Journal of Experimental Botany, Vol. 54, No. 383, pp. 727±738, February 2003
DOI: 10.1093/jxb/erg083
RESEARCH PAPER
Characterization of Pinalate, a novel Citrus sinensis
mutant with a fruit-speci®c alteration that results in yellow
pigmentation and decreased ABA content
MarõÂa-JesuÂs Rodrigo, Jose F. Marcos, Fernando AlfeÂrez, M. Dolores Mallent and Lorenzo ZacarõÂas1
Departamento de Ciencia de Alimentos, Instituto de AgroquõÂmica y TecnologõÂa de Alimentos (IATA)-CSIC,
Apartado de Correos 73, Burjassot, 46100 Valencia, Spain
Received 19 July 2002; Accepted 21 October 2002
Abstract
The characterization of a novel mutant, named
Pinalate, derived from the orange (Citrus sinensis L.
Osbeck) Navelate, which produces distinctive yellow
fruits instead of the typical bright orange colouration,
is reported. The carotenoid content and composition,
and ABA content in leaf and ¯avedo tissue (coloured
part of the skin) of fruits at different developmental
and maturation stages were analysed. No important
differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation
of linear carotenes (phytoene, phyto¯uene and zcarotene) was detected in the ¯avedo of Pinalate. As
fruit maturation progressed, the ¯avedo of mutant
fruit accumulated high amounts of these carotenes
and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental
line. Full-coloured fruit of Pinalate contained about
44% phytoene, 21% phyto¯uene, 25% z-carotene, and
10% of xanthophylls, whereas, in Navelate, 98% of
total carotenoids were xanthophylls and apocarotenoids. The ABA content in the ¯avedo of Pinalate
mature fruit was 3±6 times lower than in the corresponding tissue of Navelate, while no differences were
found in leaves. Other maturation processes were
not affected in Pinalate fruit. Taken together, the
results indicate that Pinalate is a fruit-speci®c alteration defective in z-carotene desaturase or in zcarotene desaturase-associated factors. Possible
mechanisms responsible for the Pinalate phenotype
are discussed. Because of the abnormal fruit-speci®c
carotenoid complement and ABA de®ciency, Pinalate
may constitute an excellent system for the study of
carotenogenesis in Citrus and the involvement of
ABA in fruit maturation and stress responses.
Key words: ABA, carotenoid, Citrus sinensis L. Osbeck,
colour, fruit maturation, xanthophylls, z-carotene desaturase.
Introduction
Carotenoids are terpenoids synthesized in plastids as
hydrocarbons (carotenes) and their oxygenated derivatives
(xanthophylls) (Bramley, 1997), and serve essential roles
in plants as components of the photosynthetic apparatus
and protectors against oxidation derived from excess light
energy (Demmig-Adams and Adams, 1996). They also
provide the yellow, orange or red colouration characteristic
of many ¯owers and fruits. Their importance is also
recognized as nutritional components, vitamin A precursors, in the prevention of human diseases such as cancer,
and from an industrial perspective (Bramley et al., 1993;
Mayne, 1996; Olsen, 1989; Hirschberg, 1999; Sandmann,
2001).
The biochemistry of carotenoid biosynthesis has been
well established. Genes and cDNAs encoding some of the
carotenogenic enzymes have been isolated and characterized in bacteria, algae, fungi and, more recently, in higher
plants (reviewed in Cunningham and Gantt, 1998;
Hirschberg, 2001; Sandmann, 2001). The ®rst committed
step in the carotenoid pathway (Fig. 1) is the synthesis of
phytoene, catalysed by the enzyme phytoene synthase
(PSY). Subsequently, the colourless phytoene undergoes
four consecutive symmetrical desaturation steps. In plants
and cyanobacteria these four desaturations are catalysed by
two related enzymes, postulated to act co-ordinately
1
To whom correspondence should be addressed. Fax: +34 96 363 63 01. E-mail: [email protected]
Abbreviations: ABA, abscisic acid; HPLC, high-performance liquid chromatography.
Journal of Experimental Botany, Vol. 54, No. 383, ã Society for Experimental Biology 2003; all rights reserved
728 Rodrigo et al.
Fig. 1. Schematic diagram of the biosynthetic pathway of carotenoids
in plants. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zcarotene desaturase; b-LCY, b-lycopene cyclase; e-LCY, e-lycopene
cyclase; b-CHX, b-carotene hydroxylase; e-CHX, e-carotene
hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid
dioxygenase.
(Cunningham and Gantt, 1998), phytoene desaturase
(PDS) and z-carotene desaturase (ZDS), yielding the red
carotene lycopene through the intermediate z-carotene
(pale-yellow). These desaturase reactions require plastoquinone (Norris et al., 1995) and a plastid terminal oxidase
as electron acceptors (Carol and Kuntz, 2001). In the next
step of the pathway, cyclation of lycopene yields bcarotene and/or a-carotene, and subsequent substitutions
by hydroxyl, oxo, and/or epoxy groups produce xanthophylls with bright orange/yellow colours.
Carotenoid biosynthesis in plants is connected with that
of the plant growth regulator abscisic acid (ABA), which is
produced through C15 intermediates after oxidative cleavage of speci®c xanthophylls (Marin et al., 1996; Schwartz
et al., 1997; Milborrow, 2001). Indeed, many ABAde®cient mutants identi®ed are related to carotenoid
synthesis (Taylor et al., 2000).
Carotenoid complements of fruits and ¯owers vary
considerably among species (Bramley, 1997), thus conferring their characteristic colour. The peel and pulp of
citrus fruits are among the richest source of carotenoids,
with hundreds of micrograms per gram of tissue and more
than 100 different carotenoids identi®ed (Bramley et al.,
1993; Stewart and Wheaton, 1973). Carotenoid content
and composition may vary greatly among citrus species,
and also depend on the growing conditions (Gross, 1987).
In coloured citrus fruits, such as oranges and mandarins,
epoxy and hydroxylated carotenoids are the major
components and account for up to 80% of total
carotenoids. By contrast, the content of linear carotenes
in full mature fruits is relatively low (less than 20% of total
carotenoids). Besides the common carotenoids, citrus
fruits accumulate genus-speci®c C30-apocarotenoids,
formed by cleavage of C40 precursors (Farin et al.,
1983); b-citraurin, b-citraurinene and b-apo-8¢-carotenal
being the most abundant in the peel and responsible for the
orange-reddish colouration of some citrus fruits (Gross,
1987). Despite the importance of citrus fruits as a
carotenogenic source, little is known about carotenoid
biosynthetic regulation during their maturation. Recently,
cDNAs encoding PSY, PDS and b-carotene hydroxylase
from Satsuma mandarin have been isolated. The accumulation of PSY mRNA increased in the peel and juice sacs
with the onset of coloration (Ikoma et al., 2001; Kim et al.,
2001a), whereas the levels of PDS and b-carotene
hydroxylase mRNAs remained constant once fruit is
fully developed (Kita et al., 2001; Kim et al., 2001b).
The characterization of mutants altered in the carotenoid
biosynthetic pathway is a useful experimental system to
identify molecular mechanisms regulating the process.
This approach, however, is limited to a small number of
plant species, mainly Arabidopsis and tomato (DellaPenna,
1999; Hirschberg, 2001). There is great interest in the
study of carotenoid biosynthesis in citrus fruits in order to
gain knowledge on the regulation of this process in citrus
and also as a ®rst step to address the improvement of their
nutritional and commercial quality. In this work an
interesting novel mutant of the `Navelate' orange, named
Pinalate, which produces fruits of a distinctive yellow
colour, is characterized. This remarkable phenotype suggests an alteration of the mechanism(s) regulating the
accumulation of carotenoids and may provide new insights
of how these processes are co-ordinated in citrus. It is
shown that Pinalate is partially blocked at z-carotene
desaturation. This alteration produces an accumulation of
early linear carotenes, a reduction in xanthophylls and
Pinalate, a novel Citrus with altered fruit colour 729
thus, a de®ciency in ABA content in the peel of the fruit,
whereas leaf tissue is unaffected.
Materials and methods
Plant material
Fruits of Navelate and Pinalate oranges (C. sinensis [L.] Osbeck) at
different developmental stages were harvested from trees grafted on
Citrange carrizo rootstocks. Experiments were conducted with adult
trees grown in two locations: San Pedro del Pinatar (Murcia, Spain),
where Pinalate was originally identi®ed, and The Citrus Germplasm
Bank at Instituto Valenciano de Investigaciones Agrarias (Moncada,
Valencia, Spain). In both locations, the trees of each variety were of
the same age, grown in the same orchard and subjected to standard
cultural practices.
Fruit colour was measured using a Minolta CR-330 on three
locations around the equatorial plane of the fruit. Hunter parameters
a (negative to positive correspond from green to red, respectively), b
(negative to positive, from blue to yellow, respectively) and L (0 to
100, black to white) were used and colour was expressed as the a/b
Hunter ratio, a classical relationship for colour measurement in
citrus fruits (Stewart and Whitaker, 1972). The a/b ratio is negative
for green fruits, the zero value correspond to yellow fruits at the
midpoint of the colour break period and is positive for orange fruit.
Leaves and ¯avedo tissue (the outer coloured part of the fruit peel)
were frozen in liquid nitrogen, ground to a ®ne powder and stored at
±70 °C until analysis.
Carotenoid extraction and quanti®cation
For carotenoid analysis three developmental/maturation fruit stages
were selected: (1) immature green fruit harvested in July±August
with an average diameter of 4.5260.25 cm and 3.9560.22 cm and a/
b ratios of ±0.7960.01 and ±0.7760.01 for Navelate and Pinalate,
respectively, (2) mature green fruit harvested in October±November
with an average diameter of 5.3860.35 cm and 5.2160.42 cm and a/
b ratios of ±0.4760.02 and ±0.5660.02 for Navelate and Pinalate,
respectively, and (3) full-coloured fruit harvested in February±
March, with an average diameter 5.5060.25 cm and 5.1060.32 cm
and a/b ratios of 0.6460.02 and 0.1060.01 for Navelate and
Pinalate, respectively.
Freeze-ground material (500 mg) of leaves or ¯avedo was
weighed in screw-capped Pyrex tubes (15 ml) and 2 ml of MeOH
were added. The suspension was stirred for 5 min at 4 °C. Tris-HCl
(50 mM, pH 7.5) (containing 1 M NaCl) was then added (1.5 ml) and
further stirred for 5 min at 4 °C. Chloroform (4 ml) was added to the
mixture, stirred for 5 min at 4 °C and centrifuged at 3000 g for 5 min
at 4 °C. The hypophase was removed with a Pasteur pipette and the
aqueous phase re-extracted with chloroform until it was colourless.
The pooled chloroform extracts were dried on a rotary evaporator at
40 °C. For saponi®cation the dried residue was completely dissolved
in 1.8 ml of MeOH and 200 ml of 60% (w/v) KOH. Prior to capping,
the ¯ask was gently blanketed with nitrogen, closed, and placed in
the dark overnight at room temperature. Saponi®ed carotenoids were
recovered from the upper phase after adding 2 ml of MilliQ water
and 6 ml of solution A (petroleum ether:diethyl ether, 9:1, v:v) to the
mixture. Repeated re-extractions by adding 3 ml of solution A were
carried out until the hypophase was colourless. The volume
recovered was transferred to a volumetric ¯ask and adjusted to
10 ml with solution A. An aliquot of solution A extract was used for
the quanti®cation of total carotenoid content.
The extracts were reduced to dryness by rotary evaporation at
40 °C and quantitatively transferred to a Pyrex tube (15 ml) with
acetone. In order to precipitate the sterols present in the samples, the
acetone extracts were kept overnight at ±20 °C and centrifuged at
3000 g for 15 min at 4 °C. The supernatant was transferred to a 1.5 ml
vial, dried under N2 and kept at ±20 °C until HPLC analysis. All
operations were carried out on ice under dim light to prevent photo
degradation, isomerizations and structural changes of carotenoids.
Each sample was extracted at least twice.
Absorption spectra (300±550 nm) of saponi®ed extracts (see
above) of leaves and ¯avedo from Navelate and Pinalate were
recorded at room temperature with a Diode array spectrophotometer
(model 8452A Hewlett Packard, Germany). The maximum absorbance peaks were registered and total carotenoid content was
calculated by measuring the absorbance at 450 nm according to
Davies (1976), using an extinction coef®cient of b-carotene,
E1%=2500.
HPLC analysis of carotenoids
Samples were prepared for HPLC by dissolving the dried residues in
MeOH: acetone (2:1, v:v). Volume of the sample was adjusted to
75 ml for ¯avedo extracts and to 225 ml for leaf extracts.
Chromatography was carried out with a Waters liquid chromatography system equipped with a 600E pump and a model 996
photodiode array detector, and Millenium Chromatography Manager
(version 2.0). A C30 carotenoid column (25034.6 mm, 5 mm)
coupled to a C30 guard column (2034.0 mm, 5 mm) (YMC Europe
GMBH, Germany) were used with MeOH, water and methyl tertbutyl ether (MTBE). Carotenoid pigments were analysed by HPLC
using a ternary gradient elution reported in a previous work (Rouseff
et al., 1996). Brie¯y, the initial solvent composition consisted of
90% MeOH, 5% water and 5% 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. The ®nal composition was reached at 50 min and consisted
of 50% MeOH and 50% MTBE. The initial conditions were reestablished in 2 min and re-equilibrated for 15 min before the next
injection. The ¯ow rate was 1 ml min±1, column temperature was set
to 25 °C and the injection volume was 25 ml. Each analytical
determination was replicated at least twice. The photodiode array
detector was set to scan from 250 to 540 nm.
The b-carotene, a-carotene and lycopene standards were obtained
from Sigma-Aldrich (Spain). The standards b-cryptoxanthin, lutein
and zeaxanthin were obtained from Extrasynthese (France).
ABA analysis
The quanti®cation of ABA in ¯avedo and leaf tissue was performed
by indirect enzyme-linked immunosorbent assay as reported previously (ZacarõÂas et al., 1995; Lafuente et al., 1997).
Maturity index
Juice from fruits at different developmental/maturation stages
(immature green, mature green and full-coloured) was extracted
with a household electric hand reamer, ®ltered through a metal sieve
with a pore size of 0.8 mm and analysed 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 solid content
(as °Brix) by refractrometry, using an Atago model PR32. The
maturity index is expressed as the ratio of °Brix/acidity.
Results
Identi®cation of Pinalate: a novel citrus with altered
fruit colour
A citrus mutant was originally found in an orchard in `San
Pedro del Pinatar' (Murcia, Spain) in the mid-eighties and
730 Rodrigo et al.
named Pinalate. Pinalate occurred spontaneously from the
commercial variety of orange Navelate, and the novel
tissue was identi®ed as tree branches originated from
previously grafted buds. The more remarkable phenotypic
effect of Pinalate fruits is the yellow colouration of the
¯avedo (outer coloured tissue of the fruit peel) that is
easily distinguishable from the orange colour in the
parental (wild-type) variety (Fig. 2A). The internal juice
vesicles of Pinalate are likewise yellow (not shown).
Grafting is the common propagation procedure in the
seedless Navel oranges. The Pinalate phenotype has been
propagated by grafting onto different rootstocks and
remained stable under ®eld conditions and in a germplasm
collection (IVIA, Moncada, Spain); and to the authors'
knowledge, it never reverted to the parental orangecoloured phenotype. Occasionally, individual fruits of the
outer part of the canopy showed narrow sectors of paleorange coloration in the peel (not shown). Appearance and
agronomical behaviour of Pinalate trees were normal and
indistinguishable from Navelate. During four consecutive
seasons, fruit colour was periodically measured (from June
to March) to determine changes through maturation and
ripening. Quantitative differences were observed between
seasons, probably due to environmental changes, although
the patterns of change and the differences between
Navelate and Pinalate were consistently maintained
(Fig. 2B).
The onset of fruit colouration started at the same time
(September±October) in Pinalate and Navelate, and the
time span to complete full colouration (in January±
February) is very similar in both varieties (Fig. 2B).
However, the time required to reach complete chlorophyll
disappearance (Hunter a/b ratios equal to 0) was longer in
Pinalate. In addition, Pinalate fruits reach a plateau at
lower a/b ratios when full matured, due to their distinct
yellow colour. Other parameters assayed that were indicative of the ripening process, such as shape, size, weight,
and ¯avour were not altered in Pinalate (not shown). Fruit
acidity and soluble solid content were also similar in both
phenotypes and, as a result, the internal maturity index was
not signi®cantly different (Fig. 3).
Spectrum pro®les of total carotenoid from ¯avedo of
Navelate and Pinalate fruits
Spectra of total carotenoids extracts from the ¯avedo of
Navelate and Pinalate were substantially different (Fig. 4).
Typical spectra of ¯avedo extracts from Navelate showed
two maximum peaks at 436 nm and 464 nm, while in
Pinalate extracts new maxima appeared at approximately
400 nm and 425 nm. Moreover, Pinalate spectra showed
higher absorbance values at lower wavelengths, which
might be indicative of the presence of colourless carotenes.
The total carotenoid content of ¯avedo extracts from
Navelate and Pinalate fruits at different developmental
stages was calculated by measuring the absorbance at
Fig. 2. Phenotype of the Citrus sinensis mutant Pinalate. (A) Mature
fruits are shown from C. sinensis cv. Navelate (orange fruits) and
Pinalate (yellow), assorted to emphasize colour differences. (B)
Changes in fruit colour (as evolution of Hunter a/b colour ratio)
during ripening of Navelate and Pinalate. Dotted line indicates colour
index at colour break.
Fig. 3. Internal maturation index (°Brix/acidity of the juice) during
ripening of Navelate and Pinalate fruit. Data shown are representative
of at least three independent experiments from three seasons.
Pinalate, a novel Citrus with altered fruit colour 731
Fig. 4. Representative absorbance spectra of ethereal solutions of
saponi®ed carotenoid extracts from ¯avedo of Navelate (black line)
and Pinalate (dotted line) fruits harvested in February (full-coloured
stage). Numbers above the arrows indicate maximum wavelengths of
absorbance.
450 nm, according to Davies (1976), and expressed as mg
of b-carotene g±1 of fresh weight of ¯avedo. The ¯avedo
from fruits of Navelate and Pinalate showed a similar
content of total carotenoids at immature green (48.362.1
and 42.361.1 for Navelate and Pinalate, respectively) and
mature green stage (15.962.8 and 18.363.3 for Navelate
and Pinalate, respectively). Full-coloured ¯avedo from
Navelate fruit contained 56.664.9 mg of b-carotene g±1
fresh weight, while the estimated total carotenoid content
for Pinalate ¯avedo at this stage was signi®cantly lower
(24.360.4) than in Navelate. However, this value is likely
to be underestimated, since the content of total carotenoids
is based in the absorbance of the extract at 450 nm and
Pinalate showed a clear shift of the spectrum to lower
wavelengths (Fig. 4), but it may be well indicative of the
differences in carotenoid content between both phenotypes.
Qualitative and quantitative variations of carotenoids in
the leaf and fruit from Navelate and Pinalate
HPLC carotenoid pro®les from leaves and ¯avedo from
fruits at three developmental stages (immature green,
mature green and full-coloured) from both Navelate and
Pinalate were analysed. Twenty-three carotenoid pigments
were resolved and their spectral characteristics are shown
in Table 1. Lutein (peak no. 13), zeaxanthin (peak no. 15),
b-cryptoxanthin (peak no. 19), a-carotene (peak no. 21),
and b-carotene (peak no. 22) were identi®ed by comparison of the spectra and retention time with those of
authentic standards. Sixteen more peaks were tentatively
identi®ed by matching the observed versus literature
spectral data and retention times under identical chromatographic conditions (Lee, 2001; Lee et al., 2001; Rouseff
et al., 1996). A numerical notation (%III/II), which
describes the ratio of the peak height of the longest
wavelength absorption band (band III) to that of the middle
absorption band (band II) as a percentage, was also used
for identi®cation. In general, values for lmax spectra and
%III/II obtained from this study agree well with the values
reported in the literature. Nine remaining peaks, whose
spectroscopic characteristics are also described in Table 1,
were not assigned to a de®ned carotenoid. A more
exhaustive analysis would be required to identify these
peaks.
The pro®les and composition of the carotenoids found in
leaves and ¯avedo at different developmental stages from
Navelate and Pinalate were compared. Carotenoid analysis
of leaf samples did not reveal qualitative or quantitative
signi®cant differences between Navelate and Pinalate
(Fig. 5; Table 2). Xanthophylls were represented by lutein
and neoxanthin, while carotenes by a- and b-carotene.
Smalls amounts of isolutein (lutein-5,6-epoxide) and one
unidenti®ed carotenoid (peak no. 23) were also found.
Quantitatively, around 50% of the total carotenoid content
was lutein.
When carotenoid HPLC pro®les from Navelate fruits at
different developmental stages were compared, signi®cant
quantitative and qualitative differences were observed
(Fig. 5; Table 2). In immature green Navelate fruit the
pattern and carotenoid composition was similar to that
found in leaves, being lutein and neoxanthins the main
carotenoids. The pigment pattern was modi®ed at the
beginning of chlorophyll degradation and transition from
chloroplast into chromoplast. The noticeable decrease in
total carotenoid content observed in mature green fruits
was mainly due to a large reduction in lutein and
neoxanthins. It is important to note the presence of
violaxanthin (peak no. 12) in mature green fruit, which
was not detectable in earlier stages of development.
Analysis of the HPLC pro®le of ¯avedo from full-coloured
Navelate fruit showed a remarkable increment in the
percentage of violaxanthin (more than 50% of total
carotenoid content, Table 2) as compared to the mature
green stage. At this stage, other characteristic pigments
from Citrus chromoplast were also found, as b-cryptoxanthin (peak no. 19) and some apocarotenoid-like compounds (peaks no. 7, 9, and 14), representing all together
around 12% of the total carotenoid content.
The HPLC pro®le of Pinalate extracts from ¯avedo of
immature fruit pointed to the accumulation of the linear
carotenes phytoene, phyto¯uene and z-carotene (peaks no.
11, 18 and 20, respectively), which were not detected, or
only in trace amounts, in Navelate (Fig. 5; Table 2).
Therefore, the relative proportions of the main carotenoids
lutein and neoxanthins (peaks no. 13 and 8, respectively)
were lower in Pinalate than in the corresponding tissue of
Navelate. At the mature green stage, the Pinalate
carotenoid pro®le was substantially different from
Navelate because of the presence of phytoene, phyto¯uene
732 Rodrigo et al.
Table 1. Spectroscopic characteristics of carotenoid pigments found in Citrus sinensis L. Osbeck cv. Navelate and its mutant
Pinalate
Peak no.a
Carotenoidb
Observed
Literature
lmax (nm)
Peak ratio
81
88
78
98
28
73
0
92
90
0
62
10
95
65
0
35
71
10
72
71
30
91
75
c
Reference
c
lmax (nm)
Peak ratio
401,424,451
405,430,460
416,438,467
412,434,464
75
0
87
85
Britton,
Britton,
Britton,
Britton,
276,286,297
cis326,416,440,465
421,445,474
10
98
60
Britton, 1995
Britton, 1995
Britton, 1995
428,450,478
416,440,470
276,286,297
421,445,475
331,348,367
428,450,478
331,348,367
cis296,377,398,422
26
60
10
60
90
27
90
71
Britton, 1995
Rouseff et al., 1996
Britton, 1995
Britton, 1995
Britton, 1995
Britton, 1995
Britton, 1995
Cunningham and
Schiff, 1985
Cunningham and
Schiff, 1985
Cunningham and
Schiff, 1985
Cunningham and
Schiff, 1985
Britton, 1995
Cunningham and
Schiff, 1985
1
2
3
4
5
6
7
8
8¢
9
10
11
12
13
14
15
16
11¢
17
18
19
18¢
20
Zeaxanthin*
Isolutein
Phytoene-2
a-Cryptoxanthin
Phyto¯uene-1
b-Cryptoxanthin*
Phyto¯uene-2
Cis-z-carotene-1
374,394,419
339,354,377
338,355,375
416,440,470
405,431,457
405,428,456
405,429,457
415,438,468
412,434,463
457
cis328,412,438,465
280,284,300
cis325,411,434,463
418,444,472
467
430,450,478
416,440,467
273,285,300
419,445,472
329,346,364
423,450,479
331,347,364
cis295,376,398,422
20¢
Cis-z-carotene-2
cis295,374,394,419
85
cis296,374,395,419
91
21
a-Carotene*
420,445,472
62
422,445,473
55
20¢¢
z-Carotene-3
378,400,424
102
379,400,425
105
22
20¢¢¢
b-Carotene*
z-Carotene-4
426,451,473
379,400,425
31
102
425,450,477
379,400,425
25
105
23
20¢¢¢¢
z-Carotene-5
422,446,473
380,400,426
34
102
379,400,425
105
Neochrome
8¢-Apo-b-caroten-8¢-ol
Trans-violaxanthin
Neoxanthin b
Phytoene-1
Cis-violaxanthin
Lutein*
1995
1995
1995
1995
Cunningham and
Schiff, 1985
a
Peaks are numbered by elution order.
*, identi®ed using authentic standards.
c
Peak ratio is % III/II for carotenoids (Britton, 1995).
b
and z-carotene. The proportion of these linear carotenes
accounted for up to 50% of total carotenoids, whereas in
Navelate they were not detected. By contrast, the percentage of neoxanthin, violaxanthin and lutein was signi®cantly reduced in Pinalate. In full-coloured Pinalate
¯avedo, massive amounts of phytoene were detected, as
compared with Navelate. A 4-times dilution of this sample
was necessary to obtain a linear response of the phytoene
peak area (peak no. 11¢) versus absorbance. Phyto¯uene
and z-carotene were also accumulated to a great extent
(Fig. 5; Table 2). Several isomers of phytoene, phyto¯uene
and z-carotene were identi®ed in HPLC pro®les of ¯avedo
extracts from Pinalate (see Table 1 for spectral properties).
Concomitantly with the accumulation of linear carotenes
in mature ¯avedo of Pinalate fruit, the amount of
downstream products in the carotenoid pathway, such as
the xanthophylls violaxanthin or neoxanthins, dramatically
decreased compared to Navelate. It is interesting to note
that apocarotenoid-like compounds or b-cryptoxanthin
were not identi®ed in Pinalate at any stage of development.
Consistently, peaks in the more polar diol-polyol region
(early elution times) (peaks no. 2, 3 and 4) were detected in
Pinalate chromatograms, which were not present in
Navelate.
In the context of this study, it is important to stress that
neurosporene, lycopene and their all-cis-isomers (proneurosporene and prolycopene) were not detected in either
Navelate or, most importantly, in Pinalate, as already
known for orange fruit (Gross, 1987).
ABA content in leaves and ¯avedo of fruits from
Navelate and Pinalate
Carotenoids are also precursors of the hormone ABA
(Fig. 1). To investigate whether the alteration in carotenoid
Pinalate, a novel Citrus with altered fruit colour 733
Fig. 5. HPLC pro®les of saponi®ed carotenoid extracts in leaves and ¯avedo of fruits from Navelate and Pinalate at three different development/
maturation stages: immature green (IG), mature green (MG) and full-colour (FC). All pro®les are MaxPlot chromatograms (each carotenoid shown
at its individual l maxima). Equivalent amounts of tissue were extracted and injected into HPLC, but samples from leaves are three times more
dilute than fruit samples. Note the different scale of AU for FC fruits (lower panels) due to the high concentration of violaxanthin (peak no. 12) in
Navelate and of phytoene (peak no. 11) in Pinalate extracts. Peak identi®cation is described in Table 1. AU, Absorption units.
composition found in Pinalate ¯avedo had also impaired
ABA accumulation, the content of this hormone in the
¯avedo at different ripening stages was analysed through
two consecutive seasons (Table 3). In Navelate, ABA
content increased with the onset of colouration and
remained at high levels at later stages. In Pinalate,
however, ABA content was lower and the ¯avedo of
full-coloured fruits contained between 3 and 6-times lower
734 Rodrigo et al.
Table 2. Distribution of carotenoids (as a percentage of total carotenoid content) in leaves and fruits from Navelate and Pinalate
The numbers in brackets indicate carotenoid peak number in the HPLC elution pro®le. Values are mean 6SD of at least three measurements.
Carotenoid (peak no.)
Tissue
Leaves
Peel fruit
Navelate
a
Phytoene (11)
Phyto¯uene a(18)
z-Carotene a(20)
a-Carotene (21)
Lutein (13)
b-Carotene (22)
Violaxanthin (12)
Neoxanthins (8)
Others/unidenti®ed
a
Pinalate
±
±
±
±
±
±
5.560.5
51.561.5
15.061.4
±
18.563.5
9.562.1
8.561.5
4861.4
12.061.0
±
18.061.0
13.560.5
Immature green
Mature green
Navelate
Pinalate
Navelate
Pinalate
Navelate
Pinalate
7.260.3
4.160.4
5.160.3
5.560.6
31.561.5
4.560.5
±
26.063.0
16.162.4
±
±
±
±
23.761.2
±
41.561.5
36.761.2
3.061.0
24.261.0
11.160.9
11.960.8
±
9.260.9
±
13.260.9
15.461.3
10.163.1
1.560.1
0.360.1
±
±
±
±
54.662.3
15.360.9
28.261.2
44.361.3
21.361.5
24.560.6
±
±
±
7.160.6
0.960.1
1.961.8
±
±
0.460.1
3.760.3
46.561.5
3.460.7
±
36.561.5
11.963.6
Full-colour
all isomers.
Table 3. ABA content in leaves and ¯avedo of Citrus sinensis cv. Navelate and its mutant Pinalate
Values are mean 6SD of four replicates.
Tissue
ABA (mg g±1 fr. wt.)
Experiment I
Navelate
Leaves
Peel fruit
Immature green
Mature green
Full-colour
a
Experiment II
Pinalate
Navelate
a
0.3460.11
0.3060.05
n.d.
n.d.a
0.4760.07
0.9560.11
n.d.
0.2060.03
0.1660.03
0.3860.03
0.5260.06
1.0060.23
Pinalate
n.d.
0.2660.03
0.2760.03
0.3160.04
n.d. Not determined
ABA than the parental fruit (Table 3). It is noticeable that
leaf ABA amounts were similar in both phenotypes,
suggesting, again, that the pathway is not affected in
Pinalate vegetative tissue.
Discussion
Mutants with altered carotenoid synthesis and accumulation are useful for the study of the regulation of these
processes (Bramley et al., 1993). Most of the information
on the regulation of carotenogenesis in plants arises from
studies on Arabidopsis and tomato plants, because of the
availability of collection mutants and the convenience of
these plants for genetic analysis (DellaPenna, 1999;
Hirschberg, 2001). Citrus are plants prone to develop
spontaneous mutations in the ®eld and many of the
cultivars currently available in the market have been
obtained by the agronomical and nutritional selection of
naturally occurring mutants. Navel oranges (Citrus
sinensis) are among the citrus species more sensitive to
develop spontaneous bud mutations (Saunt, 2000).
However, and despite the importance of carotenoids in
the commercial value of citrus fruits, only a few
biochemical studies on mutants with altered fruit colouration have been reported. The spontaneous Navel mutant
Cara Cara has recently been characterized and accumulates
high amounts of b-carotene, lycopene and colourless
carotenes (phytoene and phyto¯uene) in the pulp, resulting
in red pigmentation (Lee, 2001). The Sarah variety, a
Shamouti orange with pink colour, has been also described
and contains lycopene but not b-carotene (Monselise and
Halevy, 1961).
In the present work the authors have investigated the
biochemical basis of a novel fruit colour mutant of Navel
orange, named Pinalate. The most striking feature of
Pinalate is the yellow colour of the peel of mature fruits.
Data presented demonstrate that this phenotype results
from an apparent partial blockage at the z-carotene
desaturation step of the carotenogenic pathway, the
substrate z-carotene and upstream compounds, phytoene
Pinalate, a novel Citrus with altered fruit colour 735
and phyto¯uene, accumulate, whereas downstream products, such as xanthophylls and ABA, are decreased
(Fig. 5; Tables 2, 3).
Although the colour of Pinalate fruits is similar to that of
the so-called `colourless' Citrus species, such as lemon (C.
limon) and white grapefruit (C. paradisi), its pigment
complement and biochemical properties are distinctive.
Lemon accumulates phyto¯uene and z-carotene, about
18% and 17% of total carotenoids, respectively, but not
phytoene (Yokoyama and Vandercook, 1967; Gross,
1987). The peel of white grapefruit contains high relative
levels of the earlier carotenes phytoene and phyto¯uene
(accounting for up to 60±70% of total carotenoids), but the
proportion of z-carotene hardly reaches 1% (Romojaro
et al., 1979), in contrast to Pinalate composition.
Moreover, in these yellow species, ABA in the peel
increases during maturation and ripening reaching, in some
examples, concentrations even higher than that of oranges
(Aung et al., 1991). In Pinalate, by contrast, the ABA
content remains low throughout fruit maturation (Table 3).
These observations suggest differences in the regulation of
carotenoid composition between Pinalate and lemons and
white grapefruits, despite a similarity in fruit colour, and
indirectly reinforce the partial blockage of ZDS in
Pinalate.
Mutations affecting carotenoid biosynthesis in green
tissues may originate pleiotropic phenotypes. In contrast,
mutations affecting later stages of chromoplast development of fruits may provide a more unambiguous phenotype
(Bartley et al., 1994). This analyses of Pinalate did not
reveal any other perturbation in the fruit maturation
process (Fig. 3), besides those related with its altered
pigmentation and ABA de®ciency in fruits (Figs 4, 5,
Tables 2, 3), thus suggesting that the mutation in Pinalate
has not affected the general developmental regulation of
maturation. Thus, it can be speculated that reduced ABA
content in the ¯avedo may not be detrimental for fruit
development and maturation or alternatively that the low
level of ABA in Pinalate fruits is still above the threshold
required for normal development. In fruit of different
citrus species, the onset of fruit degreening has been found
to be associated with an important increase in the ABA
content (Table 3; Aung et al., 1991; Lafuente et al., 1997;
Richardson and Cowan, 1995). Since fruits of the ABAde®cient mutant exhibit a delay in the rate of degreening
(Fig. 2B) it is suggested that ABA may play a role in the
regulation of the rate of fruit colouration in citrus fruits.
To the best of these analyses, the Pinalate phenotype is
fruit-speci®c, a property exempli®ed by the carotenoid
composition and ABA content, which were substantially
distinct in the peel of Pinalate fruits, although unaffected in
leaves (Fig. 5; Tables 2, 3). Differences in carotenoid
composition are observed as early as immature green fruits
from Pinalate, where a minor accumulation of the linear
carotenes phytoene, phyto¯uene and z-carotene becomes
detectable, although the peel of immature green fruits is a
chloroplast-containing tissue. In mature green fruits, a
substantial decrease in carotenoid content, with respect to
immature green fruits, is observed in both Navelate and
Pinalate. A reduced level of carotenoids in the peel of
citrus fruits at mid-season coincides with the beginning
of the degreening process and may re¯ect the conversion
of chloroplasts to chromoplasts (Farin et al., 1983; Eilati
et al., 1975; Gross, 1987). Again, at the mature green stage,
Pinalate fruits contain a relatively high proportion of
colourless carotenes, which are not detectable in Navelate.
In Navelate fruit chromoplasts new carotenoid biosynthetic activities appeared, resulting in a massive accumulation of violaxanthin, which is the more abundant pigment
of the peel of full-coloured fruits. Other pigments,
although in minor amounts, were tentatively identi®ed as
apocarotenoids and b-cryptoxanthin, which have a signi®cant contribution to the bright-orange colour of the peel. In
full-coloured fruits of the mutant, the carotenoid biosynthetic pathway is severely altered at the level of ZDS, since
phytoene, phyto¯uene and z-carotene accumulate, and the
content of downstream products in the pathway such as
violaxanthin, neoxanthin and ABA were substantially
lower. De®ciency of the characteristic orange-red apocarotenoid-like compounds and b-cryptoxanthin is also found
in Pinalate. The biosynthesis of citrus apocarotenoids is
probably due to the enzymatic oxidative cleavage of
certain xanthophylls (Farin et al., 1983; Gross, 1987), and
therefore low xanthophyll content in Pinalate might also
prevent the formation of apocarotenoids.
The results presented show that two properties de®ne the
Pinalate phenotype: (1) an accumulation of the linear
carotenes phytoene, phyto¯uene and z-carotene, and a
reduction of xanthophylls and downstream products in the
carotenoid pathway, and (2) these alterations are fruitspeci®c, without affecting other fruit maturation events.
Taking these two properties together, several possibilities
can be suggested to explain the origin of the Pinalate
phenotype.
An interesting possibility would be the presence of two
different isoforms of ZDS in Citrus sinensis: a leafassociated isoform functional in both Pinalate and
Navelate, and a fruit isoform, which would be defective
in Pinalate. This fruit-speci®c ZDS isoform would have a
minor role in the chloroplasts of fruits, but would be
essential for the carotenoid accumulation in chromoplasts.
Similar situations have been reported for other steps of
tomato carotenoid biosynthesis, where chloroplastic and
chromoplastic isoforms of the corresponding enzymes are
differentially regulated in the chloroplast and chromoplastcontaining tissues. Tomato contains two differentially
expressed phytoene synthase genes, Psy-2 predominates in
chloroplastic tissue, while Psy-1 shows a chromoplastspeci®c expression (Fraser et al., 1994, 1999). Two blycopene cyclases have been also found in tomato, CYC-B
736 Rodrigo et al.
and LCY-B. CYC-B does play a role in chromoplastcontaining tissue, but not in vegetative tissues, whereas
LCY-B is not functional in fruits, but it is in ¯owers and
green tissues (Ronen et al., 2000). Two b-carotene
hydroxylases are also present in tomato plants, one is
expressed in green tissues while the other is exclusively
expressed in the ¯ower (Hirschberg, 2001).
Alternatively, the fruit-speci®c nature of the Pinalate
alteration might be explained by the presence of additional
regulatory factors speci®c to fruits and related to
carotenoid synthesis or accumulation, as occurs in the
tomato Delta mutant. Delta fruits accumulate d-carotene at
the expense of lycopene and strong evidence suggests that
the Del locus encodes a lycopene-e-cyclase. The amino
acid sequence of the lycopene-e-cyclase is identical
between Delta and the wild type and both are equally
functional. It is suggested that Delta contains the allele of
lycopene-e-cyclase from the green-fruited L. pennellii that
could differ from the wild-type allele in the promoter
region. In the Delta mutant lycopene-e-cyclase mRNA
fails to be down-regulated during ripening, most probably
because of the failure of the wild-type regulatory factor to
interact with the L. pennellii allele (Ronen et al., 1999).
Neither carotenoid content nor the mRNA levels of the
involved cyclase gene are affected in leaves and petals
(Ronen et al., 1999). This mutant illustrates the presence of
additional fruit-speci®c factors that might operate to
regulate, in a tissue-dependent manner, the expression of
carotenoid biosynthetic genes.
Another different possibility to explain the nature of
Pinalate would take into consideration an alteration in
desaturase-associated factors. A similar situation has been
found in mutants with a blockage in phytoene desaturation
that do not map at the pds structural gene, like immutants,
pds1 and pds2 in Arabidopsis (Norris et al., 1995; Wetzel
and Rodermel, 1998), ghost in tomato (Giuliano et al.,
1993), or võÂviparous2 in maize (Hable et al., 1998). Some
of these mutations have been shown to target additional
functions required for desaturation. For instance, pds1
codes for an enzyme required for the biosynthesis of
plastoquinones, which would act as electron carriers in the
desaturation reaction (Norris et al., 1995). Immutans and
ghost de®ne a plastid terminal oxidase (PTOX) as a
component of a redox chain in phytoene desaturation
(Carol et al., 1999; Wu et al., 1999; Josse et al., 2000).
However, alterations in these factors, necessary for
ef®cient desaturation, would probably impair both PDS
and ZDS activities, given the similarity between the
reactions catalysed by both enzymes (Cunningham and
Gantt, 1998). Since in Pinalate leaves the carotenoid
composition and ABA content are not affected and PDS
activity in fruit appears not to be impaired, it, therefore,
seems unlikely that the Pinalate phenotype is due to an
alteration in desaturase associated factors.
Recently, the isolation and characterization of a new
carotenoid isomerase (CRTISO) in Arabidopsis and
tomato has revealed a novel enzymatic activity necessary
for the cis to trans isomerizations of carotenes and the
conversion to lycopene which takes place at the level of
the z-carotene desaturation (Isaacson et al., 2002; Park
et al., 2002). Tomato, Arabidopsis, Scenedesmus, and
Synechocystis mutants which have lost or impaired
CRTISO function, accumulate poly-cis-carotenes, mainly
poly-cis lycopene, and indicates that an all-trans-conformation of lycopene is necessary for its cyclation
(Breitenbach et al., 2001; Isaacson et al., 2002; Park
et al., 2002). It also seems that isomerase activity is not
critical in photosynthetic tissues in the presence of light,
but is only required in the dark or in chromoplastcontaining tissues resulting, in some circumstances, in a
fruit-speci®c alteration. Results of HPLC analysis of
¯avedo from Pinalate do not support the possibility that
a defective isomerase may be the cause of the Pinalate
phenotype. Minor amounts of different z-carotene isomers
(Table 1; Fig. 5) have been identi®ed in Pinalate fruits.
Moreover, prolycopene or proneurosporene, which would
be the ®nal products of the pathway in the absence of
isomerase, have never been found in any of the samples
analysed. Authors believe that cis isomers tentatively
identi®ed are probably the result of a photostationary
mixture between cis and trans isomers, due to the high
accumulation of carotenes in Pinalate.
Finally, an attractive possibility is that the altered
carotene complement of Pinalate may be due to an
alteration of carotenoid-associated proteins/lipids, which
have a role in carotenoid accumulation, and also unique
mechanisms specialized for chromoplasts (Vishnevetsky
et al., 1999), which explain the fruit-speci®c nature of
Pinalate.
In conclusion, Pinalate is a fruit-speci®c alteration with
a high content of early carotenes (phytoene, phyto¯uene
and z-carotene) and a reduced proportion of downstream
xanthophylls and ABA, which result in distinctive yellowcoloured mature fruits. These alterations presume a defect
in ZDS or ZDS-associated activities. Pinalate, thus,
provides an interesting experimental system to reveal
molecular mechanisms regulating the synthesis and the
accumulation of carotenoids in Citrus. Pinalate, because of
its ABA de®ciency, also provides excellent material with
which to study the involvement of this hormone in fruit
maturation and stress responses.
Acknowledgements
We acknowledge Dr T Lafuente (IATA-CSIC) for the synthesis of
the ABA conjugate used for ABA quanti®cation, and for her help
and comments during the course of this work. We acknowledge Dr L
Navarro (IVIA, Moncada) for allowing us the use of the Citrus
Germplasm Bank of the IVIA. We also thank J Cervera and J Gil for
Pinalate, a novel Citrus with altered fruit colour 737
providing fruits from commercial orchards. The technical assistance
of A Beneyto and I Chilet is acknowledged. This work was
supported by grants ALI96-0506-C03-01 and ALI99-0954-C03-02
from `ComisioÂn Interministerial de Ciencia y TecnologõÂa' (CICYT,
Spain), GV-CAPA97-01-C2 from Conselleria de Agricultura
(Generalitat Valenciana) and a post-doctoral contract from
Ministerio of Ciencia y TecnologõÂa (to MJR).
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