Journal of Experimental Botany, Vol. 54, No. 389, pp. 1977±1984, August 2003
DOI: 10.1093/jxb/erg199
RESEARCH PAPER
Signi®cance of skin ¯avonoids for UV-B-protection in
apple fruits
Alexei Solovchenko1 and Michaela Schmitz-Eiberger2,*
1
Department of Physiology of Microorganisms, Faculty of Biology, Moscow State University, GSP-2 Moscow
119992, Russia
2
Department of Horticulture, Bonn University, Auf dem HuÈgel 6, D-53121 Bonn, Germany
Received 7 February 2003; Accepted 25 April 2003
Abstract
Introduction
An attempt has been made to assess the UV-B-protective capacity of phenolic compounds accumulated
in super®cial structures of plants using apple fruit as
a model. Two apple (Malus domestica Borkh.) cultivars (Braeburn and Granny Smith) differing in
response to high ¯uxes of solar radiation were
selected and exposed to increasing doses of UV-B
radiation. The extent of UV-B-induced damage to
photosystem II of apple skin correlated with its quercetin glycoside (but not anthocyanin) content.
Granny Smith apples did not demonstrate a pronounced response to high sunlight in terms of the
accumulation of phenolic substances in the skin and
exhibited similar patterns of Fo, Fm, and Fv/Fm
changes in the course of UV-B irradiation both on
sun-exposed and shaded surfaces of a fruit. Unlike
Granny Smith, Braeburn fruits were characterized by
a signi®cant accumulation of quercetin glycosides in
sun-exposed skin, however, shaded skin contained
these compounds in similar amounts to those in
Granny Smith. Accordingly, photosystem II in sunexposed skin of Braeburn apples was resistant to
high doses of UV-B radiation (up to 97 kJ m±2),
whereas the susceptibility of the photosynthetic
apparatus in shaded skin of Braeburn to UV-Binduced damage was much higher and similar to that
of both sun-exposed and shaded skin of Granny
Smith fruits. Anthocyanins, at least in the amounts
found in Braeburn, did not show an additional effect
in UV-B protection.
Higher plants are naturally exposed to high ¯uxes of solar
radiation and therefore they are subjected to relatively
high UV-doses (Caldwell and Flint, 1997). UV-B is the
band of lowest wavelength and highest energy that
penetrates the ozone layer of the stratosphere. It comprises about 5% of the whole UV or 0.5% of the total
energy of solar radiation (Caldwell and Flint, 1997;
Krupa et al., 1998). In spite of the relatively low
irradiance, UV-B radiation could induce severe damage
to plants via direct and indirect effects on nucleic acids,
proteins and cell membranes (Kulandaivelu and
Noorudeen, 1983; Janssen et al., 1998; SchmitzEiberger and Noga, 2001).
On a whole-plant scale, the effects of UV-B radiation
are apparent as an alteration of plant morphology, a
reduction of growth, and a decrease in yield (Tevini and
Teramura, 1989; Krupa et al., 1998). For instance,
enhanced UV-B levels, as well as high temperature, play
a key role in the development of sunscald in apples
(Andrews and Johnson, 1997; Andrews et al., 1998;
Schmitz-Eiberger and Noga, 2001).
The photosynthetic apparatus of higher plants is
particularly sensitive to damage by UV-B radiation
(Kulandaivelu and Noorudeen, 1983). The primary targets
of UV-B radiation in photosystem II are the 32 kDa D1
protein of the reaction centre and the water-oxidizing
system (Janssen et al., 1998). Damage to those components
results in a decrease in the variable ¯uorescence level
(Janssen et al., 1998; SkoÂrska, 2000) making the measurement of chlorophyll ¯uorescence parameters such as
Fo, Fm, and Fv/Fm suitable for an estimation of UV-B
induced damage to plants (SkoÂrska 2000; SchmitzEiberger and Noga, 2001).
Key words: Apple, phenolic compounds, photosystem II,
protection, UV-B radiation.
* To whom correspondence should be addressed. Fax: +49 228 735764. E-mail: [email protected]
Journal of Experimental Botany, Vol. 54, No. 389, ã Society for Experimental Biology 2003; all rights reserved
1978 Solovchenko and Schmitz-Eiberger
To cope with environmental UV-irradiation conditions,
higher plants have evolved a number of protective
mechanisms. Probably, one of the most basic and most
studied is attenuation of the radiation by super®cial
structures of plant organs (Caldwell, 1981; Flint et al.,
1985; Kolb et al., 2001). The accumulation of phenolic
compounds that selectively absorb UV radiation in the
plant cuticle and epidermis (Krauss et al., 1997; Flint et al.,
1985; Mazza et al., 2000) probably represents the most
cost-effective strategy for long-term adaptation in the case
of regular and prolonged exposure to elevated doses of
solar radiation, including its UV-B component
(Robberecht and Caldwell, 1983; Pietrini et al., 2002;
Liakoura et al., 2003).
Apple fruit are known to contain large amounts of
phenolic substances in the skin. The major ¯avonoid
classes occurring in apple skin are ¯avonols such as
quercetin 3-glycosides, monomeric and oligomeric ¯avan3-ols such as catechin, epicatechin and procyanidins and,
in red-coloured cultivars, anthocyanins such as cyanidin 3glycosides. Apple fruit also contain considerable amounts
of hydroxycinnamic acid derivatives which are mainly
represented by chlorogenic acid (Escarpa and GonzaÂlez,
1998; Awad and de Jager, 2000; Awad et al., 2000).
Flavonoids and chlorogenic acid also contribute to the
quality aspects of apple fruits. Their red coloration is
primarily due to the ¯avonoid cyanidin-3-galactoside
located in the vacuoles of skin cells (Lancaster et al.,
1994). However, different groups of phenolic compounds
demonstrate non-uniform behaviour during the adaptation
of plants to strong sunlight (Awad et al., 2000; Wang et al.,
2000; Winkel-Shirley, 2001; Ryan et al., 2002). Catechin,
as well as the phenolic acid content, does not exhibit a
distinct correlation with irradiation conditions in leaves
(Bornman et al., 1997; Burchard et al., 2000) and fruits
(Awad et al., 2000). By contrast, a dramatic induction of
synthesis and accumulation of ¯avonoids is often observed
in response to high (sun)light (Kolb et al., 2001; Reay and
Lancaster, 2001; Merzlyak and Solovchenko, 2002).
Experiments with transgenic plants demonstrated an upregulation of the genes responsible for ¯avonoid (particularly, kaempferol and quercetin) biosynthesis under elevated UV-B conditions (Wang et al., 2000; Ryan et al.,
2002). Therefore, ¯avonoids are a particularly interesting
group of phenolic substances in connection with the
investigation of the high-sunlight response. However, the
UV-protective ef®ciency of phenolic `sunscreens' induced
in the course of adaptation to high sunlight under natural
conditions is still uncertain in many cases, especially in
fruits that play a crucial role in plant reproduction.
Apple fruits represent an interesting natural system for
the investigation of photo-adaptation and photo-damage
processes in plants (Andrews and Johnson, 1997; SchmitzEiberger and Noga, 2001). During the development of fruit
on the tree, one of its surfaces (sun-exposed) is predom-
inantly exposed to direct solar radiation whereas the
opposite (shaded) surface is illuminated mainly by diffuse
sunlight. As a result, the skin of the sun-exposed surface of
apple fruit develop distinct patterns of pigment composition attributable to adaptation to high sunlight (Merzlyak
and Solovchenko, 2002). In this study, two apple cultivars
(cvs), differing in their visual response to strong sunlight,
were used. Fruits of cv. Braeburn possess red pigmentation
on sun-exposed surface due to the accumulation of
anthocyanins which is not the case for Granny Smith
apples.
The main goal of this study was to estimate the
importance of ¯avonoids accumulated in apple skin in
response to strong sunlight for the protection of the apple
photosynthetic apparatus from UV-B-induced damage.
Another aim was to discover whether the anthocyanins that
accumulated in sun-exposed skin of apple fruit have a role
in UV-protection in apple fruit.
Materials and methods
Plant material
Ripe apple (Malus domestica Borkh.) bearing no visual symptoms of
damage were used in these experiments. Two cvs, Granny Smith and
Braeburn, with different responses to high sunlight were selected for
this study. Apple fruits of both cultivars were grown at the KleinAltendorf Fruit Research Station (Germany). Fruits of both cultivars
were harvested on 12 October 2002 and stored under controlled
atmosphere conditions (1.5% O2; 3% CO2) for 3 months. On the date
of the investigation, the fruits of both cultivars had attained similar
maturity according to the Streif index measurement. The index was
calculated as fruit ®rmness/[soluble solids content3starch index
value] (DeLong et al., 1999) and was 0.2 and 0.19 for Braeburn and
Granny Smith, respectively.
Sixty fruits of each cultivar were used (a total of 120 fruits). Sunexposed and shaded sides of each fruit were estimated visually.
Small regions of fruit surface (c. 4 cm2) were selected, which was
enough to perform reliable measurement of both chlorophyll
¯uorescence and pigment content (Merzlyak and Solovchenko,
2002).
UV-B irradiation conditions
An installation of 103100 W ¯uorescent UV-B (maximum emission
energy in 280±310 nm range) lamps (`Philips', Germany) was used
as the source of UV-B radiation. A cellulose acetate ®lter was used
for the removal of shortwave UV-C radiation. Irradiance was
measured with a precalibrated spectroradiometer (`GroÈbel',
Karlsruhe, Germany). The linearity of the UV-B lamps was
controlled by the continuous measurement of UV-B levels in the
course of irradiation.
Under these experimental conditions UV-B irradiance was
11 J m±2 s±1. There were ®ve exposures from 30 min to 150 min at
30 min intervals giving a total incident UV-B dose of 97.0 kJ m±2
over 150 min, which was suf®cient to follow the UV-B induced
damage to photosystem II in real time.
Chlorophyll ¯uorescence measurements
Measurements were performed on whole fruits after exposing them
to UV-B radiation and then to dark conditions for 30 min (SchmitzEiberger and Noga, 2001). Maximum (Fm) as well as ground (Fo)
¯uorescence of apple skin were measured using a pulse-modulation
Skin ¯avonoids for UV-B protection of apple
1979
¯uorometer (`PAM±1000', Walz, Effeltrich, Germany), then the
quantum ef®ciency of open photosystem II centres was calculated as
Fv/Fm=(Fm±Fo)/Fo (Maxwell and Johnson, 2000).
Pigment analysis
An extraction procedure allowing the simultaneous assay of
chlorophylls, carotenoids, quercetin glycosides, and anthocyanins
in an extract was carried out as described by Solovchenko et al.
(2001). Apple skin discs (16 mm in diameter and c. 1 mm thickness)
were homogenized in chloroform/methanol (2/1, v/v) in the presence
of MgO. The homogenates were then ®ltered through a paper ®lter
and distilled water (1/5 of total extract volume) was added. Then
extracts were centrifuged at 3000 g for 10 min until phase separation.
Absorbance of the extracts was measured with a spectrophotometer (`Lambda 15', Perkin-Elmer, USA). Chlorophyll and
carotenoid concentrations were quanti®ed in the lower (chloroform)
phase using coef®cients reported by Wellburn (1994), and the upper
(water±methanol) phase of the extract was used for HPLC assay of
quercetin glycosides and anthocyanins. Pigment contents were
expressed on fruit surface area basis.
A Waters solvent delivery control system with a Sunchrom
Marathon sample injector and a Hewlett Packard variable wavelength UV detector were used for the identi®cation and quanti®cation of ¯avonoids. The column was a 15032 mm Phenomenex
Synergi Polar RP 80 A ®tted with a Phenomenex guard column.
Chromatograms were recorded using the Waters Millenium program. Solvents used for the elution were (A) 100 ml l±1 acetic acid in
water and (B) acetonitrile. Deaeration of the solvent system was
achieved by vacuum ®ltration through a 0.22 mm ®lter, rapid
sparging with helium (100 ml±1 for 10 min) and constant slow
bubbling of helium into capped, vented solvent reservoirs (5 ml
min±1). Samples (5 ml) were injected onto the column, which was
maintained at 30 °C using a Waters column heater. A ¯ow rate of 1
ml min±1 and a linear 20 min solvent gradient from 0±20%
acetonitrile, with a 10 min hold at the ®nal concentration was used.
The column was Returned to initial solvent composition over
1 min and re-equilibrated for 10 min before the next analysis. Eluted
components were monitored at 350 nm for ¯avonols and 530 nm for
anthocyanins. The individual compounds were identi®ed and
quanti®ed using standard solutions of quercetin and cyanidin-3glycoside (Roth, Karlsruhe, Germany).
Results
Granny Smith fruits possessed green coloration, which was
more intensive on the shaded surface. Sun-exposed
surfaces of Braeburn fruits exhibited anthocyanin pigmentation characteristic of this cultivar, whereas shaded
surfaces of these fruits were green or pale-green. The
results of pigment analysis (Figs 1, 2) showed that sunexposed and shaded apple skin of Granny Smith and
Braeburn fruits possessed different patterns of pigment
content and composition.
Pigment content and composition
As could be seen from Fig. 1, the quercetin glycoside
content of the shaded apple skin of Granny Smith was
lower than that of sun-exposed apple skin and comprised
39.963.6 and 49.565.4 nmol cm±2, respectively. Shaded
apple skin of Braeburn fruits exhibited a lower quercetin
glycoside content compared with that of Granny Smith
Fig. 1. Quercetin glycoside content of skin from sun-exposed and
shaded surfaces of Granny Smith and Braeburn apples (n=60); mean
6SE.
Fig. 2. Pigment content of sun-exposed skin compared with that of
shaded skin of Granny Smith and Braeburn apples (n=60); mean 6SE.
(30.062.8 versus 39.963.6 nmol cm±2) whereas sunexposed apple skin of this cultivar contained about a 3-fold
greater number of the ¯avonoids (109.265.3 nmol cm±2).
Anthocyanins were only found in sun-exposed apple skin
of Braeburn apples in amounts up to 6 nmol cm±2 (Fig. 2).
Granny Smith fruits had a higher total chlorophyll
content than Braeburn (Fig. 2), and shaded skin of Granny
Smith contained more chlorophylls than the sun-exposed
surface of this cv. (5.760.1 versus 4.160.4 nmol cm±2).
The reverse was true for Braeburn where the chlorophyll
content of shaded skin was lower than that of sun-exposed
skin (2.560.1 versus 1.960.1 nmol cm±2). By contrast
with pronounced difference in chlorophyll content, skin
carotenoid content was close in both cvs. A higher
carotenoid content was found in the shaded apple skin of
Granny Smith fruits compared with sun-exposed skin of
the same fruits (2.260.1 and 1.4960.1 nmol cm±2,
respectively). In the case of Braeburn, sun-exposed apple
skin contained more carotenoids than shaded apple skin
(2.260.1 and 1.560.1 nmol cm±2, respectively).
1980 Solovchenko and Schmitz-Eiberger
UV-B-screening capacity of apple skin ¯avonoids
A comparison of the extract absorbance spectra (Fig. 3)
showed that, in general, extracts obtained from sunexposed apple skin exhibited considerably higher absorbance in the UV region of the spectrum than those from
shaded skin. Broad maxima centred at 358 nm accompanied by corresponding short-wave peaks (near 260 nm)
characteristic of quercetin glycoside absorption were
present in the spectra of extracts from sun-exposed skin
in both cultivars. These spectral features were much less
pronounced in the spectra of shaded skin extracts. A slight
increase in the absorbance towards shorter wavelengths
was evidence of a contribution by other phenolics
(chlorogenic acid and proanthocyanidins) to the extract
absorption. According to the data of HPLC and spectral
analysis (not shown), ¯avonoids (represented mainly by
quercetin glycosides) were the principal phenolic constituents of apple skin extracts absorbing in the UV-A and
UV-B regions of the spectrum.
Typical spectra of apple skin extracts shown in Fig. 3
demonstrate that ¯avonoids occuring in apple skin possess
signi®cant absorption, not only in UV-A (the band of their
maximum absorption, Fig. 3, curve 1) but in UV-B as well.
In order to estimate the UV-B screening capacity of apple
skin ¯avonoids, their contribution to the extract absorption
at 300 nm was calculated and expressed on a fruit area
basis.
As can be seen from Fig. 4, there was no signi®cant
difference in absorption of UV-B radiation by skin
¯avonoids between sun-exposed and shaded surfaces of
Granny Smith fruits (Fig. 4A), which was 1.160.1 and
1.460.2, respectively. By contrast, Braeburn fruits showed
a striking difference in the ¯avonoid contribution to UV-B
absorption of the extracts between sun-exposed and shaded
sides of the fruit (Fig. 4B). The contribution of ¯avonoids
Ä (300) of the extracts made from shaded skin of
to A
Braeburn (0.960.1) was close to that of sun-exposed and
shaded skin of Granny Smith, whereas in sun-exposed skin
Fig. 3. Typical absorption spectra of the water±methanol fraction of
extracts (diluted 8-fold) obtained from (1) sun-exposed and (2) shaded
skin of Braeburn apples as well as (3) spectrum of pure rutin.
Ä (300) was approximately four times higher
of Braeburn A
(3.460.2).
Changes in chlorophyll ¯uorescence in the course of
UV-B irradiation
In order to estimate the UV-B resistance of the
photosynthetic apparatus of apple fruits with different
pigment content and composition, apple fruits were
subjected to UV-B radiation and dose curves, representing
changes in ground (Fo) and maximum (Fm) chlorophyll
¯uorescence as well as in the Fv/Fm ratio in the course of
irradiation, were obtained (Fig. 5).
In intact Granny Smith fruit, nearly the same Fo values
were recorded on sun-exposed and shaded surfaces. An
increase in UV-B dose induced a notable increase in Fo on
the shaded (but not on the sun-exposed) surface (Fig. 5A,
closed symbols). In intact Braeburn fruits, the Fo level was
higher on the sun-exposed surface than on the shaded
surface, but in the course of irradiation the difference
became insigni®cant (Fig. 5B).
Granny Smith fruits exhibited similar patterns of UVinduced Fm changes on both surfaces of a fruit (Fig. 5C):
UV-B irradiation up to 40 kJ m±2 induced a sharp decrease
in Fm, but a further increase in the dose did not induce such
large changes in Fm. The same Fm behaviour in response to
UV irradiation was recorded in the shaded surfaces of
Braeburn (Fig. 5D, closed symbols). However, on the sunexposed side of Braeburn fruits Fm was remarkably stable
in whole dose range studied (Fig. 5D, open symbols).
Fig. 4. Contribution of quercetin glycosides (at 300 nm) versus that of
chlorophylls (at 666 nm) to the absorption of extracts obtained from
shaded and sun-exposed skin of Granny Smith and Braeburn apples.
Absorbance, AÄ, was calculated as AÄ=A3V3l±13S±1, where A, V, S, and
l are absorbance, volume of extract (ml), fruit skin area (cm2) taken
for pigment extraction, and cell pathlength (cm), respectively.
Skin ¯avonoids for UV-B protection of apple
1981
The changes in Fo and Fm described above (Fig. 5A±D)
resulted in a steady decline in Fv/Fm in the course of UV-B
irradiation (Fig. 5E, F). The rate of Fv/Fm decrease was
similar in both sun-exposed and shaded surfaces of Granny
Smith (Fig. 5E). The shaded surface of Braeburn fruits
demonstrated similar dependency between UV-B dose and
Fv/Fm (Fig. 5F, closed symbols), but in sun-exposed
surfaces of these fruits Fv/Fm did not change signi®cantly
regardless of UV-B dose and remained as high as in intact
fruits (Fig. 5F, open symbols).
between Fv/Fm and skin content of quercetin glycosides in
case of intact fruits (r ~ 0, Fig. 6). In the course of UV-B
irradiation, the correlation between Fv/Fm and content of
the ¯avonoids became stronger (Fig. 6, curve 1) reaching
its maximum at c. 70 kJ m±2 (r >0.8). By contrast, a low
correlation between anthocyanins and Fv/Fm was recorded
during UV-B irradiation of the same fruits (Fig. 6, curve
2).
Role of quercetin glycosides accumulated under highsunlight conditions in UV-B protection
The experiments demonstrated that the photosynthetic
apparatus in the skin of sun-exposed surfaces of Braeburn
fruits was most resistant to UV-B induced damage
(Fig. 5D, F). The highest content of quercetin glycosides
(Fig. 1) was also recorded here. However, the sun-exposed
skin of Braeburn simultaneously contained signi®cant
amounts of anthocyanins (Fig. 2) whereas shaded apple
skin of these fruits and both surfaces of Granny Smith were
anthocyanin-free. In order to ®nd out whether anthocyanins contribute to UV-B protection of the photosynthetic
apparatus in Braeburn, the correlation coef®cient (r) for
dependency between quercetin glycoside or anthocyanin
content and Fv/Fm magnitude was calculated and plotted
versus UV-B dose (Fig. 6). No correlation was found
Plants are adapted to and respond to natural solar UV-B
radiation that varies constantly during the day (Caldwell,
1981; Ryan et al., 2002). Therefore, the estimations of UVB-protective mechanism ef®ciency using plants grown in
growth chambers under unnatural arti®cial UV and visible
irradiation conditions are often misleading (Ryan et al.,
2002). This work represents an attempt to estimate the
signi®cance of phenolic compounds, accumulated in
response to strong sunlight under natural conditions, in
the protection of the plant photosynthetic apparatus from
UV-B-induced damage.
The results of pigment analysis revealed that fruits of
Granny Smith and Braeburn exhibited different patterns of
pigment content and composition as a result of adaptation
to strong sunlight (Fig. 2). Anthocyanin-free fruits (Granny
Smith) showed a decrease in chlorophyll contents in sunexposed apple skin accompanied by a proportional
decrease in carotenoids. This was not the case in
anthocyanin-containing Braeburn fruits (Fig. 2) which
possessed higher chlorophyll and carotenoid contents in
sun-exposed apple skin. This is consistent with the results
of previous studies which showed that the adaptation of
anthocyanin-free fruits to elevated sunlight conditions is
carried out by means of lowering the chlorophyll content,
whereas anthocyanin-containing apple fruits contained
equivalent or even higher amounts of chlorophyll in sunexposed skin as compared with shaded skin (Merzlyak and
Solovchenko, 2002). Higher skin chlorophyll content in
Fig. 5. Changes in chlorophyll ¯uorescence in shaded (closed
symbols) and sun-exposed (open symbols) skin in the course of UV-B
irradiation of Granny Smith (A, C, E) and Braeburn (B, D, F) apples;
mean 6SE, n=10.
Fig. 6. Changes in correlation between Fv/Fm and (1) quercetin
glycoside or (2) anthocyanin content in the course of UV-B irradiation
of Braeburn apples (n=10).In each case, error of correlation coef®cient
was less than 0.01.
Discussion
1982 Solovchenko and Schmitz-Eiberger
anthocyanin-containing fruits was probably due to a
photoprotective function of the anthocyanins, which
reduce the danger of photo-oxidative damage by means
of limiting the amount of light quanta absorbed by the
chlorophyll (Gould et al., 2000; Merzlyak and
Chivkunova, 2000; Pietrini et al., 2002).
However, as well as the presence of anthocyanins, sunexposed skin of Braeburn fruits was characterized by a
massive accumulation of ¯avonoids, which was not
observed in the shaded skin of this cultivar (Fig. 1). As
could be seen from Fig. 1, both surfaces of Granny Smith
fruits possessed moderate skin ¯avonoid content close to
that in shaded surface of Braeburn.
The results of skin extract analysis (Fig. 3) as well as
HPLC data (not shown) revealed that quercetin glycosides
are the principal group of ¯avonoids accumulated in apple
skin in response to high sunlight. Data shown in Fig. 4
represent the contribution of quercetin glycosides to the
absorption of apple skin extracts per unit fruit surface.
Granny Smith did not demonstrate a distinct response in
terms of quercetin glycoside accumulation since the
contribution of these substances to UV-B absorption of
skin extracts was similar on both sun-exposed and shaded
surfaces (Fig. 4A). By contrast, Braeburn responded to an
enhanced level of sun radiation by a signi®cant accumulation of quercetin glycoside, resulting in a c. 2-fold
increase in their contribution to absorption of skin extract
at 300 nm (Fig. 4B). Taking into account that pigments
incorporated in turbid and high-scattering plant tissue can
absorb signi®cantly more light than equivalent amounts of
pigment in an extract due to increased optical path length
(Butler, 1964), quercetin glycosides in Braeburn skin could
absorb nearly all the incident UV-B radiation. Therefore,
apple skin ¯avonoids are able to serve as ef®cient
sunscreens which ®lter out most of the solar UV-B
radiation. It should be noted that, ¯avonoids accumulated
in fruits during on-tree ripening are not subjected to
metabolic turnover and are retained even after prolonged
storage in darkness (Awad and de Jager, 2000; Merzlyak
and Solovchenko, 2002).
Further experiments were designed to estimate the
signi®cance of other phenolic substances accumulated in
apple skin in response to high ¯uxes of sun radiation (and
hence to elevated UV-B levels) and their contribution to
enhanced resistance of apple photosynthetic apparatus.
Apple fruits with different skin phenolic content were
subjected to UV-B irradiation and UV-B-induced damage
to photosystem II was monitored by means of chlorophyll
¯uorescence measurements.
The analysis of UV-B-induced Fo, Fm and Fv/Fm
changes revealed that the resistance of the photosynthetic
apparatus of apple fruit to UV-B irradiation correlated with
skin phenolic content (Figs 5, 6). In the case of moderate
¯avonoid content (both surfaces of Granny Smith or in the
shaded surface of Braeburn) UV-B irradiation induced
severe damage to the photosynthetic machinery of apple
fruit, which was apparent as a decline in Fm and Fv/Fm
values (Fig. 5). An increase in Fo level on the shaded
surfaces of both cultivars indicates that UV-B radiation
mainly damaged the reaction centres of photosystem II in
these experiments (Maxwell and Johnson, 2000). The
extent of the UV-B induced decrease in the Fm and Fv/Fm
parameters exhibited high correlation with apple skin
quercetin glycoside content (r2 >0.8 in both cultivars, not
shown).
Granny Smith, as well as shaded surfaces of Braeburn,
characterized by similar ¯avonoid contents, also exhibited
similar patterns of Fm and Fv/Fm decrease in response to
UV-B irradiation (cf. Fig. 5C, D, E, F). According to data
of pigment analysis, sun-exposed skin of Braeburn
possessed the highest ¯avonoid content among other
variants (Fig. 1) and demonstrated remarkable UV-Bresistance of photosystem II, which did not show signs of
damage at doses up to 97 kJ m±2 (Fig. 5D, F).
The correlation between Fv/Fm and skin quercetin
glycosides content, negligible in intact fruits, signi®cantly
increases in the course of irradiation (Fig. 6, curve 1). It is
probable that, under such conditions, the integrity of
photosystem II became dependent on the screening exerted
by quercetin glycosides contained in the apple skin. This
could be a reason for an increase in the correlation between
Fv/Fm and quercetin glycoside content observed at high
UV-B doses.
Only a weak correlation was found between apple skin
anthocyanin content and Fv/Fm during UV-B irradiation
(Fig. 6, curve 2). This is in agreement with the results of
pigment analysis (Figs 1, 2). Taking into account those
data and data on the spectral properties of anthocyanins,
which characterized by low extinction coef®cients in the
UV-B (Strack and Wray, 1989), the contribution of
quercetin glycosides to the UV absorbance of apple skin
extracts must be much higher than that of anthocyanins.
These considerations support the hypothesis of a limited
signi®cance of anthocyanins in the UV-protection of plants
(Woodall and Stewart, 1998), at least when they occur in
low or moderate amounts. It should also be noted that
massive accumulation of anthocyanins in apple skin
generally takes place after the degradation of the bulk of
the chlorophyll in the course of ripening, therefore, the
Braeburn fruits used here represent the anthocyanin
content adequate for the most of the on-tree ripening
period (Saure, 1990; Merzlyak and Solovchenko 2002).
Collectively, the results obtained in this work suggest
that ¯avonoids (represented mainly by quercetin glycosides) accumulated in apple skin during acclimation to
strong sunlight can serve as an ef®cient UV-B screen. As a
consequence, they play an important role in the resistance
of the photosynthetic apparatus to the UV-B component of
sun radiation. Anthocyanins did not exhibit a detectable
synergistic effect in UV-B protection, at least in the
Skin ¯avonoids for UV-B protection of apple
amounts recorded in this study, and seemingly served for
protection from damage only by radiation in the blue-green
part of the visible spectrum (Merzlyak and Chivkunova,
2000).
Acknowledgements
The authors are grateful to Professor MN Merzlyak, Dr SI Pogosyan
and Professor Noga for helpful discussions. We also thank the
`Ministerium fuÈr Umwelt, Naturschutz, Landwirtschaft und
Verbraucher schutz', NRW, Germany, for ®nancial support.
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