Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits.

Abstract

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g(-1) and 13 μg g(-1) fresh weight (FW), respectively, and for BS 19 μg g(-1) and 0.27 μg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.

Full text

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Journal of Experimental Botany, Vol. 63, No. 1, pp. 341–354, 2012
doi:10.1093/jxb/err284Advance Access publication 11 October, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Plastid structure and carotenogenic gene expression in
red- and white-fleshed loquat (Eriobotrya japonica) fruits
Xiumin Fu1, Wenbin Kong1, Gang Peng1, Jingyi Zhou1,*, Muhammad Azam1, Changjie Xu1,†, Don Grierson1,2
and Kunsong Chen1
1Laboratory of Fruit Quality Biology/The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality
Improvement, Zhejiang University, Zijingang Campus, Hangzhou 310058, China
2Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics
LE12 5RD, UK
* Present address: Shanghai Municipal Agricultural Technology Extension and Service Center, Shanghai 201103, China.
yTo whom correspondence should be addressed. E-mail: chjxu@zju.edu.cn
Received 31 May 2011; Revised 21 July 2011; Accepted 9 August 2011
Abstract
Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing
(LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-
fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh
of LYQ was approximately 68 mg g21and 13 mg g21fresh weight (FW), respectively, and for BS 19 mg g21and 0.27 mg
g21FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening.
After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene
b-cyclase (CYCB) were higher in the peel, and CYCB and b-carotene hydroxylase (BCH) mRNAs were higher in the
flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of
plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes
were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the
globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh.
However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-
associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In
conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and
BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the
lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and
carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.
Key words: Carotenoid, chloroplast, chromoplast, colour, gene expression, fruit development, fruit ripening, loquat, plastid.
Introduction
Carotenoids are essential for plants, harvesting light for
photosynthesis, protecting plants from high light stress, and
furnishing flowers and fruits with bright yellow, red, or
orange colors that attract animals and facilitate pollination
Abbreviations: BCH, b-carotene hydroxylase; BS, Baisha; CCD, carotenoid cleavage dioxygenase; CCI, citrus colour index; CPW 18M, cell and protoplast washing
solution with 18% mannitol; CHRC, chromoplast-specific carotenoid associated protein; CRTISO, carotene isomerase; CYCB, chromoplast-specific lycopene
b-cyclase; DAFB, days after full bloom; DMAPP, dimethylallyl diphosphate; DTT, DL-dithiothreitol; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-
phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; ECH, e-carotene hydroxylase; FW, fresh weight; GAP, D-glyceraldehyde 3-phosphate;
GGPP, geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate; IDI, isopentenyl
pyrophosphate isomerase; IDS, isopentenyl pyrophosphate synthase; IPP, isopentenyl pyrophosphate; LCYB, lycopene b-cyclase; LCYE, lycopene e-cyclase; LYQ,
Luoyangqing; NCED, 9-cis-epoxycarotenoid dioxygenase; NSY, neoxanthin synthase; PAP, plastid lipid-associated protein; PDS, phytoene desaturase; PSY,
phytoene synthase; PVP, polyvinylpyrrolidone; Q-PCR, real-time quantitative PCR; TEM, transmission electron microscopy; VDE, violaxanthin de-epoxidase; ZDS,
f-carotene desaturase; ZISO, f-carotene isomerase; ZEP, zeaxanthin epoxidase.
ª 2011 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-
nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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and seed dispersal (Howitt and Pogson, 2006). Carotenoids
are also important for the nutrition of animals and humans,
providing essential nutrients and health-promoting com-
pounds. Beta-carotene, b-cryptoxanthin and a-carotene are
the primary dietary sources of vitamin A, and other
carotenoids, such as lycopene, have a strong antioxidant
function that could reduce the risk of cancer and cardiovas-
cular diseases (Hadley et al., 2002).
The valuable roles of carotenoids have been the subjects
of much research and carotenoid biosynthesis in higher
plants has been widely studied in the past two decades
(Hirschberg, 2001; Bramley, 2002; Fraser and Bramley,
2004; Taylor and Ramsay, 2005; DellaPenna and Pogson,
2006; Lu and Li, 2008). Carotenoids are synthesized from
the five-carbon intermediates isopentenyl diphosphate (IPP)
and dimethylallyl diphosphate (DMAPP). In plants, there
are two pathways for the synthesis of IPP. One is the
mevalonate pathway where IPP and DMAPP are synthe-
sized from mevalonic acid. The other, known as the
methylerythritol phosphate (MEP) pathway, occurs in
plastids and utilizes glyceraldehyde-3-phosphate (GAP) and
pyruvate for IPP and DMAPP synthesis (Fig. 1) (Cordoba
et al., 2009). Three IPPs and one DMAPP are condensed to
form C20 geranylgeranyl pyrophosphate (GGPP). Two
molecules of GGPP are converted to colourless phytoene
by a phytoene synthase (PSY). Phytoene is then converted
via f-carotene to lycopene, catalysed by phytoene desatur-
ase (PDS), f-carotene desaturase (ZDS), carotenoid isomer-
ase (CRTISO), and f-carotene isomerase (Z-ISO) (Chen
et al., 2010). Subsequently, the ends of the linear carotenoid
lycopene can be cyclized by lycopene b-cyclase (LCYB) and
lycopene e-cyclase (LCYE) to synthesize a-carotene or
cyclized by a lycopene b-cyclase (LCYB)/chromoplast-
specific lycopene b-cyclase (CYCB) (Ronen et al., 2000;
Alquezar et al., 2009) alone to synthesize b-carotene. Alpha-
carotene and b-carotene are hydroxylated to produce lutein
and zeaxanthin, respectively, catalysed by b-ring hydroxy-
lase (BCH) and ee-ring hydroxylase (ECH). Further,
zeaxanthin is transformed into violaxanthin by zeaxanthin
epoxidase (ZEP), and is reversed by violaxanthin de-
epoxidase (VDE) to give rise to the xanthophyll cycle for
plants to adapt to high light stress. Violaxanthin is
converted into neoxanthin by neoxanthin synthase (NSY).
One important product of the pathway is abscisic acid, the
formation of which is catalysed by 9-cis-epoxycarotenoid
dioxygenase (NCED).
Different carotenoid patterns are found in different plant
species and cultivars, and the mechanisms have been
investigated. It is known that lycopene is the typical
carotenoid of ripe tomato, which can be explained by the
increasing mRNA levels of PSY1 and PDS (Giuliano et al.,
1993) and decreasing expression levels of LCYE and LCYB
during ripening (Pecker et al., 1996; Ronen et al., 1999).
Capsanthin and capsorubin are the key components of
red pepper, due to high expression levels of capsanthin-
capsorubin synthase (CCS) genes (Ha et al., 2007). Differ-
ent carotenoid compositions and levels also underlie colour
differences between cultivars. For example, white pepper,
Fig. 1. Carotenoid biosynthetic pathway in higher plants. Genes
with expression levels studied are in bold letters. GAP, D-
glyceraldehyde 3-phosphate; DXS, 1-deoxy-D-xylulose 5-phos-
phate-synthase; DXR, DXP reductoisomerase; HMBPP, (E)-4-
hydroxy-3-methylbut-2-enyl diphosphate; IDS, isopentenyl pyro-
phosphate synthase; IPP, isopentenyl pyrophosphate; DMAPP,
dimethylallyl diphosphate; IDI, isopentenyl pyrophosphate isomer-
ase; GGPS, geranylgeranyl diphosphate synthase; GGPP, gera-
nylgeranyl diphosphate; PSY1, phytoene synthase; PDS, phytoene
desaturase; ZDS, f-carotene desaturase; CRTISO, carotene
isomerase; ZISO, f-carotene isomerase; LCYE, lycopene
e-cyclase; LCYB, lycopene b-cyclase; CYCB, chromoplast-specific
lycopene b-cyclase; BCH, b-carotene hydroxylase; ECH,
e-carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, viola-
xanthin de-epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-
epoxycarotenoid dioxygenase.
342 | Fu et al.

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which lacks carotenoids, has been shown to result from the
absence of expression of PSY, PDS, BCH, and CCS (Ha
et al., 2007). Compared with yellow cultivars, a higher
expression level of a carotenoid cleavage dioxygenase
(CCD) gene was observed in the white petals of chrysanthe-
mum and the white flesh of peach, where carotenoids are
thought to be produced but subsequently degraded into
colourless compounds (Ohmiya et al., 2006; Brandi et al.,
2011). Yellow and red cultivars of carrot root predomi-
nantly contained lutein (74%) and lycopene (57%), respec-
tively, which is consistent with the high expression levels of
LCYE and ZDS, respectively (Clotault et al., 2008).
Different patterns of accumulation of b-carotene resulted in
the diverse colour of Actinidia (kiwifruit) species, and
transcripts of LCYB were associated with b-carotene
accumulation (Ampomah-Dwamena et al., 2009). Some-
times selective expression of functional alleles could also
influence carotenoid accumulation. As an example, red-
fleshed Star Ruby grapefruit predominantly expressed the
dysfunctional b-LCY2b allele during fruit ripening whereas
Navel oranges preferably expressed the functional allele
b-LCY2a (Alquezar et al., 2009).
Transcriptional regulation of the carotenoid biosynthesis
pathway cannot always explain the differential accumula-
tion of carotenoids in plant tissues. Expression levels of the
carotenogenic genes in the orange cauliflower mutant (or)
were similar to those of the wild-type cauliflower (Li et al.,
2001). Expression of carotenogenesis-related genes was
observed in a white carrot root which does not contain
carotenoids (Clotault et al., 2008). Similarly, major carote-
nogenic gene expression (PSY1, PDS, and ZDS) cannot
explain the contrasting colours of different apricot varieties
(Prunus armeniaca), and Marty et al. (2005) surmise that
other types of regulation may operate, including post-
transcriptional or metabolic regulation.
Carotenoids are synthesized in plastids, and the differential
accumulation of carotenoids has been shown in some cases to
be related to plastid biogenesis and interconversion (Egea
et al., 2010). In the white carrot cultivar, Kim et al. (2010)
showed that the chromoplasts contained fewer carotenes and
were reduced in number compared with the orange variety.
The high b-carotene content in the orange curd of a cauli-
flower mutant was shown to be due to the differentiation of
proplastids and other uncoloured plastids into chromoplasts
(Paolillo et al., 2004; Lu et al., 2006). Similarly, hp1 and hp2
tomato accumulate high levels of carotenoid, due probably to
an increased plastid number and size, which provides a larger
compartment for carotenoid biosynthesis and deposition (Liu
et al., 2004; Kolotilin et al., 2007).
An increase in the size and number of plastoglobulins is
generally observed during the transition of chloroplasts to
chromoplasts and it is believed that they participate in
carotenoid sequestration (Bre ´he ´lin and Kessler, 2008; Egea
et al., 2010). In tomato, suppression of the chromoplast-
specific carotenoid associated protein (CHRC) expression
by RNAi leads to a 30% reduction in carotenoid content in
tomato flowers (Leitner-Dagan et al., 2006). Numerous
CHRC/Fib homologues have been collectively termed
plastid lipid-associated proteins (PAPs; Leitner-Dagan
et al., 2006).
Loquat (Eriobotrya japonica Lindl.), a member of the
Rosaceae, accumulates carotenoids as the main pigments in
mature fruit. The fruit can be divided into two groups, red-
and white-fleshed, according to the colour of the flesh, and
our previous study observed the differential accumulation
of carotenoids in the fruits of these two groups (Zhou et al.,
2007). The goal of the present study was to investigate the
mechanism leading to the diversity of carotenoid patterns in
Luoyangqing (LYQ, red-fleshed) and Baisha (BS, white-
fleshed) cultivars. The carotenoid content of peel and flesh
was monitored during the development of LYQ and BS,
using chemical measurements and microscopy. The mRNA
levels of 14 carotenogenic genes, indicated in Fig. 1 in bold
letters, as well as the Or gene were analysed during the
development and ripening of LYQ and BS fruits. In
addition, expression of PAP was also measured in fruit at
the fully ripe stage. The results suggested that the differen-
tial accumulation of carotenoids between cultivars was
associated with differential expression of PSY1, CYCB,
and BCH. However, microscopic and ultrastructural data
indicate that failure to develop normal chromoplast struc-
tures was more critically responsible for the lack of
carotenoid accumulation in BS flesh.
Materials and methods
Plant materials
Luoyangqing (LYQ, red-fleshed) and Baisha (BS, white-fleshed)
loquats (Eriobotrya japonica Lindl.) were sampled from an orchard
in Luqiao, Zhejiang, China. Fruit of six developmental stages, S1,
fruitlet, 45 d after full bloom (DAFB); S2, immature green, 75
DAFB; S3, mature green, 95 DAFB; S4, breaker, 105 DAFB; S5,
half ripe, 110 DAFB; S6, fully ripe, 115 DAFB, were collected
(Fig. 2). From these fruits, except those of S1 and S2, the peel was
separated from the rest of the fruit (flesh) and both tissues were
immediately frozen in liquid nitrogen, then powdered, and
carotenoids and RNA were extracted from aliquots of the same
samples.
Colour measurement
Peel colour at different developmental stages was measured using
a Hunter Lab Mini Scan XE Plus colorimeter (Hunter Associates
Laboratory, Inc., USA). The CIE L*a*b*colour scale was
adopted, and the data were expressed as L*, a*, b*, C*, Ho, as
well as the citrus colour index (CCI). The CCI is a comprehensive
indicator for colour impression [CCI¼10003a*/(L*3b*)] with
positive CCI indicating red, negative as blue-green, and zero for
an intermediate mixture of red, yellow, and blue-green (Zhou
et al., 2010). Four random measurements per fruit were made and
a mean value was obtained from six fruits per replicate.
Carotenoid extraction, quantification, and HPLC analysis
Carotenoids were extracted from fruits and analysed by HPLC,
according to a method previously described by Xu et al. (2006).
Approximately 100 mg of peel and 500 mg of flesh were extracted
with chloroform/methanol/50 mM TRIS buffer, pH 7.5. The
chloroform phase was collected by 10 min centrifugation at 10 000
g and the aqueous phases were re-extracted with chloroform. The
two combined chloroform phases were dried under nitrogen gas;
Plastid structure and gene expression in red- and white-fleshed loquat fruit | 343

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the residue was first dissolved in 20 ll of diethyl ether and then in
6% (w/v) KOH in methanol, incubated at 60 ?C for 30 min in
darkness to complete saponification. Water and chloroform were
then added, and the samples were extracted as described above,
and finally dried with nitrogen gas. Three replicates were
performed for each sample. HPLC analysis was carried out on
Waters Alliance 2695 system (Waters Corporation, USA) consist-
ing of a 2695 module and a 2996 PDA detector, equipped with
a 25034.6 mm i.d., 5 lm, YMC reverse-phase C30column and
a 2034.6 mm i.d., YMC C30guard. Chromatography was carried
out at 25 ?C with the elution programme as previously described by
Zhou et al. (2007). Compounds were detected at 450 nm (viola-
xanthin, luteoxanthin, 9-cis-violaxanthin, lutein, b-cryptoxanthin,
and b-carotene) or 400 nm (f-carotene) or 286 nm (phytoene) or 348
nm (phytofluene) and quantified according to their respective
standard curves.
Cloning of cDNAs encoding carotenogenic enzymes
Total RNA was extracted from frozen powder following our
previously published protocol (Shan et al., 2008). For the 15 target
carotenogenesis-related genes, degenerate primers were designed
based on sequences corresponding to highly conserved peptide
regions of known gene sequences of other plants from the National
Center for Biotechnology Information (NCBI) (see Supplementary
Table S1 at JXB online). The sequence of a plastid lipid-associated
protein gene (PAP) fragement was obtained from another study
involving deep sequencing, and the 3#-untranslated regions (UTR) of
the candidate sequence was amplified using the SMART? RACE
cDNA amplification Kit (Clontech) with PAPSP (5#-GAAGCTCCT-
GACATTGTCAAGAACAA-3#) as the gene-specific primer. cDNA
synthesized from LYQ at stage 6 were used as PCR templates.
Amplified PCR products of appropriate size were cloned into
pMD18-T vector (Takara, China) and were sequenced by Invitrogen
(China). Nucleotide homology to genes from other plants was
analysed using BLAST programmes from the NCBI database (http://
blast.ncbi.nlm.nih.gov/Blast.cgi).
Real-time quantitative PCR (Q-PCR) analysis
cDNA was synthesized according to a method previously de-
scribed by Wang et al. (2010). The Q-PCR reactions were
performed in a total volume of 20 ll, including 1 ll of each primer
(10 lM), 2 ll cDNA, 7 ll PCR-grade water, and 10 ll of 23
Platinum SYBR Green Supermix (Bio-Rad, USA) on an iCycler
iQ Q-PCR instrument (Bio-Rad, USA). The PCR programme was
initiated with a preliminary step of 5 min at 95 ?C, followed by 45
cycles at 95 ?C for 5 s, 58 ?C for 15 s, and 72 ?C for 10 s. A melting
curve was generated for each sample at the end of each run to
ensure the purity of the amplified products. No-template controls
for each primer pair were included in each run. The gene-specific
primers for Q-PCR were designed according to the gene sequences
obtained in this study, which have been deposited in GenBank
with accession numbers JN004208-JN004223, JN204273 (Table 1).
The expression level of actin was used to normalize the mRNA
levels for each sample, with abundance expressed as a multiple of
actin.
Light microscopy
Protoplasts were prepared as follows: peel and flesh were cut into
2–3 cm2pieces with a sterile scalpel blade. One gram of peel or
Fig. 2. Photographs showing the colours of LYQ (A) and BS (B) fruits at different stages (S1–S6) of development and ripening used
during this study. Bar, 2 cm.
344 | Fu et al.

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flesh pieces was transferred to 2 ml tubes containing 1.5 ml CPW
18M (cell and protoplast washing solution with 18% mannitol)
(pH 5.8) (Frearson et al., 1973) containing 1% cellulase, 0.3%
macerozyme, and 0.05% pectolyase. The tubes were incubated at
28 ?C on a reciprocating shaker (20–30 rpm) overnight. Proto-
plasts were washed and held in CPW 18M and observed and
photographed with an Olympus light microscope (Japan).
Plastids were isolated from 20 g flesh or 5 g peel for each cultivar
as follows: Tissues were homogenized with a Blitzkrieg Extractor
(JHBE-50S) in 100 ml of grinding buffer (0.45 M sucrose, 5 mM
EDTA, 1 mM DTT, 0.1% BSA, 0.1% PVP, 50 mM TRIS, and 10
mM Tricine, pH 7.5). The homogenate was filtered through four
layers of Miracloth. The cellular debris was eliminated by 5 min of
centrifugation at 800 g, and the chromoplast fraction was pelleted
by 20 min centrifugation at 10 000 g. The pellet was gently
resuspended in 2 ml grinding buffer, and observed and photo-
graphed with an Olympus light microscope (Japan).
Transmission electron microscopy (TEM)
Small pieces (2–3 mm2) of peel and flesh were cut and fixed for
more than 4 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer
(pH 7.0). After fixation, samples were washed three times in the
phosphate buffer; then post-fixed with 1% OsO4 in 0.1 M
phosphate buffer (pH 7.0) for 1 h and washed three times in the
phosphate buffer. Tissue samples were dehydrated by a graded
series of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) for about
15–20 min at each step, and transferred to absolute acetone for 20
min for infiltration. The first infiltration step was carried out with
a 1:1 mixture of absolute acetone and the final Spurr resin mixture
for 1 h at room temperature. The second step was the transfer to
a 1:3 mixture of absolute acetone and the final resin mixture for 3 h.
The third step was carried out in the final Spurr resin mixture
overnight. After the third infiltration step, embedded samples were
placed in capsules contained embedding medium and heated at
70 ?C for about 9 h. The samples were stained by uranyl acetate
and alkaline lead citrate for 15 min each and observed in TEM
using a Hitachi JEM-1230 (Japan).
GenBank accession numbers
Sequence data from this article have been deposited with the EMBL/
GenBank data libraries under accession numbers: JN004208 (DXS),
JN004209 (DXR), JN0042010 (PSY1), JN0042011 (PDS), JN0042012
(ZDS), JN0042013 (CRTISO), JN0042014 (LCYB), JN0042015
(CYCB), JN0042016(LCYE),
(ECH), JN0042019 (ZEP), JN0042020 (VDE), JN0042021 (CCD),
JN0042022 (Or), JN0042023 (actin) , JN204273 (PAP).
JN0042017(BCH),JN0042018
Results
Carotenoid content and composition analysis of two
loquat cultivars
The peel colour of loquat fruit changed from green to
orange (LYQ) or yellow (BS), while the flesh colour turned
from yellowish to orange in LYQ, but remained ivory white
in BS throughout development and ripening (Fig. 2). The
CIE colour values of peels are in good accordance with the
visual colour (see Supplementary Table S3 at JXB online).
The differences in carotenoid content and composition of
red- and white-fleshed loquats were first analysed for fruits at
the fully ripe stage (S6). The total content of carotenoids was
much higher in the peel than in the flesh, and in LYQ
compared with BS. The main carotenoids accumulated were
b-carotene and lutein in peel, and b-carotene as well as
b-cryptoxanthin in flesh, with a higher percentage of lutein in
BS than LYQ, both in peel and flesh (Fig. 3A).
The changes in carotenoid content and composition during
fruit development and ripening were analysed by HPLC in
two cultivars. In the present study whole fruits were analysed
at the S1 and S2 stages because the peel was hard to separate
from the rest of the fruit (flesh) at these stages, but at all
later stages the peel was separated from the flesh. The
differences in carotenoid content and composition in LYQ
and BS were quantified by HPLC (Fig. 3A, B). During the
stages S1–S2, the total carotenoid levels in LYQ was slightly
higher than in BS, but the carotenoid profiles were almost the
same, and mainly comprised the usual chloroplast-type
carotenoids, especially lutein (48.6% of total carotenoids
Table 1. Primers used for real-time quantitative PCR (Q-PCR)
GeneForward primer (5# to 3#)Reverse primer (5# to 3#) Product size (bp) GenBank accession number
DXS
DXR
PSY1
PDS
ZDS
CRTISO
LCYB
CYCB
LCYE
BCH
ECH
ZEP
VDE
CCD
Or
Actin
PAP
5#-GGTTCATCACTATTTGAAGA-3#
5#-CATCCAAACTGGAATATGGG-3#
5#-ACAGATGAGCTAGTGGATGG-3#
5#-AATGAGATGCTGACTTGGCC-3#
5#-AAGAAATGCAGTGGCTCTTGC-3#
5#-AGCATTCCAACTGTTCTTGA-3#
5#-CAAACGGTGTTAAATTTCACCA-3#
5#-AACCATGGATATCAGATTGCTC-3#
5#-ACTAGATTGTTCTTTGAGGA-3#
5#-GAGAAGGTCCGTTCGAGCT-3#
5#-CGTTCTTCAAAGGTGTGGGA-3#
5#-TACACTGGTATCGCAGATTT-3#
5#-TCTGATGTGGGAGAATTTCC-3#
5#-AAGAGCGTGCTGTCCGAAAT-3#
5#-GAACTGGATATCTTGCTTGTGC-3#
5#-AATGGAACTGGAATGGTCAAGGC-3#
5#-CGAGGTTAAACTCCAGAT-3#
5#-TCAAATTTGGCCACTCCATG-3#
5#-GCCAGAACAGATGAATCCTG-3#
5#-CATTCCTTCTACCATGTCT-3#
5#-TTGGAACCGTGTTTCTCCTG-3#
5#-CCACCTTTGGACAAGAACCA-3#
5#-TTCTTTGCCTCATAGTCCTT-3#
5#-CCACTTGGTAACCTGGATTGTA-3#
5#-GAATTACTAGTGCGCAAATAAGG-3#
5#-CAACCGGAATCCAAGACCA-3#
5#-AGGCCATCATGGACAAACAT-3#
5#-TCCAGCAGAGCAAACTGATC-3#
5#-TGAAACGCATACCACTGCAT-3#
5#-TCATGCAATTGGCAATCAAA-3#
5#-AGTAGGTTCCGATTTACCAT-3#
5#-GTAGGGCACATTACCTTTCC-3#
5#-TGCCAGATCTTCTCCATGTCATCCCA-3#
5#-GCTGAAGCGTTACATTTTACA-3#
206
182
186
263
126
122
177
134
148
172
155
128
137
119
157
227
156
JN004208
JN004209
JN004210
JN004211
JN004212
JN004213
JN004214
JN004215
JN004216
JN004217
JN004218
JN004219
JN004220
JN004221
JN004222
JN004223
JN204273
Plastid structure and gene expression in red- and white-fleshed loquat fruit | 345