Photochemistry and Photobiology, 2016, 92: 455–466
Stress Tolerance of the Endemic Antarctic Brown Alga Desmarestia anceps to UV Radiation and Temperature is Mediated by High Concentrations of Phlorotannins
Mar ıa Rosa Flores-Molina1,2*, Ralf Rautenberger2, Pamela Munoz~ 2, Pirjo Huovinen2,3 and Ivan Gomez 2,3 1Doctorado en Biolog ıa Marina, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 2Instituto de Ciencias Marinas y Limnologicas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 3Centro Fondap de Investigacion de Altas Latitudes (IDEAL), Valdivia, Chile Received 24 September 2015, accepted 19 January 2016, DOI: 10.1111/php.12580
ABSTRACT 400–700 nnm) that may impose severe limitations for photosyn- thesis. Thus, Antarctic macroalgae, are not only able to respond The endemic Antarctic brown macroalga Desmarestia anceps with the extreme seasonality in day lengths, but also exhibit a is strongly shade-adapted, but shows also a high capacity to remarkable capacity to photosynthesize efficiently at low PAR cope with different environmental stressors, e.g. UV radiation irradiances under constant low temperatures ranging between and temperature. Therefore, this species colonizes wide depth 1.8 and +2°C (reviewed in 2). gradients, which are characterized by changing environmen- Although subtidal macroalgae are usually exposed to very low tal conditions. In this study, we examine whether the differ- irradiances of solar radiation due to snow-covered sea-ice in win- ent physiological abilities allowing D. anceps to grow across a ter, many Antarctic coastal waters are highly transparent to both wide depth range is determined by high levels of phlorotan- PAR after the break-up of sea ice in spring and early summer fl nins. Photosynthesis, measured by PAM- uorometry, the before glaciers set in to melt, which results in a deep penetration contents of soluble phlorotannins, antioxidant capacities of of PAR into the water column down to 30 m (3–5). Moreover, fi eld grown were analyzed in response to different conditions at Fildes Bay on King George Island (South Shetland Islands, + of radiation (PAR and PAR UV) and temperature (2, 7 Antarctica), the penetration of ultraviolet (UV) radiation (280– ° fl and 12 C). The results show that maximal quantum of uo- 400 nm) can reach depths down to 15 m, exposing macroalgae rescence (Fv/Fm) decreased with increasing doses of UV radi- and their associated fauna and flora (i.e. epiphytes, fungi and ation, but remained unaffected by temperature. High levels bacteria) along this depth range to these potentially harmful con- fi of soluble phlorotannins were detected and con rmed by ditions (4,5). High-energy UV-B radiation (280–315 nm), which microscopic observation revealing the abundance of large is most biologically damaging, is of particular interest for marine physodes. Exposure to UV radiation and elevated tempera- biology in high latitudes because its irradiances increase under ture showed that phlorotannins were not inducible by UV stratospheric ozone depletion over Antarctica and its adjacent ° but increased at 12 C. ROS scavenging capacity was posi- regions (6–8). Scientific evidences gained throughout the past tively correlated with the contents of phlorotannins. In gen- 20 years allow to argue today that such seasonally enhanced eral, highest contents of phlorotannins were correlated with UV-B radiation plays a substantial role in the physiological and the lowest inhibition of Fv/Fm in all experimental treatments, ecological acclimation of Antarctic macroalgae, especially with highlighting the UV-protective role of these compounds in respect to photosynthetic performance, development and settle- D. anceps. ment of early life stages as well as succession processes and ver- tical zonation (2,8,9). INTRODUCTION Increasing sea surface temperatures of Antarctic waters as a result from global climate change, especially in the West Antarc- The Antarctic marine environment is characterized by extreme tic Peninsula (WAP) region, have begun to study as a factor that seasonal changes in air temperatures, light regimes and wind influences metabolic processes in the presence of UV radiation speed, as well as considerable disruptions of the upper tidal posi- (10–12). fl tions in coastal ecosystems by oating sea ice and icebergs. Due When macroalgae are exposed to both UV stress and elevated to these constraints, most of the Antarctic macroalgae grow in temperatures, their primary metabolism can be significantly fi the subtidal deeper than 5 m where they nd more stable envi- affected by structural changes or denaturation of proteins, deteri- ronmental conditions such as constant low temperatures and high oration of membrane functions, which, in turn, limits the effi- nutrient concentrations (1). However, as a consequence of inhab- ciency of photosynthetic and mitochondrial respiratory electron iting the subtidal, macroalgae adapted to predominantly occurring transport rates as well as nutrient uptake (13). The photosystem low irradiances of photosynthetically active radiation (PAR: II (PSII) reaction center complex as an essential component of the photosynthetic apparatus can be sensitive to temperatures *Corresponding author email: mrfl[email protected] (Mar ıa Rosa Flores- stress, resulting in a heat-induced decline in photosynthetic Molina) activity, as demonstrated by the sub-Antarctic brown macroalgae © 2016 The American Society of Photobiology
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Durvillaea antarctica, Lessonia spicata (studied as Lessonia upper survival temperature (UST) of 11°C (31). The thermal nigrescens) and Macrocystis pyrifera (14). Moreover, the requirements of this species related to the temperature conditions heat-induced expression of genes involved in detoxification of in its subtidal habitats in Antarctic coastal ecosystems are sum- reactive oxygen species (ROS) tries to minimize oxidative dam- marized in Fig. 1. age caused as a byproduct of stress damage (15–17). To keep Desmarestia anceps has very low light demand for growth the level of damages to photosynthetic and other cellular and photosynthesis. In fact, it shows high photosynthetic efficien- components by oxidative stress low, macroalgae have different cies (i.e. the initial slopes of photosynthesis vs irradiances antioxidant mechanisms such as increasing activities of curves), high rates of dark respiration and a positive metabolic ROS-scavenging enzymes (e.g. superoxide dismutase, ascorbate carbon balance over a depth range of 20 m. These physiological peroxidase), synthesizing antioxidants such as ascorbate and adaptations allows D. anceps to colonize water depths close to glutathione, and can regenerate metabolites efficiently to avoid 30 m (3,5,31). Due to its large size of up to 4 m in length of deficiencies in antioxidant reactions (18–20). adult sporophytes (≥2 years) and dominance in Antarctic coastal Polyphenolic compounds (also known as phlorotannins) haven ecosystems, this species can be regarded as an “ecosystem engi- been shown to be highly efficient and multifunctional “antistress neer” similar to the large kelps and kelp-like brown macroalgae agents” exclusively isolated from brown macroalgae, and can from temperate regions. However, their significantly higher con- function as herbivore deterrents (20), UV-absorbing compounds centrations of phlorotannins than temperate brown macroalgae is (21,22) and even as antioxidants (23,24). It has been demon- a characteristic feature that is strongly associated with the chemi- strated that phlorotannins are important to detoxify ROS in some cal defense against several Antarctic grazers (32). The high sub-Antarctic kelps of the genera Lessonia, Durvillaea and levels of phlorotannins measured in D. anceps (≥50 mg g 1 dry Macrocystis under combined stress of high solar radiation and weight; DW) are apparently constitutive (i.e. constantly high) temperature (25–27). Apparently, this strategy, which is based on which raise the question to its potential as mechanism of stress the induced synthesis of phlorotannins, is crucial to brown tolerance beyond grazing alone. In this respect, Antarctica macroalgae to tolerance environmental stress and, therefore, can macroalgae that are constantly exposed to low seawater tempera- be regarded as a primary defense mechanism against episodic tures have been less studied in comparison with other photosyn- stress by elevated temperature and solar radiation. thetic organisms that experience seasonal changes in temperature. Desmarestia anceps Montagne is an endemic Antarctic brown Moreover, despite the permanently low seawater temperatures macroalga that is exclusively but widely distributed in the WAP (≤2°C) and generally low irradiances of PAR and UV radiation region and the adjacent islands, where it dominates the subtidal in the subtidal, they also require efficient mechanisms to with- benthic communities between 10 and 30 m depth (4,28,29). Due stand episodes of stress by high solar radiation, including UV to its presumable origin in the cold waters of Antarctica (30), radiation. Interactive studies between temperature and UV stress this species is strongly cold-adapted, which limits its northern that were recently carried out give first hints of a link between distribution by the temperature demands for the development and high levels of soluble phlorotannins and enhanced antioxidant growth of the sporophytes, i.e.0–5°C. Moreover, it shows a low capacities in Antarctic Desmarestiales (4,12). In the context of
a b
Figure 1. (a) Photograph of the habitus of the endemic Antarctic brown macroalga Desmarestia anceps (primary stem with lateral branches and apical pieces) and (b) temperature demands for photosynthesis and growth of both its gametophytes and sporophytes in relation to environmental temperature regimes (boxed) measured on King George Island (South Shetland Islands, Antarctica). P:R ratio: photosynthesis-to-dark respiration ratio, UST: Upper survival temperature. Data were compiled from (28,45,46). Photochemistry and Photobiology, 2016, 92 457
metabolic adaptations this finding may support the hypothesis Photosynthetic measurements were performed before (t0 = 0 h) and that phlorotannins and their presumed antioxidant potential repre- after 2, 6, 24 and after 48 h of the UV 9 temperature treatments. Sam- sent a metabolic trait to respond efficiently to different environ- ples for biochemical analyses (i.e. phlorotannin contents and antioxidant capacities) were taken before (t0 = 0 h) and after 2, 6 and 48 h of the mental stressors along the vertical zonation of macroalgae (5). UV 9 temperature treatment. This material was immediately frozen and In this study, the physiological and biochemical acclimation stored in liquid nitrogen and transported in a cooled Dry Shipper of field-grown individuals of D. anceps to a combination of radi- (< 80°C) to the laboratory of the Instituto de Ciencias Marinas y Lim- ation (PAR alone and PAR + UV radiation) and temperatures nologicas of the Universidad Austral de Chile in Valdivia where they were kept at 80°C until analyses. (2, 7 and 12°C) regime were investigated to estimate its physio- Photosynthetic measurements. Photosynthetic parameters were logical acclimation potential to future climate changes. The maxi- measured using a pulse-amplitude modulated (PAM) chlorophyll a mum PSII-quantum yield (i.e.Fv/Fm) and rapid light curve fluorometer (Junior-PAM, Blue version; Walz GmbH, Effeltrich, measured by chlorophyll a fluorescence, soluble phlorotannin Germany). After macroalgal samples were taken from the experiment, and cellular antioxidant capacities were used as physiological they were kept in the dark for 10 min before minimum (F0) and maximum (F ) fluorescence were measured using a blue indicators. Based on the thermal characteristics of D. anceps m (kmax = 445 nm) measuring light and short saturation pulse (>2000 lmol 2 1 compiled in Fig. 1, we hypothesize differential responses of ris- photons m s , 0.8 s), respectively. A far-red light (kmax = 730 nm) ing seawater temperatures to UV stress: while temperatures that was applied for 5 s after the samples were placed in darkness. Variable- lay moderately above the current conditions (i.e. an increase by to-maximum fluorescence, which is an estimation of the maximum 5°C from 2°Cto7°C) can positively modulate its photosynthetic quantum yield of PSII photochemistry, was calculated after (36): response to UV radiation and improve photoprotection, consider- F =F ¼ðF F Þ=F ably higher temperatures are expected to exacerbate the negative v m o m m effects of UV radiation at 12°C. Subsequently, D. anceps was exposed to incrementally increasing actinic light intensities (30 s steps from 0 to 420 lmol photons m 2 s 1) MATERIALS AND METHODS after a short saturation pulse (>2000 lmol photons m 2 s 1, 0.8 s) was applied. Effective quantum yields of PSII photochemistry, φ(PSII), were calculated from terminal (F ) and maximum (F’ ) fluorescence measured Collection and experimental set-up. All individuals of adult sporophytes t m – at each intensity of actinic light. Electron transport rates (ETR) was cal- (18 24 individuals between 1.2 and 3.5 m in length) of D. anceps culated by multiplying the effective PSII-quantum yields (φ ) with the Montagne (Phaeophyceae, Ochrophyta) were collected from the same (PSII) corresponding intensity of actinic light (EPAR), thallus absorbance population in the lower subtidal (15 m water depth) at Fildes Bay near = ° 0 ° 0 (A 0.8514 for D. anceps) and the fraction of absorbed EPAR associated Artigas Station (62 11.51 S, 58 53.70 W) on King George Island (South to PSII (0.5) (37): Shetland Islands) in January and February 2014. After collection, the algal material was transported in light-protected plastic boxes filled with local ¼ u : seawater to the laboratory at the Antarctic Station “Base Profesor Julio ETR ðPSIIÞ EPAR 0 5 A Escudero” (62°12.090S, 58°57.770W). Apical pieces (~0.15 m in length) were cutoff lateral branches at the tip of each individual, cleaned carefully Electron transport rates were plotted against EPAR to give photosyn- using ice-cold filtered seawater (0.45 lm) and maintained in filtered thetic electron transport rate vs irradiance (ETR-E) curves. The nonlinear seawater (0.45 lm, salinity 33.8 0.2) under constant aeration at function of (38) was used to fit the data sets of ETR-E curves to estimate ° l 2 1 2 1 C and 15 mol photons m s PAR (TL-D36W/54-765 photosynthetic parameters (i.e. ETRmax, a and Ek)ofD. anceps under Daylight; Philips, Amsterdam, The Netherlands) in the experimental system the experimental treatments using the software package KaleidaGraph for 48 h prior to the experiment. Six experimental units per temperature version 4.1 (Synergy Software, Reading, PA, USA). treatment (18 units in total for three temperatures) with three replicates for The temperature coefficient (Q10 value) was calculated to measure each radiation treatment (see below). Six apical pieces per individual were changes in the metabolic rate of ETRmax between 2 and 12°CinD. an- randomly assigned to one experimental unit and to each radiation treatment ceps as follows (39): (PAR alone and PAR + UV) in a temperature-controlled set-up (Fig. 2a). Three replicates per radiation and temperature treatment were used. ð10=ð12 C 2 CÞÞ The radiation treatment was set using a combination of four fluores- Q10 ¼ðETRmax;12 C=ETRmax;2 CÞ cent tubes: two PAR-emitting tubes (TL-D36W/54-765 Daylight; Philips) which were supplemented with one UV-A (UVA-340; Q-Lab Corpora- where ETRmax,12°C and ETRmax,2°C, is the maximum electron transport tion, Westlake, OH, USA) and UV-B-emitting tube (UVB-313; Q-Lab rate at 12°C and 2°C, respectively. Corporation). All tubes were horizontally above the culture tanks contain- The percentage of the inhibition of Fv/Fm under UV stress was calcu- ing macroalgal samples of D. anceps, which covered by two different lated before and after 2, 6, 24 and 48 h of UV exposure according to the cut-off filters: Ultraphan 395 (transmission ≥395 nm; Digefra GmbH, following formula: Munich, Germany) for PAR control and Ultraphan 295 (transmission ≥295 nm; Digefra GmbH) for PAR + UV treatment. Irradiance were set % = ¼ðð = = Þ= to 0.26 W m 2 UV-B (280–315 nm), 1.51 W m 2 UV-A (315–400 nm) Fv FmInhibition Fv Fm;PAR Fv Fm;PARþUV l 2 1 and 15 mol photons m s PAR using a hyper-spectral radiometer Fv=Fm;PARÞ 100 (RAMSES-ACC2; TriOS GmbH, Oldenburg, Germany) (Fig. 2b). Under these two radiation conditions, the apical pieces were incubated to three different temperatures (2 1, 7 1, and 12 1°C) over 48 h. Microscopic observations and localization of phlorotannins. The The biologically effective dose (BED) of UV radiation was calculated spatial distribution of phlorotannins in cross-sections of fronds was by the following formula: examined using both light and fluorescence microscopy. Samples were sectioned with a microtome cryostat (Minotome, Model Mino; k¼X400nm International Equipment Company, Needham Heights, MA, USA) using â BED ¼ EkekDk Tissue-Tek , O.C.T.TM (Sakura Finetek Europe B.V., The Netherlands) k¼280nm colorless compound as a medium for sample freezing. The cross-sections (10 lm) were placed in a solution of glycerol (50% in 0.22 lm filtered where E(k) is the wavelength-dependent irradiance of UV radiation seawater) and kept in the dark at 4°C until observation under Olympus (W m 2) exposed to D. anceps and e(k) is the relative biological effec- BX51TF epifluorescence microscope (model CKX41SF; Olympus tiveness of UV radiation (dimensionless) at a given wavelength k. The Corporation, Tokio, Japan) equipped with a digital camera (QImaging weighting coefficients, e(k), for DNA damage (33) and inhibition of PSII Micro Plublisher 5.0 RTV; software QCapture pro) (5). Phenolic electron transport in chloroplasts (34) were used (for equations see 35). compounds (phlorotannins) were visualized as white to blue-green auto 458 Mar ıa Rosa Flores-Molina et al.
Figure 2. (a) Schematic drawing of the experimental set-up used for the combined exposure of Desmarestia anceps to photosynthetically active radia- tion (PAR: 400–700 nm) in absence and presence of ultraviolet radiation (UV = UV-A + UV-B: 280–400 nm) at three different temperatures (2 1, 7 1 and 12 1°C). Two PAR (white tubes) and two UV-emitting fluorescence tubes (blue tubes) were placed above all macroalgal culture tanks. Six apical pieces deriving from one individual were placed in each culture tank, which were covered by either an UV cutoff or an UV-transmitting filter (for details see Materials and Methods). Temperatures were adjusted using a temperature control unit. (b) Spectral composition of the two radiation regimes in the experiment (PAR: dashed/dotted line, PAR + UV: solid line) and the biologically weighted irradiances calculated using the action spectra for both DNA damage (33) and photoinhibition of photosynthesis (34) (inlet).
fluorescence and chlorophyll as orange-red auto fluorescence (40) with Samples were observed in a transmission electron microscope (Tecnai the U-MWBV2 mirror unit (Olympus Corporation) (excitation 400– 12; Philips), operated at 60 kV. 440 nm, detection of emission ≥475 nm). Contents of total soluble phlorotannins. The contents of the total At intracellular level, phlorotannin-containing physodes were exam- soluble phlorotannins were measured using the Folin–Ciocalteu method ined by transmission electron microscopy (TEM). Samples of approxi- after (26). Frozen samples ( 80°C) were ground to a fine powder in mately 3 mm in length were fixed in 3% (v/v) glutaraldehyde, 1% p- liquid nitrogen using mortar and pestle. Soluble phlorotannins were formaldehyde and 0.1% caffeine in filtered seawater (0.22 lm). After extracted from equal amounts of dried macroalgal powder washing with filtered seawater (0.22 lm), samples were postfixed in 2% (14.5 0.5 mg 1 DW) overnight with 70% acetone (1.5 mL) at 4°Cin osmium tetroxide and 1% potassium ferricyanide, dehydrated through a the dark. After centrifugation at 6000 g for 3 min at room temperature, series of ethanol (10–100%) and embedded in Spurr resin (41) for one the supernatant was transferred to a clean tube and evaporated by ~30% week. For staining, 4% uranyl acetate and lead citrate were used. to a volume of 1 mL at room temperature under a fume hood. Aliquots Photochemistry and Photobiology, 2016, 92 459
(100 lL) were mixed with 200 lL of 20% (w/v) NaCO3, 200 lLof ultrapure H2O, and 100 lL of 2 N Folin–Ciocalteu reagent (Sigma- Aldrich Corporation, St. Louis, MO, USA). The extracts were incubated in the dark at room temperature for 60 min and afterward centrifuged (5000 g, 3 min, room temperature). The absorbance was measured at 730 nm in a Multiskan Spectrum plate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). A standard curve of known concentrations of phloroglucinol (Sigma-Aldrich Corporation) was used for quantification of soluble phlorotannins from crude extracts (expressed in mg g DW 1). Cellular antioxidant activity. The determination of the cellular antioxidant capacity of soluble phlorotannin extracts was based on the free radical 2,2-diphenil-1-picrylhydrazylo (DPPH; Sigma-Aldrich Corporation) scavenging method of (42), modified by (26). A volume of 22 lL taken from supernatant in the soluble phlorotannin extraction were mixed in the 96-well microplate with 200 lL of 150 lM DPPH (freshly prepared in 80% acetone). Changes in absorbances of samples in lid- covered microplates were instantly measured photometrically at 520 nm over 180 min (5 min intervals) at 22°C. A standard curve of known concentrations of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid; Sigma-Aldrich Corporation) used for quantification. Cellular antioxidant capacity was defined as lmol Trolox equivalent (TE) on a DW basis (lmol TE g 1 DW). Statistical analyses. Means and standard deviations were calculated from three independent replicates per treatment (n = 3). Normal distribution of residuals were tested by the Shapiro–Wilk W-test and a Levene test was performed to test on homogeneity of variance (43). Two- way analyses of variance with repeated measures (RM-ANOVA) were conducted to identify statistically significant differences of means of Fv/Fm, contents of total soluble phlorotannins, and cellular antioxidant capacities between and within UV/temperature treatments (two levels of the between- subjects factor “light” and three levels of the between-subjects factor “temperature”) under consideration of Mauchly’s sphericity test (five and four levels of the within-subjects factor ‘time’ for Fv/Fm and phlorotannins/ antioxidant capacities, respectively, a = 0.05). A Greenhouse–Geisser correction was applied when sphericity was violated (e < 0.75). A two- way ANOVA was performed to identify statistically significant differences of means of photosynthetic parameters (i.e. ETRmax, a and Ek). A post hoc analysis (Student Newman–Keuls test; SNK) was performed to contrast the effects of radiation and temperature. To identify significant differences of Q10 between the PAR and the PAR + UV treatment, a Student’s t-test was conducted after meeting the assumptions. Nonlinear regressions were performed to estimate the relationship between soluble phlorotannins vs antioxidant capacity and soluble phlorotannins vs Fv/Fm.A5% significance level (P = 0.05) was applied in all statistical tests, which were performed using the software packages JMP 12.1 (SAS Institute Inc., Cary, NC, USA) and Statistica version 7 (StatSoft Inc., Tulsa, OK, USA).
RESULTS Figure 3. Effect of the radiation (PAR and PAR+UV) and temperature Photosynthetic responses to UV and temperature treatments on Fv/Fm of Desmarestia anceps at different times of the experiment intervals. Temperatures (a) 2°C, (b) 7°C and (c) 12°C. Data Desmarestia anceps showed significant differences in Fv/Fm val- are mean SDs (n = 3). ues between PAR and UV treatments and between incubation temperatures (2, 7 and 12°C) with time (P<0.01, -way RM- ANOVA; Fig. 3, Table S1). Initial values were close to ETRmax values based in ETR-E curves ranged between 16.8 2 1 0.78 0.02, which decreased with time in the UV treatment; at and 50.9 lmol electrons m s .At12°C, ETRmax were three ° 2°C, Fv/Fm values decreased by 37% from initial until 48 h of times higher than 2 and 7 C(P<0.05, 2-way ANOVA, SNK; fi exposure. A similar pattern was observed at 12 °C. At 7°C, Fv/ Table 1). Signi cant differences were not detected in response to > Fm values from UV treatment decreased by 50% after 24 h, radiation treatment in the three tested temperatures (P 0.05, a fi which was exacerbated after 48 h with Fv/Fm decrease of 58% 2-way ANOVA; Table S2). The initial slope ( ) showed signi - (P<0.05, 2-way RM-ANOVA; Table S1). Maximal quantum cant differences between temperatures and radiation treatments, yield measured in the PAR treatment remained constant showing lowest values at 7°C in the PAR + UV treatment (0.74 0.02) over time (Fig. 3). (i.e. 0.109 0.05 (lmol electrons m 2 s 1)(lmol photons The percentage of photoinhibition of D. anceps in the three m 2 s 1) 1)(P<0.05, 2-way ANOVA, SNK; Tables 1 and exposure temperatures showed a tendency of exponential S2). The light requirements for the saturation of photosynthesis, 2 1 increase in relation with the biologically effective dose estimated Ek, which ranged between 75 and 186 lmol photons m s using the weighting function for chloroplast photoinhibition (34). (Table 1), were significantly different between both light treat- In general, inhibition of photosynthesis was higher at 7°C com- ments (P<0.05, 2-way ANOVA; Table S2), showing higher Ek pared to 2 and 12°C (Fig. 4). under PAR + UV than under PAR alone. 460 Mar ıa Rosa Flores-Molina et al.
Significant effects of temperature and the interaction between temperature and radiation through the time on the values of phlorotannins were identified (P<0.05, 2-way RM-ANOVA; Fig. 6; Table S4). The lowest phlorotannins content were mea- sured at 7°C, in both PAR and UV treatment (P<0.05; 2-way RM-ANOVA; Fig. 7; Table S4). Conversely, the antioxidant capacity of extracts exhibited sig- nificant differences in response to radiation and time within the three tested temperatures (P<0.05, 2-way RM-ANOVA; Fig. 7; Table S5). The lowest antioxidant capacity was measured at 2°C, in both PAR and UV treatment (values between 15 and 50 mg TE g 1 DW), while the highest values were determined after 6 h under UV treatment at 7°C (128 mg TE g 1 DW) (P<0.05; 2-way RM-ANOVA; Fig. 7; Table S5). There was a nonlinear relationship (R = 0.634) between total content of soluble phlorotannins and the antioxidant capacity. This positive relationship was influenced by the values obtained at 7 and 12°C (Fig. 8a). Moreover, phlorotannins did decay Figure 4. Relationships between (a) the percentage of inhibition of Fv/ = Fm versus biologically effective dose (kJ m2) for the inhibition of photo- exponentially (R 0.571) with the reduction in Fv/Fm in treat- synthesis in isolate chloroplast (33) during 48 h of laboratory exposure at ments with UV radiation (Fig. 8b). three different temperatures (2, 7 and 12°C). Data are mean SDs (n = 3). DISCUSSION The temperature coefficient (i.e.Q10) calculated for ETRmax ° between 2 and 12 C was 1.74 for the PAR treatment and 2.00 Photosynthetic responses to UV radiation and high + P > for the PAR UV ( 0.05, t-test; Table 1 and S3). temperature regimes This study shows a pronounced, temperature-independent UV- Microscopic observations induced inhibition of the maximum PSII-quantum yields in the The cross section of an apical corticated frond of D. anceps endemic Antarctic macroalgae D. anceps. It is striking that reveals two clear regions, a medullar zone characterized by large, even when D. anceps was constantly incubated at unusually vacuolated cells with storage function and a cortex, characterized high, supra-optimal temperatures (7 and 12°C) over 48 h, Fv/ by highly pigmented cells (Fig. 5a). In the cortex of mature Fm only decreased in response to UV exposure to the same fronds, unilocular sporangia alternated with vegetative cells can level (i.e. 0.422). This remarkable UV effect is likely attribu- be observed (Fig. 5b). Based on the emission of blue autofluo- ted to a damage of the D1 protein in PSII that cannot be rescence, highest contents of phlorotannins were mainly located repaired under UV stress, although at least moderately rising in cell walls of the most external cell layers, particularly in cor- temperatures often stimulate the activity of the PSII repair tex and meristoderm. In some cases, intracellular accumulations cycle (44). In D. anceps, however, increasing seawater temper- of phlorotannins were imaged (Fig. 5c). These high intracellular atures does not seem to activate the PSII repair cycle under concentrations (see below) within vegetative cortical cells were UV stress which indicates that the PSII repair cycle is sensi- confirmed using TEM, by the presence of large and numerous tive to UV radiation as recently presumed (12). Consequently, physodes (Fig. 5d). an acclimation to UV radiation by compensating for the nega- tive UV effects on the photosynthetic apparatus does not seem to occur in D. anceps. This study confirms once more that Phlorotannin contents and cellular antioxidant capacity UV radiation can affect the PSII repair cycle, and that temper- Soluble phlorotannins showed constitutively high concentrations ature increase cannot compensate for the negative UV effects ranging between 40 and 126 mg g 1 DW (Fig. 6). on PSII (12).
Table 1. Summary of the photosynthetic parameters of maximum electron transport rate (ETRmax), initial slope of ETR-E curves (a) and light saturation points of photosynthesis (Ek) estimated as well as Q10 values of ETRmax in Desmarestia anceps.
Q10 2 1 ETRmax a (lmol electrons m s ) 2 1 2 1 1 2 1 Temperature (°C) Radiation (lmol electrons m s ) (lmol photons m s ) Ek (lmol photons m s ) PAR PAR+UV
2 PAR 26.1 4.1 a 0.267 0.05a 100 25a 1.74 2.00 PAR + UV 25.4 4.0 a 0.185 0.04a 139 29b 7 PAR 18.2 2.7 a 0.241 0.01a 75 8a PAR + UV 16.8 3.0 a 0.109 0.05b 186 106b 12 PAR 45.5 9.3 b 0.270 0.03a 168 29a PAR + UV 50.9 1.4 b 0.277 0.02a 184 9b n = 18; measured after 48 h of exposure. Superscript small case letters behind values indicate significant differences between radiation and temperature treatments, determined by the Student Newman–Keuls test. Q10 were calculated for ETRmax between 2 and 12°C. Photochemistry and Photobiology, 2016, 92 461
a b
cd
Figure 5. Ultrastructure and localization of phlorotannins in Desmarestia anceps. (a) Cross section of mature apical pieces imaged under bright field; (b) localization of phlorotannin rich regions (ph) using violet–blue light excitation where phenolic compounds are imaged as white to blue–green and chlorophyll as orange–red autofluorescence. (c) toliudine blue stained cross section of cortex showing the arrangement of vegetative cells and unilocular sporangia; (d) transmission electron microscopy (TEM) image of cortical cells showing large and abundant phlorotannin containing physodes (phy). Other structures: chloroplasts (chl); nucleus (n), cell wall (cw).
It is surprising that the photophysiology of D. anceps along fixation in the Calvin–Benson cycle are highly affected by low with other endemic Antarctic macroalgae, which were character- temperatures, which, in turn, exacerbate photoinhibition and ized to be strongly adapted to the current polar seawater temper- photodamages in photosynthesis (52,53). Nevertheless, Antarctic atures (≤2–3°C), is physiologically capable of tolerating such sublittoral macroalgae, which are constantly exposed to low tem- high temperatures not only over short periods of time (e.g.6h) peratures (Fig. 1), are physiologically well adapted to these tem- but also under UV and temperature stress over 48 h (12,13,45). peratures (e.g. by highly variable membrane lipid composition, Although heat stress-induced decreases in Fv/Fm and reductions low-energy activation for enzyme reactions and optimization of in carbon fixation results in structural changes and denaturation energy balance within photosystems) to acclimate efficiently to of proteins in PSII and the Calvin–Benson cycle, these effects UV stress (reviewed by 54). Although these mechanisms have could not be detected in D. anceps (13,46). Although strongly not been well studied in macroalgae yet, psychrophilic microal- cold-adapted endemic Antarctic macroalgae including D. anceps gae and cyanobacteria show that the reduction of the functional are thought to be sensitive to high seawater temperatures, they size of PSII by increasing the carotenoid/chlorophyll a ratio can photosynthesize well even above 2°C (12,45,47), which can seems to be essential to acclimate to high PAR or UV radiation be attributed to the adaptation of the proteins associated with the at low temperatures (55,56). A transcriptomic study of the Arctic photosynthetic process to higher temperatures (45). kelp Saccharina latissima suggest that this polar macroalga can Moreover, D. anceps can tolerate irradiances of UV radiation optimize its photosynthetic metabolism by expressing genes asso- that are higher than those occurring in their subtidal habitats in ciated with fucoxanthin-chlorophyll-binding protein, light- the field by showing slight to moderate decreases in Fv/Fm along harvesting complex as well as PSI and PSII. Compared to its with a complete recovery from UV stress within a few hours optimum temperature of photosynthesis (i.e.7°C), a higher tem- exposed to low PAR (4,11,48). Because mechanisms of photoac- perature (12°C) can mitigate the negative effects of UV radiation climation (e.g. faster repair of photodamaged D1 proteins in by the expression of genes linked to ROS scavenging (e.g. ascor- PSII) and photoprotection (e.g. dissipative energy quenching) bate peroxidase, both Fe- and Mn-containing superoxide dismu- can operate more efficiently at higher temperatures (49–51), the tase and mitochondrial thioredoxin) (17). low inhibition of photosynthesis detected at both 7 and 12°C can The photosynthetic electron transport capacity (ETRmax)in be understood as an efficient acclimation of the photosynthetic D. anceps was highest at 12°C (Table 1). The rate of enzyme- apparatus. For example, the pronounced UV-induced inhibition driven physiological processes, such as photosynthesis, strongly of Fv/Fm in both a sub-Antarctic and an Antarctic Ulva species depend on temperature as shown by the temperature coefficient at 0°C was shown to be compensated by an increased seawater Q10 (39,57). It often ranges between 2.0 and 3.0 in biological sys- temperature at 10°C (10). Although pure photochemical pro- tems, which indicates an increase in the rate of a measured meta- cesses (e.g. light absorption and excitation transfer) are indepen- bolic process by a factor of 2 to 3 in response to a 10°C rise in dent of temperature, enzyme-driven reaction such as CO2 temperature. Although photochemical processes such as excitation 462 Mar ıa Rosa Flores-Molina et al.
Figure 6. Total soluble phlorotannin contents of Desmarestia anceps in Figure 7. Variation in the cellular antioxidant capacity of extracts of response to PAR (black columns) and PAR+UV (gray columns) treat- Desmarestia anceps in response to PAR (black columns) and PAR+UV ments at different temperatures: (a) 2°C, (b) 7°C and (c) 12°C. Data are (gray columns) treatments at different temperatures: (a) 2°C, (b) 7°C and means SDs (n = 3). (c) 12°C. Data are means SDs (n = 3). transfer are independent of temperature, the enzyme-driven bio- shown that rates of both photosynthesis and dark respiration chemical reactions of the Calvin–Benson cycle are temperature- increased significantly at 20°C, even though a (i.e. photosyn- sensitive (58). A Q10 of 1.4 has been described for the photosyn- thetic efficiency) remained unchanged. Based on the temperature thetic oxygen production of D. anceps (between 0 and 10°C), demands of cultured specimens (Fig. 1), current Antarctic sea- which is in line with the endemic Antarctic brown macroalgae H. water temperatures are suboptimal for photosynthesis of the spe- grandifolious (1.4) and Ascoseira mirabilis (1.6) (45). However, in cies, although endemic Antarctic Desmarestiales have been this study, Q10 of ETRmax in D. anceps were (almost) doubled subjected to these temperatures close to 0°C for at least the past (1.74–2.00), which suggest that the photosynthetic electron trans- 15 million years (60). Apparently, they have also retained a set port capacity is differently influenced by temperature-sensitive of physiological adaptations to respond to higher temperatures, reactions (e.g. by thylakoid reactions involved in electron trans- which is reflected in USTs of gametophytes close to 10°C (31). port) than oxygen production. Tolerance to high temperatures seem to be a highly conserved, Photosynthesis of D. anceps was shown to be remarkably adaptive trait in Antarctic Desmarestiales that developed from an unaffected by the global climate change such as ocean acidifica- ancestor species of Desmarestiales, allowing closely related Des- tion and rising temperatures (12,59). For example, through O2- marestia species to spread from the southern hemisphere north- based photosynthetic measurements of macroalgal thalli exposed wards by crossing the equator and to exist in warmer waters to temperature range between 0 and 20°C (1 h) (45), it was (30). Photochemistry and Photobiology, 2016, 92 463
the intertidal brown macroalgae L. spicata: increased contents of both soluble and insoluble phlorotannins were associated with a lower UV-induced inhibition of Fv/Fm and DNA damage (21). In this study, a negative relationship between phlorotannin contents and the UV-induced inhibition of Fv/Fm was found, which was marked in algae exposed at 12°C. An increase in phlorotannins with increasing temperature as shown for the temperate brown macroalgae Pelvetia canaliculata and Asco- phyllum nodosum (both Fucales) indicates that the synthesis of phlorotannins can be beneficial to these organism, as they can be efficient chemical barriers against multiple stressors (i.e. antiherbivore resistance, antimicrobial, antifouling, protection against UVB radiation) (21–24,61,65). Phlorotannins do not only absorb harmful UV radiation to prevent cellular damage when they form part of their cell walls or exudate them into their surrounding environment (i.e. acting as UV-shielding substances), they can also be important for detoxifying ROS produced due to UV stress (8). In this study, contents of soluble phlorotannins were related to the antioxidant capacity of extracts, which has been reported for both Antarctic (4,5) and temperate brown macroalgae (23,24,26,27,68,69). When seaweeds are exposed to abiotic stress such as high tem- peratures and UV radiation protective mechanisms can be acti- vated to protect cells against oxidative damage (18,19,52,70), which could include not only phenolic compounds but also vari- ous antioxidant enzymes. In general, due to high concentrations of phlorotannins in Arctic brown macroalgae, ROS-scavenging via antioxidant enzymes can become less relevant compared to other algal groups (70). In D. anceps, ROS-scavenging by super- Figure 8. Nonlinear correlation between (a) total soluble phlorotannins and the cellular antioxidant capacity as well as (b) total soluble oxide dismutase (SOD) were higher in individuals collected from fi phlorotannins and the percentage of UV-induced inhibition of Fv/Fm in the upper subtidal (5.5 m) compared to its conspeci cs from dee- Desmarestia anceps. per growth sites (11). This pattern is similar to that one found for the antioxidant capacity of soluble phlorotannins of D. an- ceps between 10 and 30 m (5). These findings suggest that both Phlorotannins and antioxidant capacity types of ROS-scavenging mechanisms are complementary and, Although total soluble phlorotannins from D. anceps did not therefore, raise the question to a stressor-dependent activation. respond to UV radiation throughout the experiment at each For example, various gene products associated with photosyn- temperature tested, significant differences between the experi- thetic adjustments, various ROS scavengers and stress proteins mental temperatures could be detected: average contents are up-regulated in Arctic Laminariales after exposure to com- obtained at 2 and 7°C were close to 50 mg g 1 DW, while bined action of UV and temperature (16,17). those measured in conspecifics at 12°C were approximately twice as high (~100 mg g 1 DW). Although contents of soluble Ecological implications phlorotannins in Antarctic Desmarestiales seem to be normally high and noninducible by UV radiation (4,12,61), they have The ability of endemic Antarctic brown macroalgae to photosyn- UV-absorbing properties (i.e. maxima absorbance is at 195 and thesize at low temperatures and low-light conditions as well as 265 nm) simply due to their aromatic chemical structure (62) under seasonal fluctuations of light are important factors for their and, therefore, act as UV screens (5). However, their primary ecological success (2,28). However, D. anceps is able to grow at function seems to be in cell wall strengthening (63), and temperatures close to 5°C and show maximum survival tempera- wound healing (40,64). High contents of soluble phlorotannins, tures of ~12°C (Fig. 1). These findings point out its high stress a characteristic of Antarctic Desmarestiales (65–67), are in con- tolerance expressed as high photosynthetic efficiency, low inhibi- trast to other brown macroalgae species (e.g. Fucales, Laminari- tion of photosynthesis and high phlorotannin concentrations as a ales) with low contents of phlorotannins, regardless of their “safety strategy” to cope with extremely variable light conditions biogeographic distribution. For example, UV triggered increases and probably also to ameliorate the effects of potential increases in soluble phlorotannins were hitherto only detected in macroal- of temperature, at least for short periods. Due to the fact that gae with low contents (5–10 mg g 1 DW) such as the Antarc- these organisms are regarded as “ecosystem engineers” in tic fucoid Ascoseira mirabilis (12). Similarly, the intertidal Antarctic coastal ecosystems a high tolerance to stress such as kelps Lessonia spicata (studied as Lessonia nigrescens) and UV radiation can be a relevant functional trait to withstand envi- Durvillaea antarctica, from the sub-Antarctic coasts of Valdivia ronmental changes driven by climate change. In fact, Antarctic (Chile) showed a strong induction of phlorotannins when they canopy-forming species represent a relatively stable environment were exposed to UV-B radiation for 2 to 6 h (27). A putative for the development of understory species along a broad depth photoprotective role of phlorotannins has been demonstrated in profile (71). Recently, the hypothesis that facilitative interactions 464 Mar ıa Rosa Flores-Molina et al. mediated by species attaining key functional traits (e.g. physio- ture demands of marine benthic microalgae and seaweeds in polar logical capacity to endure wide ranges of light conditions or regions. Bot. Mar. 52, 593–608. € physical disturbance) define the ecosystem functioning parame- 3. Gomez, I., G. Weykam, H. Kloser and C. Wiencke (1997) Photosyn- thetic light requirements, metabolic carbon balance and zonation of ters such as biodiversity and biomass has been tested in a com- sublittoral macroalgae from King George Island (Antarctica). Mar. munity in King George Island (72). The results indicated that Ecol. Prog. Ser. 148, 281–293. sharp environmental gradients, e.g. proximity to glaciers and 4. Huovinen, P. and I. Gomez (2013) Photosynthetic characteristics and UV stress tolerance of Antarctic seaweeds along the depth gradient. depth, are less important that trait richness (e.g. the variety of – fi Polar Biol. 36, 1319 133. traits conferring stress tolerance) in con guring the mesoscale 5. Gomez, I. and P. Huovinen (2015) Lack of physiological depth pat- biomass patterns. terns in conspecifics of endemic Antarctic brown algae: a trade-off between UV stress tolerance and shade adaption? PLoS ONE 10, e0134440, 1–2. CONCLUSION 6. Farman, J., B. Gardiner and J. Shanklin (1985) Large losses of total ozone in Antartica reveal season CIOx/NOx interactions. Nature The results of this study suggest that the pronounced tolerance 315, 207–210. of D. anceps to UV radiation and high temperatures can be 7. McKenzie, R. L., L. O. Bjorn,€ A. Bais and M. Ilyas (2003) Changes strongly accounted by their constitutively high levels of soluble in biologically active ultraviolet radiation reaching the Earth’s sur- – phlorotannins. There is a trade-off between these attributes and face. Photochem. Photobiol. Sci. 2,5 15. 8. Bischof, K., I. Gomez, M. Molis, D. Hanelt, U. Karsten, U. Luder,€ M. Y. the strong shade-adaptation that allows D. anceps to colonize Roleda, K. Zacher and C. 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