Plant Physiology and Biochemistry 66 (2013) 56e62 Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy Accumulation of a bioactive triterpene saponin fraction of Quillaja brasiliensis leaves is associated with abiotic and biotic stresses Fernanda de Costa a, Anna Carolina Alves Yendo a, Juliane Deise Fleck b, Grace Gosmann c, Arthur Germano Fett-Neto a, d, * a Department of Botany, Graduate Program in Botany, Federal University of Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 9500, Porto Alegre 91501-970, RS, Brazil Graduate Program in Environmental Quality, Feevale University, RS-239 2755, Novo Hamburgo 93352-000, RS, Brazil Graduate Program in Pharmaceutical Sciences, Faculty of Pharmacy, UFRGS, Av. Ipiranga 2752, Porto Alegre 90610-000, RS, Brazil d Plant Physiology Laboratory, Graduate Program in Cell and Molecular Biology, Center for Biotechnology, UFRGS, Porto Alegre 91501-970, RS, Brazil b c a r t i c l e i n f o a b s t r a c t Article history: Received 2 January 2013 Accepted 7 February 2013 Available online 16 February 2013 The saponins from leaves of Quillaja brasiliensis, a native species from Southern Brazil, show structural and functional similarities to those of Quillaja saponaria barks, which are currently used as adjuvants in vaccine formulations. The accumulation patterns of an immunoadjuvant fraction of leaf triterpene saponins (QB-90) in response to stress factors were examined, aiming at understanding the regulation of accumulation of these metabolites. The content of QB-90 in leaf disks was significantly increased by application of different osmotic stress agents, such as sorbitol, sodium chloride and polyethylene glycol in isosmotic concentrations. Higher yields of bioactive saponins were also observed upon exposure to salicylic acid, jasmonic acid, ultrasound and UV-C light. Experiments with shoots indicated a significant increase in QB-90 yields with moderate increases in white light irradiance and by mechanical damage applied to leaves. The increased accumulation of these terpenes may be part of a defense response. The results herein described may contribute to further advance knowledge on the regulation of accumulation of bioactive saponins, and at defining strategies to improve yields of these useful metabolites. Ó 2013 Elsevier Masson SAS. All rights reserved. Keywords: Secondary metabolism Saponin Stress Quillaja brasiliensis 1. Introduction Quillaja brasiliensis (A. St.-Hill. & Tul.) Mart. (Quillajaceae) is a saponin-producing tree native of Southern Brazil [1], commonly known as soap tree, due to the capacity of its leaves and barks to Abbreviations: ABA, abscisic acid; DAB III, 10-deacetyl baccatin III; H2O2, hydrogen peroxide; HPLC, high pressure liquid chromatography; INMET, Instituto Nacional de Meteorologia (National Institute of Meteorology); INPE, Instituto Nacional de Pesquisas Espaciais (National Institute of Space Research); JA, jasmonic acid; MeJA, methyl jasmonate; MeSA, methyl salicylate; MS, Murashige and Skoog
salts; NaCl, sodium chloride; O
2 , superoxide anion; OH , hydroxyl radical; P.A.R., photosynthetic active radiation; PEG, polyethylene glycol; PS II, photosystem II; QB90, purified saponin fraction from Q. brasiliensis; R/FR, red/far red light ratio; RMS, root mean square; ROS, reactive oxygen species; SA, salicylic acid; Th, T-lymphocytes; US, ultrasound; UV, ultraviolet. * Corresponding author. Department of Botany, Graduate Program in Botany, Federal University of Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 9500, Porto Alegre 91501-970, RS, Brazil. Tel.: þ55 51 3308 7642; fax: þ55 51 3308 7309. E-mail addresses: fernandadecosta@yahoo.com.br (F. de Costa), anna.yendo@ (A.C.A. Yendo), julianefleck@yahoo.com.br (J.D. Fleck), yahoo.com.br grace.gosmann@ufrgs.br (G. Gosmann), fettneto@cbiot.ufrgs.br (A.G. Fett-Neto). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.02.003 produce persistent foam in water. The saponins of this species showed remarkable similarities to those of the barks of Quillaja saponaria Molina, a related Chilean species and one of the main sources of industrial saponins used as adjuvant in vaccine formulations. A purified saponin fraction from leaves of the Brazilian species, named QB-90, was able to stimulate both Th1 and Th2 immune response, as well as the production of cytotoxic T-lymphocytes against herpesvirus type 1 and 5 in mice, in a comparable manner to saponins from Q. saponaria (Quil-AÒ) [2,3]. Saponins are secondary metabolites widely distributed in plants, characterized by the presence of a skeleton derived from a 30-carbon 2,3-oxidosqualene precursor, to which one or more sugar residues are linked [4,5]. The complex structure and its higher variability are factors responsible for difficulties in isolating and synthesizing these molecules [6]. These obstacles, associated with the often low concentration of saponins in biomass, highlight the need for developing ways to increase their content in plants prior to extraction. Secondary metabolite accumulation is usually stimulated in response to environmental challenges mediated by biotic or abiotic stresses, such as high and low temperatures, drought, salinity, F. de Costa et al. / Plant Physiology and Biochemistry 66 (2013) 56e62 ultraviolet (UV) radiation or ozone stress, herbivory and pathogen infections. These responses are mediated by specific receptors and signaling molecules, such as jasmonic acid (JA) and its methyl ester e methyl jasmonate (MeJA), and salicylic acid (SA) and its methyl analog, methyl salicylate (MeSA), which have been shown to be take part in defense signaling against herbivore and pathogen attack [7]. Enhancement of saponin content upon jasmonate exposure can be observed in several species, including Glycyrrhiza glabra, Panax notoginseng and Centella asiatica [8e10]. Other elicitation strategies for secondary metabolites, such as different light and UV radiation treatments, have been reported in Perilla frutescens, Artemisia annua and Panax ginseng [11e13]. The effects of abiotic or biotic stresses on saponin production in Quillaja demand investigation. The commercial use of Q. saponaria bark saponins has been causing important damage to Chilean forests, with 28 thousand 30e50 year old trees being felled per year [14]. Considering that leaves of the Brazilian species have bioactive saponins of comparable function and that these organs represent a more readily sustainable source of metabolites for pharmaceutical applications, investigation on the accumulation patterns of a purified immunoadjuvant fraction (QB-90) in response to biotic and abiotic stress factors was carried out. For this purpose, leaf disks from Q. brasiliensis were exposed to: increased osmotic pressure with different osmotic stress agents, JA and SA, ultrasound (US) treatment, UV-B and UV-C radiation. In addition, Q. brasiliensis whole plants were exposed to different intensities of light and leaves were mechanically wounded to simulate herbivore attack. This investigation aimed both at better understanding the regulation of Q. brasiliensis bioactive saponin biosynthesis and at identifying possible strategies to improve the yields of these useful metabolites. 2. Results and discussion To check for the viability of leaf disks as an experimental system, chlorophyll contents were determined, since reduction of these pigments is one of the earliest indications of senescence [15,16]. Disks kept in Murashige and Skoog (MS) [17] (0.1) salts solution did not show a significant loss of chlorophyll a and b during the first four days of incubation (data not shown). Since a significant increase of senescence was not seen within the experimental period, it was possible to test different kinds of stress using the leaf disc system. 2.1. Osmotic stress treatment In preliminary experiments, the osmotic agent sorbitol was applied in different concentrations (30 mM; 100 mM and 300 mM) in Q. brasiliensis leaf disks to evaluate the impact on QB-90 content. A significant increase in saponins was observed in the fourth day of exposure to sorbitol 300 mM compared to the control (only MS) (data not shown). The treatment yielding the largest increase in saponin amount (sorbitol 300 mM) was chosen to conduct further experiments, comparing different osmotic agents. Three osmotic stress agents, sodium chloride (NaCl) 150 mM, polyethylene glycol 6000 (PEG) 35 mM and sorbitol 300 mM were tested for potential effects on QB-90 accumulation, all at isosmotic concentrations. NaCl and sorbitol are usually internalized by the cells, interfering with osmotic potential, although sorbitol often cannot be metabolized. PEG is not absorbed, acting as a typical water stress simulator molecule [18,19]. The treatments sorbitol 300 mM and PEG 35 mM stimulated QB90 biosynthesis in leaf disks of Q. brasiliensis as of the second day of exposure (Fig. 1). The highest saponin content was reached after four days of exposure, increasing more than 3-fold. This increase 57 Fig. 1. QB-90 content in leaf disks of Q. brasiliensis after 2 and 4 days of exposure to control (0.1 MS) and treatments with NaCl 150 mM, sorbitol 300 mM and PEG 35 mM. Different letters indicate significant difference by a Duncan test (P  0.05). Bars represent the means  standard error. can be explained as part of plant cell adaptation, which can alter metabolism and trigger various defense mechanisms [20] to tolerate water deficit or excessive salinity, leading to the induction of a set of genes whose products are involved in plant response to stress. The activation of genes encoding for enzymes involved in saponin biosynthesis may be part of these mechanisms. Osmotic stress can stimulate the production of valuable secondary metabolites in plants. NaCl increased accumulation of the alkaloid ajmalicin in Catharanthus roseus [21] and total alkaloids in Hyoscyamus cell cultures [22]. In Taxus chinensis, paclitaxel production was enhanced 2-fold in cells treated with sorbitol and 3fold in those treated with PEG 4000, compared to that of cells treated only with sucrose [19]. Similar result has been reported for saponins in P. ginseng, with enhancement of 31% in saponin yield by addition of sorbitol 300 mM and growth nutrients [23]. The hormone abscisic acid (ABA) increases as a result of water stress and plays important roles in regulating plant stress responses, participating in the expression of defense genes and control of secondary metabolite biosynthesis [20,24]. In line with this relationship, both osmotic stress and ABA can stimulate production of indole alkaloids in C. roseus cell culture [25]. Drought is known to inhibit photosynthetic activity due to the imbalance between light capture and its utilization. Excess light energy dissipation in PSII generates ROS (O2 , , H2O2, OH
) [26]. Mitigation of ROS-induced damage can result from enzymatic and nonenzymatic antioxidant defenses; the latter may include secondary metabolite accumulation. ROS generation is also a resource used by plant cells in defense responses to pathogen attack and treatment with elicitors. It is possible that ROS induce the accumulation of secondary metabolites by triggering the expression of various defense genes related to their biosynthesis [27]. The increase of QB-90 content induced by osmotic agent treatments could reflect a role for these metabolites in moderate water stress defense responses. This result corroborates a seasonal study conducted with leaves harvested from thirty different Q. brasiliensis adult trees grown in the field during the course of two years. The overall average variation in saponin content between seasons suggests that higher precipitation periods, such as spring, were generally associated with lower QB-90 content, whereas moderately dryer seasons, such as winter and fall, yielded the highest saponin content (Table 1). However, caution must be exercised in interpreting these data, since other factors may be simultaneously at play under field conditions, including temperature, rain distribution and irradiance. 58 F. de Costa et al. / Plant Physiology and Biochemistry 66 (2013) 56e62 Table 1 Relation between climate status (average precipitation and temperature) at the harvesting period and QB-90 yield in Q. brasiliensis leaves (Canguçu, RS, Brazil) (average of two years, 2004e2005). Monthly average Average QB-90 temperature (% dry weight) precipitation ( C) (mm) Season Months Winter Spring Summer Fall June, July 113.2 September, October 189.7 December, January 62 March, April 102.1 13.6 14.6 20.9 19.8 0.13 0.02 0.03 0.06 2.2. US stress treatment US treatment produced positive effects on QB-90 saponin accumulation at both US exposure times (1 and 2 min) compared to the respective control (Fig. 2). QB-90 content was raised by up to 2fold on day 2. US exposure triggers events that are characteristic of plant defense against pathogens, such as ROS production (oxidative burst), membrane Ca2þ influx and accumulation of defense compounds, including various secondary metabolites [28,29]. Low energy US for short periods has been a successful strategy for enhancing biosynthesis of secondary products, e.g., ginsenoside saponins of P. ginseng [30], shikonin of Lithospermum erythrorhizon [28], taxol in T. chinensis [31] and Taxus yunnanensis [29], and caffeic acid derivatives in Echinacea purpurea [32]. Our results extend the US induction of secondary metabolites to Q. brasiliensis immunoadjuvant saponins and support a potential pathogen defense role for these metabolites in planta. 2.3. UV stress treatments Although UV-C is completely absorbed by atmospheric gases, when artificially provided this highly energetic radiation (200e 280 nm) can strongly affect cellular functioning and DNA structure. UV-B (280e320 nm) is partially absorbed by atmospheric ozone, but even modest increases may lead to important biological damages [33,34]. Besides the production of antioxidant enzymes to scavenge free radicals like ROS, one of the most common protective responses is the accumulation of secondary metabolites, including terpenoids, which can absorb radiation in the ultraviolet wavelength range and scavenge some of these free-radicals [35]. Fig. 2. QB-90 content in leaf disks of Q. brasiliensis submitted to ultrasound exposure. Control (untreated disks kept in 0.1 MS), US treatments applied as a single initial exposure for different lengths of time (1 and 2 min) on disks incubated in the same medium. QB-90 yield is shown for 2 and 4 days after treatment application. Different letters indicate significant difference by a Duncan test (P  0.05). The bars represent the means  standard error. A significant increase of QB-90 with exposure to UV-C on day 2 could be observed (Fig. 3A). On day 4, there was a major decrease in QB-90 content that can be explained by a possible impairment or de-regulation of saponin metabolism, increasing catabolism. This rapid effect corroborates previous results of our group that showed significant accumulation of this fraction of saponins in whole-plants exposed to UV-C radiation on the second day of treatment (data not shown). Higher production of terpenes upon exposure to such radiation has been shown, e.g., 10-deacetyl baccatin III (DAB III) and paclitaxel in Taxus baccata [36] and artemisinin in A. annua [37]. On the other hand, QB-90 content was not affected by exposure to UV-B radiation during the same period of experiment (Fig. 3B). These results can be explained on the basis of the relatively less damaging effects of UV-B compared with UV-C, since UV-C photons are highly energetic, so that intense transduction and high levels of damage can be quickly generated [38]. It is possible that exposure for longer periods of time to UV-B radiation could have promoted QB-90 accumulation, as was shown for the monoterpene-indole alkaloid brachycerine of Psychotria brachyceras [39]. 2.4. SA stress treatment A significant increase in QB-90 content was obtained when leaf disks were submitted to 1 mM SA treatment on the second and fourth day of exposure (Fig. 4), reaching a 3-fold increase in the content of this metabolite on day 2. SA plays an important role in plant growth, development and in responses against pathogen attack or wounding [40]. SA accumulates locally at the site of infection and then it spreads to other parts of the plant, mostly as MeSA, inducing a range of defense responses, including the biosynthesis of secondary metabolites [27]. SA has been shown to elicit the accumulation of triterpenic saponins (ginsenosides) [41] and phenolic compounds [42] in suspension culture of P. ginseng, the alkaloid pilocarpine in jaborandi leaves [43], anthraquinones in callus cultures of Rubia cordifolia [44] and tropane alkaloids in hairy root cultures of Brugmansia candida [45]. 2.5. JA and mechanical damage treatment Q. brasiliensis leaf disks showed a significant increase in QB-90 content after 2 days of treatment with jasmonic acid 40 mM, which was about 2.6-fold that of control disks (Fig. 4). After that, contents dropped abruptly, reaching the same initial values of control disks. These results corroborate previous studies, in which whole plants of Q. brasiliensis treated with jasmonic acid at 400 mM had a very similar response profile (data not shown). The simulation of herbivory by applying mechanical damage to whole plant leaves led to a response profile similar to that of JA exposure, with a significant enhancement of QB-90 accumulation after 2 days from the beginning of experiment, higher than 1.5 times, followed by a decrease in content (Fig. 5). This data further support a role of JA in QB-90 accumulation. Reductions in saponin contents at later times may reflect the cessation of damage stimulation in leaves or metabolization of elicitor molecules by leaf disks. Jasmonic acid and its derivatives, such as methyl jasmonate, are key signaling molecules widely used to promote a wide range of secondary metabolites accumulation, including terpenoids, flavonoids and alkaloids, among others [27]. Numerous studies have reported activation of saponin metabolism with exogenously applied MeJA [5]. These results further support the putative role of saponins as protection metabolites against herbivores. This role is based on their action as deterrents, toxins and digestibility inhibitors [46,47]. In fact, studies report that saponins are involved in plant-phytophagous insect chemical interactions [48], such as the F. de Costa et al. / Plant Physiology and Biochemistry 66 (2013) 56e62 59 Fig. 3. QB-90 content in leaf disks of Q. brasiliensis after 2 and 4 days of exposure to white light (control) and treatments supplemented with UV-C (A) or UV-B light (B). Different letters indicate significant difference by a Duncan test (P  0.05). Bars represent the means  standard error. positive regulation of saponin accumulation in Medicago sativa L. during plant attack by the piercing-sucking aphid Acyrthosiphon pisum [49] and the generalist defoliator Egyptian cotton leafworm, Spodoptera littoralis [50]. MeJA elicitation strategy is used to enhance terpenoid accumulation such as saponins of C. asiatica, P. ginseng and G. glabra [41,51,52]. MeJA may act by regulating more than one point in the terpene biosynthetic pathway, such as the activity of glicosyltransferases in P. notoginseng, as well as of squalene synthase, squalene epoxydase and dammaranediol synthase in P. ginseng [9,53], whereas in Taxus species it would regulate geranylgeranyl diphosphate synthase and taxadiene synthase [54,55]. 2.6. Light intensity experiment Plants submitted to higher irradiance accumulated significantly more QB-90 saponins compared to control (Fig. 6). A significant decrease in saponins under lower irradiance light treatment in comparison to control could also be observed. Clearly, the higher irradiances used in these experiments were far lower than the usual sunlight irradiance of clear sky days, which are probably between 10 and 15 times higher than the ones tested herein. To analyze the role of primary metabolism on QB-90 content responses to light intensity, the concentrations of total soluble sugars were evaluated under the same treatments (data not shown). Sugar concentrations were not significantly changed by the light treatments. Therefore, the changes observed in QB-90 are likely the result of genuine effects of light on secondary metabolism Fig. 4. QB-90 content in leaf disks of Q. brasiliensis after 2 and 4 days of exposure to control conditions or to treatment with JA 40 mM or SA 1 mM treatment. Different letters indicate significant difference by a Duncan test (P  0.05). Bars represent the means  standard error. and not an indirect effect caused mainly by carbon metabolism modifications. Plants have acquired very sophisticated photoreceptors to monitor the status of various aspects of light: intensity, quality, duration and direction [56]. The stimulatory effects of light on the formation of secondary metabolites have been reported for flavones and flavonols of Petroselinum hortense [57]; anthocyanins of P. frutescens [11]; artemisinin, an anti-malarial compound isolated from A. annua [12], vindoline and other indole alkaloids of C. roseus [58]. In Grindelia chiloensis, diterpene resin acids were highly decreased in leaves with low light intensity (50% and 25% radiation condition) [59]. However, the influence of light on saponin biosynthesis is still poorly studied. Shimoyamada and Okubo [60] analyzed the effect of light irradiation on saponin content of soybean seeds. The content of soyasaponins I, II and V in germinating seeds increased due to light exposure (photoperiod of 12 h light and 12 h dark) compared with seeds germinated in darkness. In daylight, the proportion R/FR light is higher, and, in twilight, a significant drop in R/FR occurs, a situation also found in shaded places. In Q. brasiliensis, higher light intensity increased QB-90 content, whereas lower intensity light decreased it, suggesting the involvement of phytochrome in this response. Even though significant changes in soluble carbohydrate concentration were not detected among treatments, light stimulation of QB-90 production seems to be in agreement with the Carbon-Nutrient Balance Hypothesis, which predicts a reduction in allocation to terpenoids and other carbon-based secondary metabolites when carbon gain is limited relative to nutrient and light availability, as occurs under low-light conditions [61]. Further support for these results can be found in the recommended silvicultural practices for Q. brasiliensis, which classify the species as heliophile [62]. Fig. 5. Influence of mechanical damage treatment on QB-90 content of Q. brasiliensis shoots on days 2, 4 and 6 after damage. Different letters indicate significant difference by a Duncan test (P  0.05). The bars represent the means  standard error. 60 F. de Costa et al. / Plant Physiology and Biochemistry 66 (2013) 56e62 121  C for 20 min. The leaf disks were incubated in 20 mL of 0.1 MS solution with the different stress agents in Petri dishes (20 disks/dish). Control samples were maintained only with 20 mL of 0.1 MS salts. pH was adjusted to 6e7. The test was carried out in a growth room under white fluorescent tubes at 25  3  C in a 16/8 h day/night cycle [photosynthetic active radiation e P.A.R. e of approximately 45 mmol m2 s1]. Disks were collected after 2 and 4 days of incubation. After harvest, each sample was frozen in liquid nitrogen separately, and stored at 80  C until saponin extraction (item 3.3). Fig. 6. Influence of light intensity on QB-90 content of Q. brasiliensis shoots on day 30. Different letters indicate significant difference by a Duncan test (P  0.05). The bars represent the means  standard error. 2.7. Conclusion Saponin content in Q. brasiliensis proved to be a dynamic feature, modulated by abiotic external conditions, such as irradiance and water potential, and by biotic stress factors, such as herbivory and pathogen attack, mimicked by the signaling molecules JA and SA, mechanical wounding and ultrasound application. These results support a general induced defense role for these metabolites in planta. The application of mild stress treatments is apparently a useful approach to increase the flux of carbon through secondary pathways leading to saponin metabolites of interest. The results herein described may contribute to the industrial use of leaves of Q. brasiliensis as alternative to the bark of the Chilean species, affording an increase in the content of bioactive saponins. 3. Materials and methods 3.1. Plant material and foliar disk preparation Q. brasiliensis tip cuttings collected from adult plants growing at the City Botanical Garden, Porto Alegre, RS, Brazil (30 0301700 Se 51100 3600 W) were used in the experiments. Climatic data was obtained from the Instituto Nacional de Pesquisas Espaciais (INPE) and Instituto Nacional de Meteorologia (INMET) at Porto Alegre e RS, Brazil. A voucher specimen is deposited at the UFRGS University Herbarium (ICN 159894). Healthy and expanded leaves from tip cuttings were detached and surface sterilized. Sterilization was done by washing leaves for 1 min in 70% ethanol, followed by immersion for 15 min in 1.5% sodium hypochlorite with a few drops of neutral detergent. All sterilization steps were followed by four washes in sterile distilled water. Foliar disks with 1 cm of diameter were prepared with a cork borer. The foliar disks were mixed, in order to evenly distribute the variability of biological replicates, and then distributed in the various treatments. Treatments of all experiments were performed in quadruplicates (20 disks per replicate) and each experiment was independently repeated twice. 3.1.1. Osmotic stress experiments The incubations in presence of three osmotic stress agents in isosmotic concentrations, NaCl 150 mM, PEG 6000 35 mM and Dsorbitol 300 mM, were individually tested for effect on QB-90 biosynthesis. All of the solutions were previously autoclaved at 3.1.2. US stress treatments Leaf disks received a single initial application of US stress for short periods of time (1 and 2 min) in a US bath (40 kHz fixed frequency and 135 W RMS potency). Disks were exposed in batches of 80 in 50 mL of 0.1  MS solution. After treatment, they were incubated in 20 mL of previously autoclaved 0.1 MS solution in Petri dishes (20 disks/dish). pH adjustment, incubation conditions and harvest procedures were as described (item 3.1.1). 3.1.3. UV stress treatments Leaf disks were placed in Petri dishes containing filter paper (20 disks/dish) and each dish received 20 mL of 0.1 MS solution. For UV-B stress experiments, disks were treated with UV-B radiation of 47.14 kJ m2 d1 biologically effective radiation [63]. A glass plate was placed above control disks to filter out UV-B and most UV-A wavelengths. For UV-C stress experiments, disks were placed in a closed chamber divided in two compartments, one set of disks were exposed to UVC light (germicide lamp, 10.5 kJ cm2) and the other, exposed to white light only (control). pH adjustment, other incubation conditions and harvest procedures were as described (item 3.1.1). 3.1.4. SA stress treatments Leaf disks were incubated in 20 mL of 0.1 MS solutions supplemented with salicylic acid 1 mM previously autoclaved at 121  C for 20 min. Control samples were maintained in 20 mL of 0.1 MS salts only. pH adjustment, incubation conditions and harvest procedures were as described (item 3.1.1). 3.1.5. JA stress treatments Leaf disks were kept in Petri dishes (20 disks/dish) in 20 mL of previously autoclaved 0.1 MS solution. Jasmonic acid was added to the MS solution, resulting in a 40 mM final concentration. Control samples were maintained with 20 mL of 0.1 MS salts only. pH adjustment, incubation conditions and harvest procedures were as described (item 3.1.1). 3.2. Plant material and seed germination Seeds from Q. brasiliensis were collected from adult plants growing in the towns of Riozinho e RS and Canguçu e RS, Brazil (29 380 2800 Se50 270 0900 W and 31230 4200 Se52 400 3200 W, respectively). A voucher specimen is deposited at the University Herbarium (ICN 137550 and 142953 respectively). Seeds were stored at 10  2  C for 24 h before the start of the experiment. Surface sterilization was done by stirring seeds in a sequence of solutions, as follows: 20 min in DithaneÒ (antifungal) 5 g/L, 1 min in 70% ethanol, followed by immersion in 2% sodium hypochlorite with a few drops of neutral detergent for 15 min. Then, seeds were washed four times with sterile distilled water. After sterilization, seeds were germinated in vitro in 0.1 MS salts supplemented with 6 g/L agar. After one month, germinated plants were transferred to plastic pots (500 mL) containing previously autoclaved (121  C for 50 min) vermiculite and commercial sand (2/1, w/w), and received F. de Costa et al. / Plant Physiology and Biochemistry 66 (2013) 56e62 61 water and nutrient solution (0.5X MS salts) twice a week. The pots were maintained in a growth room under white fluorescent light at 28  2  C in a 16/8 h day/night cycle (P.A.R. of approximately 45 mmol m2 s1). by ANOVA followed by Duncan, P  0.05, using the statistic package SPSS 15.0. 3.2.1. Mechanical damage experiments Shoots with approximately 6 months were used in the experiments. Shoots had half of their leaves (3e4 leaves) wounded with scissors. Control samples were kept intact. Experiments were carried out in a growth room under white fluorescent light at 28  2  C in a 16/8 h day/night cycle (P.A.R. of approximately 45 mmol m2 s1). Two, 4 and 6 days after damage, samples were harvested in liquid nitrogen and stored at 80  C until analysis of QB-90 (item 3.3). All treatments were performed in quadruplicates (one shoot per sample) and the experiment was independently repeated twice. Authors would like to thank Prof. Gilson R.P. Moreira (Zoology Department, UFRGS, Brazil); Dr. Andréia Carneiro and Ari Nilson (Zoobotânica Foundation, Porto Alegre, Brazil) for assistance in locating, collecting, and identifying the plant material. This research was supported by CNPq, CAPES and FAPERGS (Brazil). 3.2.2. Light experiment Shoots with approximately 6 months were used in the experiments. Higher irradiance treatment was achieved by raising the pots closer to the fluorescent tubes (resulting in P.A.R. of 140 mmol m2 s1) and lower irradiance treatment was achieved using a black net between lamps and plants (resulting P.A.R. of 15 mmol m2 s1), taking care not to affect temperature conditions. Control plants were irradiated with white fluorescent tubes (P.A.R 45 mmol m2 s1). Treatments were applied for 30 days in a 16/8 h day/night cycle. Then, shoots were collected, washed in distilled water, frozen in liquid nitrogen, and stored at 80  C until analysis of QB-90 (item 3.3). All treatments were performed in quadruplicates (one shoot per sample) and the experiment was independently repeated twice. 3.3. Determination of QB-90 content In order to quantify the QB-90 content, samples were ground with mortar and pestle in liquid nitrogen. Then, samples were macerated with distilled water in 1/40 proportion (w/v) for 3 min, followed by ultra-sonication for 40 min. The extracts obtained were filtered; lyophilized and chemical analyses were performed using a Thermo Scientific HPLC system with a method validated according to Fleck and coworkers [64] with minor modifications. Chromatography was performed on a Waters Spherisorb C8 HPLC reverse phase column 150  4.6 mm, with corresponding guard column, using an isocratic system with acetonitrile/water/trifluoroacetic acid (65/35/0.05, v/v/v), previously filtered through Millipore membranes (0.45 mm) and degassed. Flow rate of the mobile phase was 0.8 mL/min and detection was at 214 nm. Solvents acetonitrile (Merck) and trifluoroacetic acid (Sigma) were HPLC grade, whereas all other chemicals and solvents were of analytical grade. To quantify QB-90, 20 mL of samples were injected and an external standard curve was generated using standard QB-90 purified and isolated from leaves [2]. The identification of fraction QB-90 from each sample was done by analyzing the retention time and cochromatography with QB-90 standard. The contents of QB-90 in samples were expressed as percentage of extracted dry weight. Total soluble sugars were extracted from leaves in 80% ethanol, treated with phenol-sulfuric reagent, and analyzed in a microplate spectrophotometer at 490 nm, as previously described [65]. 3.4. Experimental layout and statistics All assays herein described were performed in totally randomized layout, with biological quadruplicates and each assay was independently repeated at least twice. The results were analyzed Acknowledgments References [1] R. Reitz, A. Reis, R.M. 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