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 mem- brane 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).