Plant, Cell and Environment (2010) 33, 1–10
Abscisic acid is involved in the response of grape (Vitis
vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation
by enhancing ultraviolet-absorbing compounds,
antioxidant enzymes and membrane sterols
FEDERICO J. BERLI1, DANIELA MORENO1, PATRICIA PICCOLI1, LEANDRO HESPANHOL-VIANA2,
M. FERNANDA SILVA1, RICARDO BRESSAN-SMITH2, J. BRUNO CAVAGNARO1 & RUBÉN BOTTINI1
1
Facultad de Ciencias Agrarias-CONICET, Universidad Nacional de Cuyo, Almirante Brown 500, M5528AHB, Chacras de
Coria, Argentina and 2Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy
Ribeiro, Av. Alberto Lamego 2000, 28013-620 Campos dos Goytacazes, Brasil
ABSTRACT
We investigated the interactions of abscisic acid (ABA) in
the responses of grape leaf tissues to contrasting ultraviolet
(UV)-B treatments. One-year-old field-grown plants of
Vitis vinifera L. were exposed to photosynthetically active
radiation (PAR) where solar UV-B was eliminated by
using polyester filters, or where PAR was supplemented
with UV-B irradiation. Treatments combinations included
weekly foliar sprays of ABA or a water control. The levels
of UV-B absorbing flavonols, quercetin and kaempferol
were significantly decreased by filtering out UV-B, while
applied ABA increased their content. Concentration of two
hydroxycinnamic acids, caffeic and ferulic acids, were also
increased by ABA, but not affected by plus UV-B (+UV-B)
treatments. Levels of carotenoids and activities of the
antioxidant enzymes, catalase, ascorbate peroxidase and
peroxidase were elevated by +ABA treatments, but only
if +UV-B was given. Cell membrane b-sitosterol was
enhanced by ABA independently of +UV-B. Changes in
photoprotective compounds, antioxidant enzymatic activi-
ties and sterols were correlated with lessened membrane
harm by UV-B, as assessed by ion leakage. Oxidative
damage expressed as malondialdehyde content was
increased under +UV-B treatments. Our results suggest that
the defence system of grape leaf tissues against UV-B is
activated by UV-B irradiation with ABA acting down-
stream in the signalling pathway.
Key-words: oxidative damage; phenolic compounds.
INTRODUCTION
Solar ultraviolet (UV)-B radiation (wavelength range 280–
315 nm) is mostly attenuated by stratospheric ozone and
other atmospheric gases, so that only small amounts reach
the earth’s surface. UV-B intensity varies over time and
Correspondence: R. Bottini. Fax: +54 261 4960469; e-mail: rbottini@
fca.uncu.edu.ar
© 2009 Blackwell Publishing Ltd
doi: 10.1111/j.1365-3040.2009.02044.
pce_2044 1..10
location, mainly because of changes in the solar angle and
thickness of the ozone layer. Thus, as elevation increases the
air mass decreases and there is a greater atmospheric trans-
parency, especially with regard to shorter wavelength radia-
tion, although local environmental conditions, such as
clouds, can also modulate irradiation intensity (Madronich
et al. 1995).
Even relatively small amounts of UV-B induce diverse
morphological, physiological and biochemical responses in
higher plants. UV-B is potentially harmful depending on
the intensity, total dosage, plant species and the balance
between UV-B and photosynthetically active radiation
(PAR, 400–700 nm; Day 2001; Frohnmeyer & Staiger 2003;
Kakani et al. 2003). However, experiments with unrealistic
(i.e. different from that of natural environment) balances
between UV-B radiation, UV-A radiation (315–400 nm)
and PAR may exaggerate the effects of UV-B (Björn
1996; Caldwell & Flint 1997; Allen 1998). Also, it has been
postulated that UV-A and visible light can induce both
protective and repair mechanisms, thus reducing damage
by UV-B (Jordan et al. 1992). Nevertheless, relatively high
intensities of UV-B irradiation can result in overproduc-
tion of free radicals, namely reactive oxygen species
(ROS), which cause oxidative damage to macromolecules
such as DNA, proteins and lipids (Foyer, Lelandais &
Kunert 1994). Lipid peroxidation, a widely used stress
indicator, is promoted by UV-B and increases in mem-
brane permeability are indicative of a disruption of mem-
brane integrity (Dai et al. 1997; Alexieva et al. 2001).
UV-B can also damage thylakoids and hamper synthesis
of chlorophylls (Chl) and carotenoids (Day, Howells &
Ruhland 1996). Plant growth and harvest yield can be
reduced by ambient levels of UV-B, apparently as a result
of UV-B-induced reduction in leaf expansion (Ballaré
et al. 1996; Pinto et al. 1999). Notwithstanding the above,
photosynthetic efficiency was found to be rather insensi-
tive to solar UV-B under field conditions, or when plants
are subjected to ‘realistic’ supplemental UV-B treatments
(Searles, Caldwell & Winter 1995).
1
2 F. J. Berli et al.
Moderate levels of supplemental UV-B can stimulate
transcription of genes involved in protective responses
(Brosché & Strid 2003 and references included therein),
and relatively high levels of solar UV-B enhance the accu-
mulation of UV-absorbing compounds, mainly flavonoids
and related phenolics (Berli et al. 2008). UV-B is also
known to trigger the expression of genes encoding pheny-
lalanine ammonia lyase (PAL) and chalcone synthase
(CHS). These are regulatory enzymes of the phenylpro-
panoid and flavonoid biosynthetic pathways (Jenkins, Fugl-
evand & Christie 1997; Jansen, Gaba & Greenberg 1998;
Casati & Walbot 2003). These secondary metabolites
accumulate in the vacuoles of epidermal cells and effec-
tively absorb radiation from wavelengths in the UV-B
range (Frohnmeyer & Staiger 2003). These are thus postu-
lated to reduce UV-B transmittance and protect the photo-
synthetic apparatus in the leaf mesophyll (Burger &
Edwards 1996; Mazza et al. 2000). Also, modifications in the
plant’s architecture and morphology, such as increases in
leaf thickness, number of epidermal cell layers, epicuticular
wax and pubescence, are observed after UV-B irradiation
(Semerdjieva et al. 2003).
To cope with increased ROS, such as superoxide radicals
(O2-), hydroxyl radicals (·OH), hydrogen peroxide (H2O2),
singlet oxygen (1O2) and nitric oxide (NO·), it appears that
plant cells have developed an efficient antioxidant defence
system. This system involves both enzymatic and non-
enzymatic mechanisms. The former are represented by anti-
oxidant enzymes, such as superoxide dismutase (SOD),
ascorbate peroxidase (APX), catalase (CAT), peroxidase
(POX) and glutathione reductase (GR) (Karabal, Yücel &
Ökte 2003), along with increases in antioxidant molecules
as tocopherols, ascorbic acid, glutathione and carotenoids.
Carotenoids play an important role in the light-harvesting
complex, not only extending the range of light absorbed by
the photosynthetic apparatus, but also by photoprotecting
the photosystems (Ort 2001). They quench triplet state Chl
molecules and scavenge singlet oxygen and other toxic
ROS that are formed within the chloroplast (Ong & Tee
1992), thus dissipating the excess of excitation energy under
stress conditions (Young 1991).
Abscisic acid (ABA) is a phytohormone associated
with the plant’s responses to a range of abiotic stresses
as drought, high temperature, chilling and salinity, and
increases in ABA levels are postulated to regulate adapta-
tion to these environmental stresses (Zhu 2002; Assmann
2005). However, studies dealing with the interaction
between ABA and UV-B are scarce, particularly in regard
to the question of whether ABA can mediate the plant’s
tolerance to enhanced UV-B radiation (Duan et al. 2008). It
has been observed that ABA controls stomatal closure of
guard cells (Leung & Giraudat 1998), including grape
(Stoll, Loveys & Dry 2000). In grape applied ABA reduces
vegetative growth (Dry, Loveys & Düring 2000), but in Ilex
paraguanensis ABA enhances dry matter accumulation
(Sansberro, Mroginski & Bottini 2004). In wheat ABA
increases leaf carotenoid content and allocation of carbo-
hydrate in grains (Travaglia et al. 2007). In grape ABA
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
application was also shown to induce synthesis of polyphe-
nols (Jeong et al. 2004), and result in sugar accumulation in
the berries (Pan et al. 2005), as well as increased fruit yield
(Quiroga et al. 2009). There is also evidence that ABA
induces accumulation of ROS in plant cells as second
messengers for the activation of defensive responses
(Sakamoto, Matsuda & Iba 2008). This ABA signal also
induces the expression of genes encoding SOD, CAT and
APX (Jiang & Zhang 2001) as well as the enhancement
of the non-enzymatic defence systems (antioxidant mole-
cules, Jiang & Zhang 2002).
Malbec is the cultivar of choice for most Argentine red
wines and in the Mendoza region its cultivation extends
from altitudes of 500 m to more than 1500 m. Such varia-
tions in altitude account for different fluence rates and
dosages of UV-B reaching vineyard plants (Berli et al.
2008).
This paper presents results that support the hypothesis
that ABA is causally involved in protective responses by
leaf grape plant tissues to UV-B irradiation and that ABA
does this by enhancing both the enzymatic and non-
enzymatic response systems.
MATERIAL AND METHODS
Plant material and treatments
The experiment was carried out at Facultad de Ciencias
Agrarias, Universidad Nacional de Cuyo, Mendoza, Argen-
tina (33°0′S, 68°52′W) at an altitude of 940 m. One-year-old
plants of a selected clone of Vitis vinifera L. cv. Malbec were
planted in 2.5 L plastic pots filled with grape compost, and
cultivated under a UV protection cover (low-density poly-
ethylene; 100 mm). After 3 months under the above condi-
tions, the leaves were removed and the plants were pruned
to the fifth bud from the base of the shoot. They were
located under field conditions and allowed to break buds
and grow for 1 month in a completely randomized block
design, in a 2 ¥ 2 factorial arrangement of treatments with
five blocks (experimental unit two plants).
Minus UV-B treatments
Solar UV-B radiation was filtered to produce a minus UV-B
(-UV-B) treatment, by using a canopy of 100 mm clear
polyester (PE) filters (Oeste Aislante, Buenos Aires,
Argentina). This PE filter absorbed more than 95% of
UV-B, affecting ca. 30% of UV-A and 15% of PAR.
Plus UV-B treatments
Solar UV-B radiation was supplemented with an additional
dose of 15 mW cm-2, over a 5 h period (from 1100 to 1600 h)
centred on solar noon, using two UV-B fluorescent lamps
(TL 100 W/01; Philips, Nieuwegein, the Netherlands), sus-
pended above the plants. These lamps emitted UV-B at a
narrow spectrum of 310–315 nm and a maximum at 311 nm.
This supplemental UV-B treatment was performed in order

to mimic the UV-B irradiance received by vineyards at
ca. 1500 m, the altitude at which the most highly regarded
vineyards are located in the Mendoza region. To minimize
differences between the -UV-B and plus UV-B (+UV-B)
treatments with regard to wind, temperature or humidity
under plastic sheeting, we placed a 40 mm low-density poly-
ethylene (PET) cover over the +UV-B-treated plants. This
low-density PET transmitted most of the solar radiation
(ca. 75% of UV-B, 80% of UV-A and 85% of PAR).
ABA treatment
Starting from bud-break a 1 mm solution of ⫾-S-cis, trans
abscisic acid (90%, Kelinon Agrochemical Co., Beijing,
China) containing 0.1% Triton X-100 and a minimum
amount of 96% aqueous ethanol (to initially dissolve the
ABA, ca. 10 mL mg-1) was sprayed weekly onto the leaves
to run-off (five applications). Sprays were accomplished in
the evening to minimize photodegradation. This ABA dose
was chosen according to previous work with a range of
species (Sansberro et al. 2004; Travaglia et al. 2007) includ-
ing grape (Quiroga et al. 2009).
Control treatment
A solution containing distilled water plus 0.1% Triton
X-100 and a minimum amount of ethanol as described
above was sprayed weekly as described above.
The two oldest (1-month-old and fully expanded) leaves
from the base of the shoot of each plant were collected at
midday the day after the last ABA application. One was
cooled with ice and immediately processed to assess
protein content, antioxidant enzymatic activity and mem-
brane integrity. The other leaf was quickly frozen at -20 °C
for subsequent evaluation of photosynthetic and photopro-
tective pigments, phenolic compounds, sterols and lipid
peroxidation.
3000
2500
2000
PAR (mmol m–2 s–1)
1500
1000
500
0 0
0800 0900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
Day time (h)
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
ABA mediates grape response to solar UV-B 3
Light measurements
A LI-250 light meter with a LI-190SA quantum sensor (Li-
Cor Inc., Lincoln, NE, USA) and a PMA2200 radiometer
with a PMA2102 UV-B detector (Solar Light Company
Inc., Glenside, PA, USA) were used to measure PAR and
UV-B, respectively. Figure 1 shows the solar radiation (PAR
and UV-B) received on a typical sunny summer day at
elevations of 1450 and 940 m, plus the UV-B supplemental
irradiation to which the plants of the +UV-B treatment
were submitted. However, the UV-B fluence rates and
doses did vary somewhat among the experiments, depend-
ing on the meteorological conditions (data not shown).
ABA quantification
Each sample, consisting of 200 mg fresh weight (FW) of
grape leaf (1 month old and fully expanded), was homog-
enized in a mortar with liquid nitrogen and extracted with
2 mL of 80:19:1 (v/v) methanol : twice-distilled water : acetic
acid at 4 °C. After 12 h, 40 ng of hexa-deuterated ([2H6])-
ABA (a gift from Professor R.P. Pharis, University of
Calgary, Canada) dissolved in 2 mL of methanol was added
for ABA quantification and allowed 1 h equilibration of
the isotopes. Then, the sample was centrifuged 10 min at
9300 g and the supernatant evaporated in a rotavapour with
vacuum at 35 °C.The aqueous residue was adjusted to pH 3.0
with acetic acid and partitioned four times with equal
volumes of ethyl acetate saturated with 1% acetic acid.After
solvent evaporation in vacuum at 35 °C, the residue was
dissolved in 1 mL of water at pH 3.0 (1% acetic acid) and
passed through Sep-Pak C18 reversed phase (500 mg
of material) cartridge (Waters Associates, Milford, MA,
USA). This elution was performed at a flow rate of
0.2 mL min-1 using the following gradient: 1 mL each one of
twice-distilled water pH 3.0, hexane and 80% aqueous
methanol in 1% acetic acid. The entire eluate was collected,
and after solvent evaporation in vacuum at 35 °C, the residue
PAR 1450 m 70
PAR 940 m
Supplemented UV-B
UV-B 1450 m 60
UV-B 940 m
50
UV-B (mW cm–2)
40
30
Figure 1. Solar radiation
20 [photosynthetically active radiation
(PAR) and ultraviolet (UV)-B] registered
on 22 January 2007 at Gualtallary
10
(33°23′S, 69°15′W and 1450 m), on 26
January 2007 at Facultad de Ciencias
Agrarias (33°0′S, 68°52′W and 940 m),
and the UV-B supplemental irradiation
used to treat plants at the last location.
4 F. J. Berli et al.
was dissolved in 1 mL of water at pH 3.0. This solution was
transferred to Oasis WAX (weak anion exchanger, 60 mg of
material) cartridges (Waters Associates) in a gradient of
1 mL of each of 5% NH4OH in methanol, 5% formic acid in
methanol and finally 2% formic acid in methanol.The acidic
elute (which contains the ABA) was evaporated at 35 °C and
vacuum and then converted to the methyl-ester (Me) deriva-
tives with 10–20 mL of methanol plus 50–100 mL of fresh
ethereal CH2N2 (30 min at room temperature). After sol-
vents had been eliminated under a gentle flow of N2 at room
temperature, Me samples were dissolved in ethyl acetate,
and 1 mL was injected split–splitless into a HP-5 cross-linked
methyl silicone capillary column (30 m length, 0.25 mm
inner diameter, 0.25 mm film thickness) fitted in a capillary
gas chromatograph-electron impact mass spectrometer
(GC/MS) (Clarus 500, PerkinElmer, Shelton, CT, USA).The
GC column was eluted with He (1 mL min-1). The GC tem-
perature programme was 100 °C to 260 °C at 20 °C min-1,
then 10 min at 260 °C. The mass spectrometer was operated
with electron impact ionization energy of 70 eV.The injector
temperature was 230 °C, ion source temperature was 260 °C
and the interface temperature was 280 °C. After performing
selected ion monitoring (SIM) the amount of ABA was
calculated by comparison of the peak areas of the major ions
for the Me derivative of the deuterated internal standard
[2H6]-ABA (194/166) relative to its non-labelled counterpart
(190/162); measurements on samples from each treatment
were performed using three biological replicates.
Total UV-absorbing phenolic compounds and
anthocyanins (photoprotective pigments)
Two leaf discs (1 cm2 each) were placed in 2 mL of 99:1 (v/v)
methanol : HCl and allowed to extract 72 h in darkness and
at room temperature. Absorbance of the extracts was read
at 305 or 546 nm for determinations of total UV-absorbing
compounds or anthocyanins, respectively, according to
Mazza et al. (1999), with a Cary-50 UV-Vis spectropho-
tometer (Varian Inc., Palo Alto, CA, USA) with 1 and
10 mm optical path cells.
Characterization and quantification of phenolic
compounds and sterols
The extraction and derivation procedure was performed
according to Minuti, Pellegrino & Tesei (2006), with modi-
fications as follows. Grape leaves were frozen in liquid N2
and ground to a fine powder using a mortar and pestle.
Twenty milligrams of frozen leaf powder was placed in a
centrifuge tube and sonicated (200 W, Cleanson CS-1106,
Buenos Aires, Argentina) with 0.6 mL acidified ethyl
acetate (0.2% of 37% HCl v/v) and centrifuged for 5 min at
9300 g. Supernatant was collected and the pellets extracted
twice with 0.6 mL of acidified ethyl acetate. Then, extracts
were pooled, dried by filtering through a glass conic mini-
column packed with NaSO4, and evaporated to dryness
under a gentle N2 stream. The solid residue was diluted
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
with 30 mL of dry pyridine (Flucka, Steinheim, Germany)
and 70 mL of N,O-bis(trimethylsilyl)-trifluoroacetamide
(BSTFA) 1% TCMSi (Sigma Chem. Co., St. Louis, MO,
USA). After 75 min at 70 °C an aliquot of 1 mL of derived
extract was injected and analysed by GC/MS. The GC/MS
analysis was carried out with a PerkinElmer Model Clarus
500 with the same conditions as for ABA determinations,
except that the oven temperature programme was: initial
temperature at 80 °C for 1 min, then from 80 to 250 °C at
a rate of 20 °C min-1, and held for 1 min, then augmented
at 6 °C min-1 to 300 °C, and held for 2 min, and finally
increased at 20 °C min-1 to 320 °C, and held for 24 min. The
identity of each phenolic and sterol compounds was estab-
lished by retention times on capillary GC (see Fig. 3) and
the full mass spectra of silylated standards performed in the
range from 120 to 700 m/z (quercetin, kaempferol, caffeic
acid, p-coumaric acid and ferulic acid; Sigma Chem. Co.)
and from the National Institute of Standards and Technol-
ogy (NIST) library (sterols). The Total Ion Chromatogram
of quercetin (the most abundant metabolite studied) was
used as a reference compound for quantification. A six-
point calibration curve was obtained for quercetin, the
standard solutions contained 15% (v/v) methanol and were
subjected to the same extraction-derivation procedure
described for the samples. The quantification was per-
formed by relating the peak areas of the identified
compounds in each sample and was calculated from the
calibration plot.
Photosynthetic pigments
For determinations of Chl a, Chl b and carotenoids, one leaf
disc (1 cm2) was placed in 5 mL of dimethyl sulphoxide
(DMSO) and allowed to extract for 45 min in darkness at
70 °C. Absorbance was read at 665, 649 and 480 nm against
a blank of reagents, according to Chapelle, Kim &
McMurtrey (1992) and the formulas of Wellburn (1994)
as follows: Chl a = 12.19 OD665 - 3.45 OD649; Chl b = 21.99
OD649 - 5.32 OD665; carotenoids = (1000 OD480 - 2.14 Chl
a - 70.16 Chl b)/220; Total Chl = Chl a + Chl b.
Membrane integrity
Ten leaf discs (1 cm2 each) were washed three times with
twice-distilled water to remove surface-adhered electro-
lytes, and then placed in vials containing 30 mL of twice-
distilled water. They were incubated in darkness for 24 h
at 8 °C. The electrical conductivity of the solution (C1) was
measured, after equilibration at 25 °C, with a pH/CON
510 meter (Oakton Instruments, Vernon Hills, IL, USA).
Samples were then heated for 1 h at 80 °C, cooled and
kept in darkness for 16 h at 8 °C. Then, the electrical
conductivity of heat-killed tissues (C2) was measured at
25 °C. Membrane integrity was calculated according to
Vásquez-Tello et al. (1990) as follows: relative membrane
integrity = [1 - (C1/C2)] ¥ 100.

Lipid peroxidation
In order to evaluate oxidative damage as lipid peroxida-
tion, malondialdehyde (MDA) content was measured
following the procedure described by Beligni & Lamattina
(2002). Seventy-five milligrams of leaf tissue was sus-
pended in 1.5 mL of stock solution [15% (w/v) trichloro-
acetic acid (TCA), 0.5% (w/v) thiobarbituric acid (TBA)
and 0.25% (w/v) hydrochloric acid]. The mixture was
stirred vigorously and incubated at 95 °C for 60 min. After
centrifugation at 9300 g for 10 min, the supernatant was
collected. Absorbance of the extracts was measured at
535 nm with 1 mm optical path cell. The concentration
was calculated considering MDA molar extinction
coefficient = 1.56 ¥ 105 m-1 cm-1.
Protein content and antioxidant enzyme activity
Samples of 250 mg fresh weight leaf were homogenized
using a mortar and pestle with 5 mL of extraction solution
(100 mm potassium phosphate buffer pH 7.5; 0.1% Triton
X-100; 1 mm EDTA; 0.5 mm ascorbic acid) at 5 °C and
0.25 g of insoluble polyvinylpolypyrrolidone (PVPP), and
centrifuged for 5 min at 9300 g. Supernatant was collected
and frozen at -20 °C until assayed for proteins and antioxi-
dants. Protein content was determined according to Brad-
ford (1976) with bovine serum albumin (BSA) as standard,
measuring the absorbance at 595 nm.
The CAT activity was assayed by monitoring the con-
sumption of H2O2 at 240 nm in 2.5 mL reaction mixture
containing 100 mm potassium phosphate buffer (pH 7.5)
and 75 mL of sample. The reaction was started by adding
7.5 mL of 34.8 mm H2O2 and changes in absorbance were
followed for 60 s as stated by Azevedo et al. (1998).
The APX activity was measured by the decrease in absor-
bance of ascorbate at 290 nm in 2.5 mL reaction mixture
containing 50 mm potassium phosphate buffer (pH 7.0),
1 mm EDTA, 0.5 mm ascorbic acid and 1 mm H2O2. The
reaction was started by adding 40 mL of the sample and
changes in absorbance were followed for 60 s according to
Barka (2001).
(a) (b)
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
ABA mediates grape response to solar UV-B 5
The POX activity was assayed by monitoring the oxida-
tion of guaiacol to tetraguaiacol at 470 nm in 2.5 mL reac-
tion mixture containing 50 mm potassium phosphate buffer
(pH 6.0), 1.2 mm H2O2 and 2 mm guaiacol. The reaction was
started by adding 100 mL of the sample and changes in
absorbance were followed for 60 s as stated by Zhang &
Kirkham (1994).
Statistical analysis
Statistical analyses were performed as a randomized block
with a 2 ¥ 2 factorial arrangement of treatments with the
software Statgraphics Centurion XV version 15.0.10 (Stat-
point Technologies Inc.,Warrenton,VA, USA).The effect of
UV-B, ABA application and their interaction were deter-
minate by multifactorial anova.
RESULTS
UV-B increased the content of ABA (as assessed by
GC/MS/SIM quantification) in grape leaves 2.7-fold, ABA
treatment 3.6-fold, and combined effect of UV-B and ABA
12.5-fold (Fig. 2a).
No statistical difference was observed in the Chl content,
both total (Table 1) or Chl a and Chl b (data not shown),
and in carotenoids content. Simultaneous action of UV-B
and applied ABA showed the highest carotenoids content,
although only significant differences were obtained at
P ⱕ 0.1 for UV-B treatments (Table 1). The content of total
UV-absorbing compounds was increased by UV-B and
applied ABA with a significant interaction between UV-B
and ABA (Table 1) meaning that ABA effects are UV-B
dependent. Anthocyanin levels were also increased by
interactive action of UV-B and applied ABA (Table 1).
Figure 2b shows the different leaf pigmentation for each
treatment.
The UV-absorbing compounds characterized by GC/MS
were flavonols (quercetin and kaempferol) and the
hydroxycinnamic acids (caffeic acid, ferulic acid and
p-coumaric acid). Figure 3 shows the GC/MS profiles of
the flavonols and sterols. The quercetin concentration was
approximately 20-fold higher than kaempferol, and the
Figure 2. Abscisic acid (ABA) levels as
assessed by gas chromatograph-electron
impact mass spectrometer/selected ion
monitoring (GC/MS/SIM) in mg g-1 fresh
leaf (a) of plus ultraviolet (UV)-B
(+UV-B) (left) and minus UV-B (-UV-B)
(right) treatments (white, Control; black,
ABA treated). P(UV-B), UV-B effect;
P(ABA), effect of exogenous ABA;
P(UV-B¥ABA), UV-B ¥ ABA interaction
effect. Values are means ⫾SE, n = 3.
(b) Grape plants of +UV-B (left) and
-UV-B (right) treatments.
6 F. J. Berli et al.

Table 1. Total chlorophylls (Chl) (mg cm-2 leaf), carotenoids (mg cm-2 leaf), total ultraviolet (UV)-absorbing compounds (OD305 cm-2
leaf) and anthocyanins (OD546 cm-2 leaf) in grape leaves of plus UV-B (+UV-B) and minus UV-B (-UV-B) treatments either sprayed or
not (control) with abscisic acid (ABA)

Treatments Total Chl Carotenoids UV-absorbing compounds Anthocyanins

+UV-B ABA 30.293 ⫾ 2.860 6.862 ⫾ 0.727 0.631 ⫾ 0.050 0.049 ⫾ 0.005
Control 25.599 ⫾ 1.720 5.847 ⫾ 0.443 0.527 ⫾ 0.006 0.036 ⫾ 0.002
-UV-B ABA 28.586 ⫾ 2.915 5.515 ⫾ 0.397 0.339 ⫾ 0.007 0.036 ⫾ 0.002
Control 26.576 ⫾ 3.106 5.297 ⫾ 0.445 0.336 ⫾ 0.018 0.034 ⫾ 0.003
P(UV-B) 0.7380 0.0667 <0.00001 0.0422
P(ABA) 0.3079 0.3221 0.0511 0.0701
P(UV-B¥ABA) 0.7719 0.5540 0.0621 0.2174

P(UV-B), UV-B effect; P(ABA), effect of exogenous ABA; P(UV-B¥ABA), UV-B ¥ ABA interaction effect. Values are means ⫾SE, n = 5.

levels of both were significantly increased by UV-B and
applied ABA treatment (Table 2). There was a significant
interaction between UV-B and ABA in kaempferol
concentration, indicating that ABA promotion of
kaempferol is UV-B dependent. These results also corre-
late well with the spectrophotometric determinations of
total UV-absorbing compounds (Table 1). In samples of all
treatments the concentration of caffeic acid was approxi-
mately fivefold higher than that of p-coumaric acid, and
20-fold higher than that of ferulic acid (Table 2). Caffeic
acid and ferulic acid were significantly increased by exog-
enously applied ABA, although there was no significant
effect of ABA application on the p-coumaric acid concen-
tration. None of the hydroxycinnamic acids was affected
by filtering out UV-B.
The activity of CAT was significantly increased by
applied ABA, but there was no significant effect of filtering
out UV-B (Table 3). The activity of APX was promoted by
UV-B (relative to the treatment where UV-B was filtered
out) and there was a significant interaction between UV-B
and ABA. The activity of POX was also affected by the
interaction between UV-B and ABA. This suggests that
ABA promotion of APX and POX activities is UV-B
dependent.
The sterols b-sitosterol and stigmasterol were identified
by capillary GC/MS analysis and in all treatments the
b-sitosterol content was approximately fivefold higher than
that of stigmasterol. Membrane integrity and b-sitosterol
content were significantly improved by ABA application,
but were not affected by filtering out UV-B (Table 4).
Oxidative damage, as assessed by MDA concentration, was
significantly decreased by filtering out UV-B. Stigmasterol
content was not affected by any of the treatments (data not
shown).

Figure 3. Total ion chromatography profiles by capillary gas chromatograph-electron impact mass spectrometer (GC/MS) analysis of
flavonols and sterols, showing peaks of stigmasterol (1), b-sitosterol (2), quercetin (3) and kaempferol (4) for leaf tissue extracts
corresponding to plus ultraviolet (UV)-B (+UV-B), minus UV-B (-UV-B), control and abscisic acid (ABA) treatments.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
 ABA mediates grape response to solar UV-B 7

Table 2. Content of quercetin, kaempferol, caffeic acid, ferulic acid and p-coumaric acid, as assessed by gas chromatograph-electron
impact mass spectrometer (GC/MS) analyses, in mg g-1 fresh leaf of plus ultraviolet (UV)-B (+UV-B) and minus UV-B (-UV-B)
treatments either sprayed or not (control) with abscisic acid (ABA)

Treatments Quercetin Kaempferol Caffeic acid Ferulic acid P-coumaric acid

+UV-B ABA 2.006 ⫾ 0.229 0.120 ⫾ 0.010 0.630 ⫾ 0.085 0.025 ⫾ 0.002 0.151 ⫾ 0.089
Control 1.476 ⫾ 0.183 0.053 ⫾ 0.008 0.298 ⫾ 0.067 0.019 ⫾ 0.001 0.090 ⫾ 0.024
-UV-B ABA 0.940 ⫾ 0.084 0.036 ⫾ 0.002 0.437 ⫾ 0.074 0.027 ⫾ 0.002 0.121 ⫾ 0.072
Control 0.700 ⫾ 0.047 0.032 ⫾ 0.004 0.285 ⫾ 0.039 0.022 ⫾ 0.002 0.061 ⫾ 0.002
P(UV-B) 0.0005 <0.00001 0.1853 0.1759 0.1264
P(ABA) 0.0688 0.0003 0.0063 0.0050 0.7779
P(UV-B¥ABA) 0.4415 0.0007 0.2454 0.6293 0.8090

P(UV-B), UV-B effect; P(ABA), effect of exogenous ABA; P(UV-B¥ABA), UV-B ¥ ABA interaction effect. Values are means ⫾SE, n = 5.

Table 3. Catalase (CAT), ascorbate

Treatments CAT APX POX peroxidase (APX) and peroxidase (POX)
activities (nm min-1 mg-1 protein) in grape
+UV-B ABA 84.979 ⫾ 6.625 314.725 ⫾ 29.763 9.162 ⫾ 1.751
leaves of plus ultraviolet (UV)-B (+UV-B)
Control 46.405 ⫾ 14.297 213.761 ⫾ 15.953 5.818 ⫾ 0.779
and minus UV-B (-UV-B) treatments either
-UV-B ABA 62.880 ⫾ 5.453 181.594 ⫾ 12.512 5.611 ⫾ 0.884
sprayed or not (control) with abscisic acid
Control 44.047 ⫾ 7.416 217.294 ⫾ 35.875 6.645 ⫾ 1.208
(ABA)
P(UV-B) 0.1195 0.0090 0.2494
P(ABA) 0.0013 0.2343 0.3246
P(UV-B¥ABA) 0.1518 0.0247 0.0755

P(UV-B), UV-B effect; P(ABA), effect of exogenous ABA; P(UV-B¥ABA), UV-B ¥ ABA interaction
effect. Values are means ⫾SE, n = 5.

DISCUSSION as Pfündel (2003) reported that UV-B was responsible for

Although UV-B represents only a small portion of solar
radiation that reaches the earth surface, its impact on bio-
logical processes is considered of great importance (Björn,
Widell & Wang 2002; Jordan 2002). That is, exhaustive
research has shown that ROS production is triggered in
several cell compartments when plant tissues are exposed
to UV-B, and as a result oxidative damage of proteins and
cell membranes may occur (Scandalios 1993). Our study
was performed in a natural environment, and while an
increase in MDA content was observed, which reflects lipid
peroxidation and therefore some level of stress, membrane
damage and photooxidation of Chl in leaves exposed to
+UV-B irradiances were not observed. The maintenance of
Chl levels in the +UV-B treatments was rather unexpected,
photosystem II (PSII) inhibition (i.e. pigment bleaching and
inhibition of overall photosynthesis). However, in Pfündel’s
experiments the grape leaves had been adapted to growth
in darkness prior to UV-B treatments. Similar inhibitory
effects had been also reported in maize tissues along
with decreases in photosynthetic pigments, gas exchange,
Rubisco activity and total sugar content (Correia et al.
2005), although these UV-B effects were obtained after
nitrogen deficiency. Thus, the different results found in pre-
vious studies compared with our results may indicate dif-
ferent experimental conditions and/or genetic differences
and/or differences in developmental stages or tissues.
The increase in UV-absorbing compounds, including
anthocyanins, is another response that is postulated to
protect cell membranes, as the increased levels of phenolic

Table 4. Membrane integrity (in %), and

Treatments Membrane integrity b-Sitosterol MDA b-sitosterol (mg g-1 fresh leaf) and
malondialdehyde (MDA) (nm g-1 fresh leaf)
+UV-B ABA 96.776 ⫾ 0.529 0.3840 ⫾ 0.012 11.521 ⫾ 1.896
content in grape leaves of plus ultraviolet
Control 95.392 ⫾ 0.769 0.277 ⫾ 0.020 13.744 ⫾ 2.804
(UV)-B (+UV-B) and minus UV-B
-UV-B ABA 96.909 ⫾ 0.562 0.380 ⫾ 0.024 7.436 ⫾ 0.585
(-UV-B) treatments either sprayed or not
Control 95.328 ⫾ 0.704 0.256 ⫾ 0.014 6.250 ⫾ 0.562
(control) with abscisic acid (ABA)
P(UV-B) 0.8926 0.4949 0.0122
P(ABA) 0.0001 <0.00001 0.6805
P(UV-B¥ABA) 0.7028 0.6373 0.1934

P(UV-B), UV-B effect; P(ABA), effect of exogenous ABA; P(UV-B¥ABA), UV-B ¥ ABA interaction
effect. Values are means ⫾SE, n = 5.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
8 F. J. Berli et al.
compounds are correlated with a more efficient absorption
of harmful UV-B (Frohnmeyer & Staiger 2003; Merzlyak
et al. 2008). Such phenolics transform short-wave, high-
energy and highly destructive radiation into longer-
wavelength light and these wavelengths are less destructive
to the cellular leaf structures, including the photosynthetic
apparatus (Bilger, Johnsen & Schreiber 2001). Additionally,
phenolic compounds may enhance protection against oxi-
dative stress, as they possess chemical structures capable of
scavenging free radicals; for example, they have also been
found to be effective antioxidants (Blokhina, Virolainen &
Fagerstedt 2002). In many higher plant tissues, two classes
of phenolics appear to be involved in epidermal UV screen-
ing: the hydroxycinnamic acids and the more complex fla-
vonoids. These compounds vary from species to species and
according to developmental stages and tissues. Our results
with grape leaves showed that the UV-absorbing com-
pounds found in the leaf tissue were flavonols (quercetin
and kaempferol) and hydroxycinnamic acids (caffeic acid,
p-coumaric acid and ferulic acid). However, only quercetin
and kaempferol were significantly enhanced by +UV-B
treatments. Quercetin is a more efficient ROS-scavenger
than kaempferol, due to its increased number of hydroxyl
groups; moreover, we found that quercetin’s concentration
in grape leaf tissues was approximately 20-fold higher than
kaempferol. Such results are consistent with the finding of
increased expression of genes involved in the biosynthesis
of flavonols and phenylpropanoids in grape plants sub-
mitted to UV-B radiation (Pontin et al. 2008). The accumu-
lation of phenolic compounds that selectively and passively
absorb UV radiation in the epidermis probably represents
the most cost-effective strategy for adaptation in the case of
regular and prolonged exposure to elevated doses of UV-B,
in that it should reduce the need for maintaining con-
tinuously active avoidance and repair processes (Cockell &
Knowland 1999; Liakoura, Bornman & Karabourniotis
2003). Kolb et al. (2001) also found enhanced synthesis
of flavonoids, specifically the flavonols quercetin and
kaempferol in grape leaves exposed to UV-B. Nuñez-
Olivera et al. (2006) obtained an increase in the concen-
trations of Chl and carotenoids and a reduction in
UV-absorbing compounds in grape plants of the cultivar
Tempranillo grown under UV-B exclusion. It is worth
noting, however, that solar UV-B seems to cause some
damage in Tempranillo grapevines (purportedly suscep-
tible). This did not happen with Malbec (more tolerant),
given that our +UV-B treatments maintained Chl levels and
actually increased carotenoids levels.
Antioxidant enzymatic activity could partially explain
why membrane integrity was not affected by +UV-B when
ABA was applied, as the activities of CAT, APX and POX
were higher with this treatment. In effect, it has been
reported that POX is induced by light in grapevine (Ros
Barceló et al. 2003) and ABA may induce the biosynthesis
of these enzymes (Jiang & Zhang 2001).
While ABA can increase ROS production and induce the
activities of SOD, CAT, APX and GR in plants (Jiang &
Zhang 2001; Sakamoto et al. 2008), these studies considered
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1–10
that ABA is mainly an enhancer of oxidative damage to
plants. In contrast, our results show that increased CAT
activity was caused by ABA treatment independently of
UV-B, while the activities of APX and POX were increased
by ABA when +UV-B was present.
Cell membrane integrity, evaluated by its electrolyte
leakage, may be a good indicator of stress tolerance in
plants (Vásquez-Tello et al. 1990). In our experiments a
positive correlation was obtained between this variable and
the defence system induced by ABA; for example, mem-
brane integrity was increased after ABA application with or
without the use of +UV-B treatments. This enhancement
in membrane integrity was associated with an increase in
the membrane content of b-sitosterol. Plant membranes
include several sterols in their structure, where sitosterol
usually predominates (Hartmann 1998; Nomura et al. 1999)
and our results confirm this conclusion.
Overall, our results strongly suggest that ABA is
involved in stress tolerance of grape leaves and that they
function by enhancing the ability of epidermal tissues to
filter out UV-B. That is, they increase levels of antioxidant
enzymes and enhance the sterol-structural defence,
although these responses are, in some cases, also depen-
dent on the co-presence of high levels of UV-B irradia-
tion. In other words, the antioxidant defence system in
grape leaf tissues is initially activated by UV-B irradiation
and ABA often acts downstream in the signalling pathway
(although some responses are activated by ABA alone).
The first line of defence likely includes elevated levels of
flavonols and hydroxycinnamic acids; these filter part of
the UV-B. We found that quercetin and kaempferol were
increased by +UV-B treatments, with an additional
increase being seen when ABA was also co-applied with
the +UV-B treatments. In contrast, increases in caffeic acid
and ferulic acid concentration were triggered only by
ABA. It is postulated that a part of the UV-B-origin
energy excess is dissipated by carotenoids. In our experi-
ments carotenoids levels were enhanced by +UV-B treat-
ments alone, although one has to remember that +UV-B
treatment also increased endogenous ABA concentrations
in the leaf tissue.
The antioxidant enzymatic system (CAT, APX and POX)
is also postulated to contribute to the attenuation of UV-
B-induced stress situation, and its activity is enhanced by
exogenously applied ABA, but only under the +UV-B
treatments (APX and POX). Finally, an extra structural
resistance to UV-B-induced damages includes an enhance-
ment of membrane sterols that, in our study, are exclusively
augmented by ABA but not by UV-B itself.
ACKNOWLEDGMENTS
This work was supported by Consejo Nacional de Investi-
gaciones Científicas y Técnicas (CONICET, PID 5028 to
R.B.), Secretaría de Ciencia y Técnica de la Nación (SECyT,
PICT 08-12398 and PICT 20-20093 to R.B.), Secretaría de
Ciencia y Técnica de la Universidad Nacional de Cuyo,
Bodega Catena-Zapata, Coordenação de Aperfeiçoamento

de Pessoal de Nível Superior (CAPES, to L.H.-V.) and Con-
selho Nacional Desenvolvimento Científico e Tecnológico
(CNPq, to R.B.-S.). The authors thank the technical assis-
tance of L. Bolcato and M. Fucili.
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Received 8 May 2009; received in revised form 11 August 2009;
accepted for publication 8 September 2009