Journal of Plant Physiology 173 (2015) 41–50
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Ocean acidiﬁcation modulates the response of two Arctic kelps to ultraviolet radiation Francisco J.L. Gordillo a,∗ , José Aguilera b , Christian Wiencke c , Carlos Jiménez a a b c
Departamento de Ecología, Facultad de Ciencias, Universidad de Málaga, Bulevar Louis Pasteur s/n, 29010 Málaga, Spain Departamento de Dermatología, Facultad de Medicina, Universidad de Málaga, Bulevar Louis Pasteur s/n, 29010 Málaga, Spain Alfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, Germany
a r t i c l e
i n f o
Article history: Received 6 May 2014 Received in revised form 19 September 2014 Accepted 20 September 2014 Available online 5 October 2014 Keywords: Alaria esculenta Arctic Ocean acidiﬁcation Saccharina latissima Ultraviolet radiation
a b s t r a c t The combined effects of ocean acidiﬁcation and ultraviolet radiation (UVR) have been studied in the kelps Alaria esculenta and Saccharina latissima from Kongsfjorden (Svalbard), two major components of the Arctic macroalgal community, in order to assess their potential to thrive in a changing environment. Overall results revealed synergistic effects, however with a different amplitude in the respective species. Changes in growth, internal N, C:N ratio, pigments, optimum quantum yield (Fv/Fm) and electron transport rates (ETR) following CO2 enrichment and/or UVR were generally more pronounced in S. latissima than in A. esculenta. The highest growth rates were recorded under simultaneous CO2 enrichment and UVR in both species. UVR-mediated changes in pigment content were partially prevented under elevated CO2 in both species. Similarly, UVR led to increased photosynthetic efﬁciency (␣) and ETR only if CO2 was not elevated in A. esculenta and even under high CO2 in S. latissima. Increased CO2 did not inhibit external carbonic anhydrase (eCA) activity in the short-term but in the mid-term, indicating a control through acclimation of photosynthesis rather than a direct inhibition of eCA by CO2 . The higher beneﬁt of simultaneous CO2 enrichment and UVR for S. latissima respect to A. esculenta seems to involve higher C and N assimilation efﬁciency, as well as higher ETR, despite a more sensitive Fv/Fm. The differential responses shown by these two species indicate that ongoing ocean acidiﬁcation and UVR could potentially change the dominance at lower depths (4–6 m), which will eventually drive changes at the community level in the Arctic coastal ecosystem. These results support an existing consideration of S. latissima as a winner species in the global change scenario. © 2014 Elsevier GmbH. All rights reserved.
Introduction Benthic primary producers in the intertidal and subtidal zones have to face a number of environmental changes related to the global change scenario. One of the most relevant changes in the polar coastal ecosystem is ocean acidiﬁcation by increased dissolution of atmospheric CO2 , but it is still unknown how the interaction with other environmental factors such as the presence of solar ultraviolet radiation (UVR) will affect growth, photosynthetic performance and chemical composition of Arctic seaweeds.
Abbreviations: ␣, photosynthetic efﬁciency; A, thallus absorptance; CCM, carbon concentrating mechanisms; eCA, external carbonic anhydrase; ETR, electron transport rate; ETRm, maximum electron transport rate; Fv/Fm, optimum quantum yield; NRA, nitrate reductase activity; RGR, relative growth rate; UVR, ultraviolet radiation. ∗ Corresponding author. Tel.: +34 952 132385; fax: +34 952 137386. E-mail addresses: [email protected]
, [email protected]
(F.J.L. Gordillo). http://dx.doi.org/10.1016/j.jplph.2014.09.008 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
UVR penetration in coastal water imposes an upper depth limit for a given species, and as a result it might determine the vertical zonation pattern of benthic algal communities (Bischof et al., 2006) with far-reaching consequences for aquatic grazers and other members of these habitats. Ocean acidiﬁcation changes the water carbon chemistry, altering the form and amount of the available dissolved inorganic carbon (DIC), the major substrate for photosynthetic primary producers. The shift in DIC equilibria expected by the end of this century involves a decrease in pH to values around 7.7, a three-fold increase in dissolved free CO2 , only a small (about 10%) increase in bicarbonate and total DIC concentration, and a strong decrease in carbonate concentration to less than half, respect to preindustrial levels (Beardall et al., 2009a,b), bicarbonate remaining as the major form of inorganic C. Predictions about the ecological consequences of ocean acidiﬁcation have mainly focused on the effects on calcifying organisms, particularly those critical to the formation of habitats such as coral reefs. This focus overlooks the direct effects of CO2 on non-calcareous organisms, particularly those that
F.J.L. Gordillo et al. / Journal of Plant Physiology 173 (2015) 41–50
also play critical roles in ecosystem structure such as kelps (Connell and Russell, 2010; Russell et al., 2011; Krause-Jensen et al., 2012). Marine macroalgae in the Arctic occur commonly in the intertidal and subtidal zones of the coastal waters, and play a major role in the coastal carbon cycle, as well as serving as habitat and reproduction and nursing sites for many species. The kelps Alaria esculenta and Saccharina latissima are habitat-forming key species of the mid sublittoral zone of Kongsfjord (Svalbard), although they can also be observed in the low intertidal, and are major contributors to the coastal carbon cycle. A. esculenta dominates in the depth range 5.5–10.5 m and S. latissima in 7.5–12.5 m (Hop et al., 2012), but there is evidence that depth ranges and dominance of kelp species may change due to climatic forcings (Weslawski et al., 2010; KrauseJensen et al., 2012), although little is known on the underlying metabolic processes involved. Macroalgal growth, photosynthetic performance, chemical composition, and nutrient assimilation, have been shown to respond to increased levels of CO2 , as well as to increased UVR by separate; but simultaneous and interactive effects are poorly understood (Swanson and Fox, 2007; Gao and Zheng, 2009). CO2 can increase growth in some species despite their photosynthesis being C-saturated by means of a carbon concentrating mechanism (CCM) (e.g. Gordillo et al., 2001, 2003). Attempts to relate the effects of increased CO2 to the operation of CCMs have been generally unsuccessful (Giordano et al., 2005) and the extension and direction of changes are species-speciﬁc. Some macroalgal species have shown enhanced growth (e.g. Gordillo et al., 2001) while some rhodophytes, both calcifying (Gao and Zheng, 2009) and non-calcifying (Mercado et al., 1999; Israel et al., 1999) showed negative effects in response to elevated CO2 . There are also many species with varying CCM showing no signiﬁcant changes (Israel and Hophy, 2002). The variety in the response pattern of the different species in a community is a clear indication that, in an acidifying ocean, there will be changes in competition, settlement and dominance that will propagate to other trophic levels and affect the entire ecosystem (Connell and Russell, 2010). UVR is well known to cause deleterious effects on the physiological performance and growth of marine photosynthetic organisms (reviewed by Vincent and Roy, 1993; Helbling and Zagarese, 2003; Häder et al., 2011). Potential UVR targets include mainly nucleic acids and proteins (Vass, 1997). In plants, pigments of the photosynthetic apparatus can also be degraded by UVR exposure; but differences in the sensitivity between pigment types depend on species (Teramura, 1983; Häder and Häder, 1989; Figueroa et al., 2010). Regarding the metabolism of seaweeds these UVR effects result in the inhibition of nutrient assimilation (Gómez et al., 1998) damage to DNA (Van de Poll et al., 2002; Roleda et al., 2007) and damage to carbon assimilation mechanisms (Bischof et al., 2000), but there is still no clear insight on how these responses are to be modiﬁed in an acidiﬁed ocean scenario. In the Arctic coastal environment, potentially harmful UVR penetrates down to 6 m in the water column (Hanelt et al., 2001). The overall effect of UVR on photosynthesis and physiological performance is a balance between damage and repair (e.g. Heraud and Beardall, 2000) that can be largely modulated by interactions with other environmental factors such as PAR light, nutrient limitation and levels of dissolved CO2 (Beardall et al., 2009a,b; Häder et al., 2011). Some multifactorial analyses exist dealing with the interactions between CO2 and nutrients (Gordillo et al., 2001, 2003; Russell et al., 2009), and UVR and nutrients (Figueroa et al., 2010). However, only few have focused on the interactions between CO2 and UVR (Swanson and Fox, 2007; Gao and Zheng, 2009). Gao and Zheng showed that enhanced CO2 decreases calciﬁcation of Corallina sessilis, making it prone to UVR damage. Conversely, Swanson and Fox evidenced that, in the long term, enhanced CO2 and UVR might beneﬁt the two non-calcifying kelp species examined, S. latissima
and Nereocystis luetkeana but with species-speciﬁc differences in growth an phlorotanins production. We hypothesise that expected ocean acidiﬁcation may modify the mechanisms of acclimation to UVR, so changing the ability of a given species to thrive in a CO2 enriched environment with a signiﬁcant presence of solar UVR. The aim of this work is to elucidate to what extent the synergistic or antagonist effects of simultaneous CO2 enrichment and UVR can alter growth, photosynthetic performance and chemical composition of two common Arctic species, and shed light on the potential shifts in the community.
Materials and methods Plant material and cultivation Around 30–45 young specimens of the kelp species Alaria esculenta (L) Greville and Saccharina latissima (L) Lane et al. (formerly Laminaria saccharina (L) Lamouroux) were collected by SCUBA diving from the Kongsfjorden, Spitsbergen (78◦ 55 N 11◦ 56 E) at approximately 6 m depth in the Arctic summer (July). The blades of the sampled thalli were about 15–30 cm long. Healthy thalli free of macroscopic epibiota were selected. Between 10 and 15 discs 12 mm in diameter were cut from the meristematic part of each blade for cultivation. A number of discs up to 3.3 g in fresh weight (FW) of A. esculenta or 4.8 g FW of S. latissima were placed in 1.5 L aquaria and kept in culture for 12 days. The difference in weight accounts for the different thickness of the two species so that exposure area was kept the same for both. Every second day seawater was changed and enriched with 10 M NO3 − and 1 M PO4 3− . The aquaria were aerated at 0.5 L min−1 either with nonmanipulated ambient air (C−, ca. 380 ppm CO2 ) or with air enriched with CO2 to 1000 ppm (C+) allowing all discs to circulate without settling. The required level of CO2 was achieved by mixing air with pure CO2 from a gas tank. Final CO2 level in the mixture was continuously recorded by a CO2 sensor (Airsense, DCS, USA) prior to bubbling the cultures. Initial pH values were 8.13 ± 0.02 and 7.71 ± 0.03 for seawater from the fjord and CO2 -enriched seawater, respectively. Total alkalinity was measured by titration resulting in 2530 ± 90 mequiv. kg−1 SW, so that calculated values for pCO2 in equilibrium seawater were 359 and 1028 atm for non-enriched (C−) and CO2 -enriched (C+) seawater, respectively, at 4 ◦ C and 31.8 psu salinity. Final pH of S. latissima cultures before water change averaged 8.32 ± 0.02 and 7.79 ± 0.02 for C− and C+, respectively, corresponding to pCO2 values of 217 atm for C− and 846 atm for C+. Final pH for A. esculenta before water change averaged 8.14 ± 0.03 and 7.71 ± 0.01 for C− and C+, respectively, corresponding to pCO2 values of 351 atm for C− and 1028 atm for C+ Calculations were made using the CO2Calc software (Robbins et al., 2010). Cultures run at 4–5 ◦ C in continuous light. Temperature was regulated by submersing the aquaria in a temperature-controlled recirculating water bath. For the PAR-only treatment (UV−), light was supplied from the top by ﬂuorescent lamps (OSRAM L58/W19) at 30 mol m−2 s−1 and no further lamp or ﬁlter was applied. For UV+ treatment, the above-mentioned white light was proportionally supplemented with ultraviolet radiation (UVR) supplied by Q-Panel 340 lamps (Q-Lab, Dusseldorf, Germany) rendering 2 W m−2 of UVA and 0.2 W m−2 of UVB as measured by a Gröbel RM11 radiometer (Gröbel UV-elektronik, Ettlingen, Germany), which was intercalibrated with a Macam spectroradiometer double monocromator SR22791 (Tranent, Scotland) using the same lamp setting. The contribution of the Q-panel lamps to PAR was only 1.2 mol m−2 s−1 , so no additional cut-off ﬁlter was required. Both PAR and UV irradiance were chosen according to the average daily solar irradiance recorded at 4–6 m depth in the Kongsfjord for this
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time of the year (Bischof et al., 2002). This light setting was chosen to simulate the actual light ﬁeld proportions of PAR, UVA and UVB so that it should not be considered as an UVR-enrichment experiment, but rather a natural UVR absence/presence one. The light spectra of the light sources used are shown in Fig. 1 of the supplemental material. The resulting 300 nm normalised weighting functions and biological effective doses for chloroplast inhibition (Jones and Kok, 1966), general plant damage (Caldwell, 1971) and DNA damage (Setlow, 1974) are shown in Table 1 of the supplemental material. Supplementary Fig. I and Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2014.09.008. The different combinations of CO2 and UVR (four treatments) were assayed using three independent aquaria as triplicates for each treatment and species. Growth and biochemical composition Relative growth rate (RGR) was calculated as the intrinsic growth rate, and expressed as a percentage of daily increase in fresh weight biomass, by weighing 10 discs of alga from each culture at the beginning and at the end of the experiment (day 12), and assuming an exponential growth model
ln(FWf /FWi ) t
where FWf and FWi are ﬁnal and initial fresh weight of 10 discs, respectively, and t is time in days. Chlorophyll a and total carotenoids were extracted in N, N-dimethyl-formamide overnight (4◦ C) after re-hydrating freezedried thalli. For spectrophotometric quantiﬁcation, the equations given by Wellburn (1994) were used. Tests performed in our laboratory demonstrated that the freeze-drying process does not alter the absorbance characteristics of pigments, so that determination was not signiﬁcantly affected (t-test, P < 0.01). Total C and total N were obtained from freeze-dried material stored at −20 ◦ C until analysis. Analyses were performed by using a C:H:N elemental analyser (Perkin-Elmer 2400CHN). Photosynthetic performance Optimum, and effective quantum yield for charge separations at photosystem II (PSII) reaction centres (Fv/Fm and ФPSII , respectively) was estimated by ﬂuorescence by means of a pulse amplitude modulated ﬂuorometer (Diving-PAM, Waltz, Effeltrich, Germany) after 10 min of incubation in darkness. Fv is the maximal variable ﬂuorescence of a dark-adapted sample, and Fm the ﬂuorescence intensity with all PSII reaction centres closed. The electron transport rate (ETR) between PS II and PS I was calculated for a range of actinic light irradiance (I) from 16 to 900 mol m−2 s−1 in short (30 s) incubations producing rapid light curves: ETR = ˚PSII · I · 0.5 · A where ФPSII is the effective quantum yield (F/Fm ), 0.5 stands for the assumption of even contribution of excitons to PS I and PS II, and A is the absorptance of the thallus for PAR light as measured with the ﬂat light sensor provided by the PAM manufacturer. Although absolute values from this sensor could be not as accurate, it showed reliable for relative calculations A=1−
where I0 is the incident irradiance of PAR to the light sensor in the absence of algae, and It is the transmitted irradiance with a thallus of alga being located covering the light sensor. An intercomparison of ФPSII measurements made using a PAM 2000 and Diving PAM revealed only marginal non signiﬁcant differences (t-test, P > 0.05) for a number of green, red and brown seaweeds (not shown). The initial slope of the ETR-light curves (␣) was calculated by ﬁtting a linear equation to the ﬁrst ETRlight points, and is considered an estimation of photosynthetic efﬁciency. Maximum ETR (ETRm) was calculated by ﬁtting the rapid light curves to a rectangular hyperbolic saturation model: ETR =
ETRm · I I0.5 + I
where ETRm is the maximum ETR and I0.5 the semisaturation irradiance. Equation ﬁtting was performed using the software Kaleidagraph (Synergy Software, PA, USA). Enzymatic activities The enzymatic methods described below were applied on fresh material taken directly from the incubation aquaria. The external carbonic anhydrase (eCA EC 188.8.131.52) activity was measured potentiometrically, according to Haglund et al. (1992). The assay was carried out at 0–1 ◦ C determining the time taken for a linear drop in pH in the range 8.5–7.5 in cuvette containing 3 mL of a buffer (50 mM TRIS, 25 mM ascorbic acid, and 5 mM EDTA). Small pieces of fresh thalli weighing 150–250 mg FW were taken directly from the cultures, and immediately washed with distilled water and placed in the assay cuvette. The reaction was started by adding 1 mL of icecold CO2 -saturated distilled water. The eCA activity was calculated as (t0 /tc ) − 1, where t0 and tc are the times taken for the pH change in the absence and the presence of the alga, respectively, and then expressed as relative enzymatic activity (REA) g−1 FW. Typically, 5–6 discs were assayed. Nitrate reductase (NR, EC 184.108.40.206) activity of fresh material was measured based on the in situ method described by Corzo and Niell (1991) and according to modiﬁcations reported in Gordillo et al. (2006). With this method, the enzyme is assayed in its original cellular location preventing deactivation and denaturalization. The activity is measured as the rate of NO2 − produced. The NO2 − in the assay medium was quantiﬁed spectrophotometrically according to Snell and Snell (1949). The observed activity is a potential measure of the NR activity of the cell under the conditions prior to the assay. Typically, 3 independent replicates were assayed. Statistical analyses Replicate measurements (n = 4–8 independent thalli, as indicated elsewhere) were tested for signiﬁcance of differences by two-way ANOVAs followed by post hoc Holm–Sidak paired tests. Correlation analyses between variables were analysed by Pearson product-moment coefﬁcient. All tests were performed using the SigmaStat 3 statistical software (Systat Software Inc., Chicago USA) and the signiﬁcance level was set at P = 0.05. Results High CO2 increased growth rates of both A. esculenta and S. latissima by 57 and 151%, respectively, when UVR was present (Table 1). The increase in growth rate was signiﬁcantly larger in S. latissima than in A. esculenta. Without CO2 enrichment, UVR alone increased the growth rate of S. latissima but not of A. esculenta. The upregulation of growth by combined CO2 and UVR enrichment was higher than the sum of the effects of UVR and CO2 considered separately,
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Table 1 Relative growth rate as daily percentage of increase in FW (% day−1 ) of discs of A. esculenta and S. latissima (initial disc diameter 1.2 cm) cultured under non-manipulated (C−) and CO2 -enriched air bubbling (C+, 1000 ppm), and under PAR-only (UV−) and PAR + UV radiation (UV+). Standard deviation in brackets (n = 4). Different superscripts for signiﬁcant differences between treatments (P < 0.05).
Alaria esculenta Saccharina latissima
1.81 (0.14)a 2.06 (0.09)a
0.96 (0.04)b 2.24 (0.67)a
1.65 (0.36)a 3.23 (0.36)b
2.84 (0.23)c 5.17 (0.08)c
evidencing a synergistic effect between UVR and CO2 in the overall metabolism for both species (Table 2). Initial stress by either CO2 or UVR on photosynthesis of both species was evidenced by transient drops in optimum quantum yield (Fv/Fm) that gradually recovered to initial and control values (Fig. 1). In A. esculenta, the transient drop in Fv/Fm was observed for both C+ conditions but not for UV+C−. When UVR was combined with C+, this drop was signiﬁcantly more pronounced than in UV−C+, evidencing an additive effect of UVR and high CO2 . By day 6 of culture all treatments recovered Fv/Fm values close to the initials, but signiﬁcantly lower at high CO2 (Table 2). In S. latissima the transient drop was similar for all treatments (except the control) and recovery was completed by day 6 when either UVR or CO2 enrichment were applied separately, but not when applied simultaneously (UV+C+), which took the full 12 days to reach values close to the initials. Final values were also signiﬁcantly lower at high CO2 (Table 2). CO2 and UVR also affected the characteristics of the ETR-light curves. In general, initial slope (␣) values transiently increased under UVR and decreased by CO2 enrichment (Fig. 2A). However, ﬁnal values were the highest at combined U+C+; about 70% for A. esculenta and 200% for S. latissima, respect to their control values (UV−C−). In A. esculenta, control culture conditions did not affect ETR at culture irradiance for the ﬁrst 6 days of experiments, but a 45% decrease respect to the initial values was observed at day 12 (Fig. 2B). Respect to the control, UVR stimulated ETR under low CO2 conditions (UV+C−), while high CO2 led to a decrease in ETR. In S. latissima, CO2 enrichment resulted in a transient minimum value of 1.2 mol e− m−2 s−1 , which means a 70% decrease respect to initial values. In both species UVR stimulated ETR, reaching signiﬁcantly higher values than UV− by the end of the culture period,
regardless the CO2 condition. Final values were only affected by UVR (Table 2). Maximum ETR (ETRm), as estimated by rapid light curves ﬁttings, showed a pattern similar to that of ETR at culture irradiance (Fig. 2C). Transient upregulation was more pronounced under UVR, while they were partially prevented by high CO2 in A. esculenta. In S. latissima, CO2 enrichment led to a transient minimum in both UVR conditions. By the end of the experiments, UVR stimulated ETRm in both species, but the effect of CO2 was not signiﬁcant (Table 2). Final values were the highest at U+C−; about 80% for A. esculenta and 101% for S. latissima, respect to their control values (UV−C−). The experimental treatments affected both the chlorophyll a and the total carotenoids content in both species (Fig. 3). In A. esculenta, chlorophyll a and carotenoids gradually decreased in the control treatment (UV−C−) after 2 days of culture, presumably due to acclimation to culture conditions. Respect to the control, both UVR and high CO2 induced chlorophyll a and carotenoids degradation during the ﬁrst days of culture, the strongest decay of about 67% corresponding to UV−C+ cultures. However, during the second half of the experimental period pigment degradation ceased in UV+ cultures. Final values were signiﬁcantly affected by CO2 and UVR enrichment (Table 2), although in opposite directions, so that CO2 prevented UVR-mediated accumulation observed after day 2. In S. latissima, control culture conditions (UV−C−) did not affect the chlorophyll a and carotenoids content for the whole experimental period (Fig. 3). Respect to the control, UVR stimulated chlorophyll a and carotenoids accumulation by day 6 up to 3-fold the initial values, but only in the absence of CO2 enrichment. This stimulation effect of UVR was partially restricted under CO2 enrichment (UV+C+), reaching only a 2-fold increase respect to the initial values. In the absence of UVR, unlike A. esculenta, increased CO2 led to
Table 2 Summary of P values of two way ANOVAs of end-point measurements of Alaria esculenta and Saccharina latissima cultured under non-manipulated air (typically 380 ppm CO2 ), and CO2 -enriched air (1000 ppm CO2 ) supplied through the bubbling system, and under PAR-only or PAR + UV radiation.
Growth rate Fv/Fm ␣ ETR ETRm Chlorophyll a Total carotenoids Total C Total N C:N eCA activity NR activity *
Signiﬁcant values (P < 0.05).
A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima A. esculenta S. latissima
CO2 × UVR
0.284 0.016* 0.013* 0.037* 0.134