
SERINE/THREONINE PROTEIN KINASE
SERINE/THREONINE-PROTEIN KINASE
SERINE/THREONINE-PROTEIN KINASE HT1-LIKE
SERINE/THREONINE-PROTEIN KINASE OXI1 (AGC2-1)
ZINC FINGER SER/THR PROTEIN KINASE

gene05293
gene24830
gene23598
gene30661

LEUCINE-RICH REPEAT FAMILY PROTEIN (FARPK)
LRR RECEPTOR-LIKE SERINE/THREONINE-PROTEIN KINASE
RECEPTOR PROTEIN
RECEPTORPROTEIN KINASE (RKS1)

gene10081
gene09579
gene09873
gene22281
gene00322
gene08799
gene30404
gene27006
gene18503
gene18505
gene31679
gene30186
gene16196

26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT
E3 UBIQUITIN-PROTEIN LIGASE
F-BOX FAMILY PROTEIN
F-BOX PROTEINS
F-BOX/FBD/LRR-REPEAT PROTEIN
PROTEIN PEROXIN-4 (UBIQUITIN-CONJUGATING ENZYME E2)
RING FINGER PROTEIN
RING-H2 FINGER PROTEIN
SKP1/ASK1 PROTEIN (FASKP1)
SKP1-LIKE PROTEIN
UBIQUITIN
UBIQUITIN FUSION DEGRADATION UFD1
UBIQUITIN PROTEIN LIGASE

gene09729
gene19113

C2 DOMAIN-CONTAINING PROTEIN
C2 DOMAIN-CONTAINING PROTEIN

6.06
20.752
15.196
6.442
6.727
3.744
6.936
7.196
3.014
11.071
23.606

4.76E-04
3.78E-04
4.57E-04
5.13E-04
6.84E-04
6.19E-04
6.15E-04
4.83E-04
1.02E-03
4.55E-04
3.68E-04

13527.151
45073.96
6490.259
1227.64
14678.543
2730.391
1928.308
2113.035
5739.779
3531.296
5345.349

RECEPTORS
23.737
148.678
10.254
5.199

4.07E-04
0.00197
4.42E-03
7.36E-04

10812.41
1418.95
821.599
622.054

PROTEOLITIC PROTEIN REGULATION
3.666
2.004
170.203
127.819
2.573
2.219
4.673
29.375
104.06
67.497
20.403
4.758
33.231

1.21E-03
1.80E-03
1.88E-03
3.83E-04
5.88E-03
1.48E-03
7.27E-04
5.26E-04
1.40E-03
4.04E-04
5.18E-04
8.42E-04
1.48E-03

2713.808
2149.05
1065.77
2504.649
735.698
23217.95
2100.81
9615.67
28553.73
16857.907
3251.957
1708.866
1616.508

Arabidopsis thaliana
Prunus mume
Populus trichocarpa
Pyrus pyrifolia
Medicago truncatula
Cucumis melo
Medicago truncatula
Medicago truncatula
Hevea brasiliensis
Medicago truncatula
Artemisia annua
Arabidopsis thaliana
Medicago truncatula

2E-62

NM_125180
0.0
XP_008239171
4E-53
XM_002307964
9E-08
AB545982
4E-88
XM_003589259
2E-111 XP_008445030
2E-73
XM_003637350
8E-23
XM_003590265
1E-30
HM640272
3E-08
XM_003616811
5E-11
GQ901904
3E-151
NM_201828
4E-63
XM_003602578

CLUSTER UP(ABA+AUX0)
2.801
16.454

2.10E-03
1.44E-03

1064.908 Populus trichocarpa
2484.958 Oryza sava

0.0
4E-26

XM_006376379
NM_001054079

0
6E-10
0.0
2E-122

NM_124535
NM_001202566
XM_003555999
NM_112761

0
2E-23
4E-09

XM_002301353
AB545981
NM_118773

7E-63
1E-179

NM_001157389
XP_008219186

KINASES
gene13428
gene19366
gene20400
gene01144

LEUCINE-RICH REPEAT PROTEIN KINASE
PHOSPHORIBULOKINASE/URIDINE KINASE DOMAIN PROTEIN
SERINE/THREONINE-PROTEIN KINASE
SERINE/THREONINE-PROTEIN KINASE

2.205
2.334
3.584
4.032

gene18983
gene02131
gene04008

F-BOX FAMILY PROTEIN
F-BOX PROTEINS
RING/U-BOX DOMAIN-CONTAINING PROTEIN

7.323
2.113
3.899

gene05774
gene26891

RHODOPSIN-LIKE RECEPTOR
ODORANT RECEPTOR DOMAIN CONTAINING PROTEIN(ODR-4 HOMOLOG)

2.367
4.504

7.23E-03
2.47E-03
2.03E-03
1.90E-03

732.812
1673.079
2726.689
1051.895

Arabidopsis thaliana
Arabidopsis thaliana
Glycine max
Arabidopsis thaliana

PROTEOLITIC PROTEIN REGULATION
8.09E-04
2.72E-03
1.87E-03

889.343 Populus trichocarpa
1015.216 Pyrus pyrifolia
1072.999 Arabidopsis thaliana

RECEPTORS
1.22E-03
8.65E-03

5346.8 Zea mays
1452.98 Prunus persica

CLUSTER UP(ABA0AUX-)
gene09629
gene13280
gene21177
gene07367
gene18826
gene20494
gene07026
gene05178
gene20299

INTERACTOR OF CONSTITUTIVE ACTIVE ROPS
REMORIN
SMALL G PROTEIN FAMILY PROTEIN
SNRK2 CALCIUM SENSOR
SYNTAXIN
TRANSDUCIN FAMILY PROTEIN / WD-40 REPEAT FAMILY
TRANSDUCIN/WD-40 REPEAT-CONTAINING PROTEIN
TWO-COMPONENT RESPONSE REGULATOR ARR2 ISOFORM X1
WD REPEAT-CONTAINING PROTEIN

8.20
5.65
4.70
2.902
11.14
3.95
6.48
14.57
2.607

5.25E-04
1.03E-03
5.19E-04
3.53E-03
2.10E-04
7.62E-04
4.08E-04
4.32E-04
1.21E-03

gene15486
gene05792
gene16298
gene00913
gene16699
gene16513
gene17121
gene11097
gene11098
gene09806
gene15891
gene19993
gene29765
gene34758
gene30692

AVR9/CF-9 INDUCED KINASE
CBL-INTERACTING PROTEIN KINASE
CYCLIN-DEPENDENT KINASE
HYDROXYETHYLTHIAZOLE KINASE
KINASE
KINASE
LECTIN PROTEIN KINASE
MITOGEN-ACTIVATED PROTEIN KINASE
MITOGEN-ACTIVATED PROTEIN KINASE
PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASE
SERINE/THREONINE PROTEIN KINASE
SERINE/THREONINE-PROTEIN KINASE
SERINE/THREONINE-PROTEIN KINASE
SERINE-THREONINE PROTEIN KINASE
SPHINGOSINE KINASE

27.37
8.68
2.143
2.049
3.769
5.75
7.98
18.19
4.66
5.21
10.73
26.60
3.442
10.66
4.21

3.79E-04
4.08E-04
1.47E-03
4.14E-03
1.37E-03
9.56E-04
5.77E-04
6.97E-04
6.44E-04
9.01E-04
4.17E-04
5.69E-04
1.34E-03
1.34E-04
1.32E-03

gene03647
gene09853
gene16754
gene01188
gene16752
gene03306
gene10171
gene12032
gene32106
gene31702
gene03637
gene19216
gene23984

E3 UBIQUITIN-PROTEIN LIGASE
F-BOX FAMILY PROTEIN
F-BOX FAMILY PROTEIN
F-BOX PROTEIN
F-BOX PROTEIN
POLYUBIQUITIN
RING-H2 FINGER PROTEIN
RING-H2 FINGER PROTEIN
RING-H2 FINGER PROTEIN
RING-H2 FINGER PROTEIN
RING-H2 FINGER PROTEIN
UBIQUITIN
UBIQUITIN

gene13148
gene19496
gene30199

G-TYPE LECTIN S-RECEPTOR-LIKE SERINE/THREONINE KINASE
PROLINE-RICH RECEPTOR-LIKE PROTEIN KINASE
RECEPTOR PROTEIN KINASE

1001.321
1229.809
1163.741
1434.00
3704.822
3314.72
9900.003
2361.17
5690.84

Arabidopsis thaliana
Ricinus communis
Arabidopsis lyrata
Nicoana plumbaginifolia
Glycine max
Arabidopsis thaliana
Arabidopsis thaliana
Prunus mume
Medicago truncatula

6E-126
1E-45
2E-151
1E-175
1E-21
9E-164
3E-135
0.0

NM_129264
XP_002510796
XM_002864690
FJ882981
XM_003554411
NM_130366
NM_102297
XP_8223286

1E-149

XM_003590588

3214.94
1602.459
2370.38
1189.11
825.39
2221.244
1021.565
1113.888
2495.939
726.609
1052.508
6716.811
3815.13
3351.558
1892.259

Nicoana tabacum
Populus trichocarpa
Arabidopsis lyrata
Arabidopsis lyrata
Ricinus communis
Theobroma cacao
Populus nigra
Arabidopsis thaliana
Glycine max
Arabidopsis thaliana
Medicago truncatula
Theobroma cacao
Glycine max
Ricinus communis
Vis vinifera

5E-158
1E-120
0.0
2E-76
2E-26
0.0
0.0
1E-71
1E-94
0.0
0.0
4E-72
4E-135
1E-60
0.0

AY220481
DQ997692
XM_002873402
XM_002883451
XP_002513567
EOY11134
AB030083
NM_116919
XM_003544724
NM_001202923
XM_003604077
EOY02771
XR_137280
XM_002515360
XP_002270943

2E-47
3E-40
3E-70
4E-167
1E-83
0.0
2E-143
2E-144
9E-75
1E-34
8E-32
1E-21
0.0

XP_002283507
XM_002307964
XM_002308681
NM_104435
XP_004304799
L05361
XP_008219359
XP_ 008219359
XP_007210150
XM_003601970
XM_002892803
U84967
XM_003616928

0.0
0.0
0.0

XM_003555349
XM_003547220
XM_003629955

KINASES

PROTEOLITIC PROTEIN REGULATION
4.39
7.80
3.77
2.666
4.18
3.124
3.318
7.98
137.02
3.631
4.36
102.13
3.99

1.51E-03
2.52E-03
2.35E-03
9.90E-04
2.37E-03
3.28E-03
1.25E-03
4.73E-04
4.01E-04
1.19E-03
6.88E-04
4.17E-04
2.31E-03

2827.567
1223.653
2953.936
1194.41
1203.989
4193.87
3108.40
868.89
1200.18
10416.78
1426.00
10609.485
1545.215

Vis vinifera
Populus trichocarpa
Populus trichocarpa
Arabidopsis thaliana
Solanum lycopersicum
Arabidopsis thaliana
Prunus mume
Prunus persica
Prunus persica
Medicago truncatula
Arabidopsis lyrata
Arabidopsis thaliana
Medicago truncatula

RECEPTORS
11.27
3.615
84.48

1.83E-04
5.31E-04
3.83E-04

6464.646 Glycine max
1199.49 Glycine max
1221.866 Medicago truncatula

Reference

Funct Integr Genomics
Table 7

(continued)
Signalling and Regulatory Proteins
GENES

Putave funcon

Ripe fruit receptacles
Fold

p-value

Up regulated

a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA0AUX0)
gene28810
gene04419
gene13353
gene31011
gene24536
gene20449
gene12722
gene11610
gene32677
gene10002
gene28410
gene13840
gene23602
gene11741
gene08498
gene05851

BON1-ASSOCIATED PROTEIN
C2 DOMAIN-CONTAINING FAMILY PROTEIN
C2 DOMAIN-CONTAINING FAMILY PROTEIN
CALMODULIN PROTEIN
CYCLIN
FLOWERING TIME CONTROL PROTEIN FY (WD40 DOMAIN CONTAINING PROTEIN)
PLECKSTRIN HOMOLOGY DOMAIN-CONTAINING FAMILY PROTEIN
PROTEIN PHOSPHATASE 2C
PROTEIN PHOSPHATASE 2C
SPX DOMAIN-CONTAINING PROTEIN
SYNTAXIN
SYNTAXIN
SYNTAXIN
SYNTAXIN
TRANSDUCIN
TRANSDUCIN

3.035
3.401
3.389
3.355
3.073
4.63
2.328
2.302
2.302
2.226
2.195
2.198
2.196
2.287
2.168
3.031

9.25E-04
1.35E-03
1.67E-03
1.51E-03
2.17E-03
1.86E-02
1.48E-03
1.41E-03
2.61E-03
1.14E-03
8.07E-04
1.20E-03
1.07E-03
4.76E-03
7.33E-03
1.29E-03

gene24690
gene14737
gene04334
gene23153
gene20924

DEPHOSPHO-COA KINASE
DEPHOSPHO-COA KINASE
DEPHOSPHO-COA KINASE
PROTEIN KINASE AND PP2C-LIKE DOMAIN-CONTAINING PROTEIN
SERINE/THREONINE/TYROSINE KINASE

2.994
2.986
2.978
2.306
2.234

2.35E-03
3.00E-03
3.05E-03
1.15E-03
2.63E-03

gene00483
gene09018
gene14751
gene17739
gene09190
gene14115
gene11337
gene15211
gene30954
gene21896
gene01044
gene02007
gene15931
gene24708
gene32136
gene23698
gene24090
gene11179
gene24698
gene14941

E3 UBIQUITIN-PROTEIN LIGASE
E3 UBIQUITIN-PROTEIN LIGASE
E3 UBIQUITIN-PROTEIN LIGASE
E3 UBIQUITIN-PROTEIN LIGASE
E3 UBIQUITIN-PROTEIN LIGASE
E3 UBIQUITIN-PROTEIN LIGASE
EID1-LIKE F-BOX PROTEIN
F-BOX PROTEIN FAMILY
F-BOX PROTEIN
F-BOX/RNI PROTEIN
F-BOX/LRR-REPEAT PROTEIN
FRATAXIN, MITOCHONDRIAL PRECURSOR
PRLI-INTERACTING FACTOR
PROTEASOME ACTIVATING PROTEIN
RING FINGER PROTEIN
UBIQUITIN CARRIER PROTEIN
UBIQUITIN EXTENSION PROTEIN
UBIQUITIN FAMILY PROTEIN
UBIQUITIN-CONJUGATING ENZYME FAMILY PROTEIN
UBIQUITIN-SPECIFIC PROTEASE

2.857
2.847
2.845
3.281
2.843
3.566
2.808
2.743
2.703
2.696
2.299
2.255
2.321
2.317
2.182
2.159
2.159
2.158
2.158
2.157

gene03054

PROBABLE RECEPTOR-LIKE PROTEIN KINASE

4.444

1905.616
1347.888
955.542
1639.9
1841.267
828.33
7192.243
8840.02
1119.714
1242.786
775.611
1485.318
829.392
1129.414
994.454
3352.222

Solanum tuberosum
Populus trichocarpa
Populus trichocarpa
Medicago truncatula
Medicago truncatula
Malus domesca
Populus trichocarpa
Hevea brasiliensis
Glycine max
Vis vinifera
Ricinus communis
Populus trichocarpa
Populus trichocarpa
Glycine max
Populus trichocarpa
Arabidopsis lyrata

1E-32
0.0
0.0
1E-42
5E-111
2E-93
0.0
1E-155
2E-171
1E-126
5E-82
2E-115
2E-138
1E-111
1E-170
0.0

XM_006339390
XM_002311329
XM_006376379
XM_003602046
XM_003622816
XP_008375316
XM_002302098
EU827608
XM_003530462
XM_002283854
XM_002521518
XM_002316191
XM_002316205
NM_001254606
XM_002321649
XM_002892822

1764.206
1562.265
3340.466
870.929
7494.025

Arabidopsis thaliana
Medicago truncatula
Medicago truncatula
Medicago truncatula
Populus trichocarpa

7E-26
6E-102
8E-100
0.0
2E-51

NM_179771
XM_003593562
XM_003625853
XM_003590198
XM_006382154

0.0
3E-127
0.0
1E-123
0.0
0.0
2E-122
2E-30
3E-65
5E-174
4E-18
2E-83
5E-94
0.0
8E-18
2E-87
2E-74
0.0
8E-101
0.0

NM_001282242
XM_003615287
XM_003618721
XM_003539490
NM_120366
XP_008223431
XM_002276292
XM_002871233
XM_003624710
NM_100954
NM_117470
XP_008230187
NM_121997
NM_112178
XP_007200789
XM_003608898
XM_002322782
XM_002309840
XM_002309518
XM_002300134

0.0

XP_009376146

KINASES

PROTEOLITIC PROTEIN REGULATION
2.36E-03
8.11E-03
2.19E-03
2.98E-02
1.80E-03
1.25E-03
2.47E-03
8.60E-03
1.77E-03
2.50E-03
1.58E-03
1.14E-03
6.29E-03
1.67E-03
9.45E-03
1.08E-03
2.35E-03
2.73E-03
2.88E-03
5.05E-03

785.12
960.926
1659.639
3346.186
1102.593
3354.01
2500.017
964.801
815.817
1077.042
746.853
1081.93
806.102
7671.219
815.39
4186.952
73022.762
1394.006
15916.151
745.522

Glycine max
Medicago truncatula
Medicago truncatula
Glycine max
Arabidopsis thaliana
Prunus mume
Vis vinifera
Arabidopsis lyrata subsp. lyrata
Medicago truncatula
Arabidopsis thaliana
Arabidopsis thaliana
Prunus mume
Arabidopsis thaliana
Arabidopsis thaliana
Prunus persica
Medicago truncatula
Populus trichocarpa
Populus trichocarpa
Populus trichocarpa
Populus trichocarpa

RECEPTORS
1.14E-03

Guzmán 2012). The E3 ligases bring together a specific target
and the E2 enzyme and thereby coordinate the transfer of
ubiquitin. The ubiquitylated proteins are selectively degraded
by the 26S proteasome. The ubiquitylation is also reverted by
the action of several types of deubiquitinases (DUBs). This
ubiquitylation/deubiquitylation cycle could direct a number of
nonproteolytic events involved in chromatin structure, transcription and vesicle trafficking (Mukhopadhyay and
Riezman 2007). The individual FBX proteins and their cognate
SCF complexes have been connected to almost all facets of
plant physiology and development as sensors of light, control
of cell cycle, gene transcription, chromatin structure, stress responses, plat-pathogen defence, etc. Additionally, SCF complexes also appear to participate as hormone sensors or as component of hormone signaling pathways (auxins, GAs, ethylene,
ABA, JA and stringolactones) (Hua and Vierstra 2011). It has
been proved that SKP1 is involved in abscisic acid signaling
regulating seed germination, stomatal opening and root growth
in Arabidopsis thaliana (Li et al. 2012). SKP1 may positively
regulate ABA signaling by SCF-mediated protein degradation
(Li et al. 2012). In our arrays, we have detected two genes
corresponding to RING-finger proteins and belonging to
Cluster UP(ABA + AUX-) (gene27006 and gene30404), two
genes coding SKP1 proteins (gene18503 and gene18505),
three genes corresponding to E3-ubiquitin ligases (gene10350,
gene16196 and gene31679) and three genes (gene00322,
gene09873 and gene22281) putatively coding different F-box
proteins (Table 7; Table S21). All of these genes were expressed
at very high levels and specifically within the red ripe

34817.81 Pyrus x bretschneideri

receptacle. Similar results were obtained by Chen et al
(2015), who observed a strong up-regulation of a putative
RING-H2 finger E3 ubiquitin ligase and SKP1 genes in harvested strawberry fruits treated with ABA. All these data together suggest that these genes can play an important regulatory
role in the fruit ripening process mediated by ABA.
Similarly, included in Cluster UP(ABA + AUX0),
UP(ABA0AUX-) and UP(ABA0AUX0) appear genes coding
for the SCF complex elements (Table 7; Table S21), suggesting that proteolytic protein regulation throughout
ubiquitination is a complex process under a differential hormonal regulation along development and ripening stages.
Hormone-related genes
A number of plant regulators including auxins, ethylene, gibberellins and ABA have been proposed to control berry development and ripening (Symons et al. 2012; McAtee et al.
2013; Kuhn et al. 2014; Fortes et al. 2015). Our microarray
analysis shed light regarding the physiological role played by
these hormones in these important fruit processes.
Abscisic acid metabolism and signaling
In plants, abscisic acid (ABA) biosynthesis takes place through
the plastid-localized 2-C-methyl-D-erithritol 4-phosphate
(MEP) pathway (Cazzonelli and Pogson 2010). We show the
induction of some genes of this crucial pathway in Cluster
UP(ABA + AUX-) such as phytoene synthase (gene28765),

Funct Integr Genomics

an enzyme generally accepted as being the most important
regulatory enzyme for ABA biosynthesis, beta-carotene hydroxylase (gene13195) neoxanthin synthase (gene10665), 9cis-epoxycarotenoid dioxygenase (FaNCED1) (gene30616)
and other key regulatory enzyme as the short chain dehydrogenase (gene06565) (Table 8). Some of these genes related to the
ABA biosynthesis pathway (FaNECD1, FaPYR1 and FaABI1)
were also up-regulated in harvested strawberry fruits after exogenous ABA application (Chen et al. 2015). We have not seen
any induction of genes specifically related to the MEP pathway
in Cluster UP(ABA + AUX-). However, a 1-deoxy-D-xylulose
5-phosphate synthase (gene20709), a MEP pathway regulatory
enzyme, is present in Cluster UP(ABA0AUX-) (Table 8;
Table S22). The NCED enzyme is the key enzyme in the biosynthesis of ABA. In strawberry receptacles, the silencing of
FaNCED1 expression (gene30616) leads to a reduction of both
the ABA content and the amount of anthocyanin in ripened
fruit receptacles (Jia et al. 2011; Medina-Puche et al. 2014).

These clearly indicate that an ABA biosynthetic pathway is
present in receptacles which is induced along the ripening
and is induced by the endogenous production of ABA. In a
previous work, Symons et al. (2012) showed that there is a
weak but continuous increase in the content of ABA in
receptacles in the large green and white stages. This was
followed by a substantial increase in the content of this
hormone in the pink and red fruits (Symons et al. 2012).
An autocatalytic endogenous production of ABA along the
receptacle ripening could be necessary to explain the huge
increase in ABA production taking place in ripe receptacles. This autocatalytic biosynthesis of ABA may play an
important physiological role in the strawberry fruit ripening process.
Genes of the isoprenoid mevalonate (MVA) pathway as
3-hydroxy-3-methylglutaryl-CoA reductase (gene13004) belong
to Cluster UP(ABA + AUX-), while those coding for a farnesyl
diphosphate synthase (gene01502) or diphosphomevelonate

Table 8 Annotation of differentially regulated hormonal-related genes
sorted into the four up-regulated clusters. Magnitudes of relative
induction to ripe receptacles, p value (≤0.05) and arbitrary units of
intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.) indicates the total

signal intensity for each feature on the microarray platform. Gene ID in
purple indicates specifity of expression in ripe receptacle against to
vegetative tissues

Hormonal-related genes
GENES

Putave funcon

Ripe fruit receptacles
Fold

Up regulated

p-value

a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
AUXINS
gene13601
gene22252
gene28375
gene08921
gene08336
gene15614
gene14862

AUXIN EFFLUX CARRIER COMPONENT
AUXIN-INDUCED PROTEIN
AUXIN-INDUCED PROTEIN
AUXIN-INDUCED PROTEIN
AUXIN-RESPONSIVE PROTEIN
IAA-AMINO ACID HYDROLASE 1
SAUR FAMILY PROTEIN

3.994
17.972
12.131
4.152
2.487
2.799
4.24

6.03E-04
7.54E-04
2.02E-03
1.18E-03
2.01E-03
1.40E-03
1.36E-03

gene11424
gene11421
gene30779

1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE
1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE
ETHYLENE-FORMING-ENZYME DIOXYGENASE

27.618
29.837
39.987

1.14E-03
3.83E-04
3.77E-04

gene31337
gene18930

GA2-OXIDASE
GIBBERELLIN 20 OXIDASE

14.226
6.589

4.67E-04
5.47E-04

1085.289
40122.49
1721.586
17740.012
3174.778
3235.846
3559.66

Populus trichocarpa
Glycine max
Glycine max
Glycine max
Glycine max
Populus trichocarpa
Populus trichocarpa

0.0
0.0
5E-122
1E-158
2E-63
1E-90
4E-45

XM_002323654
XM_003531870
XM_003530787
XM_003525807
XM_003541323
XM_002308417
XM_002310820

1228.672 Ricinus communis
1678.318 Arabidopsis thaliana
51084.429 Prunus armeniaca

8E-159
1E-157
5.00E-86

XM_002514098
NM_127517
U97530

4E-161
1E-95

JF441168
XM_002284938

0.0
0.0
0.0
1E-168
0.0
3E-95
0.0
3E-87
3E-98

EF580921
ADU85829.1
KC967656
GQ338153
JX848734
JN941557
FJ784889
AB192882
FJ789568

7E-94
5E-120

XM_002893011
XM_002281543

ETHYLENE

GIBBERELLINS
7839.832 Pyrus communis
5183.161 Vis vinifera

ISOPRENOIDS, TERPENOIDS AND ABA METABOLISM
gene13004
gene30616
gene13195
gene24676
gene30669
gene10665
gene28765
gene06565
gene32435

3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASE
9-CIS-EPOXYCAROTENOID DIOXYGENASE (FANCED1)
BETA-CAROTENE HYDROXYLASE
LINALOOL SYNTHASE
LINALOOL SYNTHASE (LIS)
NEOXANTHIN SYNTHASE (NSY)
PHYTOENE SYNTHASE PROTEIN
SHORT CHAIN DEHYDROGENASE
SHORT CHAIN DEHYDROGENASE/REDUCTASE

gene04119
gene15184

S-ADENOSYL-L-METHIONINE:JASMONIC ACID CARBOXYL METHYLTRANSFERASE
JASMONATE O-METHYLTRANSFERASE

3.45
3.322
3.33
10.453
11.549
4.345
8.001
3.613
36.45

1.61E-03
2.62E-03
8.10E-04
3.79E-04
5.49E-04
5.37E-04
6.45E-04
1.14E-03
4.01E-04

107.15
203.162

3.73E-04
1.69E-03

8679.84
6688.363
3261.147
7798.971
45913.4
4320.998
17310.031
29043.42
29440.95

Malus x domesca
Fragaria x ananassa
Fragaria x ananassa
Acnidia arguta
Malus x domesca
Malus x domesca
Fragaria x ananassa
Solanum tuberosum
Nandina domesca

Jia et al., 2011

JASMONIC ACID
1193.469 Arabidopsis lyrata
40959.424 Vis vinifera

CLUSTER UP(ABA+AUX0)
gene28201

CYTOKININ-O-GLUCOSYLTRANSFERASE

gene23887
gene23886

MYRCENE SYNTHASE
TERPENE SYNTHASE

4.418
4.446

2.856

1.75E-03

13527.736 Medicago truncatula

gene12478
gene15204
gene05631

12-OXOPHYTODIENOATE REDUCTASE
CYTOKININ OXIDASE
GERMIN-LIKE PROTEIN

4.02
6.70
50.61

gene34980

AUXIN-INDUCED PROTEIN

2.874

8.50E-04

4409.95 Medicago truncatula

gene03935
gene12763

SAUR FAMILY PROTEIN
SAUR FAMILY PROTEIN

4.21
3.337

1.11E-03
3.76E-03

2563.24 Malus domesca
2069.51 Malus domesca

2E-152

XM_003615784

8E-53
2E-140

NM_001281080
JF449453

0.0
0.0
1.00E-75

NM_001246944
XM_002307645
EU116342

5E-152

XM_003626384

4E-89
2E-31

XP_008354757
XP_008376800

ISOPRENOIDS, TERPENOIDS AND ABA METABOLISM
6.76E-04
4.26E-03

2525.63 Vis vinifera
5284.76 Populus trichocarpa

CLUSTER UP(ABA0AUX-)
5.40E-04
1.85E-03
5.66E-04

1511.13 Solanum lycopersicum
1113.102 Populus trichocarpa
1728.046 Chimonanthus praecox

AUXINS

ISOPRENOIDS, TERPENOIDS AND ABA METABOLISM
gene20709
gene05525
gene23657

1-DEOXY-D-XYLULOSE 5-PHOSPHATE SYNTHASE
BETA-AMYRIN SYNTHASE
DIPHOSPHOMEVELONATE DECARBOXYLASE

6.19
217.62
3.146

3.66E-04
9.98E-04
1.84E-03

1773.386 Hevea brasiliensis
1037.386 Malus x domesca
3517.12 Hevea brasiliensis

gene01502

FARNESYL DIPHOSPHATE SYNTHASE

gene05020

GIBBERELLIN 2-OXIDASE

6.34

1.04E-03

5149.236 Panax quinquefolium

543.34

1.79E-03

gene32005
gene15694
gene17480

1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE
1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE
ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR

14.363
14.239
2.802

2.27E-03
1.02E-02
1.61E-02

gene08518

GASA PROTEIN (GASA DOMAIN CONTAINING PROTEIN)

4.349

8.95E-04

0.0
0.0
0.0
0.0

DQ473433
AB055512
AB294695
GQ401664

GIBBERELLINS
1766.437 Populus trichocarpa

3E-127

XM_002301494

0.0
4E-86
8E-59

AY706156
AJ890087
XM_003610878

CLUSTER UP(ABA0AUX0)
ETHYLENE
21539.965 Fragaria x ananassa
1129.594 Prunus domesca
5047.316 Medicago truncatula

GIBBERELLINS
7501.51 Fragaria x ananassa

2E-47

AF039183

3E-159

XM_006347691
XP_008230162

ISOPRENOIDS, TERPENOIDS AND ABA METABOLISM
gene19729
gene23889

PERAKINE REDUCTASE
TERPENE SYNTHASE DOMAIN

2.358
3.106

1.50E-03
2.71E-03

1405.768 Solanum tuberosum
823.04 Prunus mume

0.0

Medina-Escobar. 1997

Funct Integr Genomics

decarboxylase (gene23657) as well as other genes related with
the terpenoid metabolism (Table 8; Table S22) are included in
Cluster UP(ABA0AUX-).
Genes coding enzymes related to the synthesis of aromatic
terpenes, as linalool synthases (genes24676 and gene30669)
are in Cluster UP(ABA + AUX-) (Table 8; Table S22).
However, other genes potentially encoding enzymes of the
terpene metabolism as myrcene and terpene synthase genes
(gene23887 and gene23886, respectively) are included in
Cluster UP(ABA + AUX0). The gene coding for betaamyrin synthase (gene05525) responsible for the biosynthesis of pentacyclic triterpene is included in Cluster
UP(ABA0AUX-) (Table 8; Table S22). All this data
strongly suggest that there is a complex interactive regulatory network between ABA and auxins implied in the regulation of many isoprenoid and ripening-related genes. The
expression of 3-hydroxy-3-methylglutaryl-CoA reductase,
9-cis-epoxycarotenoid dioxygenase, linalool synthase,
myrcene synthase and terpene synthase were fruit specific
and strongly linked to the strawberry fruit ripening.
Recently, many of the molecular mechanisms of ABAassociated signaling components have been identified in
A. thaliana, for instance G-proteins, phospholipase C/D, protein kinases such as receptor-like kinases, SNF1-related kinases (SnRKs), calcineurin B-like protein kinases, calciumdependent protein kinases, mitogen-activated protein kinases,
protein phosphatases type 2C and different classes of transcription factors as MYB, bZIP, Apetala 2 and WRKY (Cutler et al.
2010). In our microarray analysis, we have identified genes
coding for a SnRK2 calcium sensor (gene07367) and two
mitogen-activated protein kinases (gene11097 and gene11098)
within Cluster UP(ABA0AUX-). Additionally, we have identified three genes (gene11610, gene32677 and gene23153)
coding for protein phosphatases type 2C belonging to Cluster
UP(ABA0AUX0) (Table 7; Table S21). The expression of
these genes was not influenced by the endogenous production
of ABA inside the fruit receptacle, suggesting that the function
of these putative signaling proteins is not directly related with
the ABA signaling pathway. Similarly, a great number of transcription factors pertaining to the four ripening-related clusters
have been identified in our microarray (Table 6; Table S20).
These results give us an idea of the complex hormonal regulation of the ABA-associated signaling components that are acting along the ripening of the fruit which still must be clarified.
Auxins
Auxins synthesized by achenes promote the expansion of the
receptacle cells during the early stages of fruit development,
thus determining the final size of the fruit receptacle.
However, the role played for this hormone along receptacle
ripening is really poorly understood. Recently, the auxin content of strawberry fruit along the growth and ripening

processes has been studied (Symons et al. 2012). The levels
of the auxin IAA in plants is modulated by a specific group of
amidohydrolases (ILL2 amidohydrolases) that release the active hormone from auxin–amino acid conjugated storage
forms (Bitto et al. 2009). We have detected in Cluster
UP(ABA + AUX-) two genes coding for an iaa-amino acid
hydrolase (gene15614) and a SAUR protein (gene14862)
whose expression was fruit-specific. Others auxin-related proteins coding genes were over-expressed along fruit ripening
and belong to Cluster UP(ABA + AUX-) (gene22252,
gene28375, gene08921 and gene08336) and Cluster
UP(ABA0AUX-) (gene34980) (Table 8; Table S22). The expression of all of these genes was fruit specific, indicating their
involvement in general auxin-related processes
Ethylene
It is very well known that ethylene is a plant hormone that
regulates the ripening process in climacteric soft fruits
(Cherian et al. 2014; Kumar et al. 2014). However, the physiological role played for this hormone in strawberry fruit ripening, if any, it is poorly known.
Our transcriptomic analysis showed that only a few genes
related with ethylene metabolism were ripening-related. Four
genes encode different 1-aminocyclopropane-1-carboxylate
oxidases (gene11424, gene11421, gene15694 and gene32005).
Two of these genes (gene11424 and gene11421) belong to
Cluster UP(ABA + AUX-), while gene15694 and gene32005
are included in Cluster UP(ABA0AUX0). A gene encoding an
ethylene-forming-enzyme dioxygenase (gene30779) also belongs to Cluster UP(ABA + AUX-) (Table 8; Table S22).
Only the expression of the gene coding the ethylene-forming
enzyme dioxygenase was high in fruit receptacles and fruitspecific. All these data indicate that ethylene does not play a
very relevant role in the strawberry receptacle fruit ripening
process, though a minor role cannot be disclosed as previously
proposed (Trainotti et al. 2001; Merchante et al. 2013).
Gibberellins
In strawberry, little is known about how gibberellins (GAs)
influence the strawberry fruit growth and ripening.
Gibberellin GA3 did not stimulate growth when added to
deachened fruits (Dreher and Poovaiah 1982), although their
exogenous application to strawberry fruits delayed the development of red colour (Martínez et al. 1996). A decrease in the
β-xylosidase protein and mRNA levels was described by
Bustamante et al (2009) after the application of GA3 to strawberry white fruits, and recently, it has been suggested that
gibberellins could be involved in the development and ripening of the fruit receptacle (Csukasi et al. 2011). In this study,
high levels of GA4 in fruit receptacles were found (Csukasi
et al. 2011). However, in a recent report, this latter study has

Funct Integr Genomics

been questioned supporting the proposal that the early 13hydroxylation pathway that produces GA1 is really the key
pathway that renders bioactive GAs in strawberry fruits
(Symons et al. 2012). In this report, only detectable levels of
GA1 were observed while other bioactive GAs as GA3 and
GA4 were found below detection limits. Additionally, a sharp
decrease of both IAA and GA1 levels along fruit ripening was
detected (Symons et al. 2012). These studies suggest that gibberellins could play a dual regulatory role, promoting first the
growth of receptacles in combination with auxins, and a relevant role in the ripening afterwards. Our microarray studies
have shown the induction of two biosynthetic GA genes belonging to Cluster UP(ABA + AUX-) in ripe receptacles.
These genes appear to encode a GA2-oxidase (gene31337),
a catabolic enzyme that inactivates bioactive gibberellins, and
a GA20-oxidase (gene18930), a regulatory enzyme involved
in the gibberellin biosynthesis. Both genes are transcribed at
high levels only in mature receptacles, when ABA levels in
the fruit receptacle are very high (Table 8; Table S22) on the
contrary to the GA20-oxidase gene described by Chen et al.
(2015), whose expression in harvested strawberry fruit is
slightly up-regulated for exogenous IAA but repressed for
exogenous ABA. An increase in the expression of FaGA2ox
along strawberry fruit ripening was previously reported
(Csukasi et al. 2011). Gibberellin homeostasis is maintained
by a balance between their rates of biosynthesis and deactivation. Thus, our results are partially contradictory with those
reported previously (Csukasi et al. 2011), as far as an upregulation of a regulatory gibberellin biosynthetic gene along
the receptacle ripening process was observed. Thus, our microarray study does not support an important role for GAs on
the ripening process.
Transporters
A large number of genes induced in ripen receptacle encode
proteins involved in transport processes (Fig. 3). For instance,
several genes corresponding to ATP-binding cassette (ABC)
and multi-antimicrobial extrusion protein (MATE) transporters are components of Clusters UP(ABA + AUX-),
UP(ABA0AUX-) and UP(ABA0AUX0). Glutathione Stransferases (GSTs) are also included in these three clusters
(Table 9; Table S23).
Two major transport systems have been described in the
tonoplast membrane. The primary transport system is mediated
by ABC transporters, while a secondary transport mechanism is based on the pre-existence of a H+ gradient across
the vacuolar membrane produced by V-, P-ATPases or
vacuolar H + pyrophosphatase (Martinoia et al. 2007;
Verweij et al. 2008).
Several reports have shown that different flavonoids and
phenylpropanoids, as acylated anthocyanin, are transported
inside the vacuole through MATE transporters (Gomez et al.

2009; 2011). GSTs have also been involved in the vacuolar
sequestration of anthocyanins (Conn et al. 2008; Gomez
et al. 2011). In Cluster UP(ABA + AUX-), there are two
genes (gene31672 and gene07028) coding GST and MATE
proteins whose expression is restricted to the receptacles
and very high in ripened fruits (Table 9; Table S23). This
suggests that both genes are related to the transport of
anthocyanins within the vacuole.
Primary and secondary metabolism
Several 2-oxoglutarate-dependent dioxygenases implied
in primary and secondary metabolism are highly
expressed in red ripen receptacles were found in Cluster
UP(ABA + AUX-) (gene23979 and gene31924) and Cluster
UP(ABA0AUX-) (gene11730 and gene31923) (Table 10;
Table S24). None of these orthologous dioxygenases have
been characterized, but its expression patterns clearly suggest
that these may be involved in important metabolic pathways
related to the ripening process. The physiological functions of
these enzymes need be established through further experiments. Three genes (gene23438, gene19242 and gene18910)
of Cluster UP(ABA + AUX-) encode 3-hydroxyisobutyrylCoA hydrolases highly expressed in ripe receptacles
(Table 10; Table S24). In mammals, 3-hydroxyisobutyrylCoA hydrolases are involved in the branched chain amino acid
catabolism. However, in plants, the functions of these enzymes remain unknown. In Arabidopis, mutant chy1 disrupted
in the gene CHY1 enconding a 3-hydroxyisobutyryl-CoA hydrolase, has a blockage in peroxisomal valine catabolism leading to an alteration of the peroxisomal beta-oxidation (Lange
et al. 2004). More recently, it has been showed that the chy
mutant in A. thaliana has a deficiency of benzoic acidcontaining glucosinolates in seeds (Ibdah and Pichersky
2009). The expression patterns of these 3-hydroxyisobutyrylCoA hydrolases and some 2-oxoglutarate-dependent
dioxygenases and a mono oxygenase (gene21507) which can
be potentially involved in glucosynolate metabolism (Zhang
et al. 2015) suggest an active production of these compounds
along the ripening.
Another component of Cluster UP(ABA + AUX-) is
gene24860, a putative trehalose-phosphate synthase that catalyses the production of trehalose 6-phosphate (T6P) from
glucose-6-phosphate and UDP-glucose. It is known that T6P
acts as a sensor of sucrose concentration regulating the activity
of protein kinase SnRK1 that, in turn, stimulates growth by
de-repressing the expression of many different anabolic genes
(Lawlor and Paul 2014). The concentration of T6P increases
substantially with the content of sucrose or with the increment
of sucrose flux to sink organs as fruits (Lawlor and Paul
2014). Along the strawberry fruit ripening, a raise in the concentration of sucrose, glucose and fructose has been reported
(Fait et al. 2008). This result suggests that T6P could play an

Funct Integr Genomics
signal intensity for each feature on the microarray platform. Gene ID in
purple indicates specifity of expression in ripe receptacle against to
vegetative tissues

Table 9 Annotation of differentially regulated transporters-related
genes sorted into the four up-regulated clusters. Magnitudes of relative
induction to ripe receptacles, p value (≤0.05) and arbitrary units of
intensity (a.u.i.) are given. Intensity in blue scale (a.u.i.) indicates the total
Transporters
GENES

Putave funcon

Ripe fruit receptacles

Up
regulated

Fold

p-value

a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
gene00123
gene22891
gene30234
gene21322
gene01994
gene10386
gene09427
gene10387
gene10383
gene31672
gene09426
gene18167
gene07028
gene20514
gene01912
gene30855
gene26473

ABC TRANSPORTER
ABC TRANSPORTER
ABC TRANSPORTER
ADENINE/GUANINE PERMEASE
EQUILIBRATIVE NUCLEOTIDE TRANSPORTER 3
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE-S-TRANSFERASE
MATE EFFLUX DOMAIN-CONTAINING PROTEIN
OLIGOPEPTIDE TRANSPORTER OPT FAMILY
OLIGOPEPTIDE TRANSPORTER OPT FAMILY
SUGAR TRANSPORT PROTEIN
TAMBA BLACK MULTIDRUG RESISTANCE PUMP

7.58
10.379
34.236
7.178
8.871
6.61
698.211
9.558
5.496
1215.977
68.032
19.889
56.292
3.252
2.807
5.445
11.881

4.24E-04
4.10E-04
3.64E-04
5.84E-04
6.25E-04
4.86E-04
1.44E-03
1.02E-03
6.03E-04
8.36E-04
5.91E-04
3.77E-04
4.04E-04
2.51E-03
2.26E-03
4.94E-04
8.29E-04

gene00122
gene08245
gene00933
gene13437

ATP-BINDING CASSETTE TRANSPORT FAMILY PROTEIN
BIDIRECTIONAL SUGAR TRANSPORTER SWEET1
BIDIRECTIONAL SUGAR TRANSPORTER SWEET1
CLATHRIN ADAPTOR COMPLEX SMALL CHAIN FAMILY PROTEIN

9.636
2.114
2.231
2.931

5.87E-04
4.60E-03
1.28E-03
1.34E-03

gene08088
gene08792

MITOCHONDRIAL IMPORT INNER MEMBRANE TRANSLOCASE SUBUNIT TIM44 FAMILY PROTEIN

3.618
2.347

3.71E-03
2.20E-03

gene25969
gene04460
gene23188
gene19956
gene06067
gene28763
gene21219
gene09638
gene11065
gene15073
gene03636
gene03634
gene18485
gene11300
gene06443
gene27784
gene14598
gene18781
gene08357
gene10072
gene15923
gene05814
gene12037

ABC TRANSPORTER
AMINO ACID TRANSPORTER
ANION-TRANSPORTING ATPASE FAMILY PROTEIN
CATIONIC AMINO ACID TRANSPORTER
CYCLIC NUCLEOTIDE-GATED ION CHANNEL
GLUTATHIONE S-TRANSFERASE
GLYCEROL-3-PHOSPHATE TRANSPORTER
IMPORTIN
IMPORTIN
MATE EFFLUX FAMILY PROTEIN
MATE EFFLUX FAMILY PROTEIN
MATE EFFLUX FAMILY PROTEIN
METAL TOLERANCE PROTEIN
MITOCHONDRIAL INNER MEMBRANE TRANSLOCASE
MITOCHONDRIAL PHOSPHATE TRANSPORTER
MITOCHONDRIAL SUBSTRATE CARRIER FAMILY PROTEIN
OLIGOPEPTIDE TRANSPORTER
ORGANIC ANION TRANSPORTER
PLEIOTROPIC DRUG RESISTANCE PROTEIN
POTASSIUM CHANNEL TETRAMERIZATION DOMAIN-CONTAINING PROTEIN
SOLUTE CARRIER
SUGAR TRANSPORTER
TRANSPARENT TESTA 12

gene23477
gene02466
gene28000
gene10994
gene24391
gene24391
gene11125
gene01202
gene08766
gene12262
gene13420
gene24796
gene06384
gene17615
gene12355
gene01833
gene18545
gene01868
gene06304
gene18771
gene31711
gene16819

ABC TRANSPORTER
ABC TRANSPORTER A FAMILY MEMBER 1
AMINO ACID TRANSPORTER
CATION/H(+) ANTIPORTER
COPPER TRANSPORTER
COPPER TRANSPORTER
CRCB PROTEIN
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE-S-TRANSFERASE
MAGNESIUM TRANSPORTER
MALTOSE TRANSPORTER
METAL TRANSPORTER
NUCLEOPROTEIN TPR
PEROXISOMAL MEMBRANE ABC TRANSPORTER FAMILY
RAE1 PROTEIN
RAE1 PROTEIN
SOLUTE CARRIER FAMILY
SUCROSE TRANSPORTER
TRANSLOCON AT THE OUTER MEMBRANE OF CHLOROPLASTS
VACUOLAR AMINO ACID TRANSPORTER
VACUOLAR ATP SYNTHASE SUBUNIT F

1788.617
1055.205
4836.517
739.272
919.54
77511.06
21314.41
5451.908
137548.723
10454.536
4297.895
45008.625
6939.15
1537.641
2260.423
5442.932
735.659

Vis vinifera
Nicoana tabacum
Vis vinifera
Arabidopsis thaliana
Prunus mume
Glycine max
Glycine max
Cicer arienum
Carica papaya
Malus x domesca
Lycopersicon esculentum
Rheum australe
Arabidopsis thaliana
Medicago truncatula
Populus trichocarpa
Arabidopsis thaliana
Glycine max

1E-165
0.0
2E-133
0.0
0.0
7E-62
6E-82
4E-99
1E-113
3E-115
7E-79
2E-114
1E-40
0.0
0.0
0.0
2E-162

XM_002281034
AJ831379
XM_002277136
NM_111933
XP_008230198
NM_001250784
NM_001248057
XM_004488650
AJ000923
JN573600
AY007561
EU931209
NM_124290
XM_003625124
XM_002303648
NM_116436.5
JN316209

Populus trichocarpa
Glycine max
Arabidopsis thaliana
Populus trichocarpa

0
6E-47
3E-115
2E-68

XM_006383944
XP_003523404
NM_101997
XM_002308250

1753.256 Populus trichocarpa
1576.552 Arabidopsis thaliana

4E-169
6E-142

XM_002307607
NM_179737

Arabidopsis thaliana
Populus trichocarpa
Arabidopsis lyrata
Populus trichocarpa
Arabidopsis thaliana
Pyrus communis
Glycine max
Arabidopsis thalian
Glycine max
Arabidopsis thaliana
Arabidopsis lyrata
Vis vinifera
Glycine max
Arabidopsis thaliana
Zea mays
Arabidopsis thaliana
Populus trichocarpa
Arabidopsis lyrata
Populus trichocarpa
Arabidopsis lyrata
Medicago truncatula
Ricinus communis
Pyrus x bretschneideri

0.0
7E-171
0.0
0.0
3E-45
3E-123
0.0
0.0
0.0
0.0
2E-91
0.0
0.0
2E-69
2E-160
1E-77
0.0
9E-159
3E-91
0.0
0.0
0E+00
0.0

NM_114646
XM_002323825
XM_002882617
XM_002303678
NM_124692
DQ901400
XM_003534317
NM_120385
XM_003532937
NM_103646
XM_002890044
XP_002280212
XP_003532918
AY463970
NM_001111372
NM_148200.5
XM_002303648
XM_002884794
XM_002298087
XM_002878690
XM_003596504
XP_002517216
XP_009373462

Arabidopsis thaliana
Prunus mume
Populus trichocarpa
Solanum lycopersicum
Vis vinifera
Vis vinifera
Medicago truncatula
Vis vinifera
Vis vinifera
Ricinus communis
Vis vinifera
Malus x domesca
Medicago truncatula
Ricinus communis
Theobroma cacao
Arabidopsis thaliana
Glycine max
Vis vinifera
Fragaria x ananassa
Arabidopsis thaliana
Vis vinifera
Zea mays

0.0
0.0
7E-173
0
1E-39
1E-39
1E-117
4E-102
1E-70
7E-114
5E-43
8E-171
1E-25
0.0
0.0
0.0
2E-155
5E-179
3E-05
0.0
3E-148
9E-71

NM_001204044
XP_008244242
XM_002311042
XM_004245503
HQ108189
HQ108189
XM_003601034
XM_002263350
XM_002275399
XM_002525018
XM_002282109
DQ648082
XM_003608272
XM_002518775
XM_007047913
NM_106715
NM_001255101
XM_002271889
JX013937
NM_112678
XM_002270872
NM_001156371

CLUSTER UP(ABA+AUX0)

SULFITE EXPORTER TAUE/SAFE FAMILY PROTEIN

6973.567
4009.868
17114.828
3372.606

CLUSTER UP(ABA0AUX-)
8.52
11.36
3.105
31.32
20.58
13.18
3.432
2.698
3.093
12.18
8.41
41.62
6.48
4.46
4.82
6.79
7.43
2.802
9.61
2.861
37.09
6.87
13.72

4.64E-04
5.08E-04
2.58E-03
6.09E-04
1.57E-04
1.04E-03
1.01E-03
4.24E-03
8.26E-04
3.85E-04
5.19E-03
2.05E-03
1.01E-03
3.95E-02
8.58E-04
7.55E-04
4.25E-04
1.94E-03
4.40E-04
1.08E-03
3.82E-04
6.18E-04
3.82E-04

1994.979
4799.353
1144.99
1231.89
1195.681
3014.172
7107.59
910.19
2640.33
3120.928
2051.198
808.352
6562.629
1904.287
746.01
1874.232
1786.545
1423.83
1072.145
929.08
12576.247
1700.711
1327.47

CLUSTER UP(ABA0AUX0)
5.175
2.524
4.427
3.342
4.253
4.253
3.352
2.656
2.654
2.649
2.535
2.532
2.525
2.372
2.001
9.954
56.546
2.228
2.212
2.167
2.025
2.022

important role by regulating many metabolic pathways that
are specific from ripe fruit receptacles and that can be influenced by the endogenous production of ABA. It should be
important to elucidate the physiological role that this compound plays in the ripening process.
Acetolactate synthase (ALS) is an enzyme catalyzing the
first step in the synthesis of the branched chain amino acids
valine, leucine and isoleucine. Two genes encoding putative
ALS small regulatory subunit are induced in ripe receptacles

5.73E-03
1.53E-03
1.65E-03
6.15E-04
1.84E-02
1.84E-02
5.75E-04
1.50E-03
4.75E-04
1.09E-03
4.86E-04
2.01E-03
2.58E-03
1.05E-03
1.13E-03
2.83E-04
1.46E-03
2.52E-03
8.77E-04
2.36E-02
5.72E-04
1.60E-03

1454.141
1072.99
3619.209
1139.307
998.343
998.343
2246.614
5403.135
11405.731
5785.333
911.56
1869.195
1542.737
1113.076
3820.646
3031.32
4379.698
1747.197
1347.417
920.235
6382.371
8532.878

Jia, H. 2013

(gene29713 and gene21929) and are in Cluster UP(ABA +
AUX-) (Table 10; Table S24). These genes could be involved
in the production of components of the fruit aroma. There are
other aroma-related characterized genes that are in Cluster
UP(ABA + AUX-) (Table 2; Table S16; File S1).
S-Adenosylmethionine synthetase catalyses the synthesis of S-adenosylmethionine that, in turn, is a substrate for
the O-methylation of many plant secondary metabolites as
pectins, phenylpropanoids, flavonoids, lipids, proteins,

Funct Integr Genomics
Table 10 Annotation of differentially regulated primary and secondary
metabolism-related genes sorted into the four up-regulated clusters.
Magnitudes of relative induction to ripe receptacles, p value (≤0.05)
and arbitrary units of intensity (a.u.i.) are given. Intensity in blue scale
Primary and Secondary Metabolism
GENES

Putave funcon

(a.u.i.) indicates the total signal intensity for each feature on the
microarray platform. Gene ID in purple indicates specifity of expression
in ripe receptacle against to vegetative tissues

Ripe fruit receptacles
Fold

p-value

Up regulated
a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

Reference

CLUSTER UP(ABA+AUX-)
PRIMARY METABOLISM
gene04580
gene05994
gene26175
gene25718
gene22813
gene13140

6-PHOSPHOGLUCONOLACTONASE
D-GLYCERATE 3-KINASE
ENOLASE
HEXOKINASE
PYRUVATE DEHYDROGENASE
PYRUVATE KINASE

gene27452
gene09899
gene16498
gene01062
gene23979
gene31924
gene03646
gene23438
gene19242
gene18910
gene29713
gene21929
gene26121
gene00616
gene16551
gene22147
gene19737
gene01893
gene08027
gene12578
gene22433
gene11229
gene20643
gene28508
gene22484
gene03985
gene16488
gene28822
gene07324
gene07016
gene34199
gene27856
gene05154
gene17835
gene17836
gene09927
gene32455
gene08940
gene32694
gene24141
gene31205
gene30876
gene11912
gene27838
gene16656
gene28414
gene12969
gene10008
gene21507
gene21033
gene30707
gene14267
gene09003
gene02228
gene11108
gene08702
gene12064
gene00649

2-NITROPROPANE DIOXYGENASE
2-NITROPROPANE DIOXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
3-HYDROXYISOBUTYRYL-COA HYDROLASE
3-HYDROXYISOBUTYRYL-COA HYDROLASE
3-HYDROXYISOBUTYRYL-COA HYDROLASE
ACETOLACTATE SYNTHASE SMALL SUBUNIT
ACETOLACTATE SYNTHASE SMALL SUBUNIT
ADENINE NUCLEOTIDE ALPHA HYDROLASES
ALDO-KETO REDUCTASE
ALDO-KETO REDUCTASE
ALDO-KETO REDUCTASE
ALTERNATIVE OXIDASE 4
AMINOACRYLATE HYDROLASE
ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN
ATPASE FAMILY AAA DOMAIN-CONTAINING PROTEIN
ATP-DEPENDENT DNA HELICASE 2 SUBUNIT
BETA-GALACTOSIDASE
BETA-GLUCOSIDASE
CXE CARBOXYLESTERASE
CYCLO-DOPA 5-O-GLUCOSYLTRANSFERASE
CYCLOPHILIN-LIKE PEPTIDYL-PROLYL CIS-TRANS ISOMERASE
CYTIDINE/DEOXYCYTIDYLATE DEAMINASE
CYTOCHROME C OXIDASE SUBUNIT VIB
CYTOCHROME P450
CYTOCHROME P450
CYTOCHROME P450
DIENELACTONE HYDROLASE
DIHYDROLIPOYL DEHYDROGENASE
DISTACHYON 3-HYDROXYBENZOATE 6-HYDROXYLASE
FAD/NAD(P)-BINDING OXIDOREDUCTASE
FERRIC REDUCTION OXIDASE
GLUTATHIONE PEROXIDASE
GLYCOGENIN DOMAIN-CONTAINING PROTEIN
GLYCOGENIN-LIKE STARCH INITIATION PROTEIN
HISTIDINOL PHOSPHATE PHOSPHATASE
HISTIDYL-TRNA SYNTHETASE
INORGANIC PYROPHOSPHATASE
ISOAMYLASE
ISOMERASE
L-ALLO-THREONINE ALDOLASE
L-ASPARTATE OXIDASE
LYSINE-KETOGLUTARATE REDUCTASE
METHIONINE SULFOXIDE REDUCTASE
MONOOXYGENASE
MULTIPLE INOSITOL POLYPHOSPHATE PHOSPHATASE
NAD(P)H DEHYDROGENASE B2
NAD(P)H:QUINONE OXIDOREDUCTASE
NADP-DEPENDENT MALIC ENZYME
OXYSTEROL-BINDING FAMILY PROTEIN
OXYSTEROL-BINDING FAMILY PROTEIN
PLASMA MEMBRANE H+ ATPASE
PYRIDOXINE/PYRIDOXAMINE 5'-PHOSPHATE OXIDASE
QUINONE OXIDOREDUCTASE

16.628
7.443
2.865
3.159
4.017
2.259

5.05E-04
4.75E-04
1.16E-03
1.12E-03
5.89E-04
4.20E-03

2574.234
12504.224
772.089
1131.712
8536.978
6096.448

Glycine max
Arabidopsis thaliana
Vis vinifera
Eriobotrya japonica
Glycine max
Arabidopsis thaliana

3E-138
4E-175
0.0
0.0
6E-175
0.0

XM_003516359
NM_179581
XM_002274298
JF414121
XM_003533767
NM_124670

13.142
12.848
6.495
5.862
7.136
61.726
5.751
4.742
17.182
125.628
4.588
4.555
34.36
8.169
88.176
14.878
4.583
48.071
2.792
3.713
3.793
49.103
77.276
47.098
6.68
3.266
4.703
2.863
64.066
8.67
15.193
7.079
2.233
7.659
6.921
8.354
5.496
207.743
2.206
2.728
6.326
3.98
6.333
5.105
10.403
66.513
6.519
7.247
191.25
3.135
17.292
3.061
11.371
4
2.462
4.323
7.624
4.465

3.79E-04
4.74E-04
6.96E-04
1.49E-03
4.41E-04
4.41E-04
4.69E-04
5.72E-04
3.78E-04
1.03E-03
1.18E-03
2.04E-03
4.11E-04
5.45E-04
3.66E-04
3.91E-04
2.08E-03
4.03E-04
1.59E-03
3.42E-03
8.02E-04
5.32E-04
4.64E-04
4.65E-04
8.23E-04
1.41E-03
2.65E-03
1.95E-03
5.49E-04
1.04E-03
3.78E-04
6.48E-04
2.31E-03
1.70E-03
1.69E-03
4.73E-04
5.26E-04
8.21E-04
1.34E-03
1.49E-03
7.86E-04
1.87E-03
1.11E-03
5.29E-04
4.94E-04
4.33E-04
4.33E-04
6.20E-04
6.55E-04
7.64E-04
3.83E-04
1.57E-03
6.84E-04
1.37E-03
9.77E-04
7.65E-04
1.63E-03
9.97E-04

8335.241
9265.162
801.75
1233.291
66835.624
4279.458
15418.07
4718.315
54892.377
14135.73
5118.796
2645.553
2911.479
63827.81
27282.14
200929.78
2842.268
6923.693
2574.489
1102.949
780.564
9441.474
4636.072
13047.412
1799.053
3816.298
3792.767
4691.212
2846.156
4727.742
2049.247
1722.855
1849.618
1036.006
1110.972
1293.24
13073.956
1047.09
3517.21
1421.08
8138.59
4898.77
10679.243
1430.92
24302.978
1810.436
16450.421
51846.022
2535.825
4347.381
16333.61
1552.909
5967.093
961.383
1206.215
27517.153
2502.027
1945.12

Solanum lycopersicum
Arabidopsis lyrata
Populus trichocarpa
Populus trichocarpa
Populus trichocarpa
Populus trichocarpa
Medicago truncatula
Vis vinifera
Arabidopsis thaliana
Arabidopsis lyrata
Medicago truncatula
Nicoana plumbaginifolia
Arabidopsis thaliana
Fragaria x ananassa
Cicer arienum
Fragaria x ananassa
Arabidopsis thaliana
Vis vinifera
Medicago truncatula
Vis vinifera
Glycine max
Medicago truncatula
Lotus japonicus
Malus pumila
Mirabilis jalapa
Arabidopsis thaliana
Zea mays
Arabidopsis thaliana
Populus trichocarpa
Citrus sinensis
Arabidopsis thaliana
Arabidopsis thaliana
Vis vinifera
Brachypodium distachyon
Arabidopsis thaliana
Malus domesca
Pisa straotes
Medicago truncatula
Arabidopsis thaliana
Prunus mume
Arabidopsis thaliana
Glycine max
Solanum tuberosum
Medicago truncatula
Glycine max
Arabidopsis thaliana
Gossypium hirsutum
Fragaria x ananassa
Arabidopsis lyrata
Medicago truncatula
Arabidopsis thaliana
Solanum tuberosum
Bean (P.vulgaris)
Vis vinifera
Arabidopsis lyrata
Prunus persica
Solanum lycopersicum
Glycine max

5E-113
2E-156
2E-34
6E-105
1E-98
0.0
1E-128
1E-144
8E-127
5E-111
0.0
2E-42
1E-39
0.0
8E-154
0.0
7E-139
0.0
0.0
0.0
0.0
0.0
1E-143
3E-141
2E-148
1E-57
2E-92
2E-41
1E-59
2E-78
8E-45
5E-42
0.0
6E-82
5E-72
0.0
8E-13
0.0
0.0
0.0
0.0
4E-86
0.0
9E-117
0.0
0.0
0.0
2E-116
4E-90
0.0
0.0
7E-78
0.0
0.0
0.0
0.0
0.0
3E-29

XM_004230521
XM_002866557
XM_002309556
XM_002324800
XM_002298091
XM_002330233
XM_003620308
XM_002264706
NM_128618
XM_002865047
XM_003629897
AJ234901
NM_111199
AF039182
XM_004494635
AY703448
NM_118352
XM_002279862
XM_003624606
XM_002274041
XM_003556532
XM_003630403
EU710846
DQ279907
AB182643
NM_102458
EU974505
NM_125166
XM_002299543
AF426451
NM_148854
NM_113263
XM_002276817
XM_003581183
NM_120614
XP_008373357

0.0

XM_003526736

SECONDARY METABOLISM

gene27475

QUINONE OXIDOREDUCTASE

gene21391

QUINONE OXIDOREDUCTASE

gene18229

RETINOL DEHYDROGENASE-LIKE PROTEIN

gene06906

RIPENING-RELATED P-450 MONOOXYGENASE

gene19545

13.233

EF620783
XM_003608665
NM_106363
XP_008237177
NM_111144.5
XM_003531579
AY132998
XM_003590624
XM_003521724
NM_121480
AF264147
Z69596
XM_002868137
XM_003600923
NM_116741
AB061251
J03825
XM_002283398
XM_002872324
AJ271438
XM_004236102
XM_003531961

5.39E-04

16050.211 Glycine max

5.816

4.69E-04

29626.026 Arabidopsis thaliana

3E-151

22.715

3.80E-04

3363.27 Arabidopsis thaliana

9E-146

AK221446
NM_120332

137.527

1.97E-03

2250.611 Medicago truncatula

1E-69

XM_003589612

3E-105

XM_003594254

2.11E-03

754.642 Medicago truncatula

gene09668

S-ADENOSYLMETHIONINE SYNTHETASE

5.368

7.16E-04

10902.728 Medicago truncatula

0.0

gene19647

S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE

35.764

3.98E-04

9817.183 Arabidopsis thaliana

1.00E-91

gene19469

S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE

36.527

6.24E-04

5063.202 Medicago truncatula

3E-107

XM_003624980

gene12180

S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE

11.05

4.00E-04

16487.483 Arabidopsis thaliana

7E-105

NM_ 121121

gene30813

S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE

6.439

6.13E-04

2258.405 Zea mays

gene12002

SERINE CARBOXYPEPTIDASE PROTEIN

9.123

1.05E-03

1357.057 Pisum savum

0.0

AJ251970

gene26251

S-METHYL-5-THIORIBOSE KINASE

2.287

5.15E-03

1221.528 Arabidopsis thaliana

0.0

NM_103869

gene22780

SNF5-TYPE CHROMATIN-REMODELING COMPLEX PROTEIN

3.963

5.73E-04

7E-115

HM068618

gene24860

RNA (GUANINE-9-)-METHYLTRANSFERASE DOMAIN-CONTAINING PROTEIN

TREHALOSE-PHOSPHATE SYNTHASE

gene09373

U-BOX DOMAIN-CONTAINING PROTEIN (AMIDASE CONTAINING PROTEIN)

gene20126

UDP-ARABINOSE 4-EPIMERASE

gene32119

UDP-GLUCOSE 4-EPIMERASE

7.433

921.443 Glycine max

3E-136

XM_003625634
NM_129701

EU955451

26.578

7.73E-04

1318.367 Arabidopsis thaliana

0.0

2.154
2.105

3.82E-03
3.20E-03

1692.36 Vis vinifera
2407.508 Glycine max

0.0
0.0

XM_003524972

0.0

XM_003610933

2.771

2.83E-03

986.206 Medicago truncatula

gene16762

UDP-GLUCOSE:STEROL 3-O-GLUCOSYLTRANSFERASE

12.073

4.83E-04

862.102 Withania somnifera

gene07097

UNCHARACTERIZED(NADH-UBIQUINONE REDUCTASE COMPLEX 1 MLRQ SUBUNIT)

10.098

3.90E-04

gene21168
gene04078

WD-REPEAT PROTEIN
XYLOSE ISOMERASE

12.423
4.999

1.98E-03
7.00E-04

8473.24 Morus notabilis
1747.342 Ricinus communis
1760.383 Medicago truncatula

gene21964
gene17534
gene06716
gene24970
gene03258

BIFUNCTIONAL MONODEHYDROASCORBATE REDUCTASE AND CARBONIC ANHYDRASE
CXE CARBOXYLESTERASE
CYTOCHROME P450
MANNITOL DEHYDROGENASE
QUINONE REDUCTASE

0.0

NM_106505
XM_002272055

EU342378
XP_010100274

4E-44
0.0
0.0

XM_002510961
XM_003610131

3E-144
1E-112
0.0
1E-174
2E-119

XP_008351630
DQ279909
XM_002319738
XM_003612930
XM_002867470

CLUSTER UP(ABA+AUX0)
SECONDARY METABOLISM
2.913
15.428
45.817
2.525
2.26

1.68E-03
3.80E-04
7.31E-05
1.86E-03
1.28E-03

4567.83
4299.122
3399.351
4289.28
9598.769

Malus domesca
Malus pumila
Populus trichocarpa
Medicago truncatula
Arabidopsis lyrata subsp. lyrata

Agius, F. 2003
Almeida, JR. 2007

Funct Integr Genomics
Table 10

(continued)
Primary and Secondary Metabolism
GENES

Putave funcon

Ripe fruit receptacles
Fold

p-value

Up regulated
a.u.i.

CLOSER homologs in other plant species

e-value

Best Match BlastX

2E-69
7E-168
1E-61
4E-169
2E-124
0.0
2E-149
0.0
0.0
2E-84
6E-156
0.0
3E-44
0.0
9E-45
1E-125
0.0
1E-171
0.0
0.0
0.0
5.00E-95

NM_106063
XP_002330269
XM_002298093
XM_002330233
XM_003619657
AF452450
DQ279906
EOY28772
XM_002307093
XM_002303510
GU590869
XM_002315974
NM_103710
XP_008241785
XP_003591094
NM_001202723
XP_009358973
XP_009351796
XM_003621562
FJ415190
NM_124400
NM_121121

0.0

XP_008227530

0.0
4E-138
0.0
0.0
4E-142
0.0
0.0
0
0.0
0.0
0.0
2E-115
0.0
1E-114
6E-123
0
0.0
0.0
1E-159
0.0
2E-101
1E-156
2E-111
3E-92

XP_008383990
NM_101878
XP_009352311
AHD24944
XM_004140350
XP_008218253
XP_009368303
XM_002268277
XP_008237950
XP_009366842
ABW38332
XM_003554028
XP_008350011
JN573600
XP_009365718
XM_003618131
FJ752239
NM_113698
AB426519
XM_002530324
NM_102067
XP_009345225
XP_008445030
XM_002271457
XM_002533353
NM_124635

Reference

CLUSTER UP(ABA0AUX-)
SECONDARY METABOLISM
gene20258
gene31922
gene11730
gene31923
gene17851
gene19636
gene28530
gene19490
gene06729
gene07140
gene09988
gene29956
gene17572
gene14886
gene28497
gene02043
gene17390
gene00617
gene14916
gene26275
gene09966
gene32447

2-ISOPROPYLMALATE SYNTHASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE
4,5-DOPA DIOXYGENASE EXTRADIOL PROTEIN
ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE
CARBOXYLESTERASE
CORE-2/I-BRANCHING BETA-1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE FAMILY PROTEIN
CYTOCHROME P450
CYTOCHROME P450
CYTOCHROME P450
CYTOCHROME P450
DOLICHYL-PHOSPHATE MANNOSYLTRANSFERASE POLYPEPTIDE
ESTERASE-LYPASE DOMAIN CONTAINING PROTEIN
GLUCOSE AND RIBITOL DEHYDROGENASE
MOLYBDOPTERIN BIOSYNTHESIS PROTEIN
MONOCOPPER OXIDASE
NON-FUNCTIONAL NADPH-DEPENDENT CODEINONE REDUCTASE 2-LIKE
OXIDOREDUCTASE N-TERMINAL DOMAIN CONTAINING PROTEIN
PHOSPHOGLYCERATE DEHYDROGENASE
QUINOLINATE SYNTHASE

gene16110

S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE
SENESCENCE-ASSOCIATED CARBOXYLESTERASE 101 ISOFORM X2

gene10140
gene06823
gene07049
gene30068
gene13960
gene34982
gene27413
gene33290
gene12400
gene12401
gene08033
gene10225
gene27225
gene31672
gene22014
gene25602
gene13709
gene11100
gene01456
gene14712
gene22089
gene17051
gene08799
gene34103

2-ALKENAL REDUCTASE (NADP(+)-DEPENDENT)
2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE SUPERFAMILY PROTEIN
ACYL-COA DEHYDROGENASE DOMAIN CONTAINING PROTEIN
ALCOHOL DEHYDROGENASE
ALDO-KETO REDUCTASE
ALDO-KETO REDUCTASE 1 ISOFORM X1
BETA-UREIDOPROPIONASE
BETA-UREIDOPROPIONASE
BIFUNCTIONAL ASPARTOKINASE/HOMOSERINE DEHYDROGENASE
BIFUNCTIONAL ASPARTOKINASE/HOMOSERINE DEHYDROGENASE
CYTOSOLIC FRUCTOSE-1,6-BISPHOSPHATASE
DIHYDROFOLATE REDUCTASE
GLUTAMINE AMIDOTRANSFERASE (GATASE1)-LIKE
GLUTATHIONE S-TRANSFERASE
GLUTATHIONE S-TRANSFERASE
ISOCITRATE DEHYDROGENASE
MONODEHYDROASCORBATE REDUCTASE
MONODEHYDROASCORBATE REDUCTASE
PHENYLACETALDEHYDE REDUCTASE
PHENYLACETALDEHYDE REDUCTASE
PHOSPHOGLYCERATE MUTASE PROTEIN
PROBABLE 18S RRNA (GUANINE-N(7))-METHYLTRANSFERASE
PROTEIN PEROXIN-4
PYRIDOXAL KINASE

gene17266
gene08633

RNA (GUANINE-9-) METHYLTRANSFERASE DOMAIN CONTAINING PROTEIN
UDP-SUGAR PYROPHOSPHORYLASE

2.449
18.23
14.39
32.47
2.848
7.41
3.559
4.70
355.48
57.82
35.50
8.78
3.078
2.386
12.71
6.17
2.783
41.70
3.419
4.72
17.57
22.70
4.00

3.45E-03
1.63E-03
7.39E-04
3.83E-04
2.59E-03
4.20E-04
9.22E-04
1.31E-03
1.90E-03
3.83E-04
7.16E-04
1.10E-03
1.13E-03
1.47E-03
5.16E-04
2.44E-04
1.37E-03
4.22E-04
1.17E-03
7.52E-03
8.35E-04
5.81E-04
1.10E-03

937.24
2129.666
1624.766
12119.73
17161.69
7605.394
1161.15
733.325
2966.733
3211.294
726.525
1495.191
1931.15
2904.33
772.58
2398.752
2557.98
5519.18
5612.41
4746.82
2923.164
1286.25

Arabidopsis thaliana
Populus trichocarpa
Populus trichocarpa
Populus trichocarpa
Medicago truncatula
Glycine max
Malus pumila
Theobroma cacao
Populus trichocarpa
Populus trichocarpa
Nicoana tabacum
Populus trichocarpa
Arabidopsis thalian
Prunus mume
Medicago truncatula
Arabidopsis thaliana
Pyrus x bretschneideri
Pyrus x bretschneideri
Medicago truncatula
Gossypium hirsutum
Arabidopsis thaliana
Arabidopsis thaliana

2,340.20 Prunus mume

CLUSTER UP(ABA0AUX0)
3.169
8.17
2.551
2.64
4.43
4.689
3.218
3.499
2.353
2.316
3.567
3.416
2.578
1226.608
3
2.573
2.492
2.487
2.347
2.564
4.009
2.33
2.219
2.284

3.83E-03
1.52E-03
1.22E-03
2.24E-03
6.04E-04
3.39E-03
1.17E-03
1.14E-03
1.32E-03
5.69E-04
3.46E-03
7.60E-04
1.25E-03
5.20E-05
3.42E-02
1.57E-03
2.41E-03
1.90E-03
3.57E-03
2.16E-03
7.99E-03
4.52E-03
1.48E-03
1.66E-03

2.246
2.151

1.23E-03
7.79E-03

polysaccharides, polynucleotides, etc. This methylation is fundamental for a large number of important biological functions
(Mata 2007; Hugueney et al. 2009). In our transcriptomic study,
we have detected several genes encoding both types of enzymes
(Table 10; Table S24), indicating that O-methylation process of
many metabolites, as flavonoids and phenylpropanoids, is important in ripe fruits.

Conclusion
Our data show that auxin is the main hormone regulating
the expression of genes implied in the initial development
and growth of the strawberry fruit, and that ABA is regulating the expression of genes implied in the final maturation and ripening. These data also strongly support the
proposal of (Perkins-Veazie 1995) which suggested that a
determined ratio of auxins/ABA is the main regulatory signal that triggers the events required to promote the maturation and ripening stages once the strawberry fruit is already developed. However, it cannot be ruled out that other
hormonal or physiological signals, not yet elucidated, can
influence some aspects of the strawberry fruit growth and
ripening process.

760.79
990.485
909.16
10890.92
819.10
3963.77
23434.35
16455.963
6762.14
7301.02
3544.80
18652.533
1143.48
27385.95
1377.74
7956.769
23782.809
1116.262
5706.181
714.13
1573.448
777.38
23217.95
3636.73

Malus domesca
Arabidopsis thaliana
Pyrus x bretschneideri
Rosa rugosa
Cucumis savus
Prunus mume
Pyrus x bretschneideri
Vis vinifera
Prunus mume
Pyrus x bretschneideri
Fragaria x ananassa
Glycine max
Malus domesca
Malus x domesca
Pyrus x bretschneider
Medicago truncatula
Malus x domesca
Arabidopsis thaliana
Rosa x damascena
Rosa x damascena
Arabidopsis thaliana
Pyrus x bretschneideri
Cucumis melo
Vis vinifera

1658.38 Ricinus communis
959.751 Arabidopsis thaliana

4E-112
0.0

Materials and methods
Plant material
Strawberry plants (Fragaria × ananassa Duch. BCamarosa^,
an octoploid cultivar) were grown under field conditions in
Huelva (S.W. Spain). Fragaria × ananassa fruits were harvested at different developmental stages: small-sized green
fruits (G1, 2–3 g) and red fruits (R, 6–10 g). Vegetative tissues, such as runners, roots, crowns and expanding leaves,
were also harvested. Fragaria × ananassa plants used for hormone treatments were grown in plant chambers at 25 °C,
10,000 lux and 80 % humidity and afterwards maintained in
greenhouses. All analysed tissues were immediately frozen in
liquid nitrogen after harvesting and stored at −80 °C.
Auxin treatment
Achenes of two sets of 50 full-sized green fruits (G3) each were
carefully removed from their receptacles. One set of de-achened
G3 fruits was treated with indole-3-acetic acid (IAA) embedded
in lanolin paste (1 mL) containing 1 mM of auxin IAA plus 1 %
(w/v) dimethyl sulphoxide. The other set of G3 de-achened
fruits (control group) was treated with the same lanonin mixture

Funct Integr Genomics

lacking IAA. Auxin treatments, sample collection and analysis
were performed according to Medina-Puche et al. (2014).
Nordihydroguaiaretic acid treatment
Nordihydroguaiaretic acid (NDGA) is an inhibitor of the NCED
enzyme used to block ABA biosynthesis (Creelman et al. 1992).
Strawberry fruits at mature G3 stages treated with this
compound were used in this study. The treatment was performed in accordance with Medina-Puche et al (2014).
These samples were used for ABA content measurement
and relative expression of genes.
RNA isolation
Total RNA was isolated from three independently isolated and
treated pools of strawberry fruits at different growth and ripening stages and from vegetative tissues, in accordance with
Asif et al (2000). Achenes were always removed from fruits
and only receptacle RNA was extracted and purified. The total
RNA obtained was treated with DNase I (RNase free)
(Invitrogen), following manufacturer’s instructions, and further purified using RNeasy Mini kit (Qiagen). RNA samples
were considered DNA-free when no amplicons corresponding
to the analysed genes were observed using RNA as template
in a standard PCR reaction.
Microarray probe preparation, hybridization
and statistical analysis
For transcriptomic studies, we have used a custom-made
oligo-based (60-mer) Agilent platform containing all
genes published in the strawberry genome project
(http://www.strawberry.org). The EST sequences from
Fragaria × ananassa and Fragaria vesca are 98.6 %
identical (Bombarely et al. 2010; Medina-Puche et al.
2014).
For microarray hybridization, 200 ng of total RNA of each
sample was labeled using the Low Input Quick Amp Labeling
Kit (Agilent Technologies) according to the manufacturer’s
protocol. Six hundred nanograms of Cy3-labeled aRNA was
hybridized on an 8 × 60 K Sure Print G3 custom microarray
(Agilent Technologies). The array was designed using eArray
platform using the Fragaria vesca genome as a reference.
Three biological replicates were used for each experiment.
After hybridization, arrays were washed according to the manufacturer’s protocol. Arrays were scanned with an Agilent
Microarray Scanner. Images and intensity data were obtained
using Agilent Feature Extraction v.10.7 software. After hybridization and array scanning, images of the physical hybridization were visually inspected for artefacts such as scratches,
bubbles and high regional or overall background. Thus, the

non-uniformities in a scanned image were determined as outliers and thus excluded from downstream analyses.
The microarray data were analysed with the software for
gene expression analysis ArrayStar 5 (DNASTAR). Raw expression intensities obtained from each one biological replica
were normalized through the Robust Multichip Average
(RMA) method. Moderated t test and false discovery rate
(FDR) (Benjamini and Hochberg 1995), for multiple testing
corrections, were used with an adjusted p < 0.01 to statistically
identify significant differences. The correlation coefficients (r value) and coefficients of determination (r2) for
the signals obtained in each of the biological replicates and
conditions were calculated by fitting the normalized data to
a linear mode with the lm() function implemented under
the R language.
Treemaps were performed by the Macrofocus TreeMap program, grouping genes by general functions, and representing
the fold change values by a scale of colour taking into account
that any level higher than 30-fold changes is considered saturated and therefore is drawn in the treemap with the same dark
colour. Actual fold change values shown are detailed in the
corresponding tables. There is an interactive version of the
treemap available under request that will allow the examination of the actual genes names and fold change values.
Annotation of sequences from the EST library
Blast2GO v 2.7.2 suite was used for functional annotation of
the EST sequences using BLASTX with an expected value
cutoff of 1.0E-10 (Conesa et al. 2005). Only genes that were
over- and under-expressed more than 2-fold in the microarray
analysis were used. GO data for each sequence was enriched
used ANNEX (Annotation Augmentation) and with information of the InterPro database. Graphics from GO data were
also obtained with this suite.
Gene ID shown in all of the tables and figures are those
reported in the F. vesca Genome Database (http://www.
strawberrygenome.org/).
Validation of microarray data and expression analysis
by quantitative real-time PCR
Expression analyses of genes herein analyzed and studied in all
physiological conditions were performed by quantitative realtime PCR (qRT-PCR) using an iCycler device (BioRad) as
previously described (Benítez-Burraco et al. 2003). Firststrand cDNA was obtained from 2 μg total RNA using the
iScript kit (BioRad), following manufacturer instructions. An
additional table depicts the primer sequences used for quantitative amplification (Table S1). Each reaction was performed by
triplicate, and the corresponding Ct values were normalized
using the Ct value corresponding to an interspacer 26S–18S
strawberry RNA gene (Benítez-Burraco et al. 2003; Raab

Funct Integr Genomics

et al. 2006; Encinas-Villarejo et al. 2009; Cumplido-Laso et al.
2012; Molina-Hidalgo et al. 2013). All these values were then
used to determine the relative increase or decrease of gene
expression in the samples as compared against those in control,
in accordance with Pedersen and Amtssygehus (2001).
Interspacer 26S–18S gene (primers 5′- ACC GTT GAT TCG
CAC AAT TGG TCA TCG -3′ and 5′- TAC TGC GGG TCG
GCA ATC GGA CG -3′) was selected as control gene owing to
its constitutive expression throughout all of the different
tested experimental conditions (Table S2). The efficiency
of each particular qRT-PCR and the melting curves of the
products were also analysed to ensure existence of a single
amplification peak corresponding to a unique molecular
species (Tm of interspacer 26S-18S gene was 91.5 °C).
Acknowledgments This work was supported by a grant from the
Ministerio Español de Ciencia e Innovación (MICINN) (BIO201019322). LMP and RBP are grateful for the award of a Ph.D. fellowship
within the framework of Formación del Personal Universitario (FPU)
program implemented by the Ministerio de Educación y Ciencia (MEC)
(Spain) and a post-doctoral contract from Campus de Excelencia
Internacional Agroalimentario (CEIA3) from University of Córdoba,
respectively.

References
Aharoni A, Keizer LC, Bouwmeester HJ, Sun Z, Alvarez-Huerta M,
Verhoeven HA, Blaas J, van Houwelingen AM, De Vos RC, van
der Voet H, Jansen RC, Guis M, Mol J, Davis RW, Schena M, van
Tunen AJ, O’Connell AP (2000) Identification of the SAAT gene
involved in strawberry flavour biogenesis by use of DNA microarrays. Plant Cell 12:647–662
Aharoni A, Keizer LCP, Van Den Broeck HC, Blanco-Portales R,
Muñoz-Blanco J, Bois G, Smit P, De Vos RCH, O’Connell AP
(2002) Novel insight into vascular, stress, and auxin-dependent
and -independent gene expression programs in strawberry, a nonclimacteric fruit. Plant Physiol 129:1019–1031
Asif M, Dhawan P, Nath P (2000) A simple procedure for the isolation of
high quality RNA from ripening banana fruit. Plant Mol Biol Rep
18:109–115
Benítez-Burraco A, Blanco-Portales R, Redondo-Nevado J, Bellido ML,
Moyano E, Caballero J-L, Muñoz-Blanco J (2003) Cloning and
characterization of two ripening-related strawberry (Fragaria x
ananassa cv. Chandler) pectate lyase genes. J Exp Bot 54:633–645
Benjamini Y, Hochberg H (1995) Controlling the false discovery rate: a
practical and powerful approach to multiple testing. J R Statist Soc B
1:289–300
Bitto E, Bingman CA, Bittova L, Houston NL, Boston RS, Fox BG,
Phillips GN (2009) X-ray structure of ILL2, an auxin-conjugate
amidohydrolase from Arabidopsis thaliana. Proteins 74:61–71
Blanco-Portales R, Medina-Escobar N, López-Ráez JA, González-Reyes
JA, Villalba JM, Moyano E, Caballero JL, Muñoz-Blanco J (2002)
Cloning, expression and immunolocalization pattern of a cinnamyl
alcohol dehydrogenase gene from strawberry (Fragaria x ananassa
cv. Chandler). J Exp Bot 53:1723–1734
Bombarely A, Merchante C, Csukasi F, Cruz-Rus E, Caballero JL,
Medina-Escobar N, Blanco-Portales R, Botella MA, MuñozBlanco J, Sánchez-Sevilla JF, Valpuesta V (2010) Generation and
analysis of ESTs from strawberry (Fragaria xananassa) fruits and

evaluation of their utility in genetic and molecular studies. BMC
Genomics 11:503
Böttcher C, Burbidge CA, Boss PK, Davies C (2013) Interactions between ethylene and auxin are crucial to the control of grape (Vitis
vinifera L.) berry ripening. BMC Plant Biol 13:222
Bustamante CA, Civello PM, Martínez GA (2009) Cloning of the promoter region of β-xylosidase (FaXyl1) gene and effect of plant
growth regulators on the expression of FaXyl1 in strawberry fruit.
Plant Sci 177:49–56
Castillejo C, de la Fuente JI, Iannetta P, Botella MA, Valpuesta V (2004)
Pectin esterase gene family in strawberry fruit: study of FaPE1, a
ripening-specific isoform. J Exp Bot 55:909–918
Cazzonelli CI, Pogson BJ (2010) Source to sink: regulation of carotenoid
biosynthesis in plants. Trends Plant Sci 15:266–274
Chai Y-M, Jia H-F, Li C-L, Dong Q-H, Shen Y-Y (2011) FaPYR1 is
involved in strawberry fruit ripening. J Exp Bot 62:5079–5089
Chen J-Y, Liu D-J, Jiang Y-M, Zhao M-L, Shan W, Kuang J-F, Ahn J-H,
(2011) Molecular Characterization of a Strawberry FaASR Gene in
Relation to Fruit Ripening. PLoS ONE 6(9):e24649
Chen J, Linchun M, Wenjing L, Tiejin Y, Zisheng L (2015) Transcriptome
profiling of postharvest strawberry fruit in response to exogenous
auxin and abscisic acid. Planta. doi:10.1007/s00425-015-2402-5
Cherian S, Figueroa CR, Nair H (2014) ‘Movers and shakers’ in the
regulation of fruit ripening: a cross-dissection of climacteric versus
non-climacteric fruit. J Exp Bot 65:4705–4722
Chervin C, El-Kereamy A, Roustan J-P, Latché A, Lamon J, Bouzayen M
(2004) Ethylene seems required for the berry development and ripening in grape, a non-climacteric fruit. Plant Sci 167:1301–1305
Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005)
Blast2GO: a universal tool for annotation, visualization and analysis
in functional genomics research. Bioinformatics 21:3674–3676
Conn S, Curtin C, Bézier A, Franco C, Zhang W (2008) Purification,
molecular cloning, and characterization of glutathione S-transferases
(GSTs) from pigmented Vitis vinifera L. cell suspension cultures as
putative anthocyanin transport proteins. J Exp Bot 59:3621–3634
Creelman RA, Bell E, Mullet JE (1992) Involvement of a lipoxygenase-like
enzyme in abscisic acid biosynthesis. Plant Physiol 99:1258–1260
Csukasi F, Osorio S, Gutierrez JR, Kitamura J, Giavalisco P, Nakajima M,
Fernie AR, Rathjen JP, Botella MA, Valpuesta V, Medina-Escobar N
(2011) Gibberellin biosynthesis and signalling during development
of the strawberry receptacle. New Phytol 191:376–390
Cumplido-Laso G, Medina-Puche L, Moyano E, Hoffmann T, Sinz Q,
Ring L, Studart-Wittkowski C, Luis Caballero J, Schwab W,
Munoz-Blanco J, Blanco-Portales R (2012) The fruit ripeningrelated gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis. J Exp Bot 63:4275–4290
Cutler SR, Rodríguez PL, Finkelstein RR, Abrams SR (2010) Abscisic
acid: emergence of a core signaling network. Annu Rev Plant Biol
61:651–679
Daminato M, Guzzo F, Casadoro G (2013) A shatterproof-like gene controls ripening in non-climacteric strawberries, and auxin and abscisic
acid antagonistically affect its expression. J Exp Bot 64:3775–3786
Dreher T, Poovaiah B (1982) Changes in auxin content during development in strawberry fruits. In: Hortscience, vol 17. Amer Soc horticultural science 701 North Saint Asaph street, Alexandria, VA
22314-1998, pp 475-475
Encinas-Villarejo S, Maldonado AM, Amil-Ruiz F, de los Santos B,
Romero F, Pliego-Alfaro F, Muñoz-Blanco J, Caballero JL
(2009) Evidence for a positive regulatory role of strawberry
(Fragaria x ananassa) Fa WRKY1 and arabidopsis at
WRKY75 proteins in resistance. J Exp Bot 60:3043–3065
Fait A, Hanhineva K, Beleggia R, Dai N, Rogachev I, Nikiforova VJ,
Fernie AR, Aharoni A (2008) Reconfiguration of the achene and
receptacle metabolic networks during strawberry fruit development.
Plant Physiol 148:730–750

Funct Integr Genomics
Fortes A, Teixeira R, Agudelo-Romero P (2015) Complex interplay of
hormonal signals during grape berry ripening. Molecules 20:9326
Giovannoni J (2001) Molecular biology of fruit maturation and ripening.
Annu Rev Plant Phys 52:725–749
Giovannoni JJ (2004) Genetic regulation of fruit development and ripening. Plant Cell Online 16:S170–S180
Giovannoni JJ (2007) Fruit ripening mutants yield insights into ripening
control. Curr Opin Plant Biol 10:283–289
Gomez C, Terrier N, Torregrosa L, Vialet S, Fournier-Level A, Verriès C,
Souquet J-M, Mazauric J-P, Klein M, Cheynier V, Ageorges A
(2009) Grapevine MATE-type proteins act as vacuolar H + -dependent acylated anthocyanin transporters. Plant Physiol 150:402–415
Gomez C, Conejero G, Torregrosa L, Cheynier V, Terrier N, Ageorges A
(2011) In vivo grapevine anthocyanin transport involves vesiclemediated trafficking and the contribution of anthoMATE transporters and GST. Plant J 67:960–970
Guzmán P (2012) The prolific ATL family of RING-H2 ubiquitin ligases.
Plant Signal Behav 7:1014–1021
Hua Z, Vierstra RD (2011) The cullin-ring ubiquitin-protein ligases.
Annu Rev Plant Biol 62:299–334
Hua C, Linling L, Feng X, Yan W, Honghui Y, Conghua W, Shaobing W,
Zhiqin L, Juan H, Yuping W, Shuiyuan C, Fuliang C (2013)
Expression patterns of an isoflavone reductase-like gene and its
possible roles in secondary metabolism in Ginkgo biloba. Plant
Cell Rep 32:637–650
Hugueney P, Provenzano S, Verriès C, Ferrandino A, Meudec E, Batelli
G, Merdinoglu D, Cheynier V, Schubert A, Ageorges A (2009) A
novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine. Plant Physiol 150:2057–2070
Iannetta PPM, Laarhoven L-J, Medina-Escobar N, James EK, McManus
MT, Davies HV, Harren FJM (2006) Ethylene and carbon dioxide
production by developing strawberries show a correlative pattern
that is indicative of ripening climacteric fruit. Physiol Plantarum
127:247–259
Ibdah M, Pichersky E (2009) Arabidopsis Chy1 null mutants are deficient
in benzoic acid-containing glucosinolates in the seeds. Plant Biol
(Stuttg) 11:574–581
Jia H-F, Chai Y-M, Li C-L, Lu D, Luo J-J, Qin L, Shen Y-Y (2011)
Abscisic acid plays an important role in the regulation of strawberry
fruit ripening. Plant Physiol 157:188–199
Jia H, Wang Y, Sun M, Li B, Han Y, Zhao Y, Li X, Ding N, Li
C, Ji W, Jia W (2013) Sucrose functions as a signal involved
in the regulation of strawberry fruit development and ripening.
New Phytol 198:453–465
Khan M, Xu H, Hepworth SR (2014) Blade-on-petiole genes: setting
boundaries in development and defense. Plant Sci 215–216:157–171
Klee HJ, Giovannoni JJ (2011) Genetics and control of tomato fruit ripening and quality attributes. Annu Rev Genet 45:41–59
Kong Y, Peña MJ, Renna L, Avci U, Pattathil S, Tuomivaara ST, Li X,
Reiter W-D, Brandizzi F, Hahn MG, Darvill AG, York WS, O’Neill
MA (2015) Galactose-depleted xyloglucan is dysfunctional and
leads to dwarfism in arabidopsis. Plant Physiol 167:1296–1306
Koyama K, Sadamatsu K, Goto-Yamamoto N (2010) Abscisic acid stimulated ripening and gene expression in berry skins of the cabernet
sauvignon grape. Funct Integr Genomics 10:367–381
Kuhn N, Guan L, Dai ZW, Wu B-H, Lauvergeat V, Gomès E, Li S-H,
Godoy F, Arce-Johnson P, Delrot S (2014) Berry ripening: recently
heard through the grapevine. J Exp Bot 65:4543–4559
Kumar R, Khurana A, Sharma AK (2014) Role of plant hormones and
their interplay in development and ripening of fleshy fruits. J Exp
Bot 65:4561–4575
Lange PR, Eastmond PJ, Madagan K, Graham IA (2004) An arabidopsis
mutant disrupted in valine catabolism is also compromised in peroxisomal fatty acid beta-oxidation. FEBS Lett 571:147–153

Lawlor DW, Paul MJ (2014) Source/sink interactions underpin crop
yield: the case for trehalose 6-phosphate/SnRK1 in improvement
of wheat. Front Plant Sci 5:418
Lee S, Chung EJ, Joung YH, Choi D (2010) Non-climacteric fruit ripening in pepper: increased transcription of EIL-like genes normally
regulated by ethylene. Funct Integr Genomics 10:135–146
Leshem YY, Pinchasov Y (2000) Non-invasive photoacoustic spectroscopic determination of relative endogenous nitric oxide and ethylene content stoichiometry during the ripening of strawberries
Fragaria anannasa (Duch.) and avocados Persea americana (Mill.).
J Exp Bot 51:1471–1473
Li C, Liu Z, Zhang Q, Wang R, Xiao L, Ma H, Chong K, Xu Y (2012)
SKP1 is involved in abscisic acid signalling to regulate seed germination, stomatal opening and root growth in Arabidopsis thaliana.
Plant Cell Environ 35:952–965
Manríquez D, El-Sharkawy I, Flores FB, El-Yahyaoui F, Regad F,
Bouzayen M, Latché A, Pech J-C (2006) Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression
and distinct biochemical characteristics. Plant Mol Biol 61:675–685
Martínez GA, Chaves AR, Añón MC (1996) Effect of exogenous application of gibberellic acid on colour change and phenylalanine ammonia-lyase, chlorophyllase, and peroxidase activities during ripening of strawberry fruit (Fragaria x ananassa Duch.). J Plant Growth
Regul 15:139–146
Martinoia E, Maeshima M, Neuhaus HE (2007) Vacuolar transporters and
their essential role in plant metabolism. J Exp Bot 58:83–102
Mata R (2007) Flavonoids, Chemistry, Biochemistry and Applications
By Ø. M. Andersen (University of Bergen) and K. R. Markham
(Industrial Research Ltd.). CRC Press/Taylor & Francis, Boca
Raton. 2006. xiv + 1237 pp. 7 × 101/4 in. $249.95. ISBN 0-84932021-6. J Nat Prod 70: 140-140
McAtee P, Karim S, Schaffer R, David K (2013) A dynamic interplay
between phytohormones is required for fruit development, maturation, and ripening. Front Plant Sci 4:79
Medina-Escobar N, Cárdenas J, Moyano E, Caballero JL, Muñoz-Blanco
J (1997) Cloning, molecular characterization and expression pattern
of a strawberry ripening-specific cDNA with sequence homology to
pectate lyase from higher plants. Plant Mol Biol 34:867–877
Medina-Puche L, Cumplido-Laso G, Amil-Ruiz F, Hoffmann T, Ring L,
Rodríguez-Franco A, Caballero JL, Schwab W, Muñoz-Blanco J,
Blanco-Portales R (2014) MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of
Fragaria × ananassa fruits. J Exp Bot 65:401–417
Medina-Puche L, Molina-Hidalgo FJ, Boersma M, Schuurink RC,
López-Vidriero I, Solano R, Franco-Zorrilla J-M, Caballero JL,
Blanco-Portales R, Muñoz-Blanco J (2015) An R2R3-MYB transcription factor regulates eugenol production in ripe strawberry fruit
receptacles. Plant Physiol 168:598–614
Merchante C, Vallarino JG, Osorio S, Aragüez I, Villarreal N, Ariza MT,
Martínez GA, Medina-Escobar N, Civello MP, Fernie AR, Botella
MA, Valpuesta V (2013) Ethylene is involved in strawberry fruit
ripening in an organ-specific manner. J Exp Bot 64:4421–4439
Molina-Hidalgo FJ, Franco AR, Villatoro C, Medina-Puche L, Mercado
JA, Hidalgo MA, Monfort A, Luis Caballero J, Munoz-Blanco J,
Blanco-Portales R (2013) The strawberry (Fragariaananassa) fruitspecific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes
an enzyme involved in the degradation of cell-wall middle lamellae.
J Exp Bot 64:1471–1483
Molina-Hidalgo FJ, Medina-Puche L, Gelis S, Ramos J, Sabir F, Soveral
G, Prista C, Iglesias-Fernández R, Caballero JL, Muñoz-Blanco J,
Blanco-Portales R (2015) Functional characterization of FaNIP1;1
gene, a ripening-related and receptacle-specific aquaporin in strawberry fruit. Plant Sci 238:198–211
Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions
of ubiquitin in endocytosis and signaling. Science 315:201–205

Funct Integr Genomics
Opazo MC, Lizana R, Pimentel P, Herrera R, Moya-León MA (2013)
Changes in the mRNA abundance of FcXTH1 and FcXTH2 promoted by hormonal treatments of Fragaria chiloensis fruit.
Postharvest Biol Tec 77:28–34
Pedersen S, Amtssygehus A (2001) Multiplex relative gene expression
analysis by real-time RT-PCR using the iCycler iQ™ detection system. BioRadiations (BioRad) 107:10–11
Perkins-Veazie P (1995) Growth and ripening of strawberry fruit. Hortic
Rev 17:267–297
Perkins-Veazie PM, Huber DJ, Brecht JK (1996) In vitro growth and
ripening of strawberry fruit in the presence of ACC, STS or propylene. Ann Appl Biol 128:105–116
Pillet J, Yu H-W, Chambers AH, Whitaker VM, Folta KM (2015)
Identification of candidate flavonoid pathway genes using transcriptome correlation network analysis in ripe strawberry
(Fragaria × ananassa) fruits. J Exp Bot 66:4455–4467
Raab T, López-Ráez JA, Klein D, Caballero JL, Moyano E, Schwab W,
Muñoz-Blanco J (2006) FaQR, required for the biosynthesis of the
strawberry flavour compound 4-hydroxy-2,5-dimethyl-3(2H)furanone, encodes an enone oxidoreductase. Plant Cell 18:1023–1037
Redondo-Nevado J, Moyano E, Medina-Escobar N, Caballero JL,
Muñoz-Blanco J (2001) A fruit-specific and developmentally regulated endopolygalacturonase gene from strawberry (Fragaria x
ananassa cv. Chandler). J Exp Bot 52:1941–1945
Rodríguez L, Gonzalez-Guzman M, Diaz M, Rodrígues A, IzquierdoGarcia AC, Peirats-Llobet M, Fernandez MA, Antoni R, Fernandez
D, Marquez JA, Mulet JM, Albert A, Rodríguez PL (2014) C2domain abscisic acid-related proteins mediate the interaction of
PYR/PYL/RCAR abscisic acid receptors with the plasma membrane
and regulate abscisic acid sensitivity in arabidopsis. Plant Cell 26:
4802–4820
Salvatierra A, Pimentel P, Moya-León MA, Herrera R (2013) Increased
accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene. Phytochemistry 90:25–36
Schwab W, Davidovich-Rikanati R, Lewinsohn E (2008) Biosynthesis of
plant-derived flavour compounds. Plant J 54:712–732
Seki M, Ishida J, Narusaka M, Fujita M, Nanjo T, Umezawa T, Kamiya
A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K,
Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y,
Shinozaki K (2002) Funct Integr Genomics 2:282–291
Showalter AM, Keppler B, Lichtenberg J, Gu D, Welch LR (2010) A
bioinformatics approach to the identification, classification, and
analysis of hydroxyproline-rich glycoproteins. Plant Physiol
153:485–513

Symons GM, Davies C, Shavrukov Y, Dry IB, Reid JB, Thomas MR
(2006) Grapes on steroids. Brassinosteroids are involved in grape
berry ripening. Plant Physiol 140:150–158
Symons GM, Chua Y-J, Ross JJ, Quittenden LJ, Davies NW, Reid JB
(2012) Hormonal changes during non-climacteric ripening in strawberry. J Exp Bot 63:4741–4750
Trainotti L, Spolaore S, Pavanello A, Baldan B, Casadoro G (1999) A
novel E-type endo-β-1,4-glucanase with a putative cellulosebinding domain is highly expressed in ripening strawberry fruits.
Plant Mol Biol 40:323–332
Trainotti L, Spinello R, Piovan A, Spolaore S, Casadoro G (2001) betaGalactosidases with a lectin-like domain are expressed in strawberry. J Exp Bot 52:1635–1645
Verweij W, Spelt C, Di Sansebastiano G-P, Vermeer J, Reale L,
Ferranti F, Koes R, Quattrocchio F (2008) An H+ P-ATPase
on the tonoplast determines vacuolar pH and flower colour.
Nat Cell Biol 10:1456–1462
Villarreal NM, Bustamante CA, Civello PM, Martínez GA (2010) Effect
of ethylene and 1-MCP treatments on strawberry fruit ripening. J Sci
Food Agr 90:683–689
Waters DLE, Holton TA, Ablett EM, Lee LS, Henry RJ (2005) cDNA
microarray analysis of developing grape (Vitis vinifera cv. Shiraz)
berry skin. Funct Integr Genomics 5:40–58
Wechter WP, Levi A, Harris KR, Davis AR, Fei Z, Katzir N, Giovannoni
JJ, Salman-Minkov A, Hernandez A, Thimmapuram J, Tadmor Y,
Portnoy V, Trebitsh T (2008) Gene expression in developing watermelon fruit. BMC Genomics 9:275
Wurmbach E (2009) Real-time PCR: current technology and applications, Chapter 10 validation of array data. Caister Academic Press,
London
Xu P, Cai W (2014) RAN1 is involved in plant cold resistance and development in rice (Oryza sativa). J Exp Bot 65:3277–3287
Zang A, Xu X, Neill S, Cai W (2010) Overexpression of OsRAN2 in rice
and Arabidopsis renders transgenic plants hypersensitive to salinity
and osmotic stress. J Exp Bot 61:777–789
Zhang D, Aravind L (2010) Identification of novel families and classification of the C2 domain superfamily elucidate the origin and
evolution of membrane targeting activities in eukaryotes. Gene
469:18–30
Zhang Y, Li B, Huai D, Zhou Y, Kliebenstein DJ (2015) The conserved
transcription factors, MYB115 and MYB118, control expression of
the newly evolved benzoyloxy glucosinolate pathway in arabidopsis
thaliana. Front Plant Sci 6:343