Postharvest Biology and Technology 74 (2012) 108117

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Postharvest Biology and Technology

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Stimulation of coloration and carotenoid biosynthesis during postharvest storage

of Navelina orange fruit at 12 C
Lourdes Carmona, Lorenzo Zacaras, Mara J. Rodrigo
Instituto de Agroqumica y Tecnologa de Alimentos, Consejo Superior de Investigaciones Cientcas (IATA-CSIC), Av. Agustn Escardino 7, 46980 Paterna, Valencia, Spain

a r t i c l e

i n f o

Article history:
Received 4 April 2012
Accepted 17 June 2012
Keywords:
Carotenoids
Citrus
Color
Gene expression
Orange
Storage temperature

a b s t r a c t
The effect of storage temperature on color, carotenoid content and composition, and the expression of
key carotenoid biosynthetic genes in fruit of Navelina orange were evaluated. Fruit were harvested at
two maturation stages, before color break (breaker stage) and with a light-orange coloration (colored),
and stored at 2 and 12 C and 9095% RH for up to 7 weeks. At the two maturation stages, storage
at 12 C considerably increased total carotenoid content and enhanced coloration in both avedo and
pulp. In fruit stored at 2 C, coloration and carotenoid content remained almost unchanged. The increase
in peel color during storage at 12 C was mainly related to an increment in the concentration of the
reddish C30-apocarotenoid, -citraurin, and to a minor extent to antheraxanthin. The content of cryptoxanthin, a ,-xanthophyll with pro-vitamin A activity, increased two and three times in the pulp
of breaker and colored fruit, respectively, after 7 weeks of storage at 12 C. The expression of the genes
PSY (phytoene synthase), PDS (phytoene desaturase), ZDS (-carotene desaturase), LCY1 and LCY2 (lycopene cyclase 1 and 2) and CHX (-carotene hydroxylase) increased during storage at 12 C in the
peel of fruit at both maturation stages. At 2 C, by contrast, expression of these genes was maintained or
slightly declined. The pattern of changes in gene expression in the pulp of orange fruit stored at 12 C
was dependent of the ripening stage and not always related to the increment in carotenoid content and
composition. Collectively, these results indicate that the stimulation of carotenoid biosynthesis during
storage of Navelina orange fruit at 12 C improve not only peel and pulp coloration, but also pro-vitamin A
activity of the esh, and may then be a postharvest strategy to increase the nutritional and health-related
benets of citrus fruit.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Carotenoids are a large family of isoprenoid compounds which
impart attractive colors to many fruit and vegetables (Hirschberg,
2001; DellaPenna and Pogson, 2006). Carotenoids are also components of the human diet and have important antioxidant activity
and protective effects against carcinogenesis, cardiovascular diseases and degenerative processes (Fraser and Bramley, 2004;
Lichtenstein, 2009; Nishino et al., 2009; Yamaguchi and Uchiyama,
2009). Moreover, carotenoids with at least one unsubstituted ionone ring like -carotene or -cryptoxanthin, are the precursors
of vitamin A (Melndez-Martnez et al., 2005; Edem, 2009).
Citrus fruit represent an important source of carotenoids for the
human diet due to the massive consumption worldwide as both
fresh fruit and juice (Stewart, 1977a; Melndez-Martnez et al.,
2007). The carotenoid complement in citrus fruit is rather complex,
as more than 110 different carotenes and xanthophylls have been

Corresponding author. Tel.: +34 963900022; fax: +34 963636301.

E-mail address: mjrodrigo@iata.csic.es (M.J. Rodrigo).
0925-5214/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.postharvbio.2012.06.021

reported, although some of them may be artifacts, they are responsible for the external and internal coloration of fruit of the main
citrus species (Stewart and Wheaton, 1973; Gross, 1987; Alquzar
et al., 2008a). Moreover, the external color of citrus fruit is one of
the main attributes of commercial quality and a major determinant of consumer acceptance. The characteristic color of the fruit
of diverse citrus species and varieties, from the yellow of lemons
and white grapefruit, the intense orange of mandarins and oranges,
to the red of some grapefruit and shaddocks, is each provided by
a specic carotenoid composition (Gross, 1987). In particular, the
peel and pulp color of sweet orange and mandarin is mainly determined by different ratios of ,-xanthophylls and also by some
citrus specic C30 -apocarotenoids (-citraurin) (Stewart, 1977b;
Oberholster et al., 2001; Kato et al., 2004; Rodrigo et al., 2004). A
schematic representation of the carotenoids biosynthesis pathway
is shown in Fig. 1. During maturation of oranges and mandarins, the
massive increase in ,-xanthophylls is mainly due to accumulation of 9-Z-violaxanthin and -cryptoxanthin, respectively (Kato
et al., 2004; Rodrigo et al., 2004; Alquzar et al., 2008a).
In recent years, important research has been conducted to
understand the molecular regulation of carotenoid biosynthesis

L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

2 x GGPP
PSY

phytoene
PDS

phytofluene
PDS

-carotene
ZDS
LCY
LCY

lycopene

-carotene

LCY1
LCY2

-carotene

CHX

CHX

-cryptoxanthin

-cryptoxanthin
CHX

CHX

lutein

CCD

-citraurin

zeaxanthin
VDE

ZEP

All-E-violaxanthin
All
E violaxanthin
9-Z-violaxanthin
NSY

neoxanthin
Fig. 1. Schematic diagram of the biosynthesis pathway of carotenoid in citrus fruit.
Genes for which expression has been analyzed in this work are underlined and
bold-lettered. An hypothetical step of -citraurin biosynthesis is indicated with a
discontinuous arrow. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, carotene desaturase; LCY, lycopene -cyclase; LCY1, lycopene -cyclase 1; LCY2,
lycopene -cyclase 2; CHX, -carotene hydroxylase; CHX, -carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NSY, neoxanthin
synthase; CCD, carotenoid cleavage dioxygenase.

and accumulation in citrus fruit, and the genes encoding enzymes

of the main steps of the pathway have been identied and their
expressions have been analyzed, mainly during natural ripening (Fig. 1) (Kato et al., 2004; Rodrigo et al., 2004; Liu et al.,
2007; Alquzar et al., 2008a,b; Fanciullino et al., 2008; Mendes
et al., 2011; Costa et al., 2012). The general model emerging
from these studies indicates that the massive increase in total
carotenoids and the shift from the ,-branch to the ,-branch
occurring in the peel of orange and mandarin fruit during maturation is mainly controlled by transcriptional regulation of key
genes of the pathway. During the transition from chloroplast
to chromoplast, it has been reported a signicant induction
of the expression of the genes involved in early steps of the
pathway (phytoene synthase, PSY; phytoene desaturase, PDS
; -carotene desaturase, ZDS), cyclization of lycopene (the
chromoplast-specic isoform LCY2) and the biosynthesis of
,-xanthophylls (-carotene hydroxylase; CHX) occur, concomitantly with a decrease in the expression of the lycopene
-cyclase (LCY) gene (Kato et al., 2004; Rodrigo et al., 2004;
Alquzar et al., 2009). However, physiological and molecular
studies on the changes in carotenoid content and composition
occurring during postharvest storage of citrus fruit are still scarce,
and are mainly limited to the effect of exogenous ethylene accelerating peel degreening (Fujii et al., 2007; Rodrigo and Zacaras,
2007; Matsumoto et al., 2009; Mayuoni et al., 2011).
Temperature is one of the most important environmental factors affecting citrus fruit color, and its effect during the growing

109

season has been widely reported (Eilati et al., 1969; Lee and Castle,
2001; Matsumoto et al., 2007). It is generally recognized that for citrus fruit, optimal temperatures for the transition from chloroplasts
to chromoplasts and for the induction of carotenogenesis are relatively low (Gross, 1987). The relationship between color change
and the requirement of a cold period has been established and
degradation of chlorophylls during ripening is promoted at night
temperatures below 13 C (Stearns and Young, 1942). In addition,
large differences in day/night temperatures (about 20/7 C) stimulate accumulation of xanthophylls (Young and Erickson, 1961).
Thus, under tropical climates with high day temperatures and
low differences between day/night temperatures, the peel of citrus fruit does not develop the characteristic orange coloration
and remains yellow-greenish (Young and Erickson, 1961; Reuther
and Rios-Castano, 1969; Barry and Van Wyk, 2006). In general,
high temperature promotes high concentration of chlorophylls
and impairs the increase in specic carotenoids (Stearns and
Young, 1942; Young and Erickson, 1961; Agust, 1999), mainly
,-xanthophylls and the reddish apocarotenoids, e.g. -citraurin
(Stewart and Leuenberger, 1976), whereas low temperatures produce the opposite effects, accelerating degreening and increasing
carotenoid content (Sonnen, 1977). Accordingly, it has been suggested that 1214 C might be the optimal temperatures for
-cryptoxanthin, violaxanthin and -citraurin biosynthesis in citrus fruit on the tree (Sonnen, 1977; Casas and Mallent, 1988;
Alquzar et al., 2008a). Despite this narrow temperature range for
optimal peel coloration, the pulp develops its characteristic color
independent of the eld temperature, indicating an autonomous
regulation of carotenoid biosynthesis in both fruit tissues (Tadeo
et al., 2008).
Storage at temperatures between 1 and 4 C are commonly used
to extend the commercial life and to maintain quality of citrus
fruit. Moreover, cold-quarantine treatments at temperatures
between 0.5 and 2 C are required for exportation of citrus to the
USA or Japan in order to eliminate Mediterranean fruit y (Ceratitis
capitata Wied) (El-Otmani et al., 2011). However, fruit of different
citrus species and cultivars are sensitive to developing many
chilling injury symptoms when stored at temperatures below
10 C (Lafuente and Zacaras, 2006). The relationship between
changes in fruit color and the storage temperature has only been
established in a few varieties of citrus. In Eureka and Villa franca
lemons, optimum coloration and low incidence of chilling injury
were attained at 14 C (Cohen and Schiffmann-Nadel, 1978).
An enhancement of fruit color was also obtained for the hybrid
Ortanique and Ponkan mandarins stored at temperatures around
10 C (Cohen et al., 1990; Zhu et al., 2011), and for Or and Odem
mandarins stored at 8 C, while low storage temperatures (2 and
5 C) caused a loss of peel color (Tietel et al., 2012). Moreover,
Clemenules mandarins subjected to cold shocks followed by
3 days of incubation at 20 C increased the external coloration
(Barry and Van Wyk, 2006). In Palmer Navel oranges it has been
observed that the detrimental effect of storage at sub-zero temperatures on peel color and carotenoid content could be reverted
by a subsequent storage at intermediate temperatures between
11 and 15 C but not at low (4.5 C) or high (20 C) temperatures
(Van Wyk et al., 2009). Storage of Satsuma mandarins at 5 C
slowly increased carotenoids in the avedo but reduced in the
pulp, and decreased the expression of key carotenoid biosynthetic
genes in both tissues (Matsumoto et al., 2009). Collectively, these
results indicate that peel color remains unaltered or diminished in
fruit stored at cold temperatures (<5 C) while coloration and/or
carotenoid accumulation appears to be stimulated at temperatures
between 8 and 15 C. Therefore, the objective of this study was to
investigate the physiological and molecular basis of the enhancement of coloration and carotenoid biosynthesis in both peel and
pulp of Navelina sweet orange (Citrus sinensis L. Osbeck) fruit by

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L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

prolonged storage at 12 C and to compare the changes with that

occurring under standard refrigerated storage (12 C).

prevent photodegradation, isomerizations and structural changes

of the carotenoids. Each sample was extracted at least twice.

2. Materials and methods

2.4. HPLC analysis of individual carotenoids

2.1. Plant material and storage conditions

Carotenoid composition of each sample was analyzed by

HPLC with a Waters liquid chromatography system equipped
with a 600E pump and a 996 photodiode array detector, and
Empower software (Waters, Barcelona, Spain). A C30 carotenoid
column (250 mm 4.6 mm, 5 m) coupled to a C30 guard column
(20 mm 4.0 mm, 5 m) (YMC Europe GMBH, Germany) was used.
Samples were prepared for HPLC by dissolving the dried carotenoid
extracts in CHCl3 :MeOH:acetone (3:2:1, v:v:v). A ternary gradient elution was used for carotenoid separation. The initial solvent
composition consisted of 90% MeOH, 5% water and 5% methyl tertbutyl ether (MTBE). The solvent composition changed in a linear
fashion to 95% MeOH and 5% MTBE at 12 min. During the next
8 min the solvent composition was changed to 86% MeOH and 14%
MTBE. After reaching this concentration the solvent was gradually
changed to 75% MeOH and 25% MTBE at 30 min. After 20 min, the
solvent composition changed linearly, being 50% MeOH and 50%
MTBE at 50 min. The initial conditions were re-established in 5 min
and equilibrated for 15 min before the next injection. The ow rate
was 1 mL min1 , column temperature was set to 25 C and the injection volume was 20 L. The photodiode array detector was set to
scan from 250 to 540 nm, and for each elution a Maxplot chromatogram was obtained, which plots each carotenoid peak at its
corresponding maximum absorbance wavelength.
Carotenoids were identied by their retention time, absorption and ne spectra (Rouseff et al., 1996; Britton et al., 1998;
Rodrigo et al., 2003, 2004). The carotenoid peaks were integrated
at their individual maxima wavelength and their contents were
calculated using calibration curves of -apo-8 -carotenal (a gift
from Hoffman-LaRoche) for -citraurin; -carotene (Sigma) for and -carotene; -cryptoxanthin (Extrasynthese) for - and cryptoxanthin; lutein (Sigma) for lutein and violaxanthin isomers,
and zeaxanthin (Extrasynthese) for zeaxanthin and antheraxathin.
Standards of phytoene and phytouene for quantication were
obtained from avedo extracts of Pinalate orange fruit, which
accumulates large amounts of these compounds (Rodrigo et al.,
2003), puried by TLC (Pascual et al., 1993), and calibration curves
were prepared.

In this study fruit of Navelina sweet orange (C. sinensis

L. Osbeck), a typical representative of the Navel orange group,
was selected for the following reasons: (1) the fruit is of excellent
organoleptic quality and highly appreciated by consumers for fresh
consumption and (2) the peel of mature fruit develops a brightorange color thus being especially suitable for on-peel coloration
studies. Navelina fruit were harvested at random from adult trees
grown in a commercial orchard under standard conditions. Fruit
were uniform in size and color, and free of damage and external
defects. After fruit color determination, fruit were divided into two
lots and stored at 2 C (low temperature) and 12 C (intermediate
temperature) and 9095% RH in constant darkness. Three replicate samples of 10 fruit per storage temperature were used to
follow change of fruit color during storage. For analysis of internal maturity, carotenoid content and composition, and expression
of carotenoid genes, three replicate samples of six fruit each per
temperature and storage time were used. Flavedo (the outer colored part of the fruit peel) and pulp were separated with a scalpel,
immediately frozen in liquid nitrogen, ground to a ne powder and
stored at 80 C until analysis.
2.2. Determination of color and internal maturity index
Fruit color of the peel was measured using a Minolta CR-330
chromameter on three locations around the equatorial plane of the
fruit. Hunter parameters a (negative to positive correspond from
green to red, respectively) and b (negative to positive, from blue to
yellow, respectively) were determined and coloration is expressed
as the a/b Hunter ratio, a relationship for color measurement in citrus fruit (Stewart and Wheaton, 1971). Fruit were harvested at two
different maturity stages: just before the onset of fruit coloration
(color break stage) with an a/b ratio 0.11 0.02 and colored, with
an a/b ratio 0.44 0.01. For analysis of internal maturity, juice from
fruit was extracted with a household electric hand reamer, ltered
through a metal sieve with a pore size of 0.8 mm and analyzed
immediately. The acidity of the juice was determined by titration
with 0.1 N NaOH and is expressed as mg citric acid per 100 mL
and the soluble solids content ( Brix) by refractrometry, using an
Atago model PR32. The maturity index is expressed as the ratio of
Brix/acidity.

2.5. Total RNA isolation

Plant material used for total RNA isolation was the same as that
used for pigment analysis. Total RNA extraction from peel and pulp
of Navelina orange fruit was performed as described previously
(Alquzar et al., 2008b).

2.3. Chlorophyll and total carotenoid extraction and

quantication

2.6. Quantitative RT-PCR analysis

Fruit pigments were extracted as described previously (Rodrigo

et al., 2003), from 0.5 g of avedo or 1.5 g of pulp. Briey, the chlorophyll (a + b) content was determined by measuring the absorbance
at 644 and 662 nm of the extract and calculated according to Smith
and Benitez equations (Smith and Benitez, 1955). After chlorophyll
measurement, the pigment ether solution was dried and saponied using a 10% methanolic KOH solution. The carotenoids were
subsequently re-extracted with diethyl ether until the hypophase
was colorless. An aliquot of the ether extract was used for quantication of total carotenoids content, and this was calculated by
measuring the absorbance of the saponied extract at 450 nm,
using an extinction coefcient of -carotene, E1% = 2500 (Davies,
1976). The samples were dried under N2 and kept at 20 C until
analysis. All operations were carried out on ice under dim light to

Total RNA was treated with DNase (Ambion, Huntingdon, UK)

and accurately quantied by uorometric assay with the RiboGreen
dye (Molecular Probes) following the manufacturers instructions,
in order to normalize RNA levels as described by Als et al. (2006).
Quantitative real-time PCR was performed with a LightCycler
2.0 Instrument (Roche) and data was analyzed using LightCycler
Software version 4.0. One-step RT-PCR was carried out on 100 ng
total RNA adding 2.5 units of MultiScribe Reverse Transcriptase
(Applied Biosystems), 1 unit Rnase Inhibitor (Applied Biosystems),
2 L LC FastStart DNA MasterPLUS SYBR Green I (Roche) and
0.5 M of gene specic primers in a total volume of 10 L. Primers
pairs for PSY [MJ134(sense) 5 -GGTCGTCCATTTGATATGCTTG-3
and MJ135 (antisense) 5 -CCTAAGGTCCATCCTCATTCCT-3 ],
PDS [PDS-F (sense) 5 -TCCCTTCTAAGTGTGTATGCC-3 and

L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

111

PDS-R (antisense) 5 -TGCAAGCTCCTTCATTGTAGCA-3 ], ZDS

[ZDS-F (sense) 5 -ACAATCTGTTTGAGGCGCAG-3 and ZDS-R
(antisense)
5 -CATAGGTATTGGAAACCCTTACTCC-3 ],
-LCY1
[MJ136 (sense) 5 -GAACCAGGAGCTTAGGTCTG-3 and MJ137
(antisense) 5 -GCTAGGTCTACAACAAGGCC-3 ], -LCY2 [MJ130
(sense) 5 -CCCTATTTCCATTAGGCCGC-3 and MJ138 (antisense)
5 -CACGTCATATCGAATACGATC-3 ] and -CHX [MJ126 (sense)
5 -GGCTCATAAAGCTCTGTGGC-3 and MJ127 (antisense) 5 CCAGCACCAAAACAGAGACC-3 ] were designed based on citrus
coding sequences isolated from fruit and available in the GenBank
public database (AY204550, AJ319761, AJ319762, AY094582,
AF169241 and DQ228870, respectively) and based on conserved
regions among different citrus species. The RT-PCR procedure
consisted of 48 C 30 min, 95 C 10 min followed by 35 cycles at
95 C 10 s, 55 C 5 s and 72 C 10 s. Fluorescence intensity data were
acquired during the 72 C extension step and specicity of the
reactions was checked by post-amplication dissociation curves
and sequencing the reaction products. To transform uorescence
intensity measurements into relative transcripts levels, a 10-fold
dilution series of a RNA sample was used as a standard curve.
Values were the mean of at least three independent analyses and
expression value of 1 was arbitrary assigned to the initial sample
of fruit at the color break stage.

3. Results
3.1. Changes in external fruit color, maturation index, and total
carotenoids and chlorophylls content in Navelina orange fruit
during storage at 12 C and 2 C

Fig. 2. External and internal appearance of Navelina oranges during storage at 2 C

and 12 C and 9095% RH for 7 weeks. (A) Fruit harvested at color break stage with
an a/b ratio = 0.11 0.02 and (B) colored fruit with an a/b ratio = 0.44 0.01.

External color (Hunter a/b)

during storage at 12 C of fruit harvested at the two stages followed a similar pattern. A three-fold increase in total carotenoid
was detected after 7 weeks of storage, from 22 to 62 g g1 FW in Br
fruit and from 32 to 92 g g1 FW in Co fruit (Fig. 4A and B). At 2 C,
changes in carotenoid content appeared to be dependent on the
ripening stage, since it was almost double in the avedo of Br fruit
but remained fairly constant in the Co fruit (Fig. 4A and B). In the
pulp, changes in total carotenoid content in Br and Co fruit stored at
12 C increased in a similar trend as in the peel (a two-fold increase)
but at 2 C carotenoid content barely changed (Fig. 4C and D).

1,2
1,0

1,2

1,0

0,8

0,8

0,6

0,6

0,4

0,4

0,2

0,2
2 C
12 C

0,0
-0,2
0

Weeks

0,0

External color (Hunter a/b)

To determine the inuence of the ripening stage on the development of fruit color, maturation index, and pigment contents during
storage of Navelina oranges at 12 C and 2 C, fruit were harvested
at two developmental stages: breaker (Br, a/b ratio 0.11) and
colored (Co, a/b ratio 0.44). In Fig. 2 the changes in external and
internal fruit color during 7 weeks of storage at 12 C and 2 C are
shown. A remarkable enhancement of peel and pulp color occurred
at 12 C, which was more evident in Br fruit. In fruit stored at 2 C no
visual differences were observed compared with freshly harvested
fruit. Peel color index (a/b Hunter) in fruit harvested at both stages
increased rapidly during the rst 34 weeks of storage at 12 C and
then remained nearly constant until the end of the storage period
(Fig. 3). Interestingly, the rate of coloration and then the increment
in peel color was higher in Br than in Co fruit (Fig. 2). Storage at
low temperature (2 C) only resulted in a slight increase (Br fruit)
or no change (Co fruit) in peel color index (Fig. 3), in agreement
with visual examination.
The maturation index (MI) is a critical parameter determining
the harvesting date; therefore, the inuence of storage temperature
on internal fruit quality was also evaluated. As expected, Navelina
fruit harvested at the Co stage showed a higher MI (7.6) than fruit at
the Br stage (6.1). Nevertheless, MI remaining fairly constant during the storage period and no signicant differences were observed
between fruit stored at 2 C and 12 C harvested at the two maturation stages (Table 1).
The content of chlorophylls (Chl) and total carotenoids were
analyzed in the avedo and pulp of Navelina oranges at ve-time
points during storage at 2 C and 12 C. Chl content in the avedo
of freshly harvested fruit at the Br stage was 22.14 g g1 FW and
decreased to 1015 g g1 FW after 7 weeks at 2 C. In the avedo
of fruit stored at 12 C, Chl content declined rapidly to values
below 1 g g1 FW. Chl concentration was also much lower than
1 g g1 FW in freshly harvested Co fruit and therefore was not
analyzed after subsequent storage. The changes in total carotenoid
content (expressed as g of -carotene equivalents) in the avedo

-0,2

Weeks

Fig. 3. Changes in peel color of Navelina orange during storage at 2 () and 12 C
() and 9095% RH for 7 weeks. (A) Fruit harvested at color break stage an a/b
ratio = 0.11 0.02 and (B) colored fruit with a ratio a/b = 0.44 0.01. Fruit color is
expressed as the a/b Hunter ratio. Data are means SD of three replicate samples of
10 fruit each for storage temperature.

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L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

Table 1
Changes in internal maturation index ( Brix/acidity) of Navelina orange fruit harvested at color break stage or colored stage during storage at 2 C and 12 C. Data are the
mean and SE from al least three measurements from three independent lots.
Temperature ( C)

Fruit stage

Maturation index ( Brix/acidity)

Storage time (weeks)
0

Color break

2
12

6.1 0.4

6.7 0.6
6.2 0.3

6.3 0.5
7.1 0.5

7.1 0.7
6.6 0.4

Colored

2
12

7.6 0.5

7.9 0.6
7.6 0.3

7.3 0.6
8.2 0.5

7.9 0.5
8.7 0.6

100

80

80

60

60

40

40

20

20
0

0
20

20

15

15

10

10

2 C
12 C

0
0

Weeks

Caro
otenoids (g g FW)

Carotenoids (g g FW)

100

5
0

Weeks

Fig. 4. Changes in total carotenoid content in peel (A and B) and pulp (C and D)
of Navelina orange during storage at 2 C () and 12 C () and 9095% RH for 7
weeks. (A and C) Fruit harvested at color break stage with an a/b ratio = 0.11 0.02
and (B and D) colored fruit with an a/b ratio = 0.44 0.01. Data are means SD of at
least three independent measurements.

3.2. Changes in carotenoid composition in the peel and pulp of

Navelina orange fruit during storage at 12 C and 2 C
The effect of the storage temperature on individual carotenoids
in avedo and pulp of Navelina orange harvested at two different maturity stages was also investigated. Six major carotenoids
were quantied by HPLCPDA analysis, accounting for more than
90% of total carotenoids in all the samples analyzed. Additional
carotenoids characteristic of chloroplastic tissues were also identied in the avedo (lutein, -carotene, -cryptoxanthin and
neoxanthin) and pulp (lutein and zeaxanthin) of freshly harvested
Br fruit, but their concentrations were below 0.5 g g1 FW and
remained fairly constant or disappeared during storage.
In the avedo of freshly harvested fruit at both stages, violaxanthin (sum of both isomers, all-E- and 9-Z) was the major
carotenoid, making up more than 70% of the total carotenoid content, followed by other ,-xanthophylls (-cryptoxanthin and
antheraxanthin) and the C30 apocarotenoid -citraurin (Fig. 5).
The upstream carotene phytoene was barely detectable in Br fruit
but was higher (3 g g1 FW) in Co fruit. The levels of phytouene
were very low in the avedo of fruit at the two stages. Storage
at 12 C produced a remarkable increase in the content of most
carotenoids in the avedo of fruit at both stages of maturation
(Fig. 5). After 7 weeks at 12 C, the concentration of the initial
carotenes of the pathway, phytoene and phytouene, increased to
24 and 810 g g1 FW, respectively, regardless of the initial concentration. By contrast, in the avedo of fruit stored at 2 C the
levels of phytoene and phytouene were about 3 and 1 g g1 FW,
respectively, for the same duration of storage. The concentration of
9-Z-violaxanthin, the major xanthophyll, remained constant during
storage at 2 C and slightly increased at 12 C (Fig. 5). The content

of other minor xanthophylls, such as antheraxanthin, was twotimes higher in fruit stored at 12 C than at 2 C, irrespective of the
maturity stage. The concentration of -cryptoxanthin in the avedo
was relatively low and in both maturity stages it increased during storage and with few exceptions, small differences were found
between fruit stored at 2 or 12 C (Fig. 5). One of the most significant effects of storage at 12 C was the important increase in the
content of the apocarotenoid -citraurin. After 3 weeks of storage,
the content of -citraurin in the avedo of fruit stored at 12 C was
about twice that in those kept at 2 C. By week 7, the content of the
apocarotenoid was eight and four times higher in Br and Co fruit,
respectively, stored at the higher temperature (Fig. 5). As a result
of these changes, the ratio 9-Z-violaxanthin/-citraurin which has
been described to be inversely correlated with external orange
coloration (Oberholster et al., 2001) was signicantly lower in fruit
stored at 12 C than in those at 2 C (Table 2).
The initial composition of carotenoids in the pulp was similar in
fruit at both maturity stages and the effect of the storage temperature on the carotenoid prole was analyzed. The content of colorless
carotene phytoene was very low and remained nearly constant in
both Br and Co fruit during storage at 2 and 12 C (data not shown).
Importantly, the storage temperature had a marked differential
effect on the concentration of the main ,-xanthophylls. The content of -cryptoxanthin increased seven and 14 times, with respect
to the initial levels, in the pulp of Br and Co fruit, respectively, during storage at 12 C, whereas at 2 C only a three-fold increase was
observed (Fig. 6). A similar prole was observed in the change in
antheraxanthin, showing a 4- and 11-fold increment in Br and Co
fruit, respectively, during storage at 12 C. The content of violaxanthin, which was the main carotenoid in the pulp, was also enhanced
during storage at 12 C but to a lower extent with respect to other
xanthophylls. By contrast, in fruit maintained at 2 C the content of
this xanthophyll remained constant or increased (Fig. 6). The apocarotenoid -citraurin was not detected in any of the pulp samples
analyzed, in agreement with previous data (Gross, 1987).
3.3. Expression of carotenoid biosynthesis genes in the peel and
pulp of Navelina orange fruit during storage at 12 C and 2 C
To characterize the effect of storage temperature on the regulation of carotenoid biosynthesis and whether these changes are
related to the observed effects on carotenoids concentration, the
pattern of expression of six biosynthesis genes in the peel and pulp
of fruit storage at 2 C and 12 C was analyzed. Selected genes of the
pathway were: PSY, the initial step of carotenoid production; PDS
and ZDS, two desaturases at the early steps of the pathway, -LCY1
and the chromoplast-specic -LCY2 responsible for the shift from
,-branch to the ,-branch and the formation of -carotene, and
nally, -CHX, responsible for ,-xanthophylls formation (Fig. 1).
In general, the expression of the six genes analyzed was stimulated in the avedo of fruit stored at 12 C and remained constant
or declined at 2 C, irrespective of the maturity stage (Fig. 7). In Br
fruit, the expression prole of PSY, PDS, ZDS, -LCY1 and -CHX at
12 C was very similar, with a maximum at 3 weeks of storage and

L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

113

Table 2
Relationship between coloration (Hunter a/b) and the ratio of 9-Z-violaxanthin/-citraurin in the avedo of Navelina orange fruit harvested at two stages and stored at 2 C
and 12 C.
Fruit stage

Storage temperature ( C)

Weeks

Color (Hunter a/b)

Color break

3
7
3
7

0.00
0.15
0.67
0.75

0.05
0.02
0.03
0.04

8.2
6.6
4.2
2.8

3
7
3
7

0.42
0.41
0.79
1.02

0.02
0.03
0.03
0.04

7.7
7.1
3.0
2.5

12

Colored

2
12

decreasing thereafter (Fig. 7A). The expression of -LCY2 was also

stimulated but peaked after 5 weeks of storage. In the avedo of
Co fruit, accumulation of PSY and -CHX transcripts was similar to
that reported for Br fruit (Fig. 7B). However, the transcript PDS and
ZDS remained fairly stable and -LCY1 was progressively induced
during storage while that of -LCY2 exhibited a large increment by
week 7 (Fig. 7B). The expression of the six carotenoid biosynthesis
genes, and at the two ripening stages, was sustained at levels similar
to initial levels or decreased during storage at 2 C (Fig. 7A and B).
The effect of storage temperature on the expression of
carotenoid biosynthesis genes was also examined in the pulp
of fruit at the two maturity stages. Exposure of Br fruit to 12 C
produced a transient increase in the expression of most the genes
examined (Fig. 8A). Accumulation of the transcripts PDS, ZDS,
-LCY2 and -CHX transiently increased between ve- and twofold by 1 week of storage, while accumulation of -LCY1 peaked at
3 weeks of storage at 12 C. The expression of PSY decreased during
storage at both temperatures. In freshly harvested Co fruit, the
expression of the PSY, -LCY1, -LCY2 and -CHX genes was higher
than that of Br fruit, in accordance with their advanced maturation
and coloration. In pulp of Co fruit the expression of PSY, -LCY2
and -CHX genes declined during storage at both 2 C and 12 C,
while PDS and ZDS remained fairly constant and -LCY1 transiently
increased after 1 week of storage at 12 C (Fig. 8B). In general, in
the pulp of fruit at both maturity stages the expression of all genes
decreased during storage at low temperature (2 C) (Fig. 8B).
4. Discussion
Evidence accumulated over numerous years has established
strict eld temperature requirements for optimal induction of
coloration in citrus fruit, such as night temperatures below 12 C
and marked differences between day/night temperatures (Young
and Erickson, 1961; Meredith and Young, 1969; Sonnen et al.,
1979; Agust, 1999). Under postharvest conditions, it has also been
observed that cold-shock followed by storage at intermediate
temperatures may improve external pigmentation of Palmer
Navel orange (Van Wyk et al., 2009) and short storage at low
temperatures resulted in yellowish Or and Odem mandarins,
while intermediate temperatures promoted orange color intensity
(Tietel et al., 2012). However, whether postharvest temperatures
similar to those inductive of coloration under eld conditions
have a direct effect on the carotenoid prole and/or carotenoid
biosynthesis during storage is still unknown. Results of the present
study show that storage at 12 C stimulated the expression of
carotenoid biosynthesis genes and increased carotenoid contents,
and hence enhanced fruit coloration in both avedo and pulp of
Navelina orange fruit, whereas at 2 C these parameters were
almost unaffected. Although the stimulation of gene expression
was higher in Br than in Co fruit, the relative increase in total
carotenoids and in specic carotenoids, e.g. -citraurin in the peel
or -cryptoxanthin in the pulp, were similar for both maturation
stages. Thus, carotenogenesis in citrus fruit appears to be an

Ratio 9-Z-violaxanthin/-citraurin

active process maintained under postharvest temperatures around

12 C without apparent requirement of marked alterations in the
temperature regime, as occurs under eld conditions.
The relationship between color and total carotenoids content in citrus fruit does not always show a direct correlation
(Farin et al., 1983; Fanciullino et al., 2008; Matsumoto et al.,
2009). Navelina oranges at the Br stage experienced a signicant
enhancement of external coloration after 3 weeks of storage at 12 C
(Figs. 2A and 3A) with a non-signicant increase in total carotenoids
content (Fig. 4A). This effect could be explained by the rapid loss of
chlorophyll occurring during that period which highlighted external fruit color without a clear increase on carotenoid content, as
has been reported in other varieties (Barry and Van Wyk, 2006). In
contrast, the minor increase in peel color observed in fruit stored at
2 C could be due to both effects, a slow degradation of chlorophylls
together with a minor increase in carotenoids (Figs. 3 and 4).
The improvement of coloration in orange fruit during storage
at 12 C appears to be tightly associated with the accumulation
of specic carotenoids, and interestingly, differently in the peel
and the pulp. After 7 weeks of storage at 12 C, the concentration of the deep-orange C30 apocarotenoid -citraurin and a
lesser extent the yellow-orange xanthophyll antheraxanthin in
the peel experienced a major increase than in fruit stored at 2 C
(Fig. 5). In the pulp, the major increment was observed in the
,-xanthophylls -cryptoxanthin and antheranxanthin (Fig. 6).
In general, all these changes in the carotenoid prole during
storage at 12 C are in agreement with previous data proposing that temperatures between 12 and 14 C are optimal for the
biosynthesis of -cryptoxanthin, -citraurin and violaxanthin in
avedo (Sonnen et al., 1979; Casas and Mallent, 1988) and cryptoxanthin in the pulp, which appears to be the carotenoids
with a signicant impact in the nal coloration of avedo and
pulp, respectively (Stewart and Wheaton, 1973; Farin et al., 1983;
Goodner et al., 2001; Kato et al., 2004; Alquzar et al., 2008a)
Moreover, Oberholster et al. (2001) suggested that the intensity
of external color in orange fruit is dependent not only on the
content of specic carotenoids but also on the relative balance
between 9-Z-violaxanthin and -citraurin, characteristically yellow and orange-red pigments, respectively. In the peel of Navelina
fruit stored at 12 C, the ratio 9-Z-violaxanthin/-citraurin was
always lower (<5) than in those kept at 2 C (10), in both Br
and Co fruit (Table 2). Therefore, this ratio can be used as a good
indicator of the external color quality during storage of sweet
orange and reinforces the relevance of 9-Z-violaxanthin and citraurin in the pigmentation of avedo of the fruit. Unfortunately,
the biosynthesis steps for both carotenoids have not yet been
elucidated, although an isomerase has been proposed in the conversion of all-E-violaxanthin to 9-Z-violaxanthin and an asymmetric
carotenoid cleavage dioxygenase may be involved in the formation of -citraurin from -cryptoxanthin and/or zeaxanthin (Farin
et al., 1983; Gross, 1987; Oberholster et al., 2001; Rodrigo et al.,
2004; Rodrigo and Zacaras, 2007) Whether these genes/enzymes
may be also stimulated during storage of Navelina orange fruit at

114

L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

30
25

Phytoene

Phytoene

-cryptoxanthin

-cryptoxanthin

Antheraxanthin

Antheraxanthin

Violaxanthin

Violaxanthin

20
15

10
5

0
Phytofluene

-1

9.0

Phytofluene

g g FW

6.0
3.0

2
1

1.0

0
0.5
0.0

8
-cryptoxanthin

-cryptoxanthin

1.5

1.0

-1
g g FW

0
0

0.5

3
Weeks

0.0
8

Antheraxanthin

Antheraxanthin

-citraurin

-citraurin

Violaxanthin

Violaxanthin

Weeks

Fig. 6. Changes in the composition of carotenoids in the pulp of color break

(a/b = 0.11 0.02) (A) and colored (a/b = 0.44 0.01) (B) Navelina orange fruit during storage at 2 C (white bars) and 12 C (grey bars) and 9095% RH for 1, 3, 5 and
7 weeks. Plots were arranged following the carotenoid biosynthesis sequence in
the pathway. All-E-isomer of violaxanthin is represented in black color. Data are
means SD of at least three independent measurements.

6
4
2
0
15
12
9
6
3
0
30
20

12
2

10
0

3
Weeks

Weeks

Fig. 5. Changes in the composition of carotenoids in the peel of color break

(a/b = 0.11 0.02) (A) and colored (a/b = 0.44 0.01) (B) Navelina orange fruit during storage at 2 C (white bars) and 12 C (grey bars) and 9095% RH for 1, 3, 5 and
7 weeks. Plots were arranged following the carotenoid biosynthesis sequence in
the pathway. All-E-isomer of violaxanthin is represented in black color. Data are
means SD of at least three independent measurements.

12 C remains to be determined and they may be good biochemical

and molecular targets to understand the regulation of citrus fruit
coloration and to improve external quality.
In general, carotenoid content in the pulp of fruit of most citrus species is lower than in avedo (Figs. 5 and 6) (Gross, 1987;
Xu et al., 2006; Alquzar et al., 2008b), which has been associated with a reduced expression of carotenoid biosynthetic genes
(Kato et al., 2004; Alquzar et al., 2008b; Matsumoto et al., 2009).
During storage of Navelina fruit at 12 C we have also observed

that changes in the carotenoid prole are also tissue-dependent.

For example, in avedo at the end of the storage period the contents of colorless carotenes and violaxanthin were nearly 80% that
of total carotenoids, while in the pulp, colorless carotenes were not
detected and violaxanthin accounted for 50% of total carotenoids
(Figs. 5 and 6). Moreover, during storage at 12 C the carotenoids
undergoing major increases were colorless carotenes (phytoene
and phytouene) together with -citraurin and antheraxanthin
in avedo (Fig. 5), and -cryptoxanthin and antheraxanthin in
the pulp (Fig. 6). After 7 weeks of storage the concentration of
antheraxanthin in the avedo doubled that of the pulp, whereas
the opposite condition occurred for -cryptoxanthin, indicating a
tissue-preferential accumulation of each xanthophyll. Differential
accumulation of carotenoids in the peel and in the pulp has been
also reported in fruit of other citrus varieties exposed to different
postharvest conditions (Fujii et al., 2007; Matsumoto et al., 2009;
Zhou et al., 2010). It is tempting to speculate that the higher levels of -cryptoxanthin found in the pulp may be due to the lack of
conversion into -citraurin in this tissue, contrary to the avedo
in which a signicant amount of -cryptoxanthin might be readily
catabolized to the C30 apocarotenoid. It is important to notice that
the increase in -cryptoxanthin was detected in the pulp of fruit at
both ripening stages, Br (x2) and Co (x3) (Fig. 6). This increase in
the concentration of -cryptoxanthin during fruit storage at 12 C
has not only an aesthetic interest, but more importantly an impact
in nutritional quality, as this xanthophyll has provitamin A activity
(Britton, 1995), and also provides other health benets such as antitumor activities (Nishino et al., 2009) and osteoporosis prevention
(Yamaguchi and Uchiyama, 2009). Therefore, storage at 12 C could
be a feasible and alternative postharvest strategy to enhance cryptoxanthin content in the pulp of orange fruit thereby improving
nutritional and functional value.
In order to gain insight into the molecular basis of carotenoid
accumulation during storage of Navelina fruit at 12 C, expression of six biosynthesis genes of the pathway were analyzed in

L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

PSY

115

2 C
12 C

PSY

1.5

PSY
4

PSY

1.0
3

3
2

0.5

2 C
12 C

0
PDS

0.0

PDS

0 1 2 3 4 5 6 PDS
7

PDS

1.5

Das

1.0
1

0.5

ZDS

1
05
0.5
0

0.0

LCY1

LCY1
3

mRNA re
elative abundance

1.0

1
1

ZDS

ZDS

0.0
1.5

2
1.0
1
0.5
0

0.0

0 1 2 3 4 5 6LCY1
7

LCY1

Das

10
8
6

mRNA relative abundance

1.5

mRNA relative abundance

ZDS

mRNA relative abu

undance

2
0

LCY2

LCY2

LCY2

LCY2

15

4
2

10

4
3
2

CHX

CHX

2
1
0

CHX

1.5

1.0

0.5

0.0

CHX

0.8
0.6
0.4

Weeks

Weeks

0.2
0.0
0

Weeks

Weeks

Fig. 7. Quantitative RT-PCR analysis of the expression of PSY, PDS, ZDS, -LCY1, LCY2 and -CHX genes in the peel of color break (A) and colored (B) Navelina orange
fruit during storage at 2 C () and 12 C () and 9095% RH for 7 weeks. The plots
were arranged following the carotenoid biosynthesis sequence in the pathway. The
levels of expression were normalized to the amount of RNA and the value of peel of
freshly harvested fruit at the color break stage was set to 1. The data are means SD
of three experimental replicates.

Fig. 8. Quantitative RT-PCR analysis of the expression of PSY, PDS, ZDS, -LCY1, LCY2 and -CHX genes in the pulp of color break (A) and colored (B) Navelina orange
fruit during storage at 2 C () and 12 C () and 9095% RH for 7 weeks. The plots
were arranged following the carotenoid biosynthesis sequence in the pathway. The
levels of expression were normalized to the amount of RNA and the value of the
pulp of freshly harvested fruit at the color break stage was set to 1. The data are
means SD of three experimental replicates.

the avedo and pulp, and compared with the changes occurring
in fruit stored at 2 C. PSY, PDS, ZDS, LCY1, LCY2 and CHX genes
were selected since there are involved in key regulatory steps of
the pathway during citrus fruit maturity and under some postharvest conditions (Kato et al., 2004; Rodrigo et al., 2004; Als et al.,
2006; Fanciullino et al., 2008; Alquzar et al., 2009; Matsumoto
et al., 2009; Zhou et al., 2010). In general, expression of the six
genes was higher in both peel and pulp of fruit stored at 12 C
than in those kept at 2 C (Figs. 7 and 8), in accordance with the
greater carotenoid accumulation observed in tissues stored at the
higher temperature (Figs. 5 and 6). In avedo and pulp of freshlyharvested fruit, the expression of PSY, LCY1 and LCY2 was, as
expected, higher in Co than in Br fruit, but the basal level of CHX
decreased in both maturity stages to a similar extent. It is interesting to note that changes in the expression of the six genes in

the avedo during storage at 2 C and 12 C was fairly similar at the

two maturation stages, suggesting that the response of this tissue to
postharvest temperature is comparable during these stages of fruit
coloration. The expression of PSY, which catalyzed the rst committed step of the pathway, was higher at 12 C, in good agreement
with the higher content of the colorless carotenes phytoene and
phytouene in the avedo. The expression of the genes PDS, ZDS,
LCY1, LCY2 and CHX also increased upon exposure to 12 C, even
with different trends, whereas at 2 C their expression was maintained or slightly declined. These differences in gene expression at
the two temperatures were associated with higher concentration of
the ,-xanthophylls, antheraxanthin, and the C30 apocarotenoid,
-citraurin (Fig. 5). Moreover, during the entire storage duration at
12 C the relative increment (with respect to the initial values) in
the expression of PSY and both LCYs were higher in Co than in Br

116

L. Carmona et al. / Postharvest Biology and Technology 74 (2012) 108117

fruit, as with the levels of ,-xanthophylls at the end of the storage. These results suggest that accumulation of carotenoids at 12 C
is fairly well related to the stimulation of carotenoid gene expression in the peel of orange fruit, which initiate an enhancement of
precursors to the formation of ,-xanthophylls. Thus, stimulation
of carotenogenesis during storage at 12 C is likely to be mainly
transcriptionally regulated in the peel of orange fruit.
During storage at 2 C, expression of carotenogenic genes in
the avedo of fruit at both maturity stages was very low (Fig. 7)
but the concentration of carotenoids were sustained or moderately increased (Fig. 5), indicating that these reduced levels of
gene expression may be sufcient to maintain the turnover of
carotenoids during storage at low temperature. Similar results have
been observed in the avedo of Satsuma mandarin fruit during
storage at 5 C, where the levels of individual carotenoids slightly
increased and the expression of biosynthetic genes was repressed
(Matsumoto et al., 2009).
The pattern of changes in gene expression in the pulp of orange
fruit stored at 12 C was different to that of avedo and dependent
of the maturity stage (Fig. 8). In the pulp of Br fruit the expression of most of genes showed a transient increase during storage at
12 C, whereas in Co fruit the level of transcripts declined steadily.
Despite this decline, transcript levels of PSY and LCYs after 3 weeks
of storage were higher in Co than in Br fruit (Fig. 8), and may
explain the slightly larger concentration of individual carotenoids
(Fig. 6). Moreover, the decrease in transcript accumulation during storage at 2 C was not associated with parallel changes in
carotenoid content in the pulp. These discrepancies between the
expression of carotenoid genes and evolution of carotenoid content indicated differential regulatory mechanisms with respect to
the peel and the involvement of other regulatory factors such as
post-transcriptional regulation, or other genes or enzymes such as
those of the upstream MEP pathway (Matsumoto et al., 2009).
It is worth mentioning that most of the effects induced during storage of orange fruit at 12 C on carotenoid content and
expression of biosynthesis genes resemble those induced by fruit
exposure to ethylene which enhances coloration (Stewart and
Wheaton, 1972; Eilati et al., 1975; Purvis and Barmore, 1981;
Rodrigo and Zacaras, 2007). Therefore, it is tempting to hypothesize that stimulation of carotenoid biosynthesis and accumulation
by storage at 12 C may be ethylene-mediated processes. Ethylene production in Navelina fruit was very low and remained
nearly constant (0.10.2 nL h1 g1 FW) and without signicant differences between fruit stored at 2 C and 12 C (data not shown),
suggesting that increasing sensitivity to ethylene may mediate
responses of fruit to 12 C rather than changes in ethylene production.
In conclusion, our results show that postharvest storage of
Navelina orange fruit at an intermediate temperature (12 C) stimulates coloration in peel and pulp and increases total carotenoid
content. These effects in the peel are mainly due to increasing
expression of key carotenoid biosynthesis genes. Changes in gene
expression in the pulp were different to those in the peel, indicating an inuence of the stage of maturation at harvest and the
involvement of other regulatory factors. Interestingly, accumulation of -cryptoxanthin, a carotenoid with pro-vitamin A activity,
in the esh was specically promoted at 12 C, irrespective of the
maturity stages, indicating that management and storage at this
temperature may be a promising postharvest strategy to increase
the nutritional and health-related benets of citrus fruit.

Acknowledgements
This work was supported by research grants from Ministerio de
Ciencia e Innovacin of Spain (AGL2006-09496, AGL2009-11558,

FUN-C-FOOD CSD2007-0063) and Generalitat Valenciana (PROMETEO 2010/010). The assistance of Dr. Berta Alquzar (IVIA) during
the course of this study is gratefully acknowledged. We also
acknowledged the technical support of Amparo Beneyto.

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