Responses of the macroalgae Hypnea musciformis after in vitro exposure to UV-B

Aquatic Botany 100 (2012) 8–17

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Responses of the macroalgae Hypnea musciformis after in vitro exposure to UV-B Éder C. Schmidt a,∗,1 , Beatriz Pereira b,1 , Rodrigo W. dos Santos c , Claudiane Gouveia c , Giulia Burle Costa c , Gabriel S.M. Faria c , Fernando Scherner d , Paulo A. Horta d , Roberta de Paula Martins e , Alexandra Latini e , Fernanda Ramlov f , Marcelo Maraschin f , Zenilda L. Bouzon g a Post-Graduate Program in Cell Biology and Development, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil b Scientific Initiative PIBIC-CNPq, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil c Macroalgae Laboratory, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil d Department of Botany, Federal University of Santa Catarina 88010-970, CP 476, Florianópolis, SC, Brazil e Laboratório de Bioenergética e Estresse Oxidativo, Department of Biochemistry, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil f Plant Morphogenesis and Biochemistry Laboratory, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil g Central Laboratory of Electron Microscopy, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil

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Article history: Received 3 October 2011 Received in revised form 3 March 2012 Accepted 8 March 2012 Available online 16 March 2012 Keywords: Ultraviolet radiation-B Hypnea musciformis Ultrastructure Photosynthetic performance Carotenoids Phenolic compounds Carrageenan yield Mitochondrial activity

a b s t r a c t The in vitro effects of UVBR were investigated in apical segments of Hypnea musciformis. The plants were cultivated and exposed to photosynthetically active radiation (PAR) at 80 ␮mol photons m−2 s−1 and PAR + UVBR at 1.6 W m−2 at 3 h per day for 7 days. Toluidine Blue reaction showed metachromatic granulations in vacuole, and Periodic Acid Schiff stain showed a decrease in the number of floridean starch grains. UVBR also caused changes in the ultrastructure of cortical cells, which included increased thickness of the cell wall, reduced intracellular spaces, changes in the cell contour, destruction of chloroplast internal organization, and rough endoplasmic reticula increase. The algae cultivated under PAR-only showed growth rates of 9.7% day−1 , while algae exposed to PAR + UVBR grew only 3.2% day−1 . Furthermore, compared with control algae, phycobiliprotein contents (phycoerythrin, phycocyanin, and allophycocyanin) were observed to decrease after PAR + UVBR. However, chlorophyll a levels were not significantly different (ANOVA, P = 0.52) after exposure to PAR + UVBR. As a photoprotective adaptation strategy against UVB damage, an increase of 58.9% phenolic compounds and 3.6% of carotenoids was observed. Overall, these results lead to the conclusion that both ultrastructural damage and observable changes in metabolism occurred in H. musciformis after only 3 h of daily UVB exposure over a 7-d experimental period. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The stratospheric ozone layer provides natural protection against ultraviolet radiation (UVR) exposure for all biological organisms (Madronich, 1992). It has been nearly three decades since the first reports about man-made changes in this protective barrier, which resulted from atmospheric pollutants, such as chlorofluorocarbons (CFCs), halocarbons, carbon dioxide (CO2 ), and methyl chloroform (MCF). As a consequence of ozone layer depletion, ultraviolet B radiation (UVBR) (280–320 nm) is increasingly reaching the earth’s surface (Mitchell et al., 1992; Hanelt and Roleda, 2009). UV energy induces photodamage in proteins, nucleic acids, and other compounds in biological tissues (Mitchell et al., 1992), as well as damage to cellular ultrastructure (Schmidt

∗ Corresponding author. Tel.: +55 48 3721 5149. E-mail address: [email protected] (É.C. Schmidt). 1 Eder C. Schmidt and Beatriz Pereira should be considered as first authors. 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2012.03.004

et al., 2009). Ultraviolet radiation affects all biological organisms, especially those in the aquatic ecosystem, provoking, for example, changes in macroalgae growth rates (Schmidt et al., 2009, 2010a,b). One of the strategies used by macroalgae to survive when exposed to high levels of UVR is the synthesis and accumulation of photoprotective compounds, such as mycosporine-like amino acids (MAAs) and carotenoids, which directly or indirectly absorb UVR energy (Karsten and Wiencke, 1999). The phenolic compounds are also involved in protecting the thallus against direct exposure to solar light radiation, especially UVR, as observed in the brown alga Ascophyllum nodosum (Pavia et al., 1997). Several studies suggest that changes have occurred in the concentrations of chlorophyll a in Mastocarpus stellatus and Chondrus crispus (Roleda et al., 2004), as well as Kappaphycus alvarezii (Doty) Doty ex P. Silva (Schmidt et al., 2010a,b). Phycobiliprotein content has also been altered, as demonstrated in studies by Eswaran et al. (2001) and Schmidt et al. (2010a,b) reporting on K. alvarezii. Changes in the ultrastructure of macroalgae exposed to UVBR have been reported in many studies. Some papers reported changes

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in carragenophytes subjected to UVBR, such as K. alvarezii (Schmidt et al., 2009, 2010a,b). These changes mainly occur in the chloroplasts, modifying the quantity, size, organization, as well as the number of thylakoids (Schmidt et al., 2009). Hypnea is a source of kappa carrageenan and phycocolloids throughout the world, presenting significant economic importance (Reis et al., 2008). Among the many macroalgae found in the coastal systems, Hypnea J.V. Lamouroux is the biomarker of most probable consequence, owing to its worldwide distribution in the Atlantic, Indian and Pacific Oceans. Hypnea musciformis (Wulfen) J.V. Lamouroux is the best-known species in the genus Hypnea and has been reported to occur in many tropical and subtropical shores. This alga is known as a valuable resource for the production of kappa carrageenan (Reis et al., 2008). In Brazil, it is widespread along the Brazilian coast. Despite its importance to ecology and the economy, the red macroalga H. musciformis has not been studied in the context of UVBR exposure. Thus, in this study, we investigated the in vitro effect of UVBR on this species, and we raise the following questions: (I) Do changes in cellular architecture and ultrastructure between the PAR-only (control samples) and PAR UVBR (treated plants) relate to H. musciformis UVRB sensitivity? (II) Is there a difference in the content of photosynthetic pigments, carotenoids and phenolic compounds and mitochondrial function after exposure to ultraviolet radiation-B?

2. Materials and methods 2.1. Algal material H. musciformis samples were collected from Ponta das Canas Beach (27◦ 23 34 S and 48◦ 26 11 W) in February 2010 during the summer season. This species occurs in rocky intertidal beaches and is frequently epiphytic in Sargassum cymosum. The algal samples were collected from the rocks and transported at ambient temperature in dark containers to LAMAR-UFSC (Macroalgae Laboratory, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil). At noon on sunny days during the summertime, this region receives natural solar irradiation varying from 2.2 W m−2 to 3.5 W m−2 based on a daily UVB index that varies from 9 to 14 during a typical summer season. To avoid contamination by the presence of epiphytes, the collected algae were meticulously cleaned with a brush and filtered seawater. The apical portions were maintained by immersing in seawater enriched with von Stosch medium. These segments were cultivated under the same laboratory conditions (detailed below) during 14 days (experimental acclimation period) before their utilization in the UVBR experiments.

2.2. Culture conditions The apical thalli portions were selected (±1.0 g) from the H. musciformis samples and cultivated for 7 days in beakers with 500 mL natural sterilized seawater enriched with von Stosch medium at half strength (VSES/2) with ±34 practical salinity units (p.s.u.). Culture room conditions were 24 ◦ C, continuous aeration, illumination from above with fluorescent lights (Philips C-5 Super 84 16 W/840, Brazil), photosynthetically active radiation (PAR) at 60 ␮mol photons m−2 s−1 (Li-cor light meter 250,USA) and 12 h photocycle (starting at 8 h). UVBR was provided through a Vilber Lourmat lamp (VL-6LM, Marne La Vallée, France) with peak output at 312 nm. The intensity of UVB radiation was 1.6 W m−2 (Radiometer Model IL 1400A, International Light, Newburyport, MA, USA), and plants were exposed to PAR + UVBR from 12:30 to 15:30. To

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avoid exposure to UVC radiation, a cellulose diacetate foil having a thickness of 0.075 mm was utilized. Apical thalli controls were evaluated using PAR-only, while exposed apical thalli were cultivated under PAR + UVBR. Samples for light and electron microscopy were fixed directly on day 7, the last day of experimentation, after the final exposure to UVB at 15:30 h. Twelve replicates were made for each experimental group. 2.3. Light microscope (LM) Samples approximately 5 mm in length were fixed in 2.5% paraformaldehyde in 0.1 M (pH 7.2) phosphate buffer overnight. Subsequently, the samples were dehydrated in increasing series of ethanol aqueous solutions. After dehydration, the samples were infiltrated with Historesin (Leica Historesin, Heidelberg, Germany). Sections of 5 ␮m in length were stained with different cytochemical techniques and investigated with an Epifluorescent (Olympus BX 41) microscope equipped with Image Q Capture Pro 5.1 Software (Qimaging Corporation, Austin, TX, USA). 2.4. Cytochemical staining LM sections were stained as follows: Periodic Acid-Schiff (PAS) used to identify neutral polysaccharides (Schmidt et al., 2009), Toluidine Blue (TB-O) 0.5%, pH 3.0 (Merck Darmstadt, Germany) used for acid polysaccharides through a metachromatic reaction (Schmidt et al., 2009), and Coomassie Brilliant Blue (CBB) 0.02% in Clarke’s solution (Serva, Heidelberg, Germany) used for proteins (Schmidt et al., 2009,2010c). Controls consisted of applying solutions to sections without the staining component (e.g., omission of periodic acid application in the PAS reaction). In order to reveal the floridean starch grains of polysaccharides, ultra-thin sections were treated with periodic acid and thiosemicarbazide silver proteinate (PA-TSC-SP) 1% (Electron Microscopy Sciences, Hatfield, PA, USA) according to Schmidt et al. (2009). 2.5. Confocal laser scanning microscopy (CLSM) Algae samples were investigated by a laser scanning confocal microscope (Leica TCS SP-5, Wetzlar, Germany) and an Argon laser using 440, 488 and 514 nm excitation. A Leica HCX PLAPO lambda 63×/1.4–0.6 oil immersion objective was fitted on the inverted fluorescent microscope. The autofluorescence of the chlorophyll was used for visualization of the chloroplast structure. The LAS-AF Lite program (Leica) was also used for final processing of the confocal images. 2.6. Transmission electron microscope (TEM) For observation under the transmission electron microscope (TEM), samples approximately 5 mm in length were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) plus 0.2 M sucrose overnight. The material was post-fixed with 1% osmium tetroxide for four hours, dehydrated in a graded acetone series and embedded in Spurr’s resin. Thin sections were stained with aqueous uranyl acetate followed by lead citrate. Four replicates were made for each experimental group; two samples per replication were then examined under TEM JEM 1011 (JEOL Ltd., Tokyo, Japan, at 80 kV). Similarities based on the comparison of individual treatments with replicates suggested that the ultrastructural analyses were reliable. 2.7. Growth rates (GRs) Growth rates for treatment groups and control were calculated using the following equation: GR [% day−1 ] = [(Wt /Wi ) − 1] × 100/t,

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where Wi = initial wet mass, Wt = wet mass after 7 days and t = internal time in days (Penniman et al., 1986).

2.8. Carrageenan yield Samples of PAR-only and PAR + UVBR (1.0 g fresh mass, n = 3) of H. musciformis were digested in distilled water and 6% of potassium hydroxide for 4 h at 80 ◦ C according to Hayashi et al. (2007). The digestion product was filtered under low pressure and precipitated in 0.2% of potassium chloride. The carrageenan fibers were recovered and oven-dried at 60 ◦ C during 24 h. The carrageenan yield of H. musciformis was expressed as the percentage of carrageenan from a sample of algae dry mass.

2.9. Photosynthetic performance Experiments were followed by measurements of chlorophyll fluorescence using a pulse amplitude-modulated (PAM) fluorometer (Diving-PAM underwater fluorometer; Walz, Effeltrich, Germany). The measurements were obtained through the application of a series of eight exposures to gradually increasing actinic irradiance levels using the “Rapid Light Curve” (RLC) option of the Diving-PAM. The RLC technique is a useful application for the rapid investigation of the photosynthetic apparatus and provides information on the overall photosynthetic performance of seaweeds (White and Critchley, 1999). PAM optimal configurations were previously evaluated for H. musciformis under in situ conditions, and once defined, they were kept constant (Gain = 4; Measuring intensity = 6; Saturating pulse length = 0.8 s). From each sample, a relative electron transport rate (rETR) was determined for each exposure, resulting in a rETR curve for every replicate. Since electrons leading to CO2 reduction in dark reactions of photosynthesis are derived from the splitting of water in photosystem II, ETR may be estimated from the effective quantum yield. Thus, ETR = F/Fm  × PAR × 0.5 × 0.84, where PAR is the actinic irradiance in ␮mol photons m−2 s−1 , making the assumptions that photosystem II absorbs half (0.5) of the quanta of available light and that 0.84 is an ETR-factor based on the average of light which is actually absorbed by seaweeds (Diving-PAM Underwater Fluorometer Handbook of Operation, HeinsWalz GmbH 1998). To compare RLCs using parametric statistics, two descriptive parameters were used: photosynthetic efficiency (˛) and maximum photosynthetic rate (Pmax ). These parameters were calculated by the equation of Platt et al. (1980) with the Microcal Origin 5.0 program, using rETR values obtained for each replicate. Pmax was calculated by curve fitting, using all the RLC values, while ˛ was obtained by linear fitting, using the first three points of the rETR vs. irradiance curve (Yokoya et al., 2007).

2.10. Pigments analysis The content of photosynthetic pigments (chlorophyll a and phycobiliproteins) of H. musciformis was analyzed between treatment group and control. Samples (fresh weight) were frozen by immersion in liquid nitrogen and kept at −40 ◦ C until ready for use. All pigments were extracted in quadruplicate samples.

2.10.2. Phycobiliproteins About 1 g of algae material was ground to a powder with liquid nitrogen and extracted at 4 ◦ C in darkness in 0.1 M phosphate buffer, pH 6.4. The homogenates were centrifuged at 2000 g for 20 min. Phycobiliprotein levels [allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE)] were determined by UV–vis spectrophotometry, and calculations were performed using the equations of Kursar et al. (1983). 2.10.3. Carotenoid analyses Carotenoids were extracted from samples (1.0 g fresh mass, n = 4) using hexane:acetone (1:1, v/v) containing 100 mg L−1 tertbutyl hydroxytoluene (BHT). Solutions were filtered through a cellulose membrane to remove particles, and the organosolvent extract was evaporated under an N2 flux. The residue was dissolved in hexane (3 mL). Prior to chromatographic analysis, 10% KOH in methanol (100 ␮L/mL) was added to 1 mL of the organosolvent extract in order to obtain complete carotenoid saponification, which allowed better identification of each compound by HPLC. This solution was incubated (3 h in the dark at room temperature), followed by washing with distilled-deionized water (three times). The de-esterified extract was collected, concentrated under a N2 flux and resolubilized in hexane: acetone: BHT (100 ␮L) for further chromatographic analysis, as previously described (Kuhnen et al., 2009). A concentrated sample (10 ␮L, n = 3) was injected onto the liquid chromatograph (Shimadzu LC-10A) equipped with a C18 reverse-phase column (Vydac 218TP54; 250 mm × 4.6 mm Ø, 5 ␮m, 30 ◦ C), protected by a 5 ␮m C18 reverse-phase guard column (Vydac 218GK54) and a UV–vis detector (450 nm). Elution was performed with MeOH:CH3 CN (90:10, v/v) at a flow rate of 1 mL min−1 . Carotenoid identification (␣-carotene, ˇ-carotene, lutein, zeaxanthin, and ␤-cryptoxanthin) was performed using retention times and co-chromatography of standard compounds (Sigma–Aldrich, St. Louis, MO, USA), as well as by analogy with other reports of carotenoid analysis by RP-HPLC-UV–visible under similar conditions (Hulshof et al., 2007). Carotenoid quantification was based on standard curves, employing the lutein standard curve (0.5–45 ␮g mL−1 ; y = 7044x; r2 = 0.999) for lutein, zeaxanthin and ␤-cryptoxanthin quantification and the ˇ-carotene standard curve (0.01–12 ␮g mL−1 ; y = 1019x; r2 = 0.998) for ␣- and ˇ-carotene quantification. 2.10.4. Polyphenolics Polyphenolics were extracted from samples (1.0 g fresh mass, n = 4) using methanol 80% acidified with 1% HCl. Solutions were filtered through a cellulose membrane to remove particles. This extract (10 ␮L, n = 3) was injected onto the liquid chromatograph (Shimadzu LC-10A) equipped with a C18 reverse-phase column (Shim-pack C18 ; 250 mm × 4.6 mm Ø column, 5 ␮m, 30 ◦ C), protected by a 5 ␮m C18 reverse-phase guard column and a UV–vis detector (280 ␩m). Elution was performed with water:acetic acid:n-butanol (350:1:10, v/v/v), at a flow rate at 0.8 mL min−1 . Polyphenolic identification (epicatechin and gallocatechin) was performed using retention times and co-chromatography of standard compounds (Sigma–Aldrich, St. Louis, MO, USA). Polyphenolic quantification was based on standard curves, employing the gallocatechin curve (2.5–200 ␮g mL−1 ; y = 795.09x; r2 = 0.999). 2.11. Biochemical analyses

2.10.1. Chlorophyll a (Chl a) Chlorophyll a was extracted from approximately 1 g of tissue in 3 mL of dimethylsulfoxide (DMSO, Merck, Darmstadt, FRG) at 40 ◦ C, during 30 min, using a glass tissue homogenizer (Hiscox and Israelstam, 1979). Pigments were quantified spectrophotometrically according to Wellburn (1994).

Samples (1.0 g fresh mass, n = 4) from the PAR-only and PAR + UVBR of H. musciformis groups were homogenized in 20 mM phosphate buffer, pH 7.4, and centrifuged at 1000 × g for 10 min at 4 ◦ C. The low-speed supernatants (S1) were separated and used for assessing glutathione peroxidase activity and protein content.

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2.11.1. Glutathione peroxidase assay (GPx) Glutathione peroxidase activity was measured according to Wendel (1981) using tert-butyl-hydroperoxide as substrate. The enzyme activity was determined by monitoring the NADPH disappearance at 340 nm in 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA, 1 mM glutathione, 0.2 U/mL glutathione reductase, 1 mM sodium azide, 0.2 mM tert-butylhydroperoxide, 0.2 mM NADPH and the supernatant containing 0.2–0.3 mg protein/mL. GPx activity was expressed as nmol of NADPH oxidized/mg−1 protein min−1 , using the NADPH extinction coefficient at 340 nm of 6.22 × 103 M−1 cm−1 . 2.11.2. Sample preparations for measuring the respiratory chain complex activities Samples from the PAR-only and PAR + UVBR of H. musciformis groups were homogenized in 10 volumes of 50 mM phosphate buffer, pH 7.4, containing 0.3 M sucrose, 5 mM MOPS, 1 mM EGTA and 0.1% bovine serum albumin. The homogenates were centrifuged at 1000 × g for 10 min at 4 ◦ C; the pellet was then discarded, and the supernatants were used for measuring NADH dehydrogenase activity. 2.11.3. Determination of NADH dehydrogenase activity NADH dehydrogenase activity was assessed in supernatants by the rate of NADH-dependent ferricyanide reduction at 420 nm (1 mm−1 cm−1 ), as previously described in Cassina and Radi (1996). The enzyme activity was calculated as nmol mg−1 protein min−1 . 2.11.4. Protein determination The amount of protein in the samples was determined according to Lowry et al. (1951) using bovine serum albumin as the standard.

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2.12. Data analysis Data were analyzed by unifactorial Analysis of Variance (ANOVA) and the Tukey a posteriori test. Unifactorial statistical analyses were performed using the Statistica software package (Release 6.0), considering p ≤ 0.05. Maximum photosynthetic rate (Pmax ) was calculated by the equation of Platt et al. (1980), while photosynthetic efficiency (˛) was obtained by linear fitting using the three first values of ETR. Parameter calculations were made using the Microcal Origin 5.0 program. Aside from transformation, the data did not present the necessary assumptions for parametric statistics; hence, the Mann–Whitney U test, a nonparametric test, was performed with untransformed data. Statistical tests were performed by Statsoft Statistica 6.0 software. Enzyme activity data were analyzed by using the Student’s t test for independent samples.

3. Results 3.1. Observations under LM and cytochemistry The control samples of H. musciformis stained with Toluidine Blue (TB-O) showed a metachromatic reaction in the cell wall, indicating the presence of acidic polysaccharides such as kappa carrageenan (Fig. 1A). When stained with TB-O, the PAR + UVBR of H. musciformis showed a reaction in the cell wall similar to that observed in the control plant (Fig. 1B). However, in the cytoplasm of cortical and subcortical cells of treated plants, a large quantity of metachromatic granulations was observed in the vacuoles of these cells (Fig. 1B).

Fig. 1. Light microscopy of the transversal sections of thallus exposed to PAR-only control (A, C and E) and H. musciformis exposed to PAR + UVBR (B, D and F). Section stained with TB-O. (A) The cell walls (CW) of cortical cells (CC) and subcortical cells (SC) show metachromatic reaction. Section stained with TB-O. (B) Note a large quantity of metachromatic granulations in the vacuoles of cortical and subcortical cells (arrows). Sections stained with PAS. (C) Observe the PAS-positive floridean starch grains (S) in the cells and positive reaction with the cell wall. (D) Note the reduction in quantity of floridean starch grains in plants submitted to PAR + UVBR. (E) Section stained with CBB + PAS. Note the positive reaction with numerous organelles rich in protein (blue color). Observe the marginal chloroplast position (arrows) and the pit connection (PC) between the cells. (F) Section stained with CBB + PAS. Observe the changes in morphology of cortical cells and reduction in floridean starch grains. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 2. Confocal microscopy of the transversal sections of thallus exposed to PAR-only control (A) and H. musciformis exposed to PAR + UVBR (B). (A) Observe the cortical cells (CC) with large quantity of chloroplasts (arrows) and high autofluorescence intensity. (B) Note the changes in chloroplast morphology (arrows).

Cortical and subcortical cells of control of H. musciformis stained with Periodic Acid-Schiff (PAS) showed a positive reaction in the cytoplasm, with neutral polysaccharides, especially many floridean starch grains, the main substance reserve of red algae (Fig. 1C). By PAS reaction, it was possible to detect a decrease in the density of starch grains in the cortical and subcortical cells exposed to PAR + UVBR of H. musciformis (Fig. 1D). Finally, when the cortical region of control H. musciformis was stained with Coomassie Brilliant Blue (CBB) plus PAS, the outermost cell layer reacted more intensely than other cells, indicating that the outermost cell layer presented numerous organelles rich in protein (blue color) (Fig. 1E). Moreover, in the cell wall, a

PAS-positive reaction, indicating the presence of cellulosic compounds (pink color) was observed. With CBB + PAS staining, it was possible to observe the presence of pit connections and marginal chloroplast position in the cortical and subcortical cells (Fig. 1E) as well as starch grains located in the central region (Fig. 1E). When stained with CBB + PAS, the cytoplasm of cortical and subcortical cells of H. musciformis exposed to PAR + UVBR showed a reduction in density of starch grains and changes in morphology of cortical cells (Fig. 1F). These cells lost their elongated shape, and cells with different shapes and sizes were observed (Fig. 1F). Both the mucilaginous thallus surface of the control samples and the positive reaction of PAR + UVBR-treated H. musciformis to CBB + PAS

Fig. 3. Transmission electron microscopy (TEM) micrographic images of H. musciformis control plants. (A) Detail of cortical cell with various chloroplasts (C) and large quantity of floridean starch grains (S) embedded in a thick cell wall (CW). (B) Detail of a cortical cell subjected to Thiéry test. Note the positive reaction in the starch grains. (C) Detail of the thick cell wall, showing microfibrils arranged in concentric layers. (D) Chloroplast showing internal organization with parallel and peripheral thylakoids (arrows).

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staining indicate the protein nature of the cortical region (Fig. 1E and F). 3.2. Observation under confocal laser scanning microscopy When observed under confocal microscopy, the control of H. musciformis showed cortical cells with a large quantity of chloroplasts and high autofluorescence intensity (Fig. 2A). On the other hand, plants exposed to PAR + UVBR showed changes in chloroplast morphology and reduction of autofluorescence intensity (Fig. 2B), indicating that the treatment affects chloroplast functionality. 3.3. Observations under TEM When observed by transmission electron microscopy, control samples (PAR-only) of H. musciformis showed vacuolated cortical cells, mostly filled with chloroplasts (Fig. 3A) and a large quantity of floridean starch grains in the vicinity of chloroplasts (Fig. 3A and B). These cells were surrounded by a thick cell wall (Fig. 3C)

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showing microfibrils arranged in concentric layers. The chloroplasts assumed the typical internal organization of the red algae with unstacked, evenly spaced thylakoids (Figs. 3D). The chloroplasts appeared to consist of an individual and flat thylakoid surrounded by a single peripheral thylakoid (Fig. 3D). However, after a daily 3-h exposure to PAR + UVBR for a 7-day period, H. musciformis showed ultrastructural changes involving modifications in cortical cell shape (Fig. 4A), and the cell wall become more compact in irradiated plants, a fact thought to be associated with the increase in the number of concentric microfibrils (Fig. 4B–G). In many cells, a large quantity of vesicles with fibril contents could be observed (Fig. 4D and E). These microfibrils sometimes showed an ondulate shape (Fig. 4F). An increase in the number of rough endoplasmic reticula was detected in the cytoplasm, suggesting the increased protein synthesis to be exported (Fig. 4H and I). The chloroplasts showed more visible changes in ultrastructural organization with irregular morphology. The thylakoids were disrupted, and the number of plastoglobuli was increased in the chloroplasts (Fig. 4J–L).

Fig. 4. Transmission electron microscopy (TEM) micrographic images of H. musciformis thallus exposed to PAR + UVBR. (A) Note the cortical cells (CC) with irregular shape and large quantity of starch grains (S) Thiéry test. (B) Detail of cortical cell showing thickness of cell wall (CW) (arrows). (C) Observe the cell wall with reticular aspect. (D and E) Observe a large quantity of vesicles poised to form new cell wall layers (arrows) (F) Detail of thickness in cell wall, with increase in the number microfibrils with undulate shape (arrows). (G) Note the cell wall with compact aspect. (H and I) Note the increase of rough endoplasmic reticula (Rer). (J and K) Detail of disrupted chloroplast with some intact thylakoids (arrows) and large quantity of plastoglobuli (P). (L) Detail of a chloroplast subjected to Thiéry test. Observe the slight reaction in peripheral thylakoids (arrows) and plastoglobuli.

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3.4. Growth rates After 7 days in culture, H. musciformis showed statistical differences (ANOVA, P < 0.0002) in GRs between thalli cultured under PAR-only (control condition) and thalli cultured under a combination of PAR + UVBR. The control treatment showed the highest GRs at 9.7% day−1 , compared to exposed algae which grew only 3.2% day−1 from the average 7-day cultivation. 3.5. Carrageenan yield Carrageenan yield in PAR-only of H. musciformis was 26.9%, but plants submitted to PAR + UVBR only showed a 20.9% yield. No significant differences between treatments were observed. Fig. 5. Relative electron transport rate of H. musciformis exposed to UVBR for 3 h/day over a period of 7 days. Means ± S.D., n = 4.

3.6. Pigments UVB radiation affected the content of photosynthetic pigments in H. musciformis. The amounts of photosynthetic pigments are shown in Table 1. The content of chlorophyll a was not significantly different (ANOVA, P = 0.52) between plants cultivated with PARonly and those cultivated with PAR + UVBR. However, after UVBR exposure, phycobiliprotein concentrations [allophycocyanin (APC), phycocyanin (PC) and phycoerythrin (PE)] were reduced. The values of phycobiliproteins were significantly different between control and exposed algae (ANOVA, P < 0.0005). The amount of carotenoids found in H. musciformis plants cultivated under PAR-only and PAR + UVBR was determined by HPLC. Chromatographic analysis allowed the identification of the following carotenoids: zeaxanthin (free and esterified forms), lutein trans-␤-carotene, cis-␤-carotene and an unidentified compound (3.7 min, retention time) in both treatments. Lutein was only identified in PAR-only samples of H. musciformis. Traces of ␤criptoxanthin were verified in H. musciformis cultivated under PAR + UVBR. The free and esterified zeaxanthin and the unidentified compound (3.7 min, retention time) were significantly different between PAR-only and PAR + UVBR treatments (ANOVA, P < 0.05). The contents of phenolic compounds in samples of H. musciformis treated with PAR-only and PAR + UVBR were measured by HPLC. Four compounds were detected in the algae samples, and we were able to identify epicatechin and gallocatechin. Two peaks

Table 1 Photosynthetic pigments (␮g/g FW) (Chl a: chlorophyll a; APC: allophycocyanin; PC: phycocyanin; PE: phycoerythrin), carotenoids (Rt 3.7: retention time 3.7; Lut: lutein; Free-Zea: free-zeaxanthin; Est-Zea: esterified-zeaxanthin; trans-␤-car: trans-␤carotene; cis-␤-car: cis-␤-carotene) and phenolic contents (Rt 3.8 and 4.4: retention time 3.8 and 4.4; Epi: epicatechin; Gal: gallocatechin) of PAR-only and PAR + UVBR of H. musciformis cultivated over a period of 7 days. Data are means of triplicates. Means ± S.D., n = 3. Letters indicate significant differences according to the Tukey test (p ≤ 0.05). n.d.: compound not defined. PAR-only

PAR + UVBR

Photosynthetic pigments

Chl a APC PC PE

247 ± 2.8 755 ± 1.5a 400 ± 2.5a 620 ± 2.8a

244 ± 1.8 460 ± 2.2b 260 ± 2.1b 340 ± 3.1b

Carotenoids

Rt 3.7 Lut Free-Zea Est-Zea trans-␤-car cis-␤-car

0.27 ± 0.02a 0.21 ± 0.01 1.13 ± 0.06b 0.53 ± 0.05a 0.21 ± 0.01 0.45 ± 0.06

0.19 ± 0.02b n.d. 1.54 ± 0.1a 0.43 ± 0.01b 0.23 ± 0.02 0.51 ± 0.06

Phenolic compounds

Rt 3.8 Epi Rt 4.4 Gal

n.d. 48.9 ± 8.85a 13.9 ± 0.06b 19.6 ± 2.53

66 ± 4.73 28.1± 3.77b 37 ± 0.07a n.d.

(retention times of 3.8 and 4.4 min) were also detected in the chromatograms, but not identified. 3.7. Photosynthetic performance The ETR values for H. musciformis cultivated under UVBR treatment were lower than in the control for every PAR value, showing ETR maxima of 19(±1.9) and 30 (±1.3) ␮mol−1 m−2 s−1 , respectively. For PAR levels above 610 ␮mol photons m−2 s−1 , a trend of decreasing ETR was observed for both control and UVBR treatments, but the final values rose above 940 ␮mol photons m−2 s−1 for control (Fig. 5). Maximum photosynthetic rate (Pmax ) and photosynthetic efficiency (˛) were significantly lower for plants submitted to UVBR treatment (P = 0.02). 3.8. Biochemical responses H. musciformis plants treated with PAR + UVBR showed significantly increased NADH dehydrogenase activity, when compared with PAR-only plants [t(6) = 2.72; P < 0.05] (Fig. 6A). On the other hand, GPx activity and protein content were not altered by the UVBR treatment (Fig. 6A and C). 4. Discussion The present study showed that the carragenophyte H. musciformis exposed to PAR + UVBR in vitro induced cell wall thickness, changes in cell morphology, in addition to a decrease in growth rates, photosynthetic pigments, and photosynthetic performance. These plants also showed increased contents of carotenoids and phenolic compounds, suggesting that the treatment induced the activation of antioxidant cell defense. Additionally, UVBR treatment elicited increased mitochondrial activity. It has been reported that exposure to ultraviolet radiation induces the production of reactive oxygen species (ROS) and resultant oxidative damage to biomolecules, including lipids of membranes, proteins and enzymes, and DNA (Ruhland et al., 2007). This corresponds with the increased NADH dehydrogenase activity that we observed in the treated plants, since this increased oxygen consumption finally elicits increased ROS formation. As a strategy to prevent the effects of ROS, H. musciformis plants exposed to UVBR increase the content of phenolic compounds (up to 58.9%) and carotenoids (up to 3.6%), most likely as a photoprotective mechanism against UVB damage. According to Ruhland et al. (2007), the increased concentrations of phenolic compounds in response to elevated radiation have two different functions: (1) to act as a sunscreen against potentially damaging UVB and (2) to ameliorate damage caused by increased ROS.

É.C. Schmidt et al. / Aquatic Botany 100 (2012) 8–17

Fig. 6. Biochemical responses of H. musciformis exposed to UVBR for 3 h/day over a period of 7 days. Means ± S.D., n = 4. (A) Mitochondrial NADH dehydrogenase activity of H. musciformis. (B) Glutathione peroxidase (GPx) activity in H. musciformis. (C) Protein content of H. musciformis.

According to Abdala-Díaz et al. (2006), the phenolic compounds could act as a photoprotective mechanism against higher irradiance in the ecosystems by absorbing incident photons or indirectly as a result of their antioxidant activity. The increase in NADH dehydrogenase activity observed in H. musciformis exposed to PAR + UVBR might be related to increased oxygen consumption, possibly to compensate for the potential membrane loss and energy deficit. This increase in mitochondrial activity could result in increased mitochondrial ROS formation. This idea agrees with the findings of Costa et al. (2002), who demonstrated that UVBR exposure stimulates the generation of ROS, as well as the findings of Shiu and Lee (2005) who showed

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that ROS-induced toxicity provokes an increased antioxidant enzymatic response. However, our study found that cellular antioxidant potential could be characterized by a significant increase in non enzymatic antioxidant defenses, while antioxidant enzymatic activities (GPx) were not changed by the ultraviolet irradiation. Notably, however, even when PAR + UVBR treatment resulted in reduced protein synthesis, as previously observed by our group and others (Eswaran et al., 2001; Schmidt et al., 2012), the protein content was not altered in treated H. musciformis plants, possibly resulting from the increase in rough endoplasmic reticula observed by TEM. Carotenoid patterns in H. musciformis were also altered by PAR + UVBR treatment. Changes included the absence of lutein, but slight increase in the quantity of trans-␤-carotene and cis-␤carotene, as well as increased free zeaxanthin. This latter increase could be related to the effects of ultraviolet radiation breaking the ester bonds of esterified zeaxanthin. This possibility is supported by Döhler (1998) who observed reduced lutein and zeaxanthin levels in the red macroalga Leptosomia simplex exposed to UVBR. However, in the same study, an increase in carotene levels was also observed, a phenomenon most likely related to the protection of the photosynthetic apparatus. However, Altamirano et al. (2000) observed an increase in carotenoid contents in the presence of UVBR, suggesting the activation of a protective pigment mechanism. Exposure of H. musciformis to PAR + UVBR showed a decrease in GRs, indicating that UVBR is a key factor limiting its growth. The decrease in GRs observed in H. musciformis may therefore be related to the use of energy to activate the mechanisms of adaptation and the repair of damage induced by UVBR. According to van de Poll et al. (2001), growth reduction results from the combined effects of damage to several cellular components, such as proteins from PSII reaction centers and DNA. These processes are directly affected by UVR, and the ability to repair or prevent damage eventually determines the UV tolerance of species. Thus, to assess their responses to these new light conditions, it is necessary to understand the ability of macroalgae to acclimate to UVR stress. In the present study, chlorophyll a contents of H. musciformis showed more resistance upon exposure to PAR + UVBR, when compared to PAR-only. Studies with carragenophytes have generally shown a decrease in chlorophyll a concentration after UVBR exposure, including, for example, the brown strain of K. alvarezii cultivated in vitro during 28 days of exposure (Schmidt et al., 2010b), Eucheuma strictum Schmitz cultured in vitro during 16 days of exposure (Wood, 1989), and K. alvarezii cultured in long line and incubated with UVBR during 30, 60, 90, 120, 150, and 180 min (Eswaran et al., 2001). However, other investigations with different carragenophytes have shown that UVBR stimulated the synthesis of chlorophyll a, such as the green and red strains of K. alvarezii (Schmidt et al., 2010a), M. stellatus and C. crispus (Roleda et al., 2004). The levels of phycobiliproteins (APC, PC, and PE) in H. musciformis decrease upon exposure to PAR + UVBR. Our results showed a decrease in phycobiliprotein contents similar to the findings in such carragenophytes as green, red and brown strains of K. alvarezii (Schmidt et al., 2010a,b) and K. alvarezii cultivated in long line and incubated with UVBR during 30, 60, 90, 120, 150, and 180 min (Eswaran et al., 2001). H. musciformis exposed to PAR + UVBR showed an inhibition of photosynthesis parameters. Photosystem II (PSII) is a major target of UVBR in plants and macroalgae (Vass, 1997). According to Holzinger et al. (2004), PSII photochemical efficiency in Palmaria palmata (Linnaeus) Kuntze and Odonthalia dentata (Linnaeus) Lyngbye strongly decreased to about one third of the initial value under UV. To the extent that UVBR radiation increases the difficulty of establishing a proton gradient across the thylakoid membrane,

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É.C. Schmidt et al. / Aquatic Botany 100 (2012) 8–17

photosynthetic reactions will be impaired (Poppe et al., 2002). Repair mechanisms for UVBR-induced damage of membranes or electron transport components demand increased enzymatic activity with higher nitrogen requirements (Poppe et al., 2002). The cell wall of H. musciformis treated with PAR-only and PAR + UVBR reacted positively to TB-O. This occurred because the cell wall contains sulfated polysaccharides in the molecular structure of carrageenan. The higher intensity of reaction to TB-O in plants exposed to PAR + UVBR could be related to an increase in the production of vesicles, as observed under TEM, which were subsequently incorporated into cell wall, thus causing it to thicken. Also related to cell wall thickness were the metachromatic granulations observed inside the cortical and subcortical cells of PAR + UVBR-treated H. musciformis. Especially in the vacuoles, these granulations appeared as reserves of complex polysaccharides and could be sites of accumulation of material which was subsequently incorporated into the cell wall. Overall, this finding indicates an increase in cell wall thickness, which was observed in some subcortical cells after treatment with TB-O of H. musciformis exposed to PAR + UVBR. However, no increase in carrageenan yield was observed in H. musciformis plants submitted to PAR + UVBR. A similar result was observed in K. alvarezii incubated with UVBR during 30, 60, 90, 120, 150, and 180 min (Eswaran et al., 2001). When analyzed under TEM, the PAR-only cell wall of H. musciformis showed a microfibrillar texture with microfibrils structured in concentric layers with different degrees of compression. In contrast, observations of the cell wall of plants exposed to PAR + UVBR showed an increase in that structural component. The increase in the thickness of the cell wall of H. musciformis exposed to PAR + UVBR can be interpreted as a defense mechanism against exposure to ultraviolet radiation. It is probable that the activity of Golgi bodies is more intense in plants exposed to PAR + UVBR and that this results in the large production of vesicles which then format the matrix content of the cell wall. PAS reaction, as observed under LM, indicates greater deposition of neutral polysaccharides, such as cellulose. Overall, cellular metabolism of H. musciformis exposed to PAR + UVBR is modified such that vesicles with fibril contents responsible for cell wall thickening are produced, and this, in turn, reduces the synthesis of mucilage molecules that coat the thallus. The decrease in floridean starch grains observed through TEM and LM in plants exposed to PAR + UVBR may be related to a change in the route of biosynthesis of starch enzymes of the Calvin cycle, possibly by activating the degradation pathway. The degradation pathways may be utilized to activate the biosynthesis of defense compounds. Inhibition of the synthesis of floridean starch grains by means of UDP-glucose diverts synthesis for the production of cell wall components, such as carrageenan that also uses UDPglucose as a precursor to biosynthesis of this polysaccharide. This phenomenon, as observed with TEM and LM, also results in the increase of the cell wall. In red algae, the thylakoids that are not associated with each other are free in chloroplasts. The chloroplasts of PAR-only H. musciformis have one peripheral thylakoid surrounded by parallel thylakoids. The number of parallel thylakoids is variable, and this number mainly depends on the spatial location of the cell in the algae. In contrast, the chloroplasts of H. musciformis exposed to PAR + UVBR showed significant structural changes, including modification in the quantity, size, and organization of thylakoids. When analyzed by TEM, the algae exposed to PAR + UVBR revealed an increase in the number of plastoglobuli in the chloroplast. According to Holzinger et al. (2009), when the algae are subjected to stress, nitrogen limitation and the synthesis of lipids are observed. These phenomena occur because the pathways to form protein-containing cell structures are suppressed. Similar results were reported by Schmidt et al. (2009), with the formation

of plastoglobuli in K. alvarezii after exposure to UV radiation. This increase in the number of lipids can be considered as a change in metabolism, which, in turn, results in the reduction of cell proliferation and decrease in GRs. 5. Conclusion In summary, the present study demonstrates that UVBR negatively affects various morphological, physiological and biochemical parameters in H. musciformis. In the present study, this became obvious after only 3 h of daily exposure to UVBR over a 7-d experimental period, resulting in ultrastructural damage observed primarily in the internal organization of chloroplasts, increased cell wall thickness and increased rough endoplasmic reticula of H. musciformis. Moreover, this exposure might have caused photodamage and photoinhibition of photosynthetic pigments, leading to a decrease in photosynthetic efficiency and a corresponding decrease in growth rates. A change in the carotenoidic and phenolic profiles of the UVB-treated plants compared to control plants was also confirmed, suggesting the expression of defense mechanisms associated with those antioxidant compounds. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors would like to acknowledge the staff of the Central Laboratory of Electron Microscopy (LCME), Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil, for the use of their transmission electron microscope. This study was supported in part by the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Fundac¸ão de Apoio à Pesquisa Cientifica e Tecnológica do Estado de Santa Catarina (FAPESC). The authors are grateful to CAPES for providing a scholarship to Éder C. Schmidt and CNPq-PIBIC for providing a scholarship to Beatriz Pereira. Bouzon ZL, Maraschin M, Horta P.A and Latini A are CNPq fellows. This study is part of the Ph.D. thesis of the first author. References Abdala-Díaz, R.T., Cabello-Pasini, A., Pérez-Rodríguez, E., Conde Álvarez, R.M., Figueroa, F.L., 2006. Daily and seasonal variations of optimum quantum yield and phenolic compounds in Cystoseira tamariscifolia (Phaeophyta). Mar. Biol. 148, 459–465. Altamirano, M., Flores-Moya, A., Figueroa, F.L., 2000. Long-term effects of natural sunlight under various ultraviolet radiation conditions on growth and photosynthesis of intertidal Ulva rigida (Chlorophyceae) cultivated in situ. Bot. Mar. 43, 119–126. Cassina, A., Radi, R., 1996. Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport. Arch. Biochem. Biophys. 328, 309–316. Costa, H., Gallego, S.M., Tomaro, M.L., 2002. Effect of UV-B radiation on antioxidant defense system in sunflower cotyledons. Plant Sci. 162, 939–945. Döhler, G., 1998. Effect of UV radiation on pigments of the Antarticmacroalga Leptosomia simplex L. Photosynthetica 35, 473–476. Eswaran, K., Subba-Rao, P.V., Mairh, O.P., 2001. Impact of ultraviolet-B radiation on Kappaphycus alvarezii (Solieraceae, Rhodophyta). Indian J. Mar. Sci. 30, 105–107. Hanelt, D., Roleda, M.Y., 2009. UVB radiation may ameliorate photoinhibition in specific shallow-water tropical marine macrophytes. Aqua. Bot. 91, 6–12. Hayashi, L., Paula, E.J., Chow, F., 2007. Growth rate and carrageenan analyses in four strains of Kappaphycus alvarezii (Rhodophyta, Gigartinales) farmed in subtropical waters of São Paulo State, Brazil. J. Appl. Phycol. 19, 393–399. Hiscox, J.D., Israelstam, G.F., 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot. 57, 1332–1334. Holzinger, A., Lütz, C., Karsten, U., Wiencke, C., 2004. The effect of ultraviolet radiation on ultrastructure and photosynthesis in the red macroalgae Palmaria palmata and Odonthalia dentata from Artic waters. Plant Biol. 6, 568–577.

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