J Appl Phycol (2011) 23:363–369 DOI 10.1007/s10811-010-9548-0
Effect of nitrate concentration and UVR on photosynthesis, respiration, nitrate reductase activity, and phenolic compounds in Ulva rigida (Chlorophyta) Alejandro Cabello-Pasini & Víctor Macías-Carranza & Roberto Abdala & Nathalie Korbee & Félix L. Figueroa
Received: 25 March 2010 / Revised and accepted: 11 June 2010 / Published online: 6 July 2010 # Springer Science+Business Media B.V. 2010
Abstract Seaweeds growing in the intertidal zone are exposed to fluctuating nitrate and ultraviolet radiation (UVR) levels. While it has been shown that elevated UVR levels and the decrease of nitrate concentration can reduce photosynthetic levels in seaweeds, less is known about the combined effect of nitrate levels and UVR on metabolism and photoprotection mechanisms of intertidal species. Consequently, the objective of this study was to evaluate the effect of nitrate concentration and UVR treatments on photosynthesis, respiration, nitrate reductase activity and phenolic compound levels of Ulva rigida (Chlorophyta). There was a two- to threefold increase in maximal gross photosynthesis (GPmax) and respiration rates, as nitrate increased from 0 to 50 μM NO3−. Similarly, nitrate reductase activity increased linearly from low values in algae incubated at 0 μM NO3 to high values in tissue incubated at 50 μM NO3−. Phenolic compounds in the tissue of U. rigida increased approximately 60% under 50 μM NO3− relative to those incubated at 0 μM NO3−. Algae exposed to UVR (8 h) showed a significant decrease in the effective quantum yield and respiration, however, no effect was observed in the phenolic compounds levels. Full recovery of effective quantum yield was observed after U. rigida was transferred for 48 h to low PAR. Nitrate A. Cabello-Pasini (*) : V. Macías-Carranza Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, A.P. 453, Ensenada, Baja California 22800, Mexico e-mail:
[email protected] R. Abdala : N. Korbee : F. L. Figueroa Departamento de Ecología, Facultad de Ciencias, Universidad de Málaga, Campus Universitario Teatinos, Málaga 29071, Spain
reductase also decreased after an 8-h UVR exposure, but no differences were observed among the nitrate treatments. This study shows that high nitrate levels reduced the negative effect of UVR on the effective quantum yield and increased the recovery of key metabolic enzymes. It is possible that the increase of phenolic compounds in the thallus of U. rigida under high nitrate levels provide a photoprotective mechanism when exposed to high UV levels during low tides. Keywords Nitrate . Photosynthesis . Nitrate reductase . Phenolic compounds . Photoprotection . UVR
Introduction Marine macrophytes from the intertidal are exposed to extreme fluctuations of physicochemical parameters. Midday low tides can result in an increase of photosynthetically active radiation (PAR), increased levels of UV radiation (UVR), nitrogen depletion, desiccation, high temperature stress, etc. (Lobban et al. 1985). When exposed to high PAR or UVR levels, seaweeds have to protect their photosynthetic apparatus either by autoshading or by synthesizing photoprotective compounds such as mycosporine-like amino acids in red macrolagae (Korbee et al. 2006) and phenolic compounds in brown macroalgae (Abdala-Díaz et al. 2006). In seaweeds increased UV radiation has been shown to increase DNA damage (Van de Poll et al. 2001), inhibit photosynthetic and nitrogen assimilation enzymes (Altamirano et al. 2000; Bischof et al. 2000), cause photoinhibition of photosynthesis (Häder and Figueroa 1997) and reduce growth rates (Pang et al. 2001). Because seaweeds are sessile organisms, they have acclimated (or
364
adapted) to such extreme conditions. The synthesis of UVabsorbing compounds, DNA repair mechanisms and photoprotective schemes, such as dynamic photoinhibition, allow macroalgae to survive under increasing irradiances levels (Häder and Figueroa 1997; Korbee-Peinado et al. 2004; Bischof et al. 2006). It has also been shown that the synthesis of photosynthetic accessory pigments, such as phycobiliproteins, diminishes UV harmful effects in red seaweeds (Sinha et al. 1995). Green seaweeds have also developed photoprotective mechanisms to cope with high PAR and UVR levels (Figueroa et al. 2003). For example, it was demonstrated that UVB radiation stimulated the accumulation of UV-absorbing compounds in Ulva pertusa (Han and Han 2005). Furthermore, the growth of U. rigida from Gibraltar Strait appears to be strongly impacted by high PAR levels rather than enhanced UVB radiation (Altamirano et al. 2000). In the case of U. rotundata from Cadiz Bay, detrimental effects of UVB radiation on photosynthesis and RUBISCO activities were observed in top layers of mat-like canopies, whereas, subcanopy algae were well shielded from harmfully UV radiation. In both cases, the results highlighted the synergistic effects of UV and PAR promoting canopy arrangements of Ulva in these environments (Bischof et al. 2003). Inorganic nitrogen availability plays a critical role in the physiology of marine seaweeds and the productivity of complete ecosystems (Lapointe and Duke 1984). Nitrogen depletion has been shown to increase photoinhibitory responses in photosynthesis of marine organisms, including macroalgae (Korbee-Peinado et al. 2004; Huovinen et al. 2006). On the other hand, photosynthetic pigments are generally positively correlated with nitrogen availability and rapidly respond to varying nitrogen levels (Davison et al. 2007). Biliproteins, for example, have been reported as nitrogen-storage compounds in c N-rich conditions and as nitrogen sources in N-limiting conditions (Lobban et al. 1985). In addition to PAR, UVR can impact pigment abundance depending on nitrate and ammonium levels (Döhler et al. 1995; Häder and Figueroa 1997; KorbeePeinado et al. 2004; Huovinen et al. 2006). Furthermore, key enzymes such as nitrate reductase (NR), the first enzyme of the nitrate assimilatory pathway and responsible for the reduction of nitrate to nitrite in autotrophs, is also affected by UVR (Figueroa and Viñegla 2001). The effect of UVR on NR is not the same among macroalgal species, i.e., UVA enhanced photosynthesis and stimulated both carbonic anhydrase and NR activity in Fucus spiralis whereas in Ulva olivascens UVA and UVB provoked an inhibition of NR (Viñegla et al. 2006). In red algae, photosynthesis and the accumulation of nitrogen compounds such as biliproteins and UV screening substances increased with nitrogen supply, whereas UVR stimulated photoprotection mechanisms (Korbee-Peinado et al. 2004; Korbee et al. 2005; Huovinen et al. 2006; Korbee
J Appl Phycol (2011) 23:363–369
et al. 2006). In green algae such as Dasycladus vermicularis (Pérez-Rodríguez et al. 2001) phenolic substances are accumulated under UVR but less is known on the combined effects of UVR and nitrate on photosynthesis and other metabolic processes. Consequently, the objective of this study was to test the hypothesis that nitrate reduces the negative effects of UVR on the physiology of U. rigida not only by stimulation of photosynthetic activity but also of the accumulation of UV-absorbing compounds, as it has been reported in other macroalgae (Korbee-Peinado et al. 2004).
Materials and methods Ulva rigida was collected in the intertidal zone of Malaga, Spain (36° 47′ N, 4° 19′ O) and transported in ice coolers to the laboratory in spring–Summer of 2004. Tissue samples of approximately 6 cm2 (approximately 2 g total fresh weight (FW)) were incubated for 2 weeks in six acrylic containers (2 L, 15 samples per container) with seawater containing 10 μM PO4 and 0, 5, 10, 25 and 50 μM NO3− (for the phenolics experiments incubations were conducted up to 100 μM NO3−). Nutrients were added and seawater was changed every day, except the 0 μM NO3− treatment which was changed once weekly. The NO3− concentration used in this experiments are within the range observed in the coastal waters of the Malaga, i.e., less than 5 μM NO3− (oligotrophic) to higher than 25–50 μM NO3− in coastal areas with sewage effluents (Ramírez et al. 2005). Samples were maintained at 15°C and with a photosynthetic irradiance of 100 μmol photons m−2 s−1 using cool white fluorescent bulbs (Philips CW, 40 W) and a photoperiod of 12:12 h light:dark. The thalli were kept in constant suspension by bubbling air. Thallus to volume rate was maintained constant by trimming growth in each tissue sample every other day. The experiments were run three times and physiological parameters evaluated as described below. Oxygen evolution and chlorophyll fluorescence Photosynthetic rates in U. rigida were determined using polarographically measured rates of steady-state O2 evolution (Rank Brothers, Inc., England). Tissue (n=6) of 2.5 cm2 was incubated in seawater (2.2 mM DIC) at 15°C in 5 mL jacketed chambers connected to a water-circulating bath after a 0.5 h preincubation in darkness. Halogen lamps (Quartzline, 150 W) were used as a light source, and irradiance was varied using neutral-density filters from 0 to 600 μmol photons m−2 s−1 (Lee Filters, England). Maximum oxygenic gross photosynthesis (GPmax), and the initial slope of the photosynthesis vs. irradiance curve (α) were determined by a non-linear direct fitting algorithm (Sigma
J Appl Phycol (2011) 23:363–369
Plot, Jandel Scientific) of the data to the exponential equation described by Webb et al. (1974) In vivo chlorophyll fluorescence of PSII was determined (n=6) with a portable pulse amplitude modulated fluorometer (DIVING PAM, Walz, Germany). Intrinsic fluorescence (Fo) was determined after maintaining the tissue in darkness for 1 to 2 h. A saturating actinic light pulse (9,000 μmol photons m−2 s−1, 800 ms) was applied to obtain maximum fluorescence (Fm) in the dark adapted samples. Variable fluorescence (Fv) was determined as the difference between Fm and Fo, and optimum quantum yield (Fv/Fm) was calculated as the rate of Fv to Fm (Schreiber et al. 1994). The effective quantum yield of PSII (ΔF/F′m) was determined in light adapted tissue according to Schreiber and Neubauer (1990): 0
0
0
ΔF=Fm ¼ Fm Ft =Fm 0
where Fm is the maximal fluorescence of light adapted tissue induced by a saturating actinic light pulse (9,000 μmol photons m−2 s−1, 800 ms), and Ft is the intrinsic steady-state fluorescence in the light adapted tissue. The electron transport rate (ETR) was determined according to the following formula: ETR (μmol e− m−2 s−1)=ΔF/F′m ×E× A×0.5, where A is tissue absorptance (Schreiber and Neubauer 1990), E the irradiance and 0.5 the factor that accounts for the partitioning of energy between PSI and PSII. Tissue absorptance (n≥6) was determined using an integrating sphere (LICOR-1800) between 400 and 700 nm according to the formula: A ¼ 1 T R; where T is transmittance and R is reflectance of the tissue. Simultaneous measurements of oxygen evolution and ΔF/F′m were conducted by introducing a 2 mm diameter fiber optic sensor (PAM) into the oxygen chamber. Values of ΔF/F′m were determined after light steady oxygen evolution (approx. 5 min) at each irradiance. Irradiance was increased after a 10-min period of darkness. Gross photosynthesis was calculated by subtracting the respiration measured after each irradiance period. Phenolic compound determination Phenolic compounds in the tissue of U. rigida were determined after the incubation period following the procedure described by Folin and Ciocalteu (1927). After the incubation period, tissue of 0.95 cm2 was cut using a cork borer (n=7). Disks were placed in Eppendorf vials with 1 mL of 80% (v/v) methanol, minced with small scissors and incubated at 70°C for 1 h. Samples were centrifuged at 13,000×g for 10 min. The reaction assay consisted of 200-μL sample extract, 50-μL Folin–Ciocalteu
365
reagent (3 min incubation), and 750 μL of 7.5% (w/v) Na2CO3. The reaction assay was incubated at room temperature in darkness for 2 h and the absorbance evaluated at 765 nm. The concentration of phenolic compounds in the tissue was standardized against a standard curve prepared with phloroglucinol. Nitrate reductase Nitrate reductase in tissue of U. rigida was determined modifying the protocol described by Corzo and Niell (1991) and Lartigue and Sherman (2002). Tissue was cut with a cork borer (3.43 cm2), and placed in a 1.5 mL microcentrifuge tube with 1.25 mL assay buffer (20 ppm nitrogen-free artificial seawater, pH 8.2, 2.25% (v/v) npropanol and 30 mM KNO3). The tissue was minced with small scissors and the samples incubated in darkness in a water bath at 30°C for 1 h. After the incubation period, the assay tubes were incubated for 5 min at 95°C to denature algal enzymes and to liberate nitrite from the cells. Controls were placed in 95°C water for 5 min at the beginning of the incubation period. Nitrite was determined after the samples had cooled to room temperature according to Parsons et al. (1984). Samples were centrifuged for 10 min at 12,000×g, and 1 mL of sample was reacted with 200 μL of a solution containing 2% (w/v) sulfanilamide and 0.02% (w/v) N-(1naphthyl)-ethylenediaminedihydrochloride in 1.2 N HCl. Absorbance of the samples was determined at 543 nm and the concentration of NO2 determined against a standard curve prepared with KNO2. Nitrite concentration in the controls was subtracted from the NO2 determined in the samples. Ultraviolet treatments Thalli were incubated for 2 weeks at different nitrate concentrations as described previously. Then algae were exposed for 8 h to 15.8 W m −2 UVR radiation ð15 W m2 UV A ðl ¼ 315 400 nmÞ and 0:80 W m2 UV Bðl ¼ 280 315 nmÞÞ from four Q-Panel 340 lamps (Q-Panel Co., Canada) placed perpendicular to the incubation vessels. PAR was kept at 100 μmol photons m−2 s−1 by using two cool white fluorescent bulbs. Recovery was determined by eliminating UVR and exposing the algae to100 μmol photons m−2 s−1 PAR for 48 h. Effective quantum yield, respiration, nitrate reductase activity, and phenolic compound concentration in the thallus of U. rigida was determined as described previously both during the exposure and recovery period. Statistical analysis Differences in gross photosynthesis, electron transport rate and phenolic compound concentration as a function of
366
J Appl Phycol (2011) 23:363–369
nitrate treatment were evaluated using a one-way ANOVA after testing for normality and homoscedasticity of the data (Sokal and Rohlf 1995). All pairwise multiple comparisons were conducted using Tukey's test. Minimum significance level was established at p
