Scientia Horticulturae 246 (2019) 57–67

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Arabidopsis EOD3 homologue PaCYP78A6 affects fruit size and is involved in T

sweet cherry (Prunus avium L.) fruit ripening
Xiliang Qi, Congli Liu, Lulu Song, Ming Li
⁎

Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, Henan Province, 450009, PR China

ARTICLE INFO ABSTRACT

Keywords: Sweet cherry (Prunus avium L.) is an important commercial fruit crop cultivated in temperate areas. Fruit size is
Prunus avium an influential agronomical trait in the modern horticultural breeding of sweet cherry because it directly affects
Fruit size the plant’s economic value. However, the genetic mechanisms underlying the control of fruit size are largely
PaCYP78A6 unknown. Here, we isolated and functionally characterized the sweet cherry cytochrome P450 gene CYP78A6,
Virus-induced gene silencing (VIGS)
Arabidopsis EOD3/CYP78A6 homologue gene. Expression profiles showed that PaCYP78A6 was highly expressed
Fruit ripening
in flowers and fruits. Silencing PaCYP78A6 using tobacco rattle virus-induced gene silencing (TRV-VIGS) reduced
fruit size by decreasing mesocarp cell volume and expansion during fruit growth and development. In contrast,
the overexpression of PaCYP78A6 in Arabidopsis resulted in increased silique and seed sizes, and PaCYP78A6 can
recover the phenotype of cyp78a6 mutant. Silencing of PaCYP78A6 delayed fruit ripening and downregulated
the ripening-related genes expressed in sweet cherry fruit. Moreover, PaCYP78A6 acts redundantly with
PaCYP78A9 to effect fruit size. Thus, PaCYP78A6 appears to regulate the fruit size of sweet cherry during fruit
growth and development and is involved in fruit ripening. This work provides new insights into the molecular
mechanisms of a gene affecting fruit development and ripening in P. avium.

1. Introduction such as higher germination rates, higher nutrient resources, stronger

stress-tolerance capabilities, and higher market prices (Westoby et al.,
Sweet cherry belongs to the P. genus and is an important horti- 2002; Moles et al., 2005). Increased sizes of plant reproductive organs,
cultural crop cultivated in temperate areas. It supplies nutraceutical seeds, and fruits are important quality selection traits and represent
properties, antioxidant activities, and some important natural health domestication and breeding goals of modern food and horticultural
functional components for humans worldwide and has notable eco- crops (Chakrabarti et al., 2013; Shomura et al., 2008; Zhang and
nomic value (Li et al., 2010). Large fruit, an important quality selection Whiting, 2011; Wang et al., 2012).
trait in sweet cherry during domestication and modern horticultural Plant reproductive organ growth and development is a critical,
crop breeding, demand a higher market price (Zhang et al., 2010; complex, and complicated process in plants, which mainly contains two
Whiting et al., 2006). Fruit size is regulated by multiple genetic loci in growth programs, those of the pericarp (exocarp, mesocarp, and en-
sweet cherry and other horticultural fruit trees (Whiting et al., 2006). docarp) and seed (embryo, endosperm, and maternal integument)
However, to date, our understanding of the molecular mechanisms (Tanksley, 2004; Prasad and Ambrose, 2010). Pericarp cell expansion
regulating fruit size in sweet cherry and other horticultural fruit trees is and the division of fleshy fruit are complex processes that involve in-
still quite limited, and few genes related to the molecular mechanisms teractions of numerous endogenous and environmental factors, which
have been identified. Therefore, the isolation, identification, and ultimately affect fruit size (Gillaspy et al., 1993; Taylor and Cowan,
characterization of genes associated with fruit size, and the corre- 2001; Harada et al., 2005; Olmstead et al., 2007; Zhang and Whiting,
sponding molecular mechanisms, are required for changing sweet 2011). The coordinated growth and development of the embryo, en-
cherry fruit size. dosperm, and maternal integument finally determines the seed size
Plant organ size is an important agronomical trait that significantly (Horiguchi et al., 2006; Li and Li, 2015, 2016; Si et al., 2016). In recent
affects crop yield and economic value (Chakrabarti et al., 2013). Larger years, because of the importance of plant reproductive organ size, nu-
plant organs lead to increased agricultural production and benefits, merous researchers have focused on analyzing the molecular

⁎
Corresponding author at: Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, 28 Gangwan Road, Zhengzhou, Henan Province,
450009, PR China.
E-mail addresses: [email protected] (X. Qi), [email protected] (C. Liu), [email protected] (L. Song), [email protected] (M. Li).

https://doi.org/10.1016/j.scienta.2018.10.041
Received 29 August 2018; Received in revised form 16 October 2018; Accepted 18 October 2018
Available online 30 October 2018
0304-4238/ © 2018 Elsevier B.V. All rights reserved.
X. Qi et al. Scientia Horticulturae 246 (2019) 57–67

mechanisms that determine final plant reproductive organ size at the molecular mechanisms responsible for sweet cherry fruit size, which
genomic, transcript, protein, and metabolite levels (Tanksley, 2004; can be used to increase yield.
Devoghalaere et al., 2012; Colle et al., 2017; Mu et al., 2017; Wong
et al., 2016). The size of a plant reproductive organ is influenced by 2. Materials and methods
multiple factors, including genetic and environmental factors
(Horiguchi et al., 2006; Nitsch et al., 2012; Fang et al., 2012; Yao et al., 2.1. Plant materials
2015; Qi et al., 2017). For instance, AUXINRESPONSE FACTOR2, DA1/
Big Brother, and DA1-related proteins regulate seed growth by re- Two P. avium cultivars having contrasting fruit sizes were used in
stricting cell proliferation in the maternal integuments (Jofuku et al., this study. A wild forest cherry of northern European origin, ‘Mazzard’,
2005; Li et al., 2008; Xia et al., 2013). APETALA 2 (AP2) controls seed possesses a very small fruit (∼2 g) and a landrace sweet cherry,
size by limiting cell expansion in the maternal integuments (Ohto et al., ‘Chunlu’, weighs more than 8 g. They were grown in the resource
2005). Atypical heterotrimeric G-protein gamma-subunit 3 positively garden of the National Fruit Tree Germplasm Repository, Zhengzhou
regulates seed growth by increasing maternal cell proliferation (Roy Fruit Research Institute, Chinese Academy of Agricultural Sciences
Choudhury et al., 2014). FRUITFULL (FUL) and AUXINRESPONSE (Zhengzhou, China). Arabidopsis thaliana plants were grown in a
FACTOR8 control floral organ development and organ size by pro- greenhouse at 20–22 °C, with 60%–75% relative humidity, and a 16 h/
moting microRNA172 (miR172) expression, which inhibits the trans- 8 h light/dark cycle.
lation of AP2 (Yao et al., 2015; Ripoll et al., 2015).
Cytochrome P450 monooxygenases (CYPs) are a large and highly 2.2. PaCYP78A6 gene isolation and phylogenetic analysis
conserved group of enzymes in plants that are involved in multiple
metabolic pathways, including phenylpropanoid, terpenoid, and alka- Total RNAs from cherry fruit were extracted with an EASYspin Plant
loid pathways. They play important roles in plant growth and devel- RNA rapid extraction kit according to the manufacturer’s instructions
opment, as well as defense responses. The CYP78A subfamily of genes (Yuanpinghao Bio, Tianjin, China). The purified total RNAs were used
affect the processes of organ growth and development in Arabidopsis, as the templates for reverse transcription PCR (RT-PCR) using the
rice, wheat, tomato, and soybean (Chakrabarti et al., 2013; Nagasawa FastQuant RT (With gDNase) kit (Tiangen Bio, Beijing, China). A gene
et al., 2013; Ito and Meyerowitz, 2000; Adamski et al., 2009; Sotelo- (Pav_sc0000652.1_g570.1.mk) highly homologous to Arabidopsis
Silveira et al., 2013). KLUH/CYP78A5 controls seed size by promoting AtCYP78A6 positioned at chromosome 5 was found using a BLAST al-
integument cell proliferation in Arabidopsis, tomato, wheat, and soy- gorithm-based search against the sweet cherry genomics library
bean (Chakrabarti et al., 2013; Adamski et al., 2009; Zhao et al., 2016; (http://cherry.kazusa.or.jp/map.html) (Shirasawa et al., 2017), and
Ma et al., 2016). Similarly, CYP78A3 in wheat, and CYP78A6 and was amplified using specific primers (PaCYP78A6-F and PaCYP78A6-
CYP78A9 in Arabidopsis, influence seed size by promoting embryo in- R). The amino acid sequences of PaCYP78A6 were aligned with other
tegument cell proliferation during seed growth and development (Fang CYP78 A family proteins from Triticum urartu, A. thaliana, and Oryza
et al., 2012; Ito and Meyerowitz, 2000; Sotelo-Silveira et al., 2013; Ma sativa, and the completed genome-sequenced fruit tree, and a phylo-
et al., 2015). In rice, CYP78A11 and CYP78A13 affect seed size by genetic tree was constructed using the Neighbor-Joining method with
regulating the balance between the embryo and endosperm (Miyoshi 500 bootstrap replicates in MEGA 6.0 software.
et al., 2004; Nagasawa et al., 2013; Xu et al., 2015). In sweet cherry,
only PaCYP78A9 have been identified to regulate fruit size (Qi et al., 2.3. Vector construction
2017). Further studies confirmed that CYP78A9 could be a core enzyme
by participating in the conversion of dihydrokaempferol to dihy- To generate a PaCYP78A6-overexpression vector, the full-length
droquercetin in the flavonoid biosynthesis pathway to control seed size coding sequence of PaCYP78A6 was amplified with the primers
in Arabidopsis (Sotelo-Silveira et al., 2013). While CYP78A6 on seed size PaCYP78A6-e-F and PaCYP78A6-e-R, including 17-bp-long overhangs
might be the production of a signal that promotes seed growth, not identical to the corresponding pBI121 sequence digested with XbaI or
linked to its effect on organ size (Fang et al., 2012). These studies in- BamHI, by PCR and inserted into the pBI121 vector digested with XbaI
dicated that these two genes might affect the different metabolite to and BamHI by In-Fusion cloning to generate the p35S::PaCYP78A6
control seed size. However, the roles of CYP78A6 member, Arabidopsis plasmid as described previously (Qi et al., 2017; Frandsen et al., 2012).
EOD3 homologue and a closely homolog PaCYP78A9, in controlling For PaCYP78A6 down-regulation, a TRV-VIGS vector was constructed.
organ growth and development have not been recognized in sweet A 325-bp fragment of PaCYP78A6 was amplified with the primers Pa-
cherry (Prunus avium) or other fruit trees. CYP78A6-RNAi-F and PaCYP78A6-RNAi-R by PCR and was cloned into
Here, we isolated PaCYP78A6 from ‘Chunlu’ sweet cherry, an or- the vector pTRV2 to generate pTRV2-PaCYP78A6.
tholog of Arabidopsis EOD3/CYP78A6, and that encodes sweet cherry
CYP78A6 and can rescue the phenotype of the Arabidopsis deletion 2.4. Arabidopsis transformation and TRV-Mediated gene silencing in cherry
mutant cyp78a6, while PaCYP78A9 not can rescue the defective phe- fruit
notype of mutant cyp78a6. Then we characterized PaCYP78A6 function
in the regulation of fruit size during fruit growth and development The overexpression plasmid p35S::PaCYP78A6 was transformed into
using an RNA interference (RNAi) approach. Silencing the PaCYP78A6 wild-type A. thaliana ecotype Columbia plants using the Agrobacterium
gene using tobacco rattle virus-induced gene silencing (TRV-VIGS) in tumefaciens-mediated floral-dip method. Meanwhile, the overexpression
sweet cherry fruit resulted in a reduced fruit size owing to decreased plasmids p35S::PaCYP78A6 and p35S::PaCYP78A9 were introduced
mesocarp cell volume and expansion during fruit growth and devel- into the A. thaliana homozygous cyp78a6 mutants, respectively. The
opment. In contrast, the overexpression of PaCYP78A6 in Arabidopsis empty plasmid pBI121 was introduced into wild-type A. thaliana plants
produced enlarged siliques and seeds, and PaCYP78A6 can recover the as control (col lines). The seeds of transgenic plants were selected on
phenotype of cyp78a6 mutant. In addition, silencing of PaCYP78A6 Murashige and Skoog medium supplemented with 50 mg/L kanamycin
delayed fruit ripening and downregulated the ripening-related genes and subsequently grown to form a homozygous T3 generation of
expressed in sweet cherry fruit. Moreover, PaCYP78A6 and its homolog transgenic PaCYP78A6 lines. The A. thaliana transformation and
PaCYP78A9 might play functional redundancy roles in regulating fruit screenings were performed in triplicate.
size in sweet cherry. In summary, this study provides direct evidence TRV-mediated PaCYP78A6 silencing in cherry fruits were per-
that PaCYP78A6 positively effects fruit size and involves in sweet formed with A. tumefaciens-mediated transformation as previously re-
cherry fruit ripening, and this facilitates our understanding of the ported (Qi et al., 2017; Frandsen et al., 2012; Fu et al., 2005; Shen et al.,

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Fig. 1. Phylogenetic analysis involving in

Prunus avium CYP78A6 protein and CYP78 A
orthologs from other plant species. The phylo-
genetic tree was constructed using the
Neighbor-Joining method in MEGA 6.0 with
500 bootstrap replicates. The accession num-
bers of the genes are as follows: JrCYP78A5
(Juglans regia, XM_018969352.1), ZjCYP78A5
(Ziziphus jujuba, XM_016027740.1),
VvCYP78A5 (Vitis vinifera, XM_002265274.3),
GcCYP78A3 (Glycine max, NM_001317595.1),
AtCYP78A8 (Arabidopsis thaliana, NM_100001.
2), StCYP78A3 (Solanum tuberosum, XM_
006339692.2), VvCYP78A3 (V. vinifera, XM_
002266457.2), SlCYP78A5 (S. lycopersicum,
XM_004236016.3), PpCYP78A5 (Prunus per-
sica, XM_007209835.2), AtCYP78A10 (A.
thaliana, NM_106071.1), CsCYP78A5 (Citrus
sinensis, XM_006477669.1), GcCYP78A72 (G.
max, XM_003554632.3), PbCYP78A5
(Pyrus × bretschneideri, XM_009358148.2),
AtCYP78A5 (A. thaliana, NM_101240.4),
TaCYP78A3 (Triticum aestivum, KP768392.1),
TaCYP78A5 (T. aestivum, BAK05332.1),
AtCYP78A6 (A. thaliana, NM_130231.4),
ZjCYP78A9 (Z. jujuba, XM_016033300.1),
MdCYP78A9 (Malus × domestica, XM_
008345221.2), PpCYP78A6 (P. persica,
XM_007210508.2), PaCYP78A9 (P. avium,
Pav_sc0000586.1_g760.1.mk), PmCYP78A9
(P. mume, XM_008234862.2), PaCYP78A6
(P. avium, Pav_sc0000652.1_g570.1.mk),
OsCYP78A11 (Oryza sativa, XM_015757991.1),
OSCYP78A13 (O. sativa, XM_015790749.1),
FvCYP78A5 (Fragaria vesca, XM_004298317.2),
PmCYP78A5 (P. mume, XM_008241890.2),
AtCYP78A7 (A. thaliana, NM_121034.2),
MtCYP78A29 (Medicago truncatula, DQ335794.
1), TcCYP78A9 (Theobroma cacao, XM_
007051407.2), AtCYP78A9 (A. thaliana, NM_
116053.3), NnCYP78A9 (Nelumbo nucifera,
XM_010269146.1), TcCYP78A3 (T. cacao, XM_
018117954.1). The scale bar represents 0.1 substitutions per site.

2014; Li and Li, 2015). i) Agrobacterium strains GV3101 were cultured (Pav_sc0000671.1) gene was used as the internal control. To detect the
overnight at 28 °C to OD600 of 0.8, and were resuspended in the Agro- silencing of the PaCYP78A6 gene in PaCYP78A6- and TRV::00-silenced
bacterium infiltration buffer (10 mM MgCl2, 10 mM MES, pH 5.6, sweet cherry fruit lines, the gene’s expression was quantified using
100 μM acetosyringone) to a final OD600 of 0.8-1.0. ii) Mixed Agro- semi-quantitative RT-PCR as previously described, with Histone2 as an
bacterium GV3101 strains containing the pTRV1, pTRV2 or pTRV:Pa- internal control (Qi et al., 2017). The expression level was calculated
CYP78A6 vectors were infiltrated with a needle-less syringe into the from the average of three independent biological replicates, and each
basal pedicel of cherry fruit 7 days after full bloom (DAFB) until the biological replicate was performed three times using pooled samples of
whole fruit was permeated. iii) The inoculated fruits were treated with 10 fresh transformed fruits from the same tree.
bagging for 2 days. Fruits were evaluated at 10 days post-inoculation
(dpi). The TRV-mediated gene silencing assays, involving in 30 trans-
formed fruits per strain from the same 10-year-old tree, were performed 2.6. Morphological and cytological characterization
with three biological replicates.
Plant organ morphological observations, measurements, and ex-
2.5. Semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR) aminations were performed as previously described (Adamski et al.,
analyses 2009; Sotelo-Silveira et al., 2013; Zhao et al., 2016; Disch et al., 2006).
Cytological observations and the histochemical staining of sweet cherry
Total RNAs were extracted using an EASYspin Plant RNA rapid fruit exocarp cells were performed according to methods described
extraction kit (Yuanpinghao Bio) according to the manufacturer’s previously [Chakrabarti et al., 2013; Qi et al., 2017; Rojas-Gracia et al.,
manual. The expression levels of target genes were analyzed by qRT- 2017). Serial sections (4 μm thick) were cut using a rotary microtome
PCR with target gene-specific primers (Supplementary Table 1) on an (Leica RM2016; Leica MicrosystemsIncorporated, Nussloch, Germany),
ABI7500 PCR thermocycler (Applied Biosystems, Foster City, CA, USA) and they were observed and photographed using a light microscope
using the TransStart Top Green qPCR SuperMix with SYBR Green (Microscope Nikon Eclipse Ci; Nikon, Tokyo, Japan) equipped with a
fluorescent intercalating dye (TransGen Biotech, Beijing, China) fol- Nikon DS-U3 digital camera (Nikon). Three independent biological re-
lowing the manufacturer’s instructions. The sweet cherry Histone2 plicates were performed for each sample.

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2.7. Statistical analysis during fruit growth and development (Fig. 2). In Arabidopsis, EOD3/
AtCYP78A6 was not expressed in integuments, embryos, and en-
All data are provided as the mean ± standard deviation (SD). dosperms during seed development, but EOD3/AtCYP78A6 expression
Statistically significant differences indicated in figures were based on a was detected in flowers containing stamens, carpels, sepals, and petals,
t-test or one-way analysis of variance (ANOVA) (p ≤ 0.05) using SPSS confirming that AtCYP78A6 is a temporally and spatially expressed
17.0 version (SPSS Inc., Chicago, IL, USA). Figures were constructed gene (Yao et al., 2015). Similarly, PaCYP78A6 was dominantly ex-
using Origin 9.1 (Microcal Software Inc., Northampton, MA, USA). pressed in young reproductive tissues in sweet cherry. Therefore, Pa-
CYP78A6 might play an important role during fruit growth and de-
velopment.
3. Results

3.1. Isolation of PaCYP78A6 and phylogenetic analysis of CYP78 A 3.3. Silencing of PaCYP78A6 resulted in a reduction in sweet cherry fruit
members in plants size

To isolate CYP78A6 orthologs in sweet cherry, the protein sequence To explore the function of PaCYP78A6 in influencing fruit size
of AtCYP78A6 was used as the query in a BLAST algorithm-based during fruit growth and development, the expression of PaCYP78A6 in
search against the sweet cherry genome (http://cherry.kazusa.or.jp/ the landrace sweet cherry cultivar ‘Chunlu’, with a large fruit sizes, was
map.html). A putative full-length mRNA (Pav_sc0000652.1_g570.1.mk) silenced using the TRV-VIGS technique. The TRV::PaCYP78A6 con-
was detected for the putative protein PaCYP78A6 in sweet cherry. A struct, carrying a 325-bp fragment of the PaCYP78A6 cDNA sequence
complete open reading frame of 1,629 bp encoding a polypeptide with was used to effectively silence PaCYP78A6 in sweet cherry cultivars as
542 amino acid residues was amplified using the primers PaCYP78A6-F assessed by a semi-quantitative RT-PCR assay. Semi-quantitative RT-
and PaCYP78A6-R, which were designed based on the putative mRNA PCR was carried out to detect PaCYP78A6 expression using cDNA
sequence. To evaluate the phylogenetic relationship of PaCYP78A6 samples from infiltrated fruit (TRV::PaCYP78A6 and TRV::00) at 15
with other CYP78 A family members from various plant species, a days post-inoculation (dpi). PaCYP78A6 was significantly down-
phylogenetic tree was constructed using MEGA 6.0 with CYP78As from regulated in the fruit infected with TRV::PaCYP78A6 compared with in
various plant species. PaCYP78A6 is an ortholog of PpCYP78A9, TRV::00-infected fruit, suggesting that PaCYP78A6 was efficiently si-
NnCYP78A9, and TcCYP78A3, and displayed a high identity with lenced (Fig. 3A).
Arabidopsis CYP78A8, CYP78A6, and CYP78A9 (Fig. 1). As shown in Fig. 3, the sizes of mature TRV::PaCYP78A6-infected
fruits were dramatically smaller than those of the TRV::00-infected fruit
at 25 dpi and 35 dpi (Fig. 3B, C). Furthermore, the fruit diameters,
3.2. Expression profile of PaCYP78A6 during sweet cherry fruit
weights, and the fruit flesh to fruit stone weight ratios (F/S) were
development
measured and statistically analyzed. The longitudinal diameters, hor-
izontal diameters, and weights of the TRV::PaCYP78A6-infected fruits
To determine whether PaCYP78A6 is involved in fruit development,
were reduced by 62%, 58%, and 71%, respectively, compared with
the transcript level of PaCYP78A6 was assessed by qRT-PCR from
those of the TRV::00-infected fruits at 25 dpi (Fig. 3D, E). Similarly, the
leaves, flower buds, blossoms, and fruit at different growth and devel-
F/S of TRV::PaCYP78A6-infected fruits were also significantly de-
opmental stages in two cultivars, the wild sweet cherry ‘Mazzard’, and
creased by 62%, 43%, and 76%, at 10 dpi, 15 dpi, and 25 dpi, re-
the landrace sweet cherry ‘Chunlu’. The expression levels of
spectively, compared with those of the TRV::00-infected fruits (Fig. 3F).
PaCYP78A6 in the fruit and flower were higher than those in other
Thus, PaCYP78A6 positively regulates fruit size and plays an important
tissues in both sweet cherry cultivars, and the highest expression of
role in sweet cherry fruit development.
PaCYP78A6 was detected at 25 days after full bloom (DAFB) during
fruit growth and development in the landrace sweet cherry ‘Chunlu’
(Fig. 2), suggesting the importance of PaCYP78A6 function in fruit 3.4. Silencing of PaCYP78A6 decreased mesocarp cell proliferation and
growth and development. However, during the later fruit growth and expansion in sweet cherry fruit
development period, PaCYP78A6 was expressed at relatively low levels
in both cultivars (Fig. 2). Moreover, the expression levels of PaCYP78A6 To further cytologically characterize the role of PaCYP78A6 in
in all of the tissues from the landrace sweet cherry ‘Chunlu’ were dra- regulating fruit size during growth and development in sweet cherry,
matically higher than those from the wild sweet cherry ‘Mazzard’ the cell numbers and sizes in the exocarps and mesocarps from TRV::00-
and TRV::PaCYP78A6-infected sweet cherry fruit were investigated.
The cell numbers in the mesocarps decreased by approximately 30% in
TRV::PaCYP78A6-infected fruit at 25 dpi compared with in TRV::00-
infected fruits (Fig. 4). Furthermore, the mesocarp cells in TRV::Pa-
CYP78A6-infected fruit were smaller at 25 dpi than those of TRV::00-
infected fruit (Fig. 4). We hypothesized that down-regulating Pa-
CYP78A6 expression level mainly affected cell expansion. In Arabi-
dopsis, rice, and wheat, some CYP78A family members, such as
CYP78A5, CYP78A6, CYP78A11, CYP78A13, and CYP78A9, affect the
size of the seed coats by regulating integument cell proliferation during
ovule and seed development, ultimately facilitating seed growth and
development (Miyoshi et al., 2004; Adamski et al., 2009; Fang et al.,
2012; Nagasawa et al., 2013; Sotelo-Silveira et al., 2013; Xu et al.,
2015). Likewise, in this study, the silencing of PaCYP78A6 decreased
mesocarp cell proliferation and expansion during fruit development,
Fig. 2. Expression profiles of PaCYP78A6 from leaf, flower-bud, blossom, and mainly cell expansion, leading to a reduction in fruit size. Therefore,
fruit in different cultivations of ‘Chunlu’, a landrace sweet cherry, and PaCYP78A6 probably plays an important role during fruit growth and
‘Mazzard’, a wild sweet cherry. The error bars represent the standard deviation development by affecting mesocarp cell volume and expansion, ulti-
of three independent experiments. mately determining fruit size.

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Fig. 3. Silencing of PaCYP78A6 results in small sweet cherry fruit. (A) The expression level of PaCYP78A6 as determined by semi-quantitative RT-PCR relative to
PaHistone2A in the fruit of sweet cherry TRV::PaCYP78A6- and TRV::00-silencing lines at 15 days post-inoculation (dpi). (B, C) The phenotypes of TRV::PaCYP78A6-
and TRV::00-infected fruits at 25 dpi (A) and 30 dpi (B). (D–F) The fruit diameter (D), fruit weight (E), and weight ratio of fruit flesh and fruit stone (F/S; F) in
TRV::00- and TRV::PaCYP78A6-silenced fruit at 5 dpi, 10 dpi, 15 dpi, and 25 dpi. Values represent the mean ± SD from three independent replicates. Statistical
significance was determined by Student’s t-test. (**) p < 0.01.

3.5. Fruit ripening-associated genes were downregulated in PaCYP78A6- CNR, HB1, PG, PME, XYL1, EXP1, MYB10.1, CHS, ANS, PSY1, in
silenced sweet cherry fruit TRV::PaCYP78A6-infected fruits were significantly downregulated
compared with those of TRV::00-infected control fruit (Fig. 5). Fur-
As shown in Fig. 3B, TRV::PaCYP78A6-infected sweet cherry fruit thermore, the expression levels of ANS, CHS, PG, and EXP1 in
delayed ripening in comparison with TRV::00-infected control fruit TRV::PaCYP78A6-infected fruits were reduced to 89.4%, 84.7%, 74.0%,
(Fig. 3B). To explore the underlying causes of this differences, the ex- and 82.2% of those in TRV::00-infected control fruits, respectively
pression levels of ripening-related genes, including ripening-related (Fig. 5). These results suggest that PaCYP78A6 is a necessary regulator
transcription factors [RIPENING INHIBITOR (RIN), NON-RIPENING of sweet cherry ripening.
(NOR), COLORLESS NON-RIPENING (CNR), and HD-Zip homeobox
protein (HB1)], cell wall metabolism genes [polygalacturonase (PG), 3.6. Overexpression of PaCYP78A6 increased seed size in Arabidopsis
pectin methyl esterase (PME), β-D-xylosidase (XYL1), and expansin
(EXP1)], and fruit coloring-associated genes [R2R3-MYB transcription To further characterize the function of PaCYP78A6, transgenic
factor (MYB10.1), chalcone synthase (CHS), anthocyanidin synthase Arabidopsis plants overexpressing the full-length PaCYP78A6 were
(ANS), and phytoene synthase 1 (PSY1)] were evaluated in TRV::Pa- generated under the control of the constitutive cauliflower mosaic virus
CYP78A6-infected fruit and TRV::00-infected control fruit using qRT- 35S promoter through A.-mediated transformation using the floral-dip
PCR. The expression levels of 12 ripening-related genes, RIN, NOR, method. In total, 23 independent lines of PaCYP78A6-overexpressing

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Fig. 4. Histological observations of TRV::00- and TRV::PaCYP78A6-silenced sweet cherry fruit. (A, B) Paraffin longitudinal sections of mesocarp tissues from the fresh
fruit of TRV::00-silencing (A) and TRV::PaCYP78A6-silencing (B) lines at 25 dpi stained with hematoxylin (Scale bars = 2.5 mm). (C, D) Magnified views of the
mesocarp longitudinal sections (the red virtual box area) corresponding to A and B, respectively (Scale bars = 0.40 mm). (E, F, G) Quantification of cell number (E),
cell length (F) and Cell volume (G) of the sections from the fresh fruit of TRV::00-silencing and TRV::PaCYP78A6-silencing lines at 25 dpi. Values represent the
mean ± SD from three independent replicates. **Significant differences as calculated using the Student’s t-test at p < 0.01 (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article).

Fig. 5. Expression profiles of the fruit ripening-associated genes from the sweet cherry TRV::PaCYP78A6- and TRV::00-silenced fruit at 30 dpi, including ripening-
related transcription factors RIN, NOR, CNR, and HB1, cell wall metabolism genes PG, PME, XYL1, and EXP1, and fruit coloring-associated genes MYB10.1, CHS, ANS,
and PSY1. Each value represents the mean ± SD from three independent replicates. ** indicates significant difference as calculated using Student’s t-test at
p < 0.01.

Arabidopsis plants were obtained, and the PaCYP78A6 expression levels transgenic Arabidopsis lines were examined. As shown in Fig. 6, trans-
of transgenic Arabidopsis lines were significantly higher than wild-type genic lines that overexpressed PaCYP78A6 had more and longer siliques
lines as assessed by qRT-PCR (Supplementary Figs. S1 and 6 B). Three than those of control Columbia Arabidopsis (Fig. 6A, C, F). Likewise,
independent T3 transgenic lines of PaCYP78A6-overexpressing Arabi- seed sizes from PaCYP78A6-overexpressing T3 transgenic Arabidopsis
dopsis, PaCYP78A6-4, PaCYP78A6-9, and PaCYP78A6-15, were ran- plants were increased compared with control lines (Fig. 6D, G). More-
domly selected for further phenotypic analyses. over, the expression levels of PaCYP78A6 in different tissues (leaves,
The silique and seed sizes of the PaCYP78A6-overexpressing T3 inflorescences, and siliques) from three independent PaCYP78A6-

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Fig. 6. Overexpression of PaCYP78A6 led to large siliques and seeds in Arabidopsis. (A) The phenotypes of the main inflorescence stem at stage 6.00 (The stage is in
accordance with those described by Boyes et al., 2001 (Boyes et al., 2001) in PaCYP78A6 overexpression lines and wild-type (WT) (scale bar = 10.00 mm). (B) The
expression levels of PaCYP78A6 relative to those of Histone2 in T3 lines and WT as assessed by semi-quantitative RT-PCR. (C) Silique phenotypes (scale bar =
10.00 mm). (D) Seed phenotypes (scale bar = 0.5 mm). (E) Expression levels of PaCYP78A6 in different tissues from T3 transgenic lines. Statistical significance was
determined by a one-way ANOVA, with letters indicating significant differences (p < 0.05, Duncan’s test). (F, G) Seeds (F) and siliques (G) were measured from 10
transgenic Arabidopsis plants and WT plants. Seed size is represented by the projected area of a seed. Values represent the mean ± SD from three independent
replicates. Statistical significance was determined by Student’s t-test, and ** indicates a significant differences at p < 0.01.

overexpressing T3 transgenic lines were detected. The expression level Interestingly, the fruits of TRV::PaCYP78A6;PaCYP78A9 double si-
of PaCYP78A6 was higher in siliques and inflorescences of T3 trans- lencing lines were significantly smaller than those of TRV::PaCYP78A9,
genic lines than in leaves (Fig. 6 E), consistent with the expression TRV::PaCYP78A6 single fruit lines at 25dpi, respectively (Fig. 7B). We
profile of PaCYP78A6 during sweet cherry fruit growth and develop- measured and statistically analyzed the fruit weights and diameters,
ment. Thus, the overexpression of PaCYP78A6 increased seed size. and results show that the weights, horizontal diameters, and long-
Given that the VIGS-mediated silencing of PaCYP78A6 reduced the itudinal diameters of the TRV::PaCYP78A6;PaCYP78A9 double silen-
cherry fruit size, this may indicate that PaCYP78A6 plays an indis- cing lines were decreased compared with those of the TRV::00-infected
pensable role during the growth and development of reproductive or- fruits at 25 dpi (Fig. 7C, D). In addition, The expression of PaCYP78A9
gans, and especially influences fruit size. was up-regulated in TRV::PaCYP78A6 silencing fruits compared with in
TRV::00-infected fruit. Similarly, we detected that PaCYP78A6 expres-
3.7. PaCYP78A6 acts redundantly with PaCYP78A9 in regulating fruit size sion levels were higher in TRV::PaCYP78A9 silencing fruits lines than
those in TRV::00-infected fruit (Fig. 7E, F). These findings revealed that
Previous studies showed that PaCYP78A9 gene regulated fruit size the small fruit phenotype of TRV::PaCYP78A6;PaCYP78A9 double si-
in P. avium (Qi et al., 2017), and our study ascertained that PaCYP78A6 lenced fruit lines was synergistically enhanced compared with the Pa-
influenced fruit size. We hypothesized that PaCYP78A6 may act re- CYP78A9 silenced lines, indicating that PaCYP78A6 functions re-
dundantly with PaCYP78A9 in regulating fruit size. To test this, the dundantly with PaCYP78A9 to regulate fruit growth and development
TRV-VIGS technique was used to generate the PaCYP78A6, PaCYP78A9 (Fig. 8).
single silencing sweet cherry fruit (TRV::PaCYP78A9 and TRV::Pa-
CYP78A6) and double silencing sweet cherry fruit TRV::PaCYP78A6;- 3.8. PaCYP78A6 can recover the phenotype of cyp78a6 mutant
PaCYP78A9 lines. Semi-quantitative RT-PCR results displayed that the
expression levels of PaCYP78A6 or/and PaCYP78A9 in single To further validate the function of PaCYP78A6, we overexpressed
TRV::PaCYP78A9, TRV::PaCYP78A6, and double silencing TRV::PaCY- PaCYP78A6 in homozygous cyp78a6 mutants Columbia-0 background
P78A6;PaCYP78A9 lines were significantly decreased compared with Arabidopsis (Salk_080087, obtained from the ABRC) exhibiting a phe-
the TRV::00-infected fruit at 15 dpi, respectively (Fig. 7A), confirming notype of short siliques and small seeds size, and the construct,
that PaCYP78A6 or/and PaCYP78A9 were effectively silenced. 35S::PaCYP78A9, was transformed into homozygous cyp78a6 mutants.

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Fig. 7. PaCYP78A6 acts redundantly with PaCYP78A9 in regulating fruit size. A. The phenotypes of single TRV::PaCYP78A9-, TRV::PaCYP78A6-, double silencing
TRV::PaCYP78A6;PaCYP78A9-, and TRV::00-infected fruits at 25 dpi. B. The expression level of PaCYP78A6 and PaCYP78A9 in single TRV::PaCYP78A9-,
TRV::PaCYP78A6-, double silencing TRV::PaCYP78A6;PaCYP78A9-, and TRV::00-infected fruits in sweet cherry at 15 dpi. (C, D) The fruit diameter (C) and fruit
weight (E) in single TRV::PaCYP78A9-, TRV::PaCYP78A6-, double silencing TRV::PaCYP78A6;PaCYP78A9-, and TRV::00-infected fruits in sweet cherry at 25 dpi.
Statistical significance was determined by a one-way ANOVA, with letters indicating significant differences (p < 0.05, Duncan’s test). (E) The expression level of
PaCYP78A6 in TRV::PaCYP78A9- and TRV::00-silenced fruit at 15 dpi. (F) The expression level of PaCYP78A9 in TRV::PaCYP78A6- and TRV::00-silenced fruit at 15
dpi. Values represent the mean ± SD from three independent replicates. Statistical significance was determined by Student’s t-test, and ** indicates a significant
differences at p < 0.01.

Overexpressing PaCYP78A6 in the cyp78a6 mutants caused high simi- Members of CYP78 A, a plant-specific family of cytochrome P450,
larity siliques and seeds size compared to WT plants, indicating that have distinct functions in positively regulating the growth and devel-
cyp78a6 mutants could be rescued to normal WT Arabidopsis levels. opment of reproductive organs and seeds, such as CYP78A5, CYP78A6,
However, the siliques and seeds size of cyp78a6 mutants were not and CYP78A9 in Arabidopsis (Ito and Meyerowitz, 2000; Fang et al.,
rescued by 35S::PaCYP78A9. These results suggest that the PaCYP78A6 2012; Adamski et al., 2009; Sotelo-Silveira et al., 2013), TaCYP78A3
and PaCYP78A9 genes might have distinct functions in influencing fruit and TaCYP78A5 in wheat (Ma et al., 2015, 2016), CYP78A11 and
size during fruit growth and development. CYP78A13 in rice (Nagasawa et al., 2013; Miyoshi et al., 2004), and
CYP78A5 in tomato (Chakrabarti et al., 2013). The overexpression of
CYP78 A family members in Arabidopsis or rice leads to enhanced sizes
4. Discussion of reproductive organs and seeds. Here, PaCYP78A6 was overexpressed
in Arabidopsis, resulting in larger reproductive organs and seeds in
In the present study, PaCYP78A6, a CYP78A family member, was comparison with wild-type Arabidopsis. Fleshy fruit development is a
identified and functionally characterized by overexpression in complex process that is regulated by genetic and environmental factors.
Arabidopsis and silencing using an RNAi approach. PaCYP78A6 is Once fertilization is completed, fruit development undergoes two pro-
mainly expressed in leaves, inflorescences, and fruit, particularly in the cesses, a short period of cell division followed by a longer period of cell
latter rapid growth periods. Furthermore, silencing PaCYP78A6 by expansion to determine fruit dimensions (Chakrabarti et al., 2013).
VIGS resulted in small fruit by decreasing the mesocarp cell volume and Furthermore, CYP78 A subfamily members control seed size by pro-
expansion during fruit growth and development. In contrast, over- moting the integument cell proliferation or expansion. For example,
expressing PaCYP78A6 enlarged siliques and seeds size in Arabidopsis. KLU/CYP78A5 affects seeds size by influencing integument cell pro-
In addition, the fruit ripening-associated genes were downregulated in liferation during seed development. CYP78A6 influences seeds size by
PaCYP78A6-silenced sweet cherry fruits. Thus, PaCYP78A6 positively accelerating integument cell expansion during the later seed developing
regulated sweet cherry fruit size and was involved in fruit ripening. This phase. In this study, silencing PaCYP78A6 indicated that the gene
provides new insights into the molecular basis of fruit size determina- controls fruit size by affecting mesocarp cell proliferation and
tion during the fleshy fruit development of stone fruit trees.

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Fig. 8. PaCYP78A6 can recover the phenotype of cyp78a6 Mutant, PaCYP78A9 cannot. (A) Silique phenotypes (scale bar = 10.00 mm) of WT Col, cyp78a6 mutant,
PaCYP78A9 transgenic plants in cyp78a6 mutant, and PaCYP78A6 transgenic plants in cyp78a6 mutant. (C) Seed phenotypes (scale bar = 0.1 mm). (B,D) siliques (B)
and Seeds (D) were measured from 10 transgenic Arabidopsis plants and WT plants. Values represent the mean ± SD from three independent replicates. Statistical
significance was determined by Student’s t-test, and ** indicates a significant differences at p < 0.01.

expansion during fruit growth and development, mainly the cell ex- participating in producing an undiscovered plant growth substance to
pansion. There may be some differences between the effects of control organ size (Sotelo-Silveira et al., 2013). CYP78A6 might be a
CYP78A6 on the size of reproductive organs and fruit depending on the mobile signal production to promote seed growth. However, the sig-
gene transcription level during cell proliferation or/and expansion, naling pathway and mechanisms underlying CYP78A6 controlling
which are two different processes. We hypothesize that the difference is organ size would be worthy of future study with more eff ;orts.
related to the special developmental pattern “double S model (two fruit Fruit ripening, the last stage of fruit development, is complexly
rapid development and expansion stages)” of sweet cherry fruit. Be- regulated process that includes numerous metabolic changes, such as
cause PaCYP78A6 was dominantly expressed in sweet cherry fruit pigment formation, nutritional changes, and cell wall metabolism.
during the two rapid developmental stages in fruit, PaCYP78A6 may SIKLUH in Solanum lycopersicum controls fruit mass by increasing the
play a dual role in mesocarp cell proliferation and expansion during cell layers, resulting in enlarged fruit and a concomitant delayed fruit
fruit growth and development. ripening. This suggests that the fruit ripening period was related to the
In previous reports, our results confirm that PaCYP78A9 influenced cell division stage of fruit development (Chakrabarti et al., 2013). Here,
fruit size during fruit growth and development (Qi et al., 2017). In this silencing PaCYP78A6 not only reduced cherry fruit size, but also de-
study, silencing PaCYP78A6, which is most closely related to Pa- layed fruit ripening, and downregulated the expression of ripening-re-
CYP78A9, resulted in small sweet cherry fruit. In addition, both silen- lated genes, implying that PaCYP78A6 plays an important role in fruit
cing PaCYP78A9 and PaCYP78A6 in sweet cherry fruits synergistically development and ripening. The specific high-level expressions of sev-
enhanced the smaller fruits phenotype than PaCYP78A6 silenced cherry eral transcription factors, such as the MADS-domain proteins TOMATO
fruits, suggesting that PaCYP78A6 and PaCYP78A9 may have over- AGAMOUS-LIKE1 (Itkin et al., 2009; Vrebalov et al., 2009), SEPALL-
lapping functions in fruit size regulation. Many studies have ascertained ATA1/2-like, and the AP2/ERF protein AP2a (Chung et al., 2010;
that members of the CYP78A family, CYP78A5, CYP78A6, CYP78A7, Karlova et al., 2011; Ireland et al., 2013), during fruit development also
CYP78A9, are mainly involved in controlling organ size and shape in regulate the ripening process, suggesting that fruit ripening and fruit
Arabidopsis (Ito and Meyerowitz, 2000; Miyoshi et al., 2004; Adamski development are closely related processes. In apple and strawberry, the
et al., 2009). When Arabidopsis cyp78a9 and cyp78a6 single and double strong suppression of SEPALLATA1/2-like gene expression levels re-
mutants were analyzed, the results shows that CYP78A6 acts re- sulted in small fleshy fruit and delayed ripening, suggesting that SEP-
dundantly with CYP78A9 to maternally control seed size (Fang et al., ALLATA1/2-like genes control fruit flesh development and ripening
2012). Similarly, CYP78A5 and its homolog CYP78A7 play functional (Seymour et al., 2010; Ireland et al., 2013). In tomato, miR156 (target
redundancy roles in regulating relative growth and yield via a non-cell- CNR) and miR172 (target AP2a) affect fruit formation, fruit yield, and
autonomous signal in Arabidopsis (Wang et al., 2008). In Arabidopsis, fruit ripening by fine-tuning the expression levels of CNR and AP2a
CYP78A9 could be a core enzyme in the flavonoid biosynthesis pathway (Karlova et al., 2011, 2013; 2014). Similarly, our study confirmed that

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PaCYP78A6 regulated fruit size during the fruit development process manner. Curr. Biol. 16, 272–279.
and was involved in fruit ripening in sweet cherry. We hypothesize that Fang, W.J., Wang, Z.B., Cui, R.F., Li, J., Li, Y.H., 2012. Maternal control of seed size by
EOD3/CYP78A6 in Arabidopsis thaliana. Plant J. 70, 929–939.
PaCYP78A6 might be related to fruit ripening-related gene which Frandsen, R.J., Frandsen, M., Giese, H., 2012. Targeted gene replacement in fungal pa-
regulated changes in softening, color, and flavor during fruit develop- thogens via Agrobacterium tumefaciens-mediated transformation. Plant Fungal
ment and fruit ripening, and that downregulating genes expression Pathogens: Methods and Protocols. pp. 17–45.
Fu, D., Zhu, B., Zhu, H., Jiang, W., Luo, Y., 2005. Virus-induced gene silencing in tomato
could result in delayed fruit ripening in PaCYP78A6-silenced sweet fruit. Plant J. 43, 299–308.
cherry fruit (Karlova et al., 2014; Fujisawa et al., 2013; Martel et al., Fujisawa, M., Nakano, T., Shima, Y., Ito, Y.A., 2013. Large-scale identification of direct
2011; Qin et al., 2012). Thus, the role of PaCYP78A6 in fruit devel- targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals
the regulation of fruit ripening. Plant Cell 25, 371–386.
opment and ripening remains unclear, and more efforts are needed in Gillaspy, G., Ben-David, H., Gruissem, W., 1993. Fruits: a developmental perspective.
the future to characterize its functions. Plant Cell 5, 1439–1451.
Harada, T., Kurahashi, W., Yanai, M., Wakasa, Y., Satoh, T., 2005. Involvement of cell
proliferation and cell enlargement in increasing the fruit size of Malus species. Sci.
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Horiguchi, G., Ferjani, A., Fujikura, U., Tsukaya, H., 2006. Coordination of cell pro-
In conclusion, our work clearly confirmed the important role of liferation and cell expansion in the control of leaf size in Arabidopsis thaliana. J.
PaCYP78A6 in regulating sweet cherry fruit size and its involvement in Plant Res. 119, 37–42.
Ireland, H.S., Yao, J., Tomes, S., Sutherland, P.W., Nieuwenhuizen, N., Gunaseelan, K.,
ripening during fruit development. This increased our understanding of Winz, R.A., David, K.M., Schaffer, R.J., 2013. Apple SEPALLATA1/2‐like genes
the cellular and molecular bases controlling sweet cherry fruit size, control fruit flesh development and ripening. Plant J. 73, 1044–1056.
providing new insights into the molecular mechanisms of fruit devel- Itkin, M., Seybold, H., Breitel, D., Rogachev, I., Meir, S., Aharoni, A., 2009. TOMATO
AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network. Plant J.
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provement. Ito, T., Meyerowitz, E.M., 2000. Overexpression of a gene encoding a cytochrome P450,
CYP78A9, induces large and seedless fruit in Arabidopsis. Plant Cell 12, 1541–1550.
Jofuku, K.D., Omidyar, P.K., Gee, Z., Okamuro, J.K., 2005. Control of seed mass and seed
Conflict of interest yield by the floral homeotic gene APETALA2. Proc. Natl. Acad. Sci. 102, 3117–3122.
Karlova, R., Rosin, F.M., Busscher-Lange, J., Parapunova, V., Do, P.T., Fernie, A.R.,
The authors declare that they have no conflict of interest. Fraser, P.D., Baxter, C., Angenent, G.C., de Maagd, R.A., 2011. Transcriptome and
metabolite profiling show that APETALA2a is a major regulator of tomato fruit ri-
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Author contribution statement Karlova, R., van Haarst, J.C., Maliepaard, C., van de Geest, H., Bovy, A.G., Lammers, M.,
Angenent, G.C., de Maagd, R.A., 2013. Identification of microRNA targets in tomato
fruit development using high-throughput sequencing and degradome analysis. J. Exp.
ML and XQ conceived the project. XQ designed the experiments. XQ,
Bot. 64, 1863–1878.
CL, and LS performed the experiments. XQ analyzed the results, and Karlova, R., Chapman, N., David, K., Angenent, G.C., Seymour, G.B., de Maagd, R.A.,
wrote the manuscript. ML provided scientific suggestions, and revised 2014. Transcriptional control of fleshy fruit development and ripening. J. Exp. Bot.
the manuscript. All authors reviewed and approved the final manu- 65, 4527–4541.
Li, N., Li, Y., 2015. Maternal control of seed size in plants. J. Exp. Bot. 66, 1087–1097.
script. Li, N., Li, Y., 2016. Signaling pathways of seed size control in plants. Curr. Opin. Plant
Biol. 33, 23–32.
Acknowledgments Li, Y.H., Zheng, L.Y., Corke, F., Smith, C., Bevan, M.W., 2008. Control of final seed and
organ size by the DA1 gene family in Arabidopsis thaliana. Genes Dev. 22,
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This work was supported by a grant from The Central Public-interest Li, B., Xie, Z., Zhang, A., Xu, W., Zhang, C., Liu, Q., Liu, C., Wang, S., 2010. Tree growth
Scientific Institution Basal Research Fund of Chinese Academy of characteristics and flower bud differentiation of sweet cherry (Prunus avium L.) under
different climate conditions in China. Hortic. Sci. (Prague) 37, 6–13.
Agricultural Sciences (1610192018201) and The Agricultural Science Ma, M., Wang, Q., Li, Z., Cheng, H., Li, Z., Liu, X., Song, W., Appels, R., Zhao, H., 2015.
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Appendix A. Supplementary data Martel, C., Vrebalov, J., Tafelmeyer, P., Giovannoni, J.J., 2011. The tomato MADS-box
transcription factor RIPENING INHIBITOR interacts with promoters involved in nu-
merous ripening processes in a COLORLESS NONRIPENING-dependent manner. Plant
Supplementary material related to this article can be found, in the
Physiol. 157, 1568–1579.
online version, at doi:https://doi.org/10.1016/j.scienta.2018.10.041. Miyoshi, K., Ahn, B.O., Kawakatsu, T., Ito, Y., Itoh, J.I., Nagato, Y., Kurata, N., 2004.
PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450.
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