Food Chemistry 214 (2017) 137146

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Accumulation of carotenoids and expression of carotenogenic genes in

peach fruit
Shifeng Cao a, Minhua Liang b, Liyu Shi b, Jiarong Shao b, Chunbo Song b, Kun Bian b, Wei Chen b,
Zhenfeng Yang b,
a
b

Nanjing Research Institute for Agricultural Mechanization, Ministry of Agriculture, Nanjing 210014, China
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China

a r t i c l e

i n f o

Article history:
Received 8 May 2016
Received in revised form 11 July 2016
Accepted 11 July 2016
Available online 12 July 2016
Keywords:
Peach
Carotenoid
Gene expression
Blue light
Fruit development
Harvest

a b s t r a c t
To understand better the regulatory mechanism of the carotenoid accumulation, the expression profile of
relevant carotenoid genes and metabolites were compared between two peach cultivars with different
colors during fruit development. Meanwhile, the change pattern of carotenoid content and expression
of carotenoid metabolic genes in peaches after harvest in response to blue light were also investigated.
As compared to the yellow fleshed-cultivar Jinli, lower carotenoid levels were observed in skin and pulp
in white peach cultivar Hujing, which might be explained by differentially expression of PpCCD4 gene.
With respect to Jinli, the carotenoid accumulation during fruit development in fruit skin was partially
linked with the transcriptional regulation of PpFPPS, PpGGPS, PpLCYB and PpCHYB. However, in the pulp,
the accumulation might be also associated with the increased transcriptions of PpPDS, along with the
above four genes. Blue light treatment induced carotenoid accumulation in Jinli peaches during storage.
In addition, the treated-fruit displayed higher expression of all the eight genes analysed with a lesser
extent on PpCCD4, which suggested that the much more increased carotenoid synthesis rate could result
in the higher carotenoid content in blue light-treated fruit. The results presented herein contribute to
further elucidating the regulatory mechanism of carotenoid accumulation in peach fruit.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Carotenoids are natural pigments involved in the photosynthetic process, which play indispensable roles in providing pigmentation for flowers and fruits to attract pollinators and seed
dispersers (Nisar, Li, Lu, Khin, & Pogson, 2015). Some carotenoid
compounds have been reported to exert beneficial effects on prevention of certain cancer cardiovascular diseases (Yuan, Zhang,
Nageswaran, & Li, 2015). In view of the commercial and nutritional
values of carotenoids, their biosynthetic and catabolic pathways
have been widely investigated during the past two decades
(Fig. S1) (Fraser & Bramley, 2004; Taylor & Ramsay, 2005).
Abbreviations: CCD, carotenoid cleavage dioxygenase; CHYB, b-carotene hydroxylase; DAFB, days after full bloom; DMAPP, dimethylallyl diphosphate; FPP, farnesyl
diphosphate; FPPS, farnesyl diphosphate synthase; FW, fresh weight; GGPP,
geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; IPP,
isopentenyl pyrophosphate; LCYB, lycopene b-cyclase; LED, light-emitting diode;
PCR, polymerase chain reaction; PDS, phytoene desaturase; PSY, phytoene synthase; q-PCR, quantitative real-time PCR; ZDS, f-carotene desaturase.
Corresponding author.
E-mail address: yangzf@zwu.edu.cn (Z. Yang).
http://dx.doi.org/10.1016/j.foodchem.2016.07.085
0308-8146/ 2016 Elsevier Ltd. All rights reserved.

Researches on the regulation of carotenoid accumulation during

fruit development have shown that the transcriptional level of
carotenogenic genes is principal factor controlling carotenoid accumulation in most plant species. The increased expression of
upstream genes such as phytoene synthase (PSY) and phytoene
desaturase (PDS) in carotenoid biosynthesis causes lycopene accumulation during tomato ripening (Fray & Grierson, 1993; Isaacson,
Ronen, Zamir, & Hirschberg, 2002). The massive accumulation in
a-carotene is consistent with increased transcripts of PSY and
b-carotene hydroxylase (CHYB) in pepper during maturation
(Hornero-Mndez, Gmez-Ladrn de Guevara, & MnguezMosquera, 2000; Hugueney et al., 1996). The accumulation of
b-carotene is well correlated with the expression of lycopene
b-cyclase (LCYB) in different kiwifruit species (AmpomahDwamena et al., 2009). The huge accumulation in total carotenoids
in red-fleshed loquat during maturation is found to be correlated
with the increase in gene expressions of PSY, LCYB and CHYB (Fu
et al., 2012). Therefore, differential expression of carotenogenic
genes plays key roles in determining the amount and type of
specific carotenoids.

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S. Cao et al. / Food Chemistry 214 (2017) 137146

It is well documented that the content and composition of carotenoid is developmentally regulated and affected by environmental stimuli (Cazzonelli & Pogson, 2010). Light has been reported to
be an important environmental factor which can regulate carotenoid metabolism in plants (Wu et al., 2007; Zhang et al., 2012,
2015). In tomato irradiated with red light, the accumulation of
lycopene, as well as an increase in total carotenoid content, was
observed (Alba, Cordonnier-Pratt, & Pratt, 2000; Schofield &
Paliyath, 2005). Blue light treatment induced carotenoid accumulation effectively in the juice sacs of Satsuma mandarin and Valencia
orange (Zhang et al., 2012, 2015).
The flesh color of yellow-fleshed peach fruit (Prunus persica L.
Batsch) is produced by a group of carotenoids, which is an important factor of nutritional quality and market acceptance (Cazzonelli
& Pogson, 2010; Gil, Toms-Barbern, Hess-Pierce, & Kader, 2002).
Recently, it has been reported that the carotenoid cleavage dioxygenase (PpCCD4) was the major factor in determining carotenoid
degradation in white peaches but no correlation was observed
between carotenoid accumulation and the expression levels of carotenoid biosynthetic genes (Adami et al., 2013; Brandi et al., 2011).
Despite recent efforts to understand the molecular biology of
carotenogenesis takes place in peaches, several gaps remain in
our understanding of the signals and mechanisms involved in carotenoid metabolism. To investigate further how carotenoid accumulation in peach fruit, in this study, firstly the concentration
and composition of carotenoids and the expression of several carotenoid biosynthetic genes as well as PpCCD4 in fruit peel and pulp
were comparatively analysed in two different colored-peaches
(Jinli and Hujing) during fruit development and maturation.
Then, the effect of blue light on carotenoid content and composition, and the expression of genes related to carotenoid biosynthesis
and catabolism were investigated in both peach cultivars after
harvest.
2. Materials and methods
2.1. Plant materials, sample collection, and blue light treatment
Two peach cultivars (Prunus persica), Hujing and Jinli, were
obtained from the experimental farm of Fenghua Peach Fruit
Research Institute (Ningbo, China). Trees were subjected to standard horticultural practices. Fruit ripening stages were defined
according to Tonutti, Bonghi, Ruperti, Tornielli, and Ramina
(1997) and Gabotti, Negrini, Morgutti, Nocito, and Cocucci
(2015). At the desired times, fruit (ten fruit per each of the five
plants of each cultivar considered per each ripening stage) of
Hujing and Jinli were picked and quickly transferred to the laboratory, and fruit tissues at 98 (S1), 105 (S2), 120 (S3), 127 (S4), and
134 (S5) DAFB were sampled during the spring-summer season of
2015 (Fig. S2).
For blue light treatment, fruit of each cultivar at commercial
maturity stage were selected for uniform size and color, and then
divided into two groups randomly. The blue light treatment was
the same as that in our previous publication (Gong et al., 2015).
The fruit stored at 10 C in the dark (90% relative humidity) was
considered as the control. There were three replicates of eighty
fruit each per treatment, and samples were taken initially and at
5-day intervals during storage. All samples were immediately frozen in liquid nitrogen and then stored at 80 C for RNA extraction.

carotenoids content in each sample, the HPLC analyses were carried out as previously described by Taylor and Ramsay (2005).
2.3. Total RNA extraction and cDNA synthesis
Total RNA was isolated using a Plant Total RNA Extraction Kit
(Genotheramics, Suzhou, China) according to the manufacturers
instructions. Extracted RNA was treated with RNase-free DNase
(Omega, Norcross, GA) to remove any genomic DNA according to
the instruction manual. An aliquot (2 lg) of total RNA was
reverse-transcribed with the SuperRT First Strand cDNA Synthesis
Kit (CWBIO, Beijing, China), following the manufacturers
instructions.
2.4. Quantitative real-time PCR (q-PCR) analysis
Q-PCR analysis was performed using the Mx3000P q-PCR System (Agilent Stratagene, Santa Clara, CA, USA) and the DyNAmoTM
ColorFlash SYBR Green qPCR kit (Thermo Scientific, Pittsburgh,
PA) following the manufacturers instructions. Amplifications were
performed using a total volume of 12.5 lL reaction containing
0.5 lL of the synthesized cDNA, 0.25 lL of 10 lM each forward
and reverse primers, 6.5 lL of the SYBR Green PCR Master Mix
and 5 lL of RNase-free water. The data were analysed and normalized to PpTEF2 to minimize variation in cDNA template levels. All
gene expression analyses were performed with three independent
biological replicates, and primer sequences used for real time PCR
are listed in Supplementary Table S1.
2.5. Data processing and statistical analysis
The contents of carotenoids and transcript abundance of carotenoid metabolic genes of two peach cultivars were displayed with
GraphPad Prism (v.5.01). All values are shown as the mean standard errors. Statistical analysis was performed using the SPSS package program version 16.0 (SPSS Inc., Chicago, IL). Students
unpaired T test was used to compare the means at P < 0.05.
3. Results
3.1. Carotenoid content and composition analysis in fruit peel of two
peach cultivars during fruit development
The total carotenoid content in the peel of the yellow fleshedpeaches (Jinli) and white cultivar (Hujing) at five developmental
stages was analysed. At early ripening stage S1, peel of Jinli and
Hujing had similar total carotenoid levels and accumulated only
a few carotenoid compounds at about 0.04 lg g 1 fresh weight
(FW) in both cultivars. However, a significant increase in the carotenoid content of Jinli was observed from the S2 stage, while carotenoid content in Hujing remained low. At the S5 stage, the total
carotenoid content in peach peels rose to 1.3 lg g 1 FW in Jinli
but only 0.10 lg g 1 FW Hujing (Fig. 1). HPLC measurements
showed that lutein, zeaxanthin, b-carotene and b-cryptoxanthin
were predominant in both two cultivars during ripening. All of
these carotenoids progressively increased during fruit development in Jinli but nearly remained constant in Hujing during
the whole process of fruit development (Fig. 1).

2.2. Extraction and HPLC analysis of carotenoids

3.2. Expression pattern of carotenoid metabolic genes in fruit peel of

two peach cultivars during fruit development

Extraction and purification of carotenoids in tissue samples

(2 g) were performed according to a previously described method
(Tuan et al., 2013; Wright & Kader, 1997). To determine the

The possibility that differences in carotenoid accumulation in

our peach samples could be related to the expression of carotenoid
metabolic genes was investigated. Most of the genes showed an

139

0.15

Jinli
Hujing

1.0

**

*
0.5

0.0
S1

S2

S3

S4

0.10

***

***

***

**

0.05

S5

Ripening stage

0.15

Jinli
Hujing

***

0.10

**

0.05

*
*

0.00
S2

S3

S4

S5

Jinli
Hujing

0.15
0.10

***

***

S1

S2

S3

S4

S5

Ripening stage

0.06

Jinli
Hujing

***
***

0.04

**
0.02

0.00
S1

S2

S3

S4

S5

Ripening stage

Ripening stage

0.20

0.05

Jinli
Hujing

0.00

S1

-Carotene content ( g/g)

***

***

Lutein content ( g/g)

1.5

-Cryptoxanthin content ( g/g)

Zeaxanthine content ( g/g)

Total carotenoids content ( g/g)

S. Cao et al. / Food Chemistry 214 (2017) 137146

***

**
*

0.00
S1

S2

S3

S4

S5

Ripening stage
Fig. 1. Carotenoid content and composition in fruit peel of two peach cultivars during fruit development. All data is presented as a mean of three biological replicates and
error bars represent standard errors. Asterisks (*) indicate significant differences between the two cultivars at different stages (Students unpaired T test; P < 0.05, P < 0.01,
and P < 0.001).

increasing transcript level during fruit development in peels of

Jinli fruit, which was consistent with the accumulation of the total
carotenoids. More FFPS transcripts were detected in peels of Jinli
as compared to Hujing. However, Hujing showed more transcripts of PpPSY, PpPDS, PpZDS and PpCCD4 during fruit development with a few exceptions (Fig. 2). From stage S1 to S2, the
expression of PpGGPS was lower in the peel of Jinli than in
Hujing, but the gene expression level was approximately twice
as great in Jinli as in Hujing thereafter. The expression levels of
PpLCYB and PpCHYB increased with fruit development in the peel
of Hujing and peaked at stage S2 followed by a dramatically
decrease. Interestingly, they were higher in Hujing than in Jinli
from S1 to S3 but higher expressions were observed in Jinli at
stages S4 and S5 (Fig. 2).

3.3. Carotenoid content and composition analysis in pulp of two peach

cultivars during fruit development
The changes and profiles in pulp of Jinli fruit were similar to
those in peels. The total carotenoid accumulated with ripening
principally due to the increased lutein, zeaxanthin, b-carotene
and b-cryptoxanthin during ripening. In the pulp of Jinli, the total
carotenoid content was usually higher than in the peel. However,
almost no carotenoids were detected in the pulp of the white cultivar Hujing (Fig. 3).

3.4. Expression pattern of carotenoid metabolic genes in pulp of two

peach cultivars during fruit development
In pulp of Jinli, expression of most carotenogenic genes
increased gradually from stage S1 to reach a maximum at stage
S4. Abundance of PpFPPS and PpCHYB was higher in pulp of Jinli
than in Hujing tissue during fruit development but higher transcripts of PpPSY, PpZDS and PpCCD4 were observed in Hujing in
this process with an exception of PpPSY expression at stage S2. In
regard of PpGGPS and PpPDS genes, their expression was higher
in the flesh of Jinli than in Hujing during ripening with exceptions
of PpGGPS at stage S5 and PpPDS at stage S1. Meanwhile, the transcript of PpLCYB was more abundant in Jinli at stages S2, S4 and S5
(Fig. 4).
3.5. Effect of blue light treatment on carotenoid content and
composition
In the present study, the effect of blue light treatment on the
accumulation of the main carotenoids in peach fruit of Jinli and
Hujing after harvest was also investigated. During the whole storage, the total carotenoid content and b-cryptoxanthin level in Jinli
increased with storage time. In respect to zeaxanthin and
b-carotene in Jinli, their levels were increased firstly and peaked
at day 15 and day 10, respectively, followed by a decline thereafter.
Lutein content in Jinli decreased during the first 5 days of storage

PpFPPS

***
***

0.6
0.4

Jinli
Hujing

**

*
*

0.2
0.0

Expression relative to PpTEF2

S1

S2

S3

S4

S5

Ripening stage
6

PpPSY

Jinli
Hujing

***

**
*

0
S1

S2

S3

S4

S5

Expression relative to PpTEF2

0.8

Expression relative to PpTEF2

S. Cao et al. / Food Chemistry 214 (2017) 137146

Expression relative to PpTEF2

140

2.0

PpGGPS

***

1.5

***
1.0

**
*

0.5

S1

S2

0.15

**

**

0
S1

S2

S3

S4

S5

Ripening stage
10

PpCHYB

8
6

Jinli
Hujing

***

**

**
*

0
S1

S2

S3

S5

PpPDS

Jinli
Hujing

**
*
0.05

0.00

S4

S5

Ripening stage

Expression relative to PpTEF2

**

S4

0.10

S1
0.8

S2

S3

S4

S5

Jinli
Hujing

PpLCYB

***

0.6

**
0.4

**
0.2

0.0

Expression relative to PpTEF2

Expression relative to PpTEF2

Expression relative to PpTEF2

Jinli
Hujing

S3

Ripening stage

Ripening stage

***

PpZDS

0.0

Ripening stage
3

Jinli
Hujing

S1

S2

S3

S4

S5

Ripening stage
15

PpCCD4

**

Jinli
Hujing

10

**
**
*

0
S1

S2

S3

S4

S5

Ripening stage

Fig. 2. Relative transcripts of eight carotenogenic genes in fruit peel of two peach cultivars during fruit development. The expression level of PpTEF2 (JQ732180) was used to
normalize the mRNA levels for each sample. All data is presented as a mean of three biological replicates and error bars represent standard errors. Asterisks (*) indicate
significant differences between the two cultivars at different stages (Students unpaired T test; P < 0.05, P < 0.01, and P < 0.001).

and increased during the remaining days. The contents of all these
four carotenoids could be induced by blue light treatment and as a
result, the total carotenoid content was higher in the light treatedJinli fruit than in control fruit (Fig. 5). No significant changes in
levels of total carotenoid as well as each carotenoid such as lutein,
zeaxanthin and b-cryptoxanthin were observed in Hujing fruit
during storage with a slight decrease in b-carotene content. Blue
light did not have any influence on carotenoid changes and profiles
in this cultivar (Fig. 5).

3.6. Effect of blue light treatment on gene expression related to

carotenoid metabolism
Most of genes including PpPSY, PpPDS, PpZDS and PpCHYB
expressed higher in Jinli than in Hujing during storage. However,
the higher carotenoid degradative PpCCD4 gene was detected in
Hujing (Fig. 6). In the control fruit of both two cultivars without
blue light treatment, no significant change in PpFPPS but a decrease
of PpGGPS was observed during storage. Blue light induced the

141

20
15

***

***

***

**

10
5
0

*
S1

S3

S4

Zeaxanthine content ( g/g)

Jinli
Hujing

***
***

1.0

**
*

0.0
S1

S2

S3

S4

S5

Ripening stage
-Carotene content ( g/g)

***

**

1.5

**

**

1.0
0.5

S5

2.0

0.5

Jinli
Hujing

2.0

0.0
S2

Ripening stage

1.5

Lutein content ( g/g)

2.5

Jinli
Hujing

S1

-Cryptoxanthin content ( g/g)

Total carotenoids content ( g/g)

S. Cao et al. / Food Chemistry 214 (2017) 137146

S2

S3

S4

S5

Ripening stage
0.8

Jinli
Hujing

0.6

***

***

**
0.4

0.2
0.0
S1

S2

S3

S4

S5

Ripening stage

Jinli
Hujing

***

***
**

*
1

0
S1

S2

S3

S4

S5

Ripening stage
Fig. 3. Carotenoid content and composition in fruit pulp of two peach cultivars during fruit development. All data is presented as a mean of three biological replicates and
error bars represent standard errors. Asterisks (*) indicate significant differences between the two cultivars at different stages (Students unpaired T test; P < 0.05, P < 0.01,
and P < 0.001).

expression of PpFPPS and inhibited the decline of PpGGPS expression. Consequently, higher transcripts of these two genes were
detected in the blue light treated-peaches regardless of the cultivars. The other six genes such as PpPSY, PpPDS, PpZDS, PpLCYB,
PpCHYB and PpCCD4 in both cultivars showed a similar change pattern during storage. The expression in peaches increased during
the first 5 or 10 days and decreased thereafter with exceptions of
PpZDS and PpLCYB in Hujing. The transcripts of all these genes
were more abundant in light treated-fruit than that in control peaches irrespective of the cultivars (Fig. 6).
4. Discussion
In the white cultivar Hujing in present study, less or no carotenoids were observed in the skin or flesh, however, the transcripts
for all carotenoid biosynthetic genes investigated in this study
were detected, which indicated that the transcriptional regulation
of these genes was not the major reason for the low level of caro-

tenoids in the white peaches. It is well documented that the

biosynthetic or degradative enzyme involved in carotenoid metabolism can regulate carotenoid accumulation (Nisar et al., 2015).
In peach fruit, PpCCD4 has been reported to associate with the
white color by transforming carotenoids into colorless compounds
(Adami et al., 2013; Brandi et al., 2011). Similarly, higher expression of CmCCD4 was related to the much less carotenoid accumulation in white chrysanthemum petals (Ohmiya, Kishimoto, Aida,
Yoshioka, & Sumitomo, 2006). Our results confirmed that the level
of PpCCD4 transcripts in white fleshed-cultivar Hujing was higher
than in Jinli, the yellow one regardless of fruit skin and flesh
which suggested that the biosynthetic carotenoids were gradually
degraded by the highly active PpCCD4 during fruit development,
further resulting into almost undetectable carotenoids levels in
the white fleshed-peaches. Together with the previous reports,
our results here demonstrated that the differential expression of
PpCCD4 plays a determining role in the carotenoid content in white
fleshed peaches.

0.15

PpFPPS

Jinli
Hujing

**

**
0.10

0.05

0.00
S1

S2

S3

S4

S5

Expression relative to PpTEF2

S. Cao et al. / Food Chemistry 214 (2017) 137146

Expression relative to PpTEF2

142

0.5

PpGGPS

0.3

0.2
0.1
0.0
S1

S2

PpPSY

Jinli
Hujing

0
S1

S2

S3

S4

S5

0.15

Jinli
Hujing

**

0.10

1.5

**

*
*

1.0
0.5
0.0
S1

S2

S3

S4

0.00

S5

S1

S2

***

0.5

***

Jinli
Hujing

**

1.0

0.0
S1

S2

S3

S3

S4

S5

0.3

S4

Jinli
Hujing

PpLCYB

**
0.2

**

**

*
0.1

0.0
S1

S2

S3

S4

S5

Ripening stage

S5

Ripening stage

Expression relative to PpTEF2

Expression relative to PpTEF2

PpCHYB

1.5

**

0.05

Ripening stage
2.0

**

**

Expression relative to PpTEF2

Expression relative to PpTEF2

2.0

S5

Ripening stage
Jinli
Hujing

PpZDS

S4

PpPDS

Ripening stage
2.5

S3

Ripening stage
Expression relative to PpTEF2

Expression relative to PpTEF2

Ripening stage

Jinli
Hujing

**

**

0.4

20

PpCCD4

***

15

Jinli
Hujing

10

**
**

*
0
S1

S2

S3

S4

S5

Ripening stage

Fig. 4. Relative transcripts of eight carotenogenic genes in fruit pulp of two peach cultivars during fruit development. The expression level of PpTEF2 (JQ732180) was used to
normalize the mRNA levels for each sample. All data is presented as a mean of three biological replicates and error bars represent standard errors. Asterisks (*) indicate
significant differences between the two cultivars at different stages (Students unpaired T test; P < 0.05, P < 0.01, and P < 0.001).

In the yellow colored cultivar Jinli, the composition and content of carotenoids, was resulted from the difference in carotenogenesis gene transcripts partially. FPPS and GGPS are the early
enzymes in carotenoid biosynthesis, which was reported to be
the key steps in some plants (Fraser & Bramley, 2004; Taylor &
Ramsay, 2005). In apple, MdGGPS expression took an important
part in carotenoid content in different apple cultivars
(Ampomah-Dwamena et al., 2012). Tobacco plant overexpressing
a FPPS gene from Saccharomyces cerevisiae displayed a clear

increase of carotenoid biosynthesis (Daudonnet, Karst, & Tourte,

1997). In our present study, it is quite evident that the upregulation of FPPS and GGPS, contributing to the formation of larger quantity of carotenoid precursors, might induce carotenoid
biosynthesis and accumulation in yellow fleshed-peaches irrespective of the fruit tissues, which suggested that enhanced expression
of key biosynthetic genes may be an important reason for the elevated carotenoids in yellow fleshed-peaches. Similar relationship
was observed between GGPS transcripts and carotenoid content

143

CK-Jingli
Blue light-Jinli
CK-Hujing
Blue light-Hujing

10

**
**

*
*

Zeaxanthine content ( g/g)

*
**

**

0
0

10

15

20

Storage time (d)

0.6

**
0.4

0.2

0.0
0

10

15

20

Storage time (d)

-Carotene content ( g/g)

Lutein content ( g/g)

15

-Cryptoxanthin content ( g/g)

Total carotenoids content ( g/g)

S. Cao et al. / Food Chemistry 214 (2017) 137146

10

15

20

Storage time (d)

6

***
***

**

0
0

10

15

20

Storage time (d)

2.0

**
1.5

*
*

1.0

0.5

0.0
0

10

15

20

Storage time (d)

Fig. 5. Effect of blue LED light on the carotenoid content and composition in two peach cultivars during storage. All data is presented as a mean of three biological replicates
and error bars represent standard errors. Asterisks (*) indicate significant differences between light-treated and control samples (Students unpaired T test; P < 0.05,

P < 0.01, and P < 0.001).

in watermelon (Lv, Li, Liu, Gu, & Zhao, 2015). In addition, it must be
pointed that higher transcripts of PpPDS were also observed from
the early fruit stage (S2) to ripening (S5) in the pulp of yellow peaches in comparison with Hujing. In citrus, the expression of PDS
gene was revealed to link with b-carotene accumulation directly
(Ha, Kim, Park, Lee, & Cho, 2007). High concentration of carotenoids was related to the high PDS transcripts as well in pepper
(Ha et al., 2007). These results combined to suggest that the
increased of PpPDS in the pulp in Jinli might lead to the high level
of carotenoids in this cultivar to some extent.
Cyclisation of lycopene is a critical regulatory point of branching in carotenoid synthesis (Fraser & Bramley, 2004; Taylor &
Ramsay, 2005). LCYB and CHYB play important roles in regulating
the biosynthesis of b-carotene, lutein, zeaxanthin and
b-cryptoxanthin. In chrysanthemum, the differential expression
of LCYB led to the change in carotenoid composition between
petals and leaves (Kishimoto & Ohmiya, 2006). The increased LCYB
and CHYB expression was resulted into carotenoid accumulation in

kiwifruit, (Ampomah-Dwamena et al., 2012). In squash fruit, high

transcript level of CHYB was observed in the high carotenoid cultivars in comparison with the low carotenoid ones (Nakkanong,
Wang, & Zhang, 2012). In this study, PpLCYB expression in Jinli
pulp and peel was more strongly expressed than that in the white
cultivar at the late development stages, possibly contributing to
b-carotene accumulation in Jinli. Meanwhile, the higher expression levels of PpCHYB in flesh and skin of Jinli compared with
Hujing might partially explain the higher levels of lutein, zeaxanthin and b-cryptoxanthin. In contrast, although the transcripts of
PpCCD4 showed an increasing trend during Jinli ripening, its
expression level was much lower than in Hujing. Therefore, the
lower rate of carotenoid degradation with CCD4 gene may be
another factor for the increased carotenoid accumulation in Jinli
as compared to Hujing.
Taken together, our results suggested that no matter the fruit
tissues (peel and pulp) used, the changes in carotenoid profiles in
the yellow fleshed-peaches were linked obviously, but not in

0.4

CK-Jingli
Blue light-Jinli
CK-Hujing
Blue light-Hujing

0.3
0.2

*
0.1

PpFPPS

**
**

**

0.0
0

10

15

20

Expression relative to PpTEF2

S. Cao et al. / Food Chemistry 214 (2017) 137146

Expression relative to PpTEF2

144

0.4

*
*

0.3

*
*

10

0
0

10

15

20

*
*

6
4
2

10

15

20

0
0

0.0
0

0.3

*
*

*
*

2
1
0
0

10

15

15

20

**

*
**

10

15

0.1

0.0
0

20

0.3

PpLCYB

**
**

0.2

*
*

0.1

0.0
0

10

15

20

Ripening stage

*
*

10

**
0.2

20

Storage time (d)

Expression relative to PpTEF2

Expression relative to PpTEF2

PpCHYB

PpPDS

Storage time (d)

5

Storage time (d)

Expression relative to PpTEF2

Expression relative to PpTEF2

PpZDS

0.1

Storage time (d)

10

Storage time (d)

Expression relative to PpTEF2

Expression relative to PpTEF2

PpPSY

*
*

0.2

Storage time (d)

15

PpGGPS

PpCCD4

**

4
3

2
1

10

15

0
0

20

Storage time (d)

Fig. 6. Effect of blue LED light on the expression of carotenogenic genes in two peach cultivars during storage. The expression level of PpTEF2 (JQ732180) was used to
normalize the mRNA levels for each sample. All data is presented as a mean of three biological replicates and error bars represent standard errors. Asterisks (*) indicate
significant differences between light-treated and control samples (Students unpaired T test; P < 0.05, P < 0.01, and P < 0.001).

complete parallel, with the expression pattern of certain genes

such as FPPS and GGPS in the methylerythritol phosphate pathway
and PpLCYB and PpCHYB in the carotenoid biosynthetic pathway

during fruit development. However, in terms of the flesh in yellow

fleshed-fruit, in addition to the higher transcripts of the above four
genes, the higher expression of PpPDS and lower transcripts of

S. Cao et al. / Food Chemistry 214 (2017) 137146

PpCCD4 genes, as compared to the white cultivar, also coincided

with the higher level of carotenoids, suggesting that both of the
two genes might be involved in carotenoid accumulation in pulp
of yellow fleshed-fruit during maturation. It is noteworthy that
total carotenoids in the flesh in yellow peaches showed higher
levels compared with those in the peel during development,
although the gene expression profile in the flesh was similar to that
in peel. This suggested that in peach, other factors in addition to
gene expression levels, govern carotenoids accumulation, which
needs to be further studied.
During storage, carotenoid accumulated in yellow fleshedpeaches, however, no significant change was observed in the white
cultivar. Similarly to the white fleshed-peaches during maturation,
in the white peaches during storage, the discrepancy between carotenoid level and expression profile of carotenoid biosynthetic
genes indicated that high PpCCD4 gene expression during storage
might be also concomitant with the low level of carotenoids in this
accession. However, the pattern of changes in gene expression in
yellow fleshed-peaches during storage was different from that in
fruit during development. As compared with the white cultivar,
besides the higher transcription of PpCHYB and lower expression
of PpCCD4 gene, the yellow fruit also experienced higher transcripts of PpPSY and PpZDS, two important genes for the linear carotenoid formation, which was also probably responsible for the
higher concentration of carotenoids in this cultivar during storage.
Blue light plays an essential role in chloroplast development,
stomata opening, and shoot growth (Wu et al., 2007; Zhang
et al., 2012, 2015), which has been reported as an effective carotenoid accumulation inducer in plants. Blue light irradiation
enhanced the expression levels of genes involved in the carotenoid
biosynthesis in sprouts of Tartary buckwheat (Tuan et al., 2013).
Blue light treatment could also induce carotenoid accumulation
via regulating the expression of carotenoid biosynthetic genes in
the juice sacs of Satsuma mandarin and Valencia orange (Zhang
et al., 2012, 2015). In yellow-fleshed peaches, blue light treatment
during storage not only induced carotenoid level but also brought
about important changes in the gene expression involved in carotenoid metabolism. Blue light up-regulated the expression of all
the genes analysed with a lesser extent on PpCCD4, which indicated that as compared to the control fruit, the much more
increased carotenoid synthesis rate could result into the higher
carotenoid content in blue light-treated fruit. With respect to
white peaches, it is noticeable that the expression of all the genes
analysed was stimulated by blue light as well, however, no significant difference on carotenoid composition and content was
observed between control and light-treated peaches. The results
confirmed here again that in white fleshed-peaches, alteration in
the pattern of these biosynthetic genes not to be the main factor
responsible for the low carotenoid content. The trace level of carotenoids observed in white peaches treated with blue light might be
linked to the high transcription of carotenoid degradative gene
during storage.
In conclusion, accumulation of carotenoids was observed in
peel and flesh of yellow fleshed-peaches during fruit maturation.
The expression of PpFPPS, PpGPPS, PpLCYB and PpCHYB might be
contributed to the carotenoid accumulation in the peel of this
accession during fruit maturation. Besides these four genes, PpPDS
might also play an important role in carotenoid biosynthesis in the
pulp. However, in the white cultivar, the transcript of PpCCD4 was
likely to be the major determinant in carotenoid content. Our
results also showed blue light was effective in inducing the carotenoid accumulation in yellow fleshed-peaches after harvest. Results
on expression profiles of genes in carotenoid metabolism indicated
that the much more increased carotenoid synthesis rate induced by
blue light could result into the higher carotenoid content in treated
fruit. Future researches on the protein expression and post-

145

translational control, as well as the potential role of allelic

differences and plastid biogenesis, will be needed to understand
carotenoid metabolism in peaches.
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (31371866 and 31571905), the Natural
Science Foundation of Zhejiang Province (LQ15C200004) and
Natural Science Foundation of Ningbo (2015A610262).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
07.085.
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