Antioxidant Content in Two CAM Bromeliad Species As a Response to Seasonal Light Changes in a Tropical Dry Deciduous Forest

ARTICLE IN PRESS

Journal of Plant Physiology 167 (2010) 792–799

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Journal of Plant Physiology

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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 Garcıá-Sosa b, Luis Manuel Penã-Rodrı´guez b,n a Unidad de Recursos Naturales, Centro de Investigacioń Cientı´fica de Yucatań, Calle 43 No. 130, Col. Chuburna´,Me´rida, Yucatań 97200, Me´xico b Unidad de Biotecnologıá, Centro de Investigacioń Cientı´fica de Yucatań, Calle 43 No. 130, Col. Chuburna´,Me´rida, Yucatań 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; Hernańdez 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 (Hernańdez et al., 2006; Herna´ ndez et al., 2006; Hernańdez 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 Penã-Rodrı´guez). maintaining daytime carbon concentrations high. Thus, constant

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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 metabolites, 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-Garcıá 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 plug and then through filter terrestrial CAM bromeliads is a very plastic light-harvesting paper (Whatman no.1). The solvent was evaporated under system, which acclimates rapidly to contrasting light environ- reduced pressure at 35 1C, using a rotary evaporator (BUCHI¨ RE ments (Maxwell et al., 1992, 1995; Martin, 1994). The present 111, Flawil, Switzerland) to yield the corresponding ethanolic study evaluated the production of antioxidant metabolites in the crude extract. Portions (500 mg) of the crude ethanolic extract leaves of two CAM bromeliads with different growth habitat, were fractionated by either successive sonication with n-hexane Tillandsia brachycaulos Schltdl. and Bromelia karatas L., in a and ethyl acetate (3 h, 100 mL) in an ultrasonic bath (Cole-Parmer tropical dry deciduous forest in Yucatań, Me´xico. Field measure- Instrument Company, Illinois, USA), or by successive liquid–liquid ments of the light environment were made above individuals in partition of an aqueous (3:2 water/methanol) suspension with n- two light microhabitats for both species during the dry and rainy hexane (2:1, 1:1, 1:1, v/v), ethyl acetate (2:1, 1:1, 1:1, v/v) and seasons. Both T. brachycaulos and B. karatas exhibit the CAM water-saturated n-butanol (1:4 v/v), to produce the correspond- photosynthetic pathway (Martin, 1994; Graham and Andrade, ing low, medium, and high-polarity fractions. Both the ethanolic 2004; Cervantes et al., 2005). T. brachycaulos is an epiphytic crude extracts and the purified fractions were analyzed by thin bromeliad commonly found in tropical dry forests, moist forests, layer chromatography (TLC) and evaluated for their content of and semi-arid shrublands in southern Me´xico and Central antioxidant metabolites. Chromatographic analyses were per- America (Mondrago´ n et al., 2004; Ramı´rez et al., 2005). B. karatas formed on 5 5cm2, 0.2 mm-thick silica gel plates (E.M. Merck is a terrestrial bromeliad which can be found in tropical dry DC Alufolien). Elution was carried out using a mixture of forests, tropical thorn forests, semideciduous tropical forests, and dichloromethane/methanol/water (14:7:1) as the eluant. The coastal dunes (Ramı´rez et al., 2005). resulting chromatograms were visualized using a solution of 4% phosphomolybdic acid, containing a trace of ceric sulfate, in 5% sulfuric acid, followed by gentle heating. Materials and methods Antioxidant activity was determined by evaluating the reduction of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH, Sigma- Study site Aldrich Chemie, Steinheim, Germany), following the procedures below: Measurements and sampling were carried out at the Dzibil- Qualitative assay: crude extracts and purified fractions were chaltu´ n National Park (211050N, 891990W, 10 m elevation) in spotted on a TLC plate and eluted as described previously. The Yucatań, Me´xico, in the dry (April 2007) and rainy (September resulting chromatograms were sprayed with DPPH (0.2% weight/ 2007) seasons. The site is a subtropical-dry to tropical-arid forest, volume) and the presence of antioxidant metabolites was ARTICLE IN PRESS

794 C. Gonza´lez-Salvatierra et al. / Journal of Plant Physiology 167 (2010) 792–799 detected 24 h later by observing yellow spots against a purple The average proportion of total daily PPF received by shaded and background (Nanjo et al., 1996). exposed plants of T. brachycaulos during the dry season was Quantitative assay: to prepare the calibration curve, a 0.1 mM 3571.3% and 78.572.5%, respectively (Figs. 1B and F), and methanol solution of DPPH and ﬁve dilutions (0.038, 0.217, 1.195, 14.372.3% and 65.571.2% in the rainy season (Figs. 1A and E). 6.59, and 36.36 mg/mL) of L-ascorbic acid (Sigma, Sigma-Aldrich During the dry season, shaded and exposed plants of B. karatas Chemie, Steinheim, Germany) in methanol were prepared averaged 65.2472.3% and 80.6771.5% of total daily PPF, (Molyneux, 2003). The initial DPPH concentration was calculated respectively (Figs. 1D and H), while during the rainy season from the calibration curve using the equation determined by shaded and exposed plants averaged 30.8671.4% and linear regression. The antioxidant activity measurements were 80.1570.95% of total PPF, respectively (Figs. 1C and G). In carried out by adding aliquots of DPPH to a methanol solution of general, shaded plants of B. karatas and of T. brachycaulos received the crude extracts and puriﬁed fractions. After 30 min, the an average of 2.5 and 1.5 times higher PPF, respectively, in the dry absorbance was measured at 517 nm using a spectrophotometer than in the rainy season. (model SM 110215, Dubuque, Iowa USA), and the percentage of remaining DPPH was calculated using the formula %

(DPPH)rim=[(Abs DPPH 0.1 mM)t=0/(Abs DPPH sample)30]n100 (Brand-Williams et al., 1995; Sańchez-Moreno et al., 1999). Antioxidant activity Spectrometric measurements were made using methanol as a blank; DPPH solutions were freshly prepared in methanol every Qualitative assay: the degree of antioxidant activity of the day and kept protected from light. Ascorbic acid (1%) was used as samples tested was determined from observation of the intensity positive control. An equal volume of the solvent employed to of yellow-colored spots on a purple background, when TLC- dissolve the samples tested, was added to the control tubes. The chromatograms were sprayed with a DPPH solution. Testing of the assay was run three times with the same leaf sample. ethanolic extracts from the two bromeliads showed a strong antioxidant activity in both species. Fractionation of the extracts Assay for total anthocyanins allowed the localization of the potentially active components in the high-polarity fractions (Table 1), suggesting that most Tissue-specific localization: five exposed and five shaded plants antioxidant metabolites were polar and hydrophilic. were processed during dry and rainy seasons. The localization of Quantitative assay: the antioxidant activity of the extracts of anthocyanins was estimated on hand-cut transverse sections of T. brachycaulos and B. karatas and their corresponding fractions fresh leaf tissue stained with 1 M HCl, as reported by Gould et al. confirmed that their highest antioxidant content was similar in (2002), and observed using a light microscope with an integrative both the crude extracts and high-polarity fractions of the two camera (Nikon, Coolpix L12). Red regions of the laminae were species. The quantitative evaluation of the antioxidant activity of sectioned and the histological location of the red-pigmentation the various organic extracts allowed the determination of the noted under bright field microscopy. seasonal effects in shaded and exposed plants (Fig. 2). While Extraction and determination: the extraction of anthocyanins exposed plants of T. brachycaulos showed significant differences in was carried out following Neff and Chory (1998). Fresh leaves antioxidant activity between seasons (Po0.001), shaded plants (0.05 g) collected from exposed and shaded plants (n=5), during did not (P40.10; Fig. 2A). During the dry season, exposed plants dry and rainy seasons; the leaves were ground in liquid nitrogen, of T. brachycaulos exhibited a higher antioxidant activity resuspended in methanol-1% HCl, and left in the dark for 24 h at (Po0.005; Kolmogorov–Smirnov test) than shaded plants 4 1C. Solids were separated by centrifugation (5000g for 5 min) (Fig. 2A). For B. karatas, a higher antioxidant activity was found and the absorbance of the supernatant was read at 530 nm using a in exposed plants during both dry and rainy seasons (Po0.025; spectrophotometer (Beckman Coulter DU650). Total anthocyanins Fig. 2B), while shaded plants showed significant differences in content was determined according to the equation given by Yang antioxidant activity between dry and rainy seasons (Po0.005). et al. (2008).

Statistical analysis Assay for total anthocyanins The qualitative DPPH assay was performed in triplicate and all data were tested for differences in concentration of antioxidant Conventional microscopy of HCl-treated leaf cross-sections metabolites in terms of season (dry/rainy) and PPF (shaded/ showed red areas suggestive of anthocyanins accumulation in exposed), using the Kolmogorov–Smirnov test comparing two both the adaxial and abaxial epidermis of T. brachycaulos (Figs. 3A independent samples. Differences between treatments of the and B) and in the adaxial epidermis of exposed plants of B. karatas anthocyanin assay were established using a two-way ANOVA. A during the dry season (Figs. 3C and D). Red areas in leaves were simple regression analysis was performed to describe the larger and darker in exposed plants of T. brachycaulos than in correlation between incident PPF and total anthocyanin content B. karatas. Although exposed plants of T. brachycaulos showed during rainy and dry seasons for both species (STATISTICA significant seasonal differences in anthocyanin content (Po0.05; software, version 7). Fig. 4A), no significant differences were found in shaded plants during the rainy or dry seasons (P40.05). In general, B. karatas showed a lower anthocyanin content than that of T. brachycaulos Results although exposed plants did show seasonal differences (Fig. 4; Po0.05). A high linear correlation was found between total Light characterization of microsites anthocyanin content and total daily PPF received by T. brachycaulos (Fig. 5A; r2=0.75, P=0.001, and r2=0.89, P=0.001 Integrated PPF above the canopy during clear days was for dry and rainy seasons, respectively). For B. karatas, a similar 40.772.16 mol m2 d1 in the dry season (9 h light period) and correlation was not significant (Fig. 5B; r2=0.40, P=0.054, and 47.373.96 mol m2 d1 in the rainy season (12 h light period). r2=0.60, P=0.009 for dry and rainy seasons, respectively). ARTICLE IN PRESS

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Fig. 1. Percentage of total daily photosynthetic photon ﬂux (PPF) received relative to the top canopy in Dzibilchaltu´ n, Me´xico, for a clear day during dry (April 18, 2007) and rainy (September 15, 2007) seasons. Individual plants of T. brachycaulos (A–B and E–F) and B. karatas (C–D and G–H) in two light conditions: shaded (A–D) and exposed (E–H) plants. Black line: above canopy PPF; gray line: below canopy PPF. Integrated PPF received at the canopy was 47.3 and 40.7 mol m2 s1 for the rainy and dry seasons, respectively (data taken from the meteorological station of Dzibilchaltu´ n National Park).

Table 1 Qualitative antioxidant activity of crude extracts and puriﬁed fractions from T. brachycaulos and B. karatas.

Sample evaluated Tillandsia brachycaulos Bromelia karatas

Crude extract nnn nnn Sonication fractions Hexane nn Ethyl acetate nn nn Residue nnn nnn Liquid–liquid fractions Hexane nn Ethyl acetate nn nn Butanol nnn nnn

Low activity: n, medium activity: nn, high activity: nnn

Discussion activity occurred in both the crude extracts and the high-polarity fractions (Fig. 2). Since none of the purified fractions showed a Although the integrated daily PPF above the canopy was stronger antioxidant activity than that of the originating crude slightly higher during the rainy season (mainly because the days extracts, all of the evaluations of antioxidant activity were carried were longer), shaded plants of B. karatas, and shaded and exposed out using crude extracts. The polar nature of the metabolites plants of T. brachycaulos, received more PPF during the dry season responsible for the antioxidant activity detected in the extracts of than during the rainy season because of leaf shedding by host T. brachycaulos and B. karatas suggests that this activity is due to trees. Many tropical rosette plants, often possessing CAM, are able the presence of phenolic products (Brand-Williams et al., 1995), to tolerate extremely high and rapidly changing light intensities including flavonoids and anthocyanins (Sańchez-Moreno et al., (Merzlyak et al., 2005). In this particular tropical dry forest, 1999; Espıń et al., 2000). T. brachycaulos grows and reproduces better in partially shaded The production of antioxidant metabolites was very similar for microsites within the canopy, where the individuals can capture both exposed and shaded plants of both species during the rainy more water and dissipate the heat more effectively (Cervantes season, which indicates that these species do not increase their et al., 2005; Andrade et al., 2009). It has been reported that CAM production of antioxidant metabolites under higher PPF, when prevents ROS production, controlling photoinhibition and oxida- water is not a limiting factor. However, the higher antioxidant tive stress (Niewiadomska and Borland, 2008). Nevertheless, activity of exposed and shaded plant species during the dry during the dry season, T. brachycaulos shows a decrease in season suggests that in both T. brachycaulos and B. karatas, photosynthetic efficiency and photoinhibition (Graham and antioxidant defenses are more active under water deficit condi- Andrade, 2004; Cervantes et al., 2005), indicating that tions, as a part of CAM activity to reduce photooxidative damage. T. brachycaulos is susceptible to over-excitation of photosystem These results are in agreement with previous reports from a wide II (PSII) and over-reduction of the redox-elements. variety of light- and water-stressed plants in several ecosystems, The qualitative evaluation of the antioxidant activity, using the where higher amounts of antioxidant metabolites may prevent reduction of the DPPH radical as an indicator, showed that the irreversible oxidative damage (Frankel and Berenbaum, 1999). antioxidant activity detected in the leaf crude extracts of the two The increase in antioxidant activity detected in the leaves of bromeliads was mainly due to their polar components (Table 1). exposed plants of T. brachycaulos during the dry season can be This was confirmed by the quantitative determination of the explained by the fact that under water deficit, the chloroplasts antioxidant activity, which showed that the strongest antioxidant contain greater amounts of ascorbate and display a high ARTICLE IN PRESS

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Fig. 2. Metabolites with antioxidant activity (mg/mL) for T. brachycaulos (A) and B. karatas (B) under two light conditions: exposed and shaded plants, during dry and rainy seasons in Dzibilchaltu´ n, Me´xico. Data are means7SE (n=27); Po0.05. enzymatic activity involved in ascorbate metabolism (Mittler, and this can explain the lower antioxidant activity values shown by 2002; Potters et al., 2002). In addition to reducing hydrogen the extracts of B. karatas, when compared to those of T. brachycaulos. peroxide, ascorbate also functions in the de-epoxidation of the The production of different types of flavonoids has been xanthophyll cycle and contributes to the dissipation of thermal reported in a number of species of Bromeliaceae (Saito and energy (Jung and Niyogi, 2006). Harborne, 1983) as a response to high PPF and other stressful Although ecophysiological information about B. karatas is scarce, conditions (Benzing, 2000). In most cases this response involves data from other species having a similar ecology, structure, and the accumulation of anthocyanins (Chalker-Scott, 1999; Gould, physiology (e.g. B. humilis, Medina et al., 1986; Ananas species, 2004; Gould et al., 2002). For T. brachycaulos, anthocyanins were Borland and Griffiths, 1989; Medina et al., 1993; B. karatas, Reyes- located in a single layer under the epidermis on both sides of the Garcıá and Griffiths, 2009) are in agreement with our results. In the leaves (Fig. 3A); additionally, concentration was higher in exposed case of antioxidant activity, the similar values of shaded plants of plants during the dry season, supporting the hypothesis of their B. karatas during both dry and rainy seasons, can be explained by the sunscreen role (Fig. 4A). These results suggest that anthocyanins, fact that this terrestrial bromeliad commonly occurs in more either on their own or combined with other pigments (e.g. exposed locations (Fig. 1) and has the ability to store water between photosynthetic pigments), prevent an excessive light–chlorophyll seasons (Reyes-Garcıá and Griffiths, 2009). An effective photopro- interaction and play a crucial role in the prevention of oxidative tection requires a regulated interaction between the use of absorbed damage. The high correlation between incident daily PPF and total light and its dissipation, particularly in CAM plants, where CO2 anthocyanin content (Fig. 5A) suggests that these molecules are supply to the mesophyll and Rubisco activity vary considerably involved in photoprotection as part of a short-term defense. This through a diurnal course (Borland et al., 2000). Thus, for B. karatas, would diminish the effect of light-derived reactions by preventing CAM and water availability confer enough antioxidant protection over-energization and over-reduction in the photosynthetic ARTICLE IN PRESS

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Fig. 3. Optical microscopy photograph (40 , ocular lens) of transverse leaf sections of exposed (A and C) and shaded (B and D) plants of T. brachycaulos (A–B) and B. karatas (C–D) during the dry season. ad: adaxial epidermis; ab: abaxial epidermis. Arrows indicate zones with high anthocyanins concentration.

electron transport and in this way protect the photosynthetic apparatus from the effects of photoxidative stress (Gould et al., 2000; Steyn et al., 2002). Although the red-pigmentation in the adaxial surface of exposed leaves of B. karatas also suggested high anthocyanin content, our results showed a low concentration of these pigments, coinciding with reports for other CAM species (Vogt et al., 1999; Close and Beadle, 2003; Merzlyak et al., 2005). For some plant species seemingly devoid of anthocyanins, it has been documented that strong sunlight or a combination of high PPF and water deficit induces a red-pigmentation of the leaves. This pigmentation has been attributed to an accumulation of carotenoids and other flavonoids in the epidermal cells of the plants, to provide protection against radiation in the UV and the shortwave part of the visible spectrum (e.g. in Aloe arborescens is a keto-carotenoid: rhodoxantin; Merzlyak et al., 2005). The low level of anthocyanins visualized under the epidermis of exposed plants of B. karatas during the dry season (Fig. 3B), suggests that these pigments have a limited photoprotective role in this species. However, although anthocyanin content in exposed plants of B. karatas during the dry season was significantly different from that in the rainy season, the linear regression showed that the red coloration was not related to high PPF (Fig. 4B), suggesting that the increase in anthocyanin content might be related to water availability (Fig. 5B; Steyn et al., 2002). The accumulation of red pigments as a response to a combination of high PPF and water deficit conditions appears to contribute to the tolerance of both bromeliads to prolonged dry periods, since the pigments can absorb a significant portion of light energy (Hatier and Gould, 2007). The data presented here confirms B. karatas as a CAM plant that Fig. 4. Total anthocyanins concentration for leaves of T. brachycaulos (A) and B. karatas (B) under two light conditions: exposed and shaded, during the rainy showed a significantly lower concentration of radiation-protecting and dry seasons. Data are means7SE (n=5); Po0.05. anthocyanins compared to T. brachycaulos, which produces these ARTICLE IN PRESS

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Fig. 5. Correlation between incident photosynthetic photon flux (PPF) and total anthocyanins for leaves of T. brachycaulos (A) and B. karatas (B) during the dry and rainy seasons. Open symbols: exposed plants; closed symbols: shaded plants; squares: dry season; circles: rainy season. Regression lines, determination coefficients and P values are indicated in the figures (significant at Po0.005). pigments during the dry season and which may confer enhanced 24588 (to JLA) and FOMIX-Yucatań no. 66262 (to LMPR). C. photoprotection. These results indicate that, in T. brachycaulos, Gonza´lez-Salvatierra was recipient of a PhD fellowship from activation of additional mechanisms of photoprotection, when Consejo Nacional de Ciencia y Tecnologıá, Me´xico (CONACYT- compared to those of the terrestrial bromeliad, is correlated with 172810). the epiphytic environment. However, both CAM species under high PPF and water deficit conditions may prevent oxidative damage by increasing their antioxidant content, as part of a defense mechanism References to survive and grow in this tropical dry deciduous forest.

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