J. Plant Physiol. 160.

451 – 459 (2003)

 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

Carotenoid levels during the period of growth and ripening in fruits of

different olive varieties (Hojiblanca, Picual and Arbequina)

María Roca, María Isabel Mínguez-Mosquera*

Departamento de Biotecnología de Alimentos, Instituto de la Grasa, (CSIC), Avda. Padre García Tejero, 4, Sevilla 41012, Spain

Received February 12, 2002 · Accepted July 30, 2002

Summary
During fruit growth and development, carotenoid accumulation follows the same qualitative pattern in
three olive varieties (Olea europaea L.). In the stage of ripening, the Arbequina variety is differen-
tiated from Hojiblanca and Picual by its possession of esterified xanthophylls. The Chl a/b ratio is
higher in Arbequina than in Hojiblanca and Picual throughout the life cycle of the fruit, while the per-
centage of lutein is always lower, and that of β-carotene higher. Independent of the high (Hojiblanca
and Picual) or low (Arbequina) pigment content, the chlorophyll/carotenoid ratio (a + b)/(x + c) is sim-
ilar for the three varieties. There is evident carotenoid breakdown at the onset of ripening in the fruits
of the Hojiblanca and Picual varieties, while in Arbequina there is a new period of carotenoid
accumulation. As ripening proceeds in Arbequina fruits, a slow carotenoid-breakdown process is ini-
tiated.

Key words: carotenoids – chlorophylls – growth – Olea europaea – olive varieties – ripening

Abbreviations: x + c = total carotenoids. – Chl = chlorophyll. – a + b = total chlorophylls

Introduction
The carotenoids are found linked non-covalently to the protein
complexes in the thylakoid membranes, and are fundamen-
tally important functional and structural components of the
photosynthetic apparatus (Grossman et al. 1995). The chloro-
plasts of higher plants typically accumulate lutein, β-caro-
tene, violaxanthin, and neoxanthin (in that order of abun-
dance) as major carotenoids (Lichtenthaler 1969, 1987). In
general, carotenoid levels are directly proportional to the
amount of chlorophyll in the photosynthetic tissues (Lichten-
thaler and Calvin 1964). There is a specific binding of β-caro-
* E-mail corresponding author: minguez@cica.es
tene to the Chl a pigment proteins of PSI and PSII, and of
lutein and neoxanthin together with Chl a and Chl b in the
light-harvesting chlorophyll a/b complexes of PSII (Lichten-
thaler et al. 1982, Lichtenthaler 1987). Thus, the levels of lutein
and neoxanthin are related to those of chlorophyll b, and lev-
els of β-carotene to those of chlorophyll a (Juhler et al. 1993).
There are three typical patterns of carotenoid content var-
iation through the growth cycle exhibited in fruits (Gross
1987): One curve shows a minimum carotenoid level in mid-
cycle, observed originally by Laval-Martin (1975) in «cherry»
tomatoes and later in other fruits such as kumquat (Huyskens
et al. 1985); the second shows a continuous increase, as in
citrus fruits (Stewart 1977); and the last is characterised by a
continuous decrease. The latter comprises three subdivi-

0176-1617/03/160/05-451 $ 15.00/0
452 María Roca, María Isabel Mínguez-Mosquera
sions: fruits that contain other pigments when ripe, such as
cherries (Okombi et al. 1980) or olives (Mínguez-Mosquera
and Gallardo-Guerrero 1995); fruits with low carotenoid con-
tent, such as grapes (Banet et al. 1981); and specific curves,
like that of a peach variety (Lessertois and Monéger 1978),
which show the total disappearance of carotenoids.
The effect of the process of fruit growth and ripening on the
carotenoid levels has not been investigated widely in non-
carotenogenic fruits, except for a study in two varieties of the
kiwi (Watanabe and Takahashi 1999), differentiated one from
the other by the high and low content in both chlorophylls and
carotenoids. For fruits such as the mango (Ketsa et al. 1999)
or pepper (Hornero-Méndez et al. 2000), in which all the va-
rieties are carotenogenic, the scientific documentation is
abundant. In the olive, although the content of carotenoids is
low, their presence is important because they are liposoluble
and, together with the chlorophylls, supply colour to virgin
olive oil. Varieties can be differentiated by their high (Hoji-
blanca and Picual) or low (Arbequina) pigment content (Roca
and Mínguez-Mosquera 2001). The latter variety is remark-
able for the esterified xanthophylls identified in its oils (Gan-
dul-Rojas and Mínguez-Mosquera 1996). Previous studies
have shown that, during the fruits’ colour change from green
to yellowish, there is an unusual carotenogenesis, besides a
process of carotenoid esterification (Roca and Mínguez-Mos-
quera 2001). This indicates a different pigment accumulation
in the Arbequina variety. This study attempts to elucidate
whether, in fact, the accumulation and/or breakdown of chlo-
rophylls and carotenoids during the life cycle of olive fruits is
different in the variety Arbequina than that in Hojiblanca and
Picual.
Materials and Methods
Raw material
The study was carried out with olives (Olea europaea L.) of the Hoji-
blanca, Picual, and Arbequina varieties, from four olive trees per var-
iety, chosen in the plot owned by the Agricultural Experimental Station
at Cabra (Córdoba, Spain). The experiment was begun the second
week in July. Sampling was done every seven days until the second
week in December for the Hojiblanca and Picual varieties, and until
the beginning of February for the Arbequina variety. For each sam-
pling, 2 kg of olives were picked from the whole perimeter of all 4 olive
trees selected per variety for the study. Sampling was always per-
formed between 9 and 10 a.m., from branches within arm’s reach. The
number of samplings was 23 for the first two varieties and 32 for the
Arbequina variety, which ripens later.
Extraction and identification of pigments
Samples were made from a triturate homogenised from 100 pitted
fruits of the most representative size by accurately weighing approx-
imately 4–15 g for each analysis, depending on the degree of ripe-
ness of the fruits. Pigment extraction was performed with N,N-
dimethylformamide under a green light according to the method of
Mínguez-Mosquera and Garrido-Fernández (1989). All analyses were
performed in triplicate.
Pigment separation and quantification
This was carried out by HPLC using an HP 1100 Hewlett Packard liq-
uid chromatograph fitted with an HP 1100 automatic injector and
Diode Array Detector. Data were collected and processed with an LC
HP ChemStation (Rev. A.05.04). A stainless steel column (25 ×
0.46 cm), packed with 5 µm C18 Spherisorb ODS-2 (Teknokroma, Bar-
celona, Spain), was used. The column was protected with a pre-col-
umn (1 × 0.4 cm i.d.) packed with the same material. The solution of
pigments in acetone was centrifuged at 13,000 ×g (MSE Model Micro
Centaur) prior to injection into the chromatograph (20 µL). Separation
was performed using an elution gradient (flow-rate 2 mL min –1) with
the mobile phases (A) water/ion-pair-reagent/methanol (1 : 1 : 8 v/v/v)
and (B) acetone/methanol (1 : 1 v/v). The ion pair reagent was
0.05 mol/L tetrabutylammonium acetate (Fluka, Chemie AG Switzer-
land) and 1 mol/L ammonium acetate (Fluka) in water. The gradient
scheme has been described in detail in a previous work (Mínguez-
Mosquera et al. 1992). Detection was performed simultaneously at
430, 450, and 666 nm. External standard calibration was used for
quantitation. All standards were purified by TLC using different
eluents described in a previous publication (Mínguez-Mosquera et al.
1993, Mínguez-Mosquera and Gandul-Rojas 1995).
Results
1. Fruit development and changes in moisture
The olive is a drupe (apricot-, plum-, cherry-, etc. type), and
its growth is defined by a double sigmoid, in which three
stages are distinguished. Figure 1 shows the change in fruit
moisture and weight, differentiating mesocarp and endocarp,
for each olive variety: Hojiblanca (Fig. 1 a), Picual (Fig. 1 b),
and Arbequina (Fig. 1c).
The period of growth and development of the olive is rela-
tively prolonged. In all varieties, phase I is characterised by
rapid fruit growth. During this period, the weight of the meso-
carp increases and the endocarp reaches its final weight, its
tissues becoming fully developed with cell division almost fin-
ished. At the same time, the moisture content increases in the
first weeks and then falls very slightly. During the second
phase, mesocarp growth is very slow (practically constant),
while the moisture falls slightly. In the third and final growth
phase, fruit weight increases considerably due to the in-
crease in size of the pulp cells. At the end of this period, the
fruit size is fixed, epidermal colour begins to change, and the
seed reaches maturity. The moisture falls in all three varieties.
Using the parameters described above, the growth period
can be established as 12 weeks in the Arbequina variety and
16 weeks in the Hojiblanca and Picual varieties. The period of
ripening then begins in each variety, with a colour change in
the fruit skin coinciding with the start of the synthesis of
anthocyanin compounds, which spread over the fruit surface
 Carotenoid levels during growth-ripening of different olive varieties 453

2. Chlorophyll and carotenoid profile during the life

cycle of olive fruits of different varieties
Figure 2 shows the change in the chlorophyll (Fig. 2 a) and
carotenoid (Fig. 2 b) fractions during the stages of growth and
ripening in the fruits of the Hojiblanca, Picual, and Arbequina
varieties. In the last variety, the onset of ripening involves es-
terification of xanthophylls, which are evaluated with their re-
spective precursors. The fruits of the Hojiblanca variety show
the highest content in chlorophyll and carotenoid pigments,
closely followed by those of the Picual variety. The concentra-
tions for the Arbequina variety are much lower.
In the fruits of the Hojiblanca and Picual varieties, the con-
tent of both pigment families increases noticeably during the
rapid growth stage, and then falls slowly or remains constant
during the rest of the growth period. At the onset of ripening,
the concentration of chlorophylls and carotenoids falls
sharply, and continues to fall throughout this stage. All these
changes in pigment content are much more attenuated in the
fruits of the Arbequina variety. The curve is quite different,
above all with regard to the carotenoid fraction. Careful ob-
servation of the experimental points for the Arbequina variety
shows how the growth and ripening phases are separated by

Figure 1. Development (open symbols) and ripening (closed sym-

bols) of olive fruits in the Hojiblanca (a), Picual (b) and Arbequina (c)
varieties. Changes in mesocarp (䉭) weight, endocarp (䊊) weight and
moisture content (䊐). (Coefficient of variation < 3 % in all cases).

and then into the flesh of the ripe fruits. In the Arbequina var-
iety, the colour change at the onset of ripening is different;
yellowish tones appear that last until week 25. Anthocyanin
synthesis is slower in this variety, and the fruit surface is not
covered until the beginning of February. Although the flower-
ing period is the same in the three varieties, Arbequina takes
longer to ripen (Barranco 1997).
The olive fruits differ in size and shape as well. Fruit weight
Figure 2. Changes in the total Chl (a) and carotenoid content (b)
ranges between the 1.72 g for the spherical fruits of the Arbe- during development and ripening in the olive fruits of Hojiblanca
quina variety and the 4.40 g for the ellipsoidal fruits of Hoji- (䊐, ——), Picual (䉭, - - - -) and Arbequina (䊊, · · · · ·) varieties. Open
blanca. The fruits of Picual, with a somewhat lower weight and closed symbols as Fig. 1. (Coefficient of variation < 6 % in all
(3.00 g), have a small peak at the apex. cases).
454 María Roca, María Isabel Mínguez-Mosquera
Figure 3. Changes in the total carotenoid content, differentiating
between the development and the ripening stages, in the olive fruits
of Arbequina variety. (Coefficient of variation < 6 % in all cases).
Figure 4. Changes in Chl a/b (a) and the pigment ratio (a + b)/(x + c) (b)
during development and ripening of olive fruits in the Hojiblanca,
Picual and Arbequina varieties. Symbols as Fig. 2. (Coefficient of var-
iation < 3 % in all cases).
a lag phase, during which new regulatory phenomena induce
transformation of pigments. The lag phase corresponds to the
disintegration of the grana system as the chlorophylls disap-
pear. This is confirmed if the fit is made differentiating these
two stages (Fig. 3). It is not found in the other two varieties.
The fruits of the Arbequina variety consistently have the
highest values for the Chl a/b ratio (Fig. 4 a), exceeding 4.0
units in the growth phase, and even exceeding 5.0 at certain
points during ripening. In the fruits of the Hojiblanca and
Picual varieties, this ratio fluctuates between 3.5 and 4.0 dur-
ing the growth stage, and falls slightly during ripening. In con-
trast, the (a + b)/(x + c) ratio (Fig. 4 b) is always higher in Hoji-
blanca and Picual (4.0 – 5.0) than in Arbequina (3.5 – 4.0) dur-
ing the fruit growth period. At the onset of ripening, the value
of the ratio falls sharply for Hojiblanca and Picual (reaching
1.0), and remains between 2.0 and 3.0 for Arbequina.
3. Carotenoid accumulation
The carotenoids present in olive fruits are lutein, β-carotene,
violaxanthin, neoxanthin, antheraxanthin, and β-crypto-
xanthin. The first four typically make up more than 95 % of the
carotenoids present in the olive, and are the characteristic
carotenoids in the base composition of the chloroplast,
largely conserved during evolution (Gross 1987). In the Arbe-
quina variety, esters of violaxanthin and neoxanthin also have
been found, but in this study, these esterified xanthophylls
have been determined jointly with their corresponding free
precursors. When the light intensity is high, violaxanthin is
transformed via antheraxanthin into zeaxanthin (Schindler
and Lichtenthaler 1996). However, zeaxanthin was not
detected at any point during fruit growth and ripening, prob-
ably because the illumination during sampling was below the
threshold for the transformation.
Fruit growth phase
The major chloroplast pigment of the three olive varieties
throughout the life cycle of the fruit is lutein (Fig. 5), the only
representative of the β, ε series. In the three varieties, its con-
tent increases progressively during the first weeks of fruit
growth, and then remains constant, at different levels de-
pending on variety, until the fruit is fully developed.
Figure 5. Changes in lutein content during development and ripening
in the olive fruits of Hojiblanca, Picual, and Arbequina varieties. Sym-
bols as Fig. 2. (Coefficient of variation < 6 % in all cases).
 Carotenoid levels during growth-ripening of different olive varieties
Figure 6. Changes in β-carotene (䊐, — —), violaxanthin (䊊, ——),
neoxanthin (䉭, - - - -), antheraxanthin (䉫, · · · · ·) and β-cryptoxanthin
(insert d in each figure) during development and ripening of olive
fruits in the Hojiblanca (a), Picual (b) and Arbequina (c) varieties.
Open and closed symbols as Fig. 1. (Coefficient of variation < 6 % in
all cases).
The rest of the carotenoids belong to the β, β series (Fig. 6)
and, as with lutein, their content increases markedly in the
first weeks of the growth period. For each particular caroten-
oid, this increase depends on the availability of its precursor.
The balance set up between them differentiates the fruits of
the Arbequina variety.
455
Fruit ripening phase
In this phase, the content of all the carotenoids begins to fall
relatively rapidly, coinciding with the progressive synthesis of
anthocyanin compounds. At the same time, in fruits whose
ripe colour is due to anthocyanin compounds, the skin colour
changes gradually from green to black. This does not happen
in the Arbequina variety, whose colour, at the onset of ripen-
ing, becomes yellow, showing an evident delay in anthocya-
nin synthesis, which is almost never complete.
During the course of fruit ripening, it is notable that
whereas in the Hojiblanca and Picual varieties there is a pro-
gressive decrease in content of all the carotenoids, both for
the β, ε series (Fig. 5) and for the β, β series (Fig. 6), in Arbe-
quina the content of all the carotenoids increases at the start
of this period. Later, in the ripe fruit with black coloration, the
content of all the carotenoids begins to fall gradually, as hap-
pens earlier in the Hojiblanca and Picual varieties.
The kinetic study of the disappearance of each carotenoid
during the ripening stage (Table 1) shows that the highest
rates are for the carotenoids present in the fruits of the Picual
variety, followed very closely by those of Hojiblanca. The Ar-
bequina variety has much lower values, indicating differences
with the other varieties. β-cryptoxanthin is not included in the
kinetic study because it shows only trace levels of content
during the ripening period, and is thus of lesser interest.
The slopes (Table 1) provided by the kinetic equations
enable the rates of carotenoid disappearance to be ranked
from lowest to highest, and the behaviours to be compared,
both within a single variety and among varieties. In Hojiblanca
and Picual, there is a parallelism in the order of disappear-
ance of each carotenoid. For both varieties, lutein is the most
slowly degraded, followed closely by antheraxanthin. Some
time later, and in equal measure, are β-carotene and neoxan-
thin and; lastly, violaxanthin is the carotenoid that disappears
most rapidly. Although the slopes are very similar, the higher
values are always those of the Picual variety.
The carotenoids present in the Arbequina variety show a
different behaviour, not only in the lower value of their slopes,
but also in the similarity among them, indicating a complete
parallelism in the disappearance of these compounds. Also
noteworthy is that in this variety the correlation coefficient
found for the carotenoids (except lutein) is lower than 0.9. The
explanation for the exception may be that during part of the ri-
pening phase, the carotenoid content continues to increase,
and the fit corresponds to degradation kinetics.
Consequently, the data treatment is repeated to include
only the ripeness states involving a fall in content; that is, from
week 23 to 32. Although the new results give slopes of higher
values, these continue to be lower than those for the fruits of
the Hojiblanca and Picual varieties. The new correlation coef-
ficients are improved considerably for antheraxanthin, β-caro-
tene, and neoxanthin. That for lutein remains the same, and
that for neoxanthin decreases. This is possibly because the
content of lutein is constant until practically the end of the ri-
456 María Roca, María Isabel Mínguez-Mosquera

Table 1. Slope (B) and correlation coefficient (R) obtained to first-order degradation rate of carotenoids during the ripening of Hojiblanca, Picual
and Arbequina varieties.

Hojiblanca variety Picual variety Arbequina variety

Weeks 17 – 23 Weeks 17 – 23 Weeks 13 – 32 Weeks 23 – 32

B R B R B R B R

Lutein – 0.15 0.95 – 0.21 0.98 – 0.06 0.93 – 0.12 0.93

Antheraxanthin – 0.23 0.99 – 0.28 0.98 – 0.06 0.86 – 0.21 0.98
ß-carotene – 0.25 0.98 – 0.33 0.99 – 0.07 0.90 – 0.20 0.99
Neoxanthin – 0.26 0.96 – 0.33 0.99 – 0.06 0.89 – 0.13 0.83
Violaxanthin – 0.29 0.97 – 0.34 0.99 – 0.06 0.81 – 0.19 0.94

Table 2. Percentage composition of individual carotenoids of fruits of the Arbequina, Picual and Hojiblanca varieties of olives.

Carotenoids (%)

Variety Lutein ß-carotene Violaxanthin Neoxanthin Antheraxanthin

Growth
Arbequina 52.74 23.00 12.47 8.21 3.23
Picual 54.80 21.54 11.70 8.94 2.79
Hojiblanca 54.04 21.69 11.93 9.37 2.82
Ripening
Arbequina 52.10 19.53 13.42 6.71 6.91
Picual 72.64 13.44 4.90 5.73 2.97
Hojiblanca 69.64 15.34 5.17 6.68 3.01

pening period, and that of neoxanthin even increases during
this stage. The order in the rate of carotenoid disappearance
for the Arbequina variety reveals lutein as being the slowest
to disappear, as in Hojiblanca and Picual, but in this case ac-
companied by neoxanthin. Higher slopes are found for viola-
xanthin, β-carotene and antheraxanthin, in that order, al-
though with values very similar to each other.
of neoxanthin, which in the growth phase was slightly lower
than that in Hojiblanca and Picual, falls during ripening, be-
coming practically equal to that in those varieties. These dif-
ferences are a consequence of the carotenogenic process
taking place in Arbequina, and of the different rates of carot-
enoid disappearance in this variety.

Changes in the percentage composition
For comparison, Table 2 shows (in percentage terms) the in-
dividual contribution of each carotenoid to the fruits of the
three varieties, considering the whole growth and ripening
phases. The percentage contribution of each carotenoid in
Hojiblanca and Picual is similar, in both growth and ripening,
although during the latter, lutein increases, β-carotene, viola-
xanthin and neoxanthin decrease, and antheraxanthin re-
mains constant. In Arbequina, however, the percentage of lu-
tein remains constant during ripening (and continues to be
lower than in Hojiblanca and Picual), while that of β-carotene
falls, although its percentage continues to be higher than in
the other two varieties. Violaxanthin and antheraxanthin in-
crease with ripening, augmenting the differences found with
Hojiblanca and Picual during growth. Lastly, the percentage
Discussion
Presumably due to their functionality, the ratio between chlo-
rophylls and carotenoids is usually around 4 : 1, and is con-
stant both for the whole thylakoid system and when grana
and stroma lamellas are separated (Juhler et al. 1993). This
means that, independent of high or low pigment content, the
ratio remains constant, as demonstrated in the olive fruit. Al-
though the (a + b)/(x + c) ratio is slightly lower in fruits of the Ar-
bequina variety, there is an almost perfect correlation be-
tween the two pigment families: the fruits of the Hojiblanca
and Picual varieties are characterised by high content in both
chlorophylls and carotenoids, whereas fruits of the Arbequina
variety show a low chlorophyll and carotenoid content.
The grana complex is rich in chlorophyll b and xantho-
phylls, as it contains approximately 85 % of the PSII, 36 % of
the PSI, and the LHCII, while the stroma lamellas are rich in
 Carotenoid levels during growth-ripening of different olive varieties
chlorophyll a and β-carotene, as they contain some 64 % of
the PSI and 15 % of the PSII (Juhler et al. 1993). Figure 4 b
shows that Arbequina fruits are richer in chlorophyll a than
those of Hojiblanca and Picual, in which there is proportio-
nally more chlorophyll b. Consequently, the percentage con-
tent of lutein in the fruits of Hojiblanca and Picual (Table 2) is
always higher than in those of Arbequina, while in the fruits of
the latter variety, the percentage of β-carotene is higher than
in Hojiblanca and Picual. The percentage of neoxanthin, like
that of lutein, is lower in Arbequina than in Hojiblanca and
Picual, but during ripening, it is the major presence in Arbe-
quina, possibly due to the carotenogenic process during
ripening in this variety. This indicates that the distribution of
the thylakoid system differs with variety: in Arbequina fruits;
the thylakoids must be less packed so that the grana system
is less substantial than in Hojiblanca and Picual.
As stated in the introduction, the curve of change in total
carotenoids can differ depending on the fruit species. Figure
2 shows the same phenomenon among different varieties of a
single species. The curves for the fruits of the Hojiblanca and
Picual varieties are similar to each other and different from
those of the fruits of the Arbequina variety. These profiles indi-
cate that in the former varieties, the degradation of pigment
continues to the end of the ripening stage. However, in the
fruits of the Arbequina variety, at the start of the ripening pe-
riod and consequent disintegration of the grana system, bio-
synthetic reactions are re-activated, leading to an increase in
carotenoid content. This means that carotenoid accumulation
and the regulatory signals that control it proceed differently in
the varieties of olive studied. In the classification of Gross
(1987), the varieties Hojiblanca and Picual fit the model show-
ing a continuous decrease in pigments during ripening, typi-
cal of fruits that synthesise another type of pigment when they
ripen. In contrast, the fruits of Arbequina show a minimum
value in the middle of their life cycle, first observed by Laval-
Martin et al. (1975) in «cherry» tomatoes, and later in other
fruits such as the mandarin (Noga 1981, Farin et al. 1983).
With regard to individual carotenoids, the lutein content re-
mains practically constant during the growth period in the
fruits of the three varieties. In percentage terms, this is the
major carotenoid of the four basic ones constituting the «uni-
versal chloroplast», confirming its vital function in photosyn-
thesis. Recent studies with mutants (DellaPenna 1999) have
demonstrated that there is a marked loss in photosynthesis
when lutein is absent from the chloroplast, although it can be
partly substituted by violaxanthin. Figure 4 shows how the
content of lutein remains constant throughout the growth
stage, implying that during this phase, the biosynthetic path-
way remains active.
Because β-carotene is the precursor of the β, β carotenoid
series, the fluctuations in its curve will be reflected in the pig-
ments. In the Hojiblanca variety, β-carotene reaches its max-
imum content in week 5, but antheraxanthin, violaxanthin, and
neoxanthin do so during the first three weeks, showing that
during this period the biosynthetic pathway is directed to-
457
wards the formation of these pigments. The subsequent de-
crease in the content of antheraxanthin results in the mainte-
nance of the content of violaxanthin and neoxanthin. The con-
tent of neoxanthin is maintained at the expense of its respec-
tive precursors. The same phenomenon is seen in Picual, but
with antheraxanthin having a greater role as intermediary in
favour of violaxanthin and neoxanthin in the first stage, and of
neoxanthin in the second. In Arbequina, the change in carot-
enoids up to week 7 of fruit growth coincides with that in
Picual: antheraxanthin is again intermediary in the synthesis
of the other pigments. Its concentration then increases pro-
gressively, while that of violaxanthin decreases in favour of
neoxanthin. Thus, during this time there is a net synthesis of
β-carotene, affecting directly and differently the carotenoids
originated. In Hojiblanca and Picual, β-cryptoxanthin acts
solely as an intermediary, so that its content, at trace level,
fluctuates during the growth stage, while in the Arbequina
variety it is practically absent. The constant transformation of
certain pigments into others demonstrates the biosynthetic
activity of carotenoids during the fruit growth stage in the
three varieties.
This variety-specific similarity in biosynthetic activity during
the growth period is not observed during the catabolic stage
of ripening. In the Hojiblanca and Picual varieties, all the ca-
rotenoids show a sharp and continuous decrease during ri-
pening. In contrast, carotenoid accumulation is different in Ar-
bequina fruits. The curve for lutein indicates that synthesis of
the compound remains active at the beginning of this stage,
then falls slightly, and is thereafter constant. Such a pattern
indicates that the pathways that originate and degrade lutein
are still balanced, and thus the synthesis of this pigment con-
tinues, confirming an earlier study (Roca and Mínguez-Mos-
quera 2001). It shows that the level at which mechanisms in-
volved in lutein accumulation during ripening are expressed
in Hojiblanca and Picual is different to that in Arbequina.
There is a similar effect for the carotenoids of the β, β path-
way in the Arbequina variety. Once ripening starts, anthera-
xanthin in this variety reaches its maximum content in week
17. This increase, at the expense of β-carotene, results di-
rectly in a new synthesis of violaxanthin and neoxanthin. Al-
though the antheraxanthin content then falls, its value is still
higher than that seen during the growth period. It fluctuates
until week 23, resulting in a decrease in β-carotene content
and the maintenance of violaxanthin, since the neoxanthin
content decreases progressively during this period. It seems
that the step from violaxanthin to neoxanthin is interrupted or
inhibited in this stage, and then activated again at the same
time as the first signs of anthocyanin synthesis appear (week
26). Lastly, the slight decrease or maintenance in the content
of antheraxanthin and β-carotene is seen as an increase in
the respective content of violaxanthin and neoxanthin. As a
response to this new biosynthesis of carotenoids at the onset
of ripening, there is an increase that may quadruple the con-
tent of β-cryptoxanthin, which remains constant during the
whole of this phase and then falls in the black fruit. The bio-
458 María Roca, María Isabel Mínguez-Mosquera
synthesis of β-cryptoxanthin in carotenogenic fruits is a gen-
eralised phenomenon. In the skin of ripe persimmon on the
tree, a chromoplast pattern is synthesised in which the carot-
enoids of the β, β pathway are dominant. Particularly notewor-
thy is the synthesis of β-cryptoxanthin, which increases from
1.7 % to 42 % (Ebert and Gross 1985). A similar pattern is
found in persimmon pulp, where β-cryptoxanthin becomes
the major pigment (48 %) in the ripe fruit. Following the onset
of ripening, there is a general decrease in individual carot-
enoid content, although carotenoid accumulation seems to
be different in Arbequina fruits.
If the Arbequina carotenoids, although showing degrada-
tion kinetics of very low slopes (Table 1), had the same rate of
carotenoid degradation as in the Hojiblanca and Picual varie-
ties, it could have been postulated that the differences were
due to a factor uniformly affecting the accumulation of all the
carotenoids in Arbequina. However, not only are the degrada-
tion rates lower, but also the pattern of carotenoid disappear-
ance (according to the kinetic slopes) differentiates the Arbe-
quina variety from Hojiblanca and Picual. Even when the ca-
rotenogenic factor is ignored, the ranking for carotenoid deg-
radation rate in Arbequina does not coincide with that for the
Hojiblanca and Picual varieties. Therefore, there must be
some factor/s besides the carotenogenic process, individu-
ally and differently affecting the processes of formation and
degradation of each carotenoid since the balance between
anabolic and catabolic reactions depends on the variety in
question.
Acknowledgements. We express our sincere gratitude to the Comi-
sion Interministerial de Ciencia y Tecnología of the Spanish Govern-
ment (CICYT) for supporting this research project, AGL 2000 – 0699
and Junta de Andalucía.
References
Banet E, Romojaro F, Llorente S (1981) Evolution of photosynthetic
pigments in flavedo and pulp of Marsh grapefruit. An Edaf Agrobiol
40: 259 – 267
Barranco D (1997) Variedades y patrones. In: Barranco D, Fernández-
Escobar D, Rallo L (ed) El cultivo del olivo. Mundi-Prensa Edicio-
nes, Madrid pp 59–79
DellaPenna D (1999) Carotenoid synthesis and function in
plants : insights from mutant studies in Arabidopsis thaliana. In:
Frank HA, Young AJ, Britton G, Codgell RJ (ed) The photochem-
istry of carotenoids. Kluwer Academic Publishers, Dordrecht pp
21– 37
Ebert G, Gross J (1985) Carotenoid changes in the peel of ripening
persimmon (Dyospyros kaki). Cv. Triumph. Phytochem 24: 29 – 32
Farin D, Ikan R, Gross J (1983) The carotenoid pigments in the juice
and flavedo of a mandarin hybrid (Citrus reticulata) cv. Michal dur-
ing ripening. Phytochem 22: 403 – 408
Gandul-Rojas B, Mínguez-Mosquera MI (1996) Chlorophyll and carot-
enoid composition in virgin olive oils from various spanish olive va-
rieties. J Sci Food Agric 72: 31– 39
Gross J (1987) Pigments in Fruits. Academic Press, London
Grossman AR, Bhaya D, Apt KE, Kehoe DM (1995) Light-harvesting
complexes in oxygenic photosynthesis: Diversity, control, and evo-
lution. Ann Rev Genetics 29: 231– 288
Hornero-Méndez D, Gómez-Ladrón de Guevara R, Mínguez-Mos-
quera MI (2000) Carotenoid biosynthesis changes in five red pep-
per (Capsicum anuum L.) cultivars during ripening. Cultivar selec-
tion for breeding. J Agric Food Chem 48: 3857– 3864
Huyskens S, Timberg R, Gross J (1985) Pigment and plastid ultra-
structural changes in kumquat (Fortunella margarita) Nagami dur-
ing ripening. J Plant Physiol 118: 61–72
Juhler RK, Andreasson E, Yu SG, Albertson PA (1993) Composition of
photosynthetic pigments in thylakoid membrane vesicles from
spinach. Photosynth Res 35: 171–178
Ketsa S, Phakawatmongkol W, Subhadrabhandhu S (1999) Peel enzy-
matic activity and colour changes in ripening mango fruit. J Plant
Physiol 154: 363 – 366
Laval-Martin D, Quennemet J, Monéger R (1975) Pigment evolution in
Lycopersicum esculentum fruits during growth and ripening.
Phytochem 14: 2357– 2362
Lessertois D, Monéger R (1978) Evolution des pigments pendant la
croissance et la maturation du fruit de Prunus persica. Phytochem
17: 411– 415
Lichtenthaler HK (1969) Light-stimulated synthesis of plastid quinones
and pigments in etiolated barley seedlings. Biochim Biophys 184:
164–172
Lichtenthaler HK (1987) Chlorophylls and carotenoids, the pigments of
photosynthetic biomembranes. In: Douce R, Packer L (eds) Meth-
ods in enzymology. Academic Press Inc., New York pp 350 – 382
Lichtenthaler HK, Calvin M (1964) Quinone and pigment composition
of chloroplasts and quantasome aggregates from Spinacia olera-
cea. Biochim Biophys Acta 79: 30 – 40
Lichtenthaler HK, Prenzel U, Kuhn G (1982) Carotenoid composition
of chlorophyll-carotenoid-proteins from radish chloroplasts. Z Na-
turforsch 37c: 10–12
Mínguez-Mosquera MI, Gallardo-Guerrero L (1995) Disappearance of
chlorophylls and carotenoids during ripening of the olive. J Sci
Food Agric 69: 1– 6
Mínguez-Mosquera MI, Garrido-Fernández J (1989) Chorophyll and
carotenoid presence in olive fruit (Olea europaea). J Agric Food
Chem 37: 1–7
Mínguez-Mosquera MI, Gandul-Rojas B, Gallardo-Guerrero L (1992)
Rapid method of quantification of chlorophylls and carotenoids in
virgin olive oil by high-performance liquid chromatography. J Agric
Food Chem 40: 60 – 63
Mínguez-Mosquera MI, Gallardo-Guerrero L, Gandul-Rojas B (1993)
Characterization and separation of oxidized derivatives of pheo-
phorbides a and b by TLC and HPLC. J Chromatogr 633: 295 – 299
Mínguez-Mosquera MI, Gandul-Rojas B (1995) High performance liq-
uid chromatographic study of alkaline treatment of chlorophyll. J
Cromatograph 690: 161–176
Noga G (1981) Ursachen der Rauhschaligkeit und mangelhaften
Ausfärbung bei Satsumamandarinen (Citrus unshiu Marc). Disser-
tation, Friedrich Wilhelms Universität, Bonn
Okombi G, Billot J, Hartmann C (1980) Les caroténoides de la cerise
(Prunus avium) cv. Bigarreau Napoléon. Evolution au cours de la
croissance et de la maturation. Fruits 35: 313 – 320
 Carotenoid levels during growth-ripening of different olive varieties
Roca M, Mínguez-Mosquera MI (2001) Changes in chloroplast pig-
ments of olive varieties during fruit ripening. J Agric Food Chem
49: 832 – 839
Roca M, Mínguez-Mosquera MI (2001) Unusual carotenogenesis in
fruits with pronounced anthocyanic ripening (Olea europaea var.
Arbequina). J Agric Food Chem 49: 4414 – 4419
Schindler C, Lichtenthaler HK (1996) Photosynthetic CO2 assimilation,
chlorophyll fluorescence and zeaxanthin accumulation in field-
459
grown maple trees in the course of a sunny and a cloudy day. J
Plant Physiol 148: 399 – 412
Stewart I (1977) Provitamin A and carotenoid content of citrus juices. J
Agric Food Chem 25: 1132–1137
Watanabe K, Takahashi B (1999) Chlorophyll and carotenoid pigments
in green-and yellow-fleshed kiwifruit during fruit development and
storage. J Japan Soc Hort Sci 68: 1038–1045