ARTICLE IN PRESS Journal of Plant Physiology 167 (2010) 792–799 Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.de/jplph Antioxidant content in two CAM bromeliad species as a response to seasonal light changes in a tropical dry deciduous forest Claudia Gonza´lez-Salvatierra a,b, Jose´ Luis Andrade a, Fabiola Escalante-Erosa b, Karlina Garcı´a-Sosa b, Luis Manuel Pen˜a-Rodrı´guez b,n a Unidad de Recursos Naturales, Centro de Investigacio´n Cientı´fica de Yucata´n, Calle 43 No. 130, Col. Chuburna´,Me´rida, Yucata´n 97200, Me´xico b Unidad de Biotecnologı´a, Centro de Investigacio´n Cientı´fica de Yucata´n, Calle 43 No. 130, Col. Chuburna´,Me´rida, Yucata´n 97200, Me´xico article info abstract Article history: Plants have evolved photoprotective mechanisms to limit photodamage; one of these mechanisms Received 16 October 2009 involves the biosynthesis of antioxidant metabolites to neutralize reactive oxygen species generated Received in revised form when plants are exposed to excess light. However, it is known that exposure of plants to conditions of 6 January 2010 extreme water stress and high light intensity results in their enhanced susceptibility to over-excitation Accepted 6 January 2010 of photosystem II and to photooxidative stress. In this investigation we used the 2,2-diphenyl-1- picrylhydrazyl reduction assay to conduct a broad survey of the effect of water availability and light Keywords: exposure conditions on the antioxidant activity of the leaf extracts of two bromeliad species showing Anthocyanins crassulacean acid metabolism. One of these was an epiphyte, Tillandsia brachycaulos, and the other a Antioxidant activity terrestrial species, Bromelia karatas. Both species were found growing wild in the tropical dry deciduous Bromeliaceae forest of Dzibilchaltu´ n National Park, Me´xico. The microenvironment of T. brachycaulos and B. karatas Crassulacean acid metabolism Light microenvironments experiences significant diurnal and seasonal light variations as well as changes in temperature and water availability. The results obtained showed that, for both bromeliads, increases in antioxidant activity occurred during the dry season, as a consequence of water stress and higher light conditions. Additionally, in T. brachycaulos there was a clear correlation between high light intensity conditions and the content of anthocyanins which accumulated below the leaf epidermis. This result suggests that the role of these pigments is as photoprotective screens in the leaves. The red coloration below the leaf epidermis of B. karatas was not due to anthocyanins but to other unidentified pigments. & 2010 Elsevier GmbH. All rights reserved. Introduction et al., 2008). These radicals represent a threat to the cell because they react with proteins, lipids, and DNA, causing rapid cell Plant adaptations to avoid damage from excess light include damage and destroying chloroplast pigments and membrane morphological, biochemical, and physiological features. The lipids (Chalker-Scott, 1999; Gould et al., 2002). To prevent potential for light acclimation is species specific and involves damage by ROS, plants use both enzymes and metabolites with major structural and functional changes in the photosynthetic antioxidant activity (Shao et al., 2008; Herna´ndez et al., 2009). apparatus (Lambreva et al., 2006; Luttge,¨ 2008). During photo- Antioxidant enzymes such as superoxide dismutase, catalase and synthesis, excessive amounts of light may cause over-energizing ascorbate peroxidase are capable of removing, neutralizing or (photooxidative stress) and damage of the photosynthetic scavenging oxy-intermediates in leaves (Gould et al., 2002; apparatus, leading to photoinhibition (Herna´ndez et al., 2006; Herna´ ndez et al., 2006; Herna´ndez et al., 2009). Metabolites with Lambreva et al., 2006; Jung and Niyogi, 2006). Although excess antioxidant activity include carotenoids, anthocyanins, flavonoids, energy is dissipated as heat or fluorescence (Demmig-Adams and and molecules such as ascorbic acid (vitamin C), tocopherol Adams III, 1996), under high photosynthetic photon flux (PPF) (vitamin E), and ferrodoxin (Krieger-Liszkay and Trebst, 2006). conditions a number of toxic radicals known as reactive oxygen Most of the antioxidant metabolites accumulate primarily in the species (ROS) are produced (Karpinski et al., 1999; Krieger-Liszkay epidermis to both scavenge ROS and, presumably, screen solar light in the absence of a photon-scattering indumentum (Steyn et al., 2002; Merzlyak et al., 2005; Tanaka et al., 2008). Abbreviations: CAM, crassulacean acid metabolism; DPPH, 2, 2-diphenyl-1- It has been proposed that crassulacean acid metabolism picrylhydrazyl radical; TLC, thin layer chromatography; PPF, photosynthetic (CAM), a photosynthetic pathway in which most of the carbon photon flux; PSII, photosystem II; ROS, reactive oxygen species n Corresponding author. Tel.: +52 999 9428330; fax: +52 999 9813900. acquisition occurs during the nightime, prevents ROS production E-mail address: [email protected] (L. Manuel Pen˜a-Rodrı´guez). maintaining daytime carbon concentrations high. Thus, constant 0176-1617/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.01.001 ARTICLE IN PRESS C. Gonza´lez-Salvatierra et al. / Journal of Plant Physiology 167 (2010) 792–799 793 daytime CO2 assimilation prevents the over-energization of the with a maximum canopy height of 8 m (Mondrago´ n et al., 2004; photosynthetic machinery, controlling photoinhibition and oxi- Cervantes et al., 2005). The rainfall pattern is markedly seasonal: dative stress under moderate levels of light intensity and water- mean annual rainfall and temperature are 700 mm and 25.8 1C, limiting conditions (Borland et al., 2000; Osmond and Forster,¨ respectively (Thien et al., 1982). A marked dry season (March– 2006; Niewiadomska and Borland, 2008). However, it has been May; where most trees are leafless) occurs between an early dry reported that CAM plants exposed to a combination of prolonged, season when scattered rains occur (November–February) and a extreme water deficit, and high PPF are susceptible to over- rainy season (June–November; Orellana, 1999). excitation of photosystem II (PSII) and over-reduction of the redox-elements, as a consequence of sustained electron transport behind closed stomata (Miszalski and Niewiadomska, 2001; Light characterization of microsites Luttge,¨ 2004). CAM plants possess effective antioxidative response systems with a diurnal pattern of expression and regulation of Integrated total PPF data above the canopy by the seasons and antioxidant metabolites (e.g. anthocyanins and flavonoids) and days of measurement were obtained from the meteorological antioxidant enzymes (e.g. catalase, superoxide dismutase) with a station of Dzibilchaltu´ n National Park. The light environment of circadian pattern of expression and tight regulation (S´lesak et al., microsites was characterized using hemispherical photography. A 2002; Niewiadomska et al., 2004). Also, according to Niewia- digital picture (Nikon Coolpix 4300, Japan) was taken from above domska and Borland (2008), there is little evidence to support each plant with a fish-eye lens (Nikon FC-E8 0.21 x, Japan), during oxidative stress or oxidative damage in CAM plants and they the dry and rainy seasons to quantify the fraction of the gap mention that an interplay between various processes that distribution above the plant, and hence the directional distribu- increase enzyme activity and the abundance of metabolites tion of direct and diffuse PPF in the understory. Images were involved in the destruction and scavenging of ROS optimizes processed using WINPHOT version 5.0 (ter Steege, 1996). Six photosynthetic performance, in line with the dynamic shifts in individuals growing under the shaded lower canopy or on CO2 and O2 concentrations that occur during the diurnal phases exposed sites were located and selected for both species. of CAM. Individuals of each species were grouped according to the total The family Bromeliaceae appears to be unique amongst the PPF received daily during the dry season. Plot sites varied from monocotyledons in the variety and abundance of antioxidant fully exposed to direct sunlight (80–90% total daily PPF) to shaded metabolites (e.g. flavones and flavonols; Williams, 1978; Saito and (20–30% total daily PPF); three replicates were taken per species Harborne, 1983; Benzing, 2000). In most bromeliads, their gradual and light environment. adaptation to high PPF environments may be favored by the development of reflective cuticles and photoprotective metabo- lites, leading to an enhancement of photosynthesis and antiox- Antioxidant activity idant defenses (Maxwell et al., 1995; Benzing, 2000; Steyn et al., 2002; Gould, 2004). This is particularly important in a tropical dry Leaf extraction: leaves from ten individuals of T. brachycaulos deciduous forest, where bromeliads are exposed to multiple stress and five individuals of B. karatas were collected during each factors during the dry season, including a combination of water season (dry and rainy), under both light regimes. All leaves from deficit, high temperature, and high PPF (Benzing, 2000; Graham each species were mixed together, ground and then extracted and Andrade, 2004; Cervantes et al., 2005; Reyes-Garcı´a and with ethanol at room temperature for 24 h. The resulting slurry Griffiths, 2009). An additional adaptation of some epiphytic and was filtered, first through a cotton