Abscisic Acid Is Involved in the Response of Grape (Vitis Vinifera L.) Cv

Plant, Cell and Environment (2009) doi: 10.1111/j.1365-3040.2009.02044.x

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 sterolspce_2044 1..10

FEDERICO J. BERLI1, DANIELA MORENO1, PATRICIA PICCOLI1, LEANDRO HESPANHOL-VIANA2, M. FERNANDA SILVA1, RICARDO BRESSAN-SMITH2, J. BRUNO CAVAGNARO1 & RUBÉN BOTTINI1

1Facultad 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 location, mainly because of changes in the solar angle and thickness of the ozone layer.Thus, as elevation increases the We investigated the interactions of abscisic acid (ABA) in air mass decreases and there is a greater atmospheric trans- the responses of grape leaf tissues to contrasting ultraviolet parency, especially with regard to shorter wavelength radia- (UV)-B treatments. One-year-old field-grown plants of tion, although local environmental conditions, such as Vitis vinifera L. were exposed to photosynthetically active clouds, can also modulate irradiation intensity (Madronich radiation (PAR) where solar UV-B was eliminated by et al. 1995). using polyester filters, or where PAR was supplemented Even relatively small amounts of UV-B induce diverse with UV-B irradiation. Treatments combinations included morphological, physiological and biochemical responses in weekly foliar sprays of ABA or a water control. The levels higher plants. UV-B is potentially harmful depending on of UV-B absorbing flavonols, quercetin and kaempferol the intensity, total dosage, plant species and the balance were significantly decreased by filtering out UV-B, while between UV-B and photosynthetically active radiation applied ABA increased their content. Concentration of two (PAR, 400–700 nm; Day 2001; Frohnmeyer & Staiger 2003; hydroxycinnamic acids, caffeic and ferulic acids, were also Kakani et al. 2003). However, experiments with unrealistic increased by ABA, but not affected by plus UV-B (+UV-B) (i.e. different from that of natural environment) balances treatments. Levels of carotenoids and activities of the between UV-B radiation, UV-A radiation (315–400 nm) antioxidant enzymes, catalase, ascorbate peroxidase and and PAR may exaggerate the effects of UV-B (Björn peroxidase were elevated by +ABA treatments, but only 1996; Caldwell & Flint 1997; Allen 1998). Also, it has been if +UV-B was given. Cell membrane b-sitosterol was postulated that UV-A and visible light can induce both enhanced by ABA independently of +UV-B. Changes in protective and repair mechanisms, thus reducing damage photoprotective compounds, antioxidant enzymatic activi- by UV-B (Jordan et al. 1992). Nevertheless, relatively high ties and sterols were correlated with lessened membrane intensities of UV-B irradiation can result in overproduc- harm by UV-B, as assessed by ion leakage. Oxidative tion of free radicals, namely reactive oxygen species damage expressed as malondialdehyde content was (ROS), which cause oxidative damage to macromolecules increased under +UV-B treatments. Our results suggest that such as DNA, proteins and lipids (Foyer, Lelandais & the defence system of grape leaf tissues against UV-B is Kunert 1994). Lipid peroxidation, a widely used stress activated by UV-B irradiation with ABA acting down- indicator, is promoted by UV-B and increases in mem- stream in the signalling pathway. brane permeability are indicative of a disruption of membrane integrity (Dai et al. 1997; Alexieva et al. 2001). Key-words: oxidative damage; phenolic compounds. UV-B can also damage thylakoids and hamper synthesis of chlorophylls (Chl) and carotenoids (Day, Howells & INTRODUCTION Ruhland 1996). Plant growth and harvest yield can be reduced by ambient levels of UV-B, apparently as a result Solar ultraviolet (UV)-B radiation (wavelength range 280– of UV-B-induced reduction in leaf expansion (Ballaré 315 nm) is mostly attenuated by stratospheric ozone and et al. 1996; Pinto et al. 1999). Notwithstanding the above, other atmospheric gases, so that only small amounts reach photosynthetic efficiency was found to be rather insensi- the earth’s surface. UV-B intensity varies over time and tive to solar UV-B under field conditions, or when plants Correspondence: R. Bottini. Fax: +54 261 4960469; e-mail: rbottini@ are subjected to ‘realistic’ supplemental UV-B treatments fca.uncu.edu.ar (Searles, Caldwell & Winter 1995). © 2009 Blackwell Publishing Ltd 1 2 F. J. Berli et al.

Moderate levels of supplemental UV-B can stimulate application was also shown to induce synthesis of polyphe- transcription of genes involved in protective responses nols (Jeong et al. 2004), and result in sugar accumulation in (Brosché & Strid 2003 and references included therein), the berries (Pan et al. 2005), as well as increased fruit yield and relatively high levels of solar UV-B enhance the accu- (Quiroga et al. 2009). There is also evidence that ABA mulation of UV-absorbing compounds, mainly flavonoids induces accumulation of ROS in plant cells as second and related phenolics (Berli et al. 2008). UV-B is also messengers for the activation of defensive responses known to trigger the expression of genes encoding pheny- (Sakamoto, Matsuda & Iba 2008). This ABA signal also lalanine ammonia lyase (PAL) and chalcone synthase induces the expression of genes encoding SOD, CAT and (CHS). These are regulatory enzymes of the phenylpro- APX (Jiang & Zhang 2001) as well as the enhancement panoid and flavonoid biosynthetic pathways (Jenkins, Fugl- of the non-enzymatic defence systems (antioxidant mole- evand & Christie 1997; Jansen, Gaba & Greenberg 1998; cules, Jiang & Zhang 2002). Casati & Walbot 2003). These secondary metabolites Malbec is the cultivar of choice for most Argentine red accumulate in the vacuoles of epidermal cells and effec- wines and in the Mendoza region its cultivation extends tively absorb radiation from wavelengths in the UV-B from altitudes of 500 m to more than 1500 m. Such varia- range (Frohnmeyer & Staiger 2003). These are thus postu- tions in altitude account for different fluence rates and lated to reduce UV-B transmittance and protect the photo- dosages of UV-B reaching vineyard plants (Berli et al. synthetic apparatus in the leaf mesophyll (Burger & 2008). Edwards 1996; Mazza et al. 2000).Also, modifications in the This paper presents results that support the hypothesis plant’s architecture and morphology, such as increases in that ABA is causally involved in protective responses by leaf thickness, number of epidermal cell layers, epicuticular leaf grape plant tissues to UV-B irradiation and that ABA wax and pubescence, are observed after UV-B irradiation does this by enhancing both the enzymatic and non- (Semerdjieva et al. 2003). enzymatic response systems. To cope with increased ROS, such as superoxide radicals - (O2 ), hydroxyl radicals (·OH), hydrogen peroxide (H2O2), 1 MATERIAL AND METHODS singlet oxygen ( O2) and nitric oxide (NO·), it appears that plant cells have developed an efficient antioxidant defence Plant material and treatments system. This system involves both enzymatic and non- enzymatic mechanisms.The former are represented by anti- The experiment was carried out at Facultad de Ciencias oxidant enzymes, such as superoxide dismutase (SOD), Agrarias, Universidad Nacional de Cuyo, Mendoza, Argen- ′ ′ ascorbate peroxidase (APX), catalase (CAT), peroxidase tina (33°0 S, 68°52 W) at an altitude of 940 m. One-year-old (POX) and glutathione reductase (GR) (Karabal, Yücel & plants of a selected clone of Vitis vinifera L. cv. Malbec were Ökte 2003), along with increases in antioxidant molecules planted in 2.5 L plastic pots filled with grape compost, and as tocopherols, ascorbic acid, glutathione and carotenoids. cultivated under a UV protection cover (low-density poly- Carotenoids play an important role in the light-harvesting ethylene; 100 mm). After 3 months under the above condi- complex, not only extending the range of light absorbed by tions, the leaves were removed and the plants were pruned the photosynthetic apparatus, but also by photoprotecting to the fifth bud from the base of the shoot. They were the photosystems (Ort 2001). They quench triplet state Chl located under field conditions and allowed to break buds molecules and scavenge singlet oxygen and other toxic and grow for 1 month in a completely randomized block ROS that are formed within the chloroplast (Ong & Tee design, in a 2 ¥ 2 factorial arrangement of treatments with 1992), thus dissipating the excess of excitation energy under five blocks (experimental unit two plants). stress conditions (Young 1991). Abscisic acid (ABA) is a phytohormone associated Minus UV-B treatments with the plant’s responses to a range of abiotic stresses as drought, high temperature, chilling and salinity, and Solar UV-B radiation was filtered to produce a minus UV-B increases in ABA levels are postulated to regulate adapta- (-UV-B) treatment, by using a canopy of 100 mm clear tion to these environmental stresses (Zhu 2002; Assmann polyester (PE) filters (Oeste Aislante, Buenos Aires, 2005). However, studies dealing with the interaction Argentina). This PE filter absorbed more than 95% of between ABA and UV-B are scarce, particularly in regard UV-B, affecting ca. 30% of UV-A and 15% of PAR. 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 Plus UV-B treatments guard cells (Leung & Giraudat 1998), including grape Solar UV-B radiation was supplemented with an additional (Stoll, Loveys & Dry 2000). In grape applied ABA reduces dose of 15 mWcm-2, over a 5 h period (from 1100 to 1600 h) vegetative growth (Dry, Loveys & Düring 2000), but in Ilex centred on solar noon, using two UV-B fluorescent lamps paraguanensis ABA enhances dry matter accumulation (TL 100 W/01; Philips, Nieuwegein, the Netherlands), sus- (Sansberro, Mroginski & Bottini 2004). In wheat ABA pended above the plants. These lamps emitted UV-B at a increases leaf carotenoid content and allocation of carbo- narrow spectrum of 310–315 nm and a maximum at 311 nm. hydrate in grains (Travaglia et al. 2007). In grape ABA This supplemental UV-B treatment was performed in order © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment ABA mediates grape response to solar UV-B 3 to mimic the UV-B irradiance received by vineyards at Light measurements ca. 1500 m, the altitude at which the most highly regarded A LI-250 light meter with a LI-190SA quantum sensor (Li- vineyards are located in the Mendoza region. To minimize Cor Inc., Lincoln, NE, USA) and a PMA2200 radiometer differences between the -UV-B and plus UV-B (+UV-B) with a PMA2102 UV-B detector (Solar Light Company treatments with regard to wind, temperature or humidity Inc., Glenside, PA, USA) were used to measure PAR and under plastic sheeting, we placed a 40 mm low-density poly- UV-B, respectively.Figure 1 shows the solar radiation (PAR ethylene (PET) cover over the +UV-B-treated plants. This and UV-B) received on a typical sunny summer day at low-density PET transmitted most of the solar radiation elevations of 1450 and 940 m, plus the UV-B supplemental (ca. 75% of UV-B, 80% of UV-A and 85% of PAR). irradiation to which the plants of the +UV-B treatment were submitted. However, the UV-B fluence rates and ABA treatment doses did vary somewhat among the experiments, depending on the meteorological conditions (data not shown). Starting from bud-break a 1 mm solution of Ϯ-S-cis, trans abscisic acid (90%, Kelinon Agrochemical Co., Beijing, China) containing 0.1% Triton X-100 and a minimum ABA quantification amount of 96% aqueous ethanol (to initially dissolve the Each sample, consisting of 200 mg fresh weight (FW) of -1 ABA, ca.10mLmg ) was sprayed weekly onto the leaves grape leaf (1 month old and fully expanded), was homog- to run-off (five applications). Sprays were accomplished in enized in a mortar with liquid nitrogen and extracted with the evening to minimize photodegradation. This ABA dose 2 mL of 80:19:1 (v/v) methanol : twice-distilled water : acetic was chosen according to previous work with a range of 2 acid at 4 °C. After 12 h, 40 ng of hexa-deuterated ([ H6])- species (Sansberro et al. 2004; Travaglia et al. 2007) includ- ABA (a gift from Professor R.P. Pharis, University of ing grape (Quiroga et al. 2009). 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 Control treatment 9300 g and the supernatant evaporated in a rotavapour with A solution containing distilled water plus 0.1% Triton vacuum at 35 °C.The aqueous residue was adjusted to pH 3.0 X-100 and a minimum amount of ethanol as described with acetic acid and partitioned four times with equal above was sprayed weekly as described above. volumes of ethyl acetate saturated with 1% acetic acid.After The two oldest (1-month-old and fully expanded) leaves solvent evaporation in vacuum at 35 °C, the residue was from the base of the shoot of each plant were collected at dissolved in 1 mL of water at pH 3.0 (1% acetic acid) and midday the day after the last ABA application. One was passed through Sep-Pak C18 reversed phase (500 mg cooled with ice and immediately processed to assess of material) cartridge (Waters Associates, Milford, MA, protein content, antioxidant enzymatic activity and mem- USA). This elution was performed at a flow rate of brane integrity. The other leaf was quickly frozen at -20 °C 0.2 mL min-1 using the following gradient: 1 mL each one of for subsequent evaluation of photosynthetic and photopro- twice-distilled water pH 3.0, hexane and 80% aqueous tective pigments, phenolic compounds, sterols and lipid methanol in 1% acetic acid.The entire eluate was collected, peroxidation. and after solvent evaporation in vacuum at 35 °C,the residue

3000 PAR 1450 m 70 PAR 940 m Supplemented UV-B 60 2500 UV-B 1450 m UV-B 940 m 50

) 2000 UV-B ( –1 s

–2 40 m

1500 W cm mol m m 30 –2 )

PAR ( PAR 1000 Figure 1. Solar radiation 20 [photosynthetically active radiation (PAR) and ultraviolet (UV)-B] registered 500 on 22 January 2007 at Gualtallary 10 (33°23′S, 69°15′W and 1450 m), on 26 January 2007 at Facultad de Ciencias 0 0 Agrarias (33°0′S, 68°52′W and 940 m), 0800 0900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 and the UV-B supplemental irradiation Day time (h) used to treat plants at the last location. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment 4 F. J. Berli et al. was dissolved in 1 mL of water at pH 3.0. This solution was with 30 mL of dry pyridine (Flucka, Steinheim, Germany) transferred to Oasis WAX (weak anion exchanger, 60 mg of and 70 mL of N,O-bis(trimethylsilyl)-triﬂuoroacetamide material) cartridges (Waters Associates) in a gradient of (BSTFA) 1% TCMSi (Sigma Chem. Co., St. Louis, MO,

1 mL of each of 5% NH4OH in methanol, 5% formic acid in USA). After 75 min at 70 °C an aliquot of 1 mL of derived methanol and finally 2% formic acid in methanol.The acidic extract was injected and analysed by GC/MS. The GC/MS elute (which contains theABA) was evaporated at 35 °C and analysis was carried out with a PerkinElmer Model Clarus vacuum and then converted to the methyl-ester (Me) deriva- 500 with the same conditions as for ABA determinations, tives with 10–20 mL of methanol plus 50–100 mL of fresh except that the oven temperature programme was: initial ethereal CH2N2 (30 min at room temperature). After sol- temperature at 80 °C for 1 min, then from 80 to 250 °C at -1 vents had been eliminated under a gentle flow of N2 at room a rate of 20 °C min , and held for 1 min, then augmented temperature, Me samples were dissolved in ethyl acetate, at 6 °C min-1 to 300 °C, and held for 2 min, and finally and 1 mL was injected split–splitless into a HP-5 cross-linked increased at 20 °C min-1 to 320 °C, and held for 24 min. The methyl silicone capillary column (30 m length, 0.25 mm identity of each phenolic and sterol compounds was estab- inner diameter, 0.25 mm film thickness) fitted in a capillary lished by retention times on capillary GC (see Fig. 3) and gas chromatograph-electron impact mass spectrometer the full mass spectra of silylated standards performed in the (GC/MS) (Clarus 500,PerkinElmer,Shelton,CT,USA).The range from 120 to 700 m/z (quercetin, kaempferol, caffeic GC column was eluted with He (1 mL min-1). The GC tem- acid, p-coumaric acid and ferulic acid; Sigma Chem. Co.) perature programme was 100 °C to 260 °C at 20 °C min-1, and from the National Institute of Standards and Technol- then 10 min at 260 °C.The mass spectrometer was operated ogy (NIST) library (sterols). The Total Ion Chromatogram with electron impact ionization energy of 70 eV.The injector of quercetin (the most abundant metabolite studied) was temperature was 230 °C, ion source temperature was 260 °C used as a reference compound for quantification. A six- and the interface temperature was 280 °C.After performing point calibration curve was obtained for quercetin, the selected ion monitoring (SIM) the amount of ABA was standard solutions contained 15% (v/v) methanol and were calculated by comparison of the peak areas of the major ions subjected to the same extraction-derivation procedure for the Me derivative of the deuterated internal standard described for the samples. The quantification was per- 2 [ H6]-ABA (194/166) relative to its non-labelled counterpart formed by relating the peak areas of the identified (190/162); measurements on samples from each treatment compounds in each sample and was calculated from the were performed using three biological replicates. calibration plot.

Total UV-absorbing phenolic compounds and Photosynthetic pigments anthocyanins (photoprotective pigments) For determinations of Chl a,Chlb and carotenoids, one leaf Two leaf discs (1 cm2 each) were placed in 2 mL of 99:1 (v/v) disc (1 cm2) was placed in 5 mL of dimethyl sulphoxide methanol : HCl and allowed to extract 72 h in darkness and (DMSO) and allowed to extract for 45 min in darkness at at room temperature. Absorbance of the extracts was read 70 °C. Absorbance was read at 665, 649 and 480 nm against at 305 or 546 nm for determinations of total UV-absorbing a blank of reagents, according to Chapelle, Kim & compounds or anthocyanins, respectively, according to McMurtrey (1992) and the formulas of Wellburn (1994) Mazza et al. (1999), with a Cary-50 UV-Vis spectropho- as follows: Chl a = 12.19 OD - 3.45 OD ;Chlb = 21.99 tometer (Varian Inc., Palo Alto, CA, USA) with 1 and 665 649 OD - 5.32 OD ; carotenoids = (1000 OD - 2.14 Chl 10 mm optical path cells. 649 665 480 a - 70.16 Chl b)/220; Total Chl = Chl a + Chl b.

Characterization and quantiﬁcation of phenolic compounds and sterols Membrane integrity The extraction and derivation procedure was performed Ten leaf discs (1 cm2 each) were washed three times with according to Minuti, Pellegrino & Tesei (2006), with modi- twice-distilled water to remove surface-adhered electro-

ﬁcations as follows. Grape leaves were frozen in liquid N2 lytes, and then placed in vials containing 30 mL of twice- and ground to a ﬁne powder using a mortar and pestle. distilled water. They were incubated in darkness for 24 h

Twenty milligrams of frozen leaf powder was placed in a at 8 °C. The electrical conductivity of the solution (C1)was centrifuge tube and sonicated (200 W, Cleanson CS-1106, measured, after equilibration at 25 °C, with a pH/CON Buenos Aires, Argentina) with 0.6 mL acidified ethyl 510 meter (Oakton Instruments, Vernon Hills, IL, USA). acetate (0.2% of 37% HCl v/v) and centrifuged for 5 min at Samples were then heated for 1 h at 80 °C, cooled and 9300 g. Supernatant was collected and the pellets extracted kept in darkness for 16 h at 8 °C. Then, the electrical twice with 0.6 mL of acidified ethyl acetate. Then, extracts conductivity of heat-killed tissues (C2) was measured at were pooled, dried by filtering through a glass conic mini- 25 °C. Membrane integrity was calculated according to column packed with NaSO4, and evaporated to dryness Vásquez-Tello et al. (1990) as follows: relative membrane under a gentle N2 stream. The solid residue was diluted integrity = [1 - (C1/C2)] ¥ 100. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment ABA mediates grape response to solar UV-B 5

Lipid peroxidation The POX activity was assayed by monitoring the oxida- tion of guaiacol to tetraguaiacol at 470 nm in 2.5 mL reac- In order to evaluate oxidative damage as lipid peroxidation mixture containing 50 mm potassium phosphate buffer tion, malondialdehyde (MDA) content was measured (pH 6.0), 1.2 mm H2O2 and 2 mm guaiacol. The reaction was following the procedure described by Beligni & Lamattina started by adding 100 mL of the sample and changes in (2002). Seventy-ﬁve milligrams of leaf tissue was sus- absorbance were followed for 60 s as stated by Zhang & pended in 1.5 mL of stock solution [15% (w/v) trichloro- Kirkham (1994). acetic acid (TCA), 0.5% (w/v) thiobarbituric acid (TBA) and 0.25% (w/v) hydrochloric acid]. The mixture was Statistical analysis stirred vigorously and incubated at 95 °C for 60 min. After centrifugation at 9300 g for 10 min, the supernatant was Statistical analyses were performed as a randomized block collected. Absorbance of the extracts was measured at witha2¥ 2 factorial arrangement of treatments with the 535 nm with 1 mm optical path cell. The concentration software Statgraphics Centurion XV version 15.0.10 (Stat- was calculated considering MDA molar extinction point Technologies Inc.,Warrenton,VA, USA).The effect of coefﬁcient = 1.56 ¥ 105 m-1 cm-1. UV-B, ABA application and their interaction were deter- minate by multifactorial anova.

Protein content and antioxidant enzyme activity RESULTS Samples of 250 mg fresh weight leaf were homogenized UV-B increased the content of ABA (as assessed by using a mortar and pestle with 5 mL of extraction solution GC/MS/SIM quantification) in grape leaves 2.7-fold, ABA (100 mm potassium phosphate buffer pH 7.5; 0.1% Triton treatment 3.6-fold, and combined effect of UV-B and ABA X-100; 1 mm EDTA; 0.5 mm ascorbic acid) at 5 °C and 12.5-fold (Fig. 2a). 0.25 g of insoluble polyvinylpolypyrrolidone (PVPP), and No statistical difference was observed in the Chl content, centrifuged for 5 min at 9300 g. Supernatant was collected both total (Table 1) or Chl a and Chl b (data not shown), and frozen at -20 °C until assayed for proteins and antioxi- and in carotenoids content. Simultaneous action of UV-B dants. Protein content was determined according to Brad- and applied ABA showed the highest carotenoids content, ford (1976) with bovine serum albumin (BSA) as standard, although only significant differences were obtained at measuring the absorbance at 595 nm. P Յ 0.1 for UV-B treatments (Table 1).The content of total The CAT activity was assayed by monitoring the con- UV-absorbing compounds was increased by UV-B and sumption of H2O2 at 240 nm in 2.5 mL reaction mixture applied ABA with a significant interaction between UV-B containing 100 mm potassium phosphate buffer (pH 7.5) and ABA (Table 1) meaning that ABA effects are UV-B and 75 mL of sample. The reaction was started by adding dependent. Anthocyanin levels were also increased by

7.5 mL of 34.8 mm H2O2 and changes in absorbance were interactive action of UV-B and applied ABA (Table 1). followed for 60 s as stated by Azevedo et al. (1998). Figure 2b shows the different leaf pigmentation for each The APX activity was measured by the decrease in absor- treatment. bance of ascorbate at 290 nm in 2.5 mL reaction mixture The UV-absorbing compounds characterized by GC/MS containing 50 mm potassium phosphate buffer (pH 7.0), were ﬂavonols (quercetin and kaempferol) and the

1mm EDTA, 0.5 mm ascorbic acid and 1 mm H2O2.The hydroxycinnamic acids (caffeic acid, ferulic acid and reaction was started by adding 40 mL of the sample and p-coumaric acid). Figure 3 shows the GC/MS proﬁles of changes in absorbance were followed for 60 s according to the ﬂavonols and sterols. The quercetin concentration was Barka (2001). approximately 20-fold higher than kaempferol, and the

(a) (b)

Figure 2. Abscisic acid (ABA) levels as assessed by gas chromatograph-electron impact mass spectrometer/selected ion monitoring (GC/MS/SIM) in mgg-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 ϮSE, n = 3. (b) Grape plants of +UV-B (left) and -UV-B (right) treatments. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment 6 F. J. Berli et al.

-2 -2 -2 Table 1. Total chlorophylls (Chl) (mgcm leaf), carotenoids (mgcm leaf), total ultraviolet (UV)-absorbing compounds (OD305 cm -2 leaf) and anthocyanins (OD546 cm 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 Ϯ 2.860 6.862 Ϯ 0.727 0.631 Ϯ 0.050 0.049 Ϯ 0.005 Control 25.599 Ϯ 1.720 5.847 Ϯ 0.443 0.527 Ϯ 0.006 0.036 Ϯ 0.002 -UV-B ABA 28.586 Ϯ 2.915 5.515 Ϯ 0.397 0.339 Ϯ 0.007 0.036 Ϯ 0.002 Control 26.576 Ϯ 3.106 5.297 Ϯ 0.445 0.336 Ϯ 0.018 0.034 Ϯ 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 ϮSE, n = 5. levels of both were significantly increased by UV-B and out UV-B (Table 3). The activity of APX was promoted by applied ABA treatment (Table 2). There was a significant UV-B (relative to the treatment where UV-B was filtered interaction between UV-B and ABA in kaempferol out) and there was a significant interaction between UV-B concentration, indicating that ABA promotion of and ABA. The activity of POX was also affected by the kaempferol is UV-B dependent. These results also corre- interaction between UV-B and ABA. This suggests that late well with the spectrophotometric determinations of ABA promotion of APX and POX activities is UV-B total UV-absorbing compounds (Table 1). In samples of all dependent. treatments the concentration of caffeic acid was approxi- The sterols b-sitosterol and stigmasterol were identified mately fivefold higher than that of p-coumaric acid, and by capillary GC/MS analysis and in all treatments the 20-fold higher than that of ferulic acid (Table 2). Caffeic b-sitosterol content was approximately fivefold higher than acid and ferulic acid were significantly increased by exog- that of stigmasterol. Membrane integrity and b-sitosterol enously applied ABA, although there was no significant content were significantly improved by ABA application, effect of ABA application on the p-coumaric acid concen- but were not affected by filtering out UV-B (Table 4). tration. None of the hydroxycinnamic acids was affected Oxidative damage, as assessed by MDA concentration, was by filtering out UV-B. significantly decreased by filtering out UV-B. Stigmasterol The activity of CAT was significantly increased by content was not affected by any of the treatments (data not applied ABA, but there was no significant effect of filtering 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 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 Ϯ 0.229 0.120 Ϯ 0.010 0.630 Ϯ 0.085 0.025 Ϯ 0.002 0.151 Ϯ 0.089 Control 1.476 Ϯ 0.183 0.053 Ϯ 0.008 0.298 Ϯ 0.067 0.019 Ϯ 0.001 0.090 Ϯ 0.024 -UV-B ABA 0.940 Ϯ 0.084 0.036 Ϯ 0.002 0.437 Ϯ 0.074 0.027 Ϯ 0.002 0.121 Ϯ 0.072 Control 0.700 Ϯ 0.047 0.032 Ϯ 0.004 0.285 Ϯ 0.039 0.022 Ϯ 0.002 0.061 Ϯ 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 ϮSE, 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 Ϯ 6.625 314.725 Ϯ 29.763 9.162 Ϯ 1.751 leaves of plus ultraviolet (UV)-B (+UV-B) Control 46.405 Ϯ 14.297 213.761 Ϯ 15.953 5.818 Ϯ 0.779 and minus UV-B (-UV-B) treatments either -UV-B ABA 62.880 Ϯ 5.453 181.594 Ϯ 12.512 5.611 Ϯ 0.884 sprayed or not (control) with abscisic acid Control 44.047 Ϯ 7.416 217.294 Ϯ 35.875 6.645 Ϯ 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 ϮSE, n = 5.

DISCUSSION as Pfündel (2003) reported that UV-B was responsible for photosystem II (PSII) inhibition (i.e. pigment bleaching and Although UV-B represents only a small portion of solar inhibition of overall photosynthesis). However, in Pfündel’s radiation that reaches the earth surface, its impact on bio- experiments the grape leaves had been adapted to growth logical processes is considered of great importance (Björn, in darkness prior to UV-B treatments. Similar inhibitory Widell & Wang 2002; Jordan 2002). That is, exhaustive effects had been also reported in maize tissues along research has shown that ROS production is triggered in with decreases in photosynthetic pigments, gas exchange, several cell compartments when plant tissues are exposed Rubisco activity and total sugar content (Correia et al. to UV-B, and as a result oxidative damage of proteins and 2005), although these UV-B effects were obtained after cell membranes may occur (Scandalios 1993). Our study nitrogen deﬁciency.Thus, the different results found in pre- was performed in a natural environment, and while an vious studies compared with our results may indicate dif- increase in MDA content was observed, which reﬂects lipid ferent experimental conditions and/or genetic differences peroxidation and therefore some level of stress, membrane and/or differences in developmental stages or tissues. damage and photooxidation of Chl in leaves exposed to The increase in UV-absorbing compounds, including +UV-B irradiances were not observed. The maintenance of anthocyanins, is another response that is postulated to Chl levels in the +UV-B treatments was rather unexpected, 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 Ϯ 0.529 0.3840 Ϯ 0.012 11.521 Ϯ 1.896 content in grape leaves of plus ultraviolet Control 95.392 Ϯ 0.769 0.277 Ϯ 0.020 13.744 Ϯ 2.804 (UV)-B (+UV-B) and minus UV-B -UV-B ABA 96.909 Ϯ 0.562 0.380 Ϯ 0.024 7.436 Ϯ 0.585 (-UV-B) treatments either sprayed or not Control 95.328 Ϯ 0.704 0.256 Ϯ 0.014 6.250 Ϯ 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 ϮSE, n = 5. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment 8 F. J. Berli et al. compounds are correlated with a more efficient absorption that ABA is mainly an enhancer of oxidative damage to of harmful UV-B (Frohnmeyer & Staiger 2003; Merzlyak plants. In contrast, our results show that increased CAT et al. 2008). Such phenolics transform short-wave, high- activity was caused by ABA treatment independently of energy and highly destructive radiation into longer- UV-B, while the activities of APX and POX were increased wavelength light and these wavelengths are less destructive by ABA when +UV-B was present. to the cellular leaf structures, including the photosynthetic Cell membrane integrity, evaluated by its electrolyte apparatus (Bilger, Johnsen & Schreiber 2001).Additionally, leakage, may be a good indicator of stress tolerance in phenolic compounds may enhance protection against oxi- plants (Vásquez-Tello et al. 1990). In our experiments a dative stress, as they possess chemical structures capable of positive correlation was obtained between this variable and scavenging free radicals; for example, they have also been the defence system induced by ABA; for example, mem- found to be effective antioxidants (Blokhina, Virolainen & brane integrity was increased after ABA application with or Fagerstedt 2002). In many higher plant tissues, two classes without the use of +UV-B treatments. This enhancement of phenolics appear to be involved in epidermal UV screen- in membrane integrity was associated with an increase in ing: the hydroxycinnamic acids and the more complex fla- the membrane content of b-sitosterol. Plant membranes vonoids.These compounds vary from species to species and include several sterols in their structure, where sitosterol according to developmental stages and tissues. Our results usually predominates (Hartmann 1998; Nomura et al. 1999) with grape leaves showed that the UV-absorbing com- and our results confirm this conclusion. pounds found in the leaf tissue were flavonols (quercetin Overall, our results strongly suggest that ABA is and kaempferol) and hydroxycinnamic acids (caffeic acid, involved in stress tolerance of grape leaves and that they p-coumaric acid and ferulic acid). However, only quercetin function by enhancing the ability of epidermal tissues to and kaempferol were significantly enhanced by +UV-B filter out UV-B. That is, they increase levels of antioxidant treatments. Quercetin is a more efficient ROS-scavenger enzymes and enhance the sterol-structural defence, than kaempferol, due to its increased number of hydroxyl although these responses are, in some cases, also depen- groups; moreover, we found that quercetin’s concentration dent on the co-presence of high levels of UV-B irradia- in grape leaf tissues was approximately 20-fold higher than tion. In other words, the antioxidant defence system in kaempferol. Such results are consistent with the finding of grape leaf tissues is initially activated by UV-B irradiation increased expression of genes involved in the biosynthesis and ABA often acts downstream in the signalling pathway of flavonols and phenylpropanoids in grape plants sub- (although some responses are activated by ABA alone). mitted to UV-B radiation (Pontin et al. 2008). The accumu- The first line of defence likely includes elevated levels of lation of phenolic compounds that selectively and passively flavonols and hydroxycinnamic acids; these filter part of absorb UV radiation in the epidermis probably represents the UV-B. We found that quercetin and kaempferol were the most cost-effective strategy for adaptation in the case of increased by +UV-B treatments, with an additional regular and prolonged exposure to elevated doses of UV-B, increase being seen when ABA was also co-applied with in that it should reduce the need for maintaining con- the +UV-B treatments. In contrast, increases in caffeic acid tinuously active avoidance and repair processes (Cockell & and ferulic acid concentration were triggered only by Knowland 1999; Liakoura, Bornman & Karabourniotis ABA. It is postulated that a part of the UV-B-origin 2003). Kolb et al. (2001) also found enhanced synthesis energy excess is dissipated by carotenoids. In our experi- of flavonoids, specifically the flavonols quercetin and ments carotenoids levels were enhanced by +UV-B treat- kaempferol in grape leaves exposed to UV-B. Nuñez- ments alone, although one has to remember that +UV-B Olivera et al. (2006) obtained an increase in the concen- treatment also increased endogenous ABA concentrations trations of Chl and carotenoids and a reduction in in the leaf tissue. UV-absorbing compounds in grape plants of the cultivar The antioxidant enzymatic system (CAT,APX and POX) Tempranillo grown under UV-B exclusion. It is worth is also postulated to contribute to the attenuation of UV- noting, however, that solar UV-B seems to cause some B-induced stress situation, and its activity is enhanced by damage in Tempranillo grapevines (purportedly suscep- exogenously applied ABA, but only under the +UV-B tible). This did not happen with Malbec (more tolerant), treatments (APX and POX). Finally, an extra structural given that our +UV-B treatments maintained Chl levels and resistance to UV-B-induced damages includes an enhance- actually increased carotenoids levels. ment of membrane sterols that, in our study, are exclusively Antioxidant enzymatic activity could partially explain augmented by ABA but not by UV-B itself. why membrane integrity was not affected by +UV-B when ABA was applied, as the activities of CAT, APX and POX ACKNOWLEDGMENTS were higher with this treatment. In effect, it has been reported that POX is induced by light in grapevine (Ros This work was supported by Consejo Nacional de Investi- Barceló et al. 2003) and ABA may induce the biosynthesis gaciones Científicas y Técnicas (CONICET, PID 5028 to of these enzymes (Jiang & Zhang 2001). R.B.), Secretaría de Ciencia y Técnica de la Nación (SECyT, While ABA can increase ROS production and induce the PICT 08-12398 and PICT 20-20093 to R.B.), Secretaría de activities of SOD, CAT, APX and GR in plants (Jiang & Ciencia y Técnica de la Universidad Nacional de Cuyo, Zhang 2001; Sakamoto et al. 2008), these studies considered Bodega Catena-Zapata, Coordenação de Aperfeiçoamento © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment ABA mediates grape response to solar UV-B 9 de Pessoal de Nível Superior (CAPES, to L.H.-V.) and Con- Casati P. & Walbot V. (2003) Gene expression profiling in response selho Nacional Desenvolvimento Científico e Tecnológico to ultraviolet radiation in Zea mays genotypes with varying (CNPq, to R.B.-S.). The authors thank the technical assis- flavonoid content. Plant Physiology 132, 1739–1754. Chapelle E.W., Kim M.S. & McMurtrey J. (1992) Ratio analysis of tance of L. Bolcato and M. 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