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Article

Response of Juvenile Saccharina japonica to the Combined Stressors of Elevated pCO2 and Excess Copper

by
Wenze Zhang
1,2,3,
Lianghua He
1,2,
Jiangqi Pan
1,2,
Yuhong Zhou
1,2,
Ruxiang Ge
1,2,
Sufang Li
4,
Yunyun Shi
1,2,
Xinhua Chen
1,2,* and
Yaoyao Chu
1,2,*
1
College of Marine Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Key Laboratory of Marine Biotechnology of Fujian Province, Institute of Oceanology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
Department of Aquaculture and Aquatic Sciences, Kunsan National University, Gunsan 54150, Republic of Korea
4
Laboratoire Génie des Procédés et Matériaux (LGPM), CentraleSupélec, Université Paris-Saclay, 91190 Gif-sur-Yvette, France
*
Authors to whom correspondence should be addressed.
Plants 2023, 12(5), 1140; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12051140
Submission received: 3 January 2023 / Revised: 19 February 2023 / Accepted: 27 February 2023 / Published: 2 March 2023
(This article belongs to the Special Issue Physiological and Biochemical Adjustments in Response to Abiotic Stressors Associated with Climate Change)
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Abstract

:
Coastal macroalgae may be subjected to global and local environmental stressors, such as ocean acidification and heavy-metal pollution. We investigated the growth, photosynthetic characteristics, and biochemical compositions of juvenile sporophytes of Saccharina japonica cultivated at two pCO2 levels (400 and 1000 ppmv) and four copper concentrations (natural seawater, control; 0.2 μM, low level; 0.5 μM, medium level; and 1 μM, high level) to better understand how macroalgae respond to ongoing environmental changes. The results showed that the responses of juvenile S. japonica to copper concentrations depended on the pCO2 level. Under the 400 ppmv condition, medium and high copper concentrations significantly decreased the relative growth rate (RGR) and non-photochemical quenching (NPQ) but increased the relative electron transfer rate (rETR) and chlorophyll a (Chl a), chlorophyll c (Chl c), carotenoid (Car), and soluble carbohydrate contents. At 1000 ppmv, however, none of the parameters had significant differences between the different copper concentrations. Our data suggest that excess copper may inhibit the growth of juvenile sporophytes of S. japonica, but this negative effect could be alleviated by CO2-induced ocean acidification.

1. Introduction

Copper is an essential micronutrient for algae, as it can be involved in many metabolic processes, such as electron transport in photosynthesis and enzymatic reactions in which it acts as a cofactor [1,2]. Total copper concentrations in natural seawater vary around the world but have been reported to be less than 1 μg/L [2,3]. However, as a result of industrial activities and wastewater emissions, the concentration of this metal in coastal seawater has increased several-fold, exceeding the tolerance capability of some seaweeds [4,5,6]. As a result, it has been widely reported that excessive copper inhibited algal growth and photosynthesis, as well as reduced chlorophyll content [6,7,8]. For example, the growth rates of Ulva compressa [3], Ulva reticulata [9], and Porphyra haitanensis [10] were significantly reduced when algae were subjected to high copper concentrations. Toxic effects of copper on the photosynthetic activities of macroalgae, such as Gracilaria domingensis [11], Ulva prolifera [12], Ecklonia cava, and Ulva pertusa [13], have been recorded. In these studies, decreases in the maximum quantum yield (Fv/Fm), maximum electron transport rate (rETRmax), and pigment content were also found.
Since the industrial revolution, the oceans have absorbed approximately one-third of atmospheric CO2 emitted to the atmosphere, resulting in a drop in seawater pH, a phenomenon known as ocean acidification (OA) [14,15]. The growing atmospheric CO2 concentration is anticipated to be between 800 and 1000 ppm by the end of this century [14]. Numerous studies have found that CO2-induced OA affects algal photosynthetic activity, which in turn affects growth [16,17,18]. However, because of the difference in inorganic carbon acquisition mechanisms, macroalgae respond to OA in species-specific ways [16,18,19,20]. The majority of macroalgae use HCO3− and/or CO2 in photosynthesis via carbon concentration mechanisms (CCMs) [21], while a few species only use CO2 via passive diffusion [22,23]. The latter does not require additional energy for carbon acquisition, implying that OA may benefit these species by lowering the energy costs of photosynthesis [24]. As such, OA can display divergent impacts on macroalgae (i.e., negative, neutral, and positive).
Macroalgae in coastal oceans may face a simultaneous shift in several environmental drivers, including heavy-metal pollution and ocean acidification. The simultaneous action of ocean acidification and heavy-metal exposure may have complex and diverse consequences [25,26]. Furthermore, because OH− and CO32− complex with metal ions in seawater and decreased pH results in lower OH− and CO32−, OA affects their bioavailability and toxicity by increasing heavy metals’ solubility [27,28,29,30]. Copper pollution is a common form of heavy-metal pollution in coastal areas. Several studies on the coupled effects of OA and copper pollution on macroalgae have recently been published [6,12,29], and the combined effects may differ from the individual effects of each stressor, showing synergistic or antagonistic effects. For example, an increased pCO2 level reduced the inhibition rate of copper on the growth of U. prolifera [12] and weakened its inhibition of the maximum photosynthetic efficiency (Fv/Fm) of Sargassum fusiforme [6]. In contrast, copper and OA synergistically reduced the growth and photosynthetic performance of Ulva linza [29]. These various responses reflect specific-species capabilities. However, to date, few experiments have been conducted to evaluate the potential combined impacts of OA and copper on kelp species.
Saccharina japonica, a brown seaweed, is a foundational species in subtidal and intertidal habitats in the northwestern Pacific region, including China, Japan, and Korea [31,32]. It not only has significant ecological effects on marine ecosystems by providing habitats, shelters, and breeding areas for a variety of marine organisms [33] but also has significant economic value, such as providing food and raw materials for chemical products, pharmaceuticals, and cosmetics [34,35]. The individual effects of OA and copper on S. japonica have been studied [19,36,37]. These studies revealed that OA could significantly alter meiospore germination, fecundity, the reproductive success of the microscopic stage, and the growth and photosynthetic physiology of sporophytes of S. japonica [19,36,37,38]. Furthermore, investigations regarding the effect of copper on S. japonica showed a reduction in the development of female gametophytes and the growth of young sporophytes [39]. These results indicated that the growth and physiology of the microscopic stage or sporophytes of S. japonica would be significantly affected by OA or copper. Nevertheless, it is not well known how juvenile sporophytes of S. japonica respond to OA and copper in isolation and combination.
Therefore, in this study, we cultured juvenile sporophytes of S. japonica with four levels of copper (natural seawater; 0.2 μM; 0.5 μM; and 1 μM) and two levels of pCO2 (400 and 1000 ppmv). The aim was to investigate the combined effect of OA and copper on the growth, photosynthetic performance, and biochemical composition of S. japonica and illustrate how OA and excess copper affect the physiology of this alga.

2. Results

2.1. Carbonate Parameters

The effects of pCO2 and copper conditions on the seawater carbonate parameters were determined (Table 1). A two-way ANOVA analysis (p = 0.05) showed that pCO2 had a significant effect on all parameters except for total alkalinity (TA), whereas copper concentrations had no significant effect on any of the parameters. The post hoc Duncan comparison (p = 0.05) showed that, compared to 400 ppmv, the average pH and CO32− at the 1000 ppmv level decreased from 7.87–7.90 to 7.53–7.60 and from 80.1–84.8 μmol kg−1 to 38.5–44.7 μmol kg−1 at the four copper concentrations, respectively. However, HCO3− and DIC increased from 2020.9–2029.0 μmol kg−1 to 2105.6–2128.2 μmol kg−1 and from 2132–2138 μmol kg−1 to 2205–2232 μmol kg−1, respectively.

2.2. Relative Growth Rates

The relative growth rates (RGRs) were significantly affected by pCO2 and copper individually and interactively (Figure 1; Table S1). At the lower pCO2 level, there were no significant differences between the control and Lcu, while the RGRs significantly decreased from 21.69% d−1 (control) to 19.81% d−1 (Mcu) and 16.22% d−1 (Hcu), respectively. At the higher pCO2 level, no significant differences were found at different copper concentrations. Additionally, the RGRs at the higher pCO2 level were significantly higher than those at the lower pCO2 level under both Mcu and Hcu conditions. However, no significant differences were found between the two pCO2 levels in control and Lcu conditions.

2.3. Chlorophyll Fluorescence

The Fv/Fm values were significantly affected by the pCO2 level and copper (Figure 2, Table S1). However, there was no significant interaction between pCO2 and copper. At the lower pCO2 level, the Fv/Fm values increased with the rise in copper concentration, but the Fv/Fm values were only significantly increased by 6.24% at Hcu compared to those in the control. In contrast, the Fv/Fm values were not significantly changed by different copper concentrations at the higher pCO2 level. Meanwhile, the higher pCO2 concentration significantly decreased the Fv/Fm values by 8.07% and 7.65% at Mcu and Hcu, respectively, compared to the lower pCO2 level. However, no significant differences were found in Fv/Fm between the two pCO2 concentrations in control and Lcu conditions.
The relative electron transport rate (rETR) values were significantly affected by the pCO2 level, but they were not significantly influenced by copper (Figure 2, Table S1). A significant interaction between pCO2 and copper was detected. At the lower pCO2 level, with increasing copper concentration, the rETR values increased by 59.73% at Hcu compared to those in the control. At the higher pCO2 level, no significant differences were found in rETR values in any copper treatments. Similarly, there were no significant differences between the two pCO2 levels in the control. However, under Lcu, Mcu, and Hcu conditions, the rETR values were significantly decreased by 39.40%, 36.45%, and 50.28%, respectively, at the higher pCO2 level compared to those at the lower pCO2 level.
The non-photochemical quenching (NPQ) values were significantly affected by the pCO2 level, but they were not significantly influenced by copper (Figure 2, Table S1). A significant interaction between pCO2 and copper was detected. At the lower pCO2 level, with increasing copper concentration, the NPQ values only significantly decreased by 49.71% at Hcu compared to the control. At the higher pCO2 level, there were no significant differences at different copper concentrations. Similarly, NPQ values had no significant differences between the two pCO2 concentrations in control and Lcu conditions. However, under Mcu and Hcu conditions, the NPQ values at the higher pCO2 level were significantly increased by 48.79% and 166.37%, respectively, compared to those at the lower pCO2 level.

2.4. Pigment Contents

The contents of chlorophyll a (Chl a), chlorophyll c (Chl c), and carotenoid (Car) were significantly affected by pCO2 and copper individually and interactively (Figure 3; Table S1). At the lower pCO2 level, the contents of Chl a, Chl c, and Car increased with increasing copper concentrations. Compared with the control, Chl a and Car, respectively, were significantly increased by 73.45 and 77.56% at HCu, and Chl c was significantly increased by 27.22% and 47.80% at MCu and HCu, respectively. However, at the higher pCO2 level, the contents of Chl a, Chl c, and Car had no significant differences at any of the copper concentrations. Moreover, the contents of Chl a and Car at the higher pCO2 level significantly decreased by 34.91% and 33.22%, respectively, compared to the lower pCO2 level under the HCu condition. At the same time, higher pCO2 significantly decreased Chl c by 18.34% and 24.41% at Cu and HCu, respectively, compared to the lower pCO2 level.

2.5. Soluble Carbohydrate Contents

The soluble carbohydrate contents were significantly affected by pCO2 and copper individually and interactively (Figure 4, Table S1). At the lower pCO2 level, with the rise in copper concentration, the soluble carbohydrate contents at MCu and HCu were significantly increased by 273.44% and 718.75% compared to those in the control. However, at the higher pCO2 level, the soluble carbohydrate contents did not significantly differ at any of the copper concentrations. Additionally, under the HCu condition, the soluble carbohydrate contents at the higher pCO2 level were significantly decreased by 64.89% compared to those at the lower pCO2 level.

3. Discussion

3.1. The Effect of Copper on Growth and Photosynthetic Physiology at Lower pCO2 Level

Copper is not only an essential trace element for macroalgae growth but also a highly toxic element at higher concentrations. In this study, the growth of juvenile S. japonica sporophytes was progressively reduced in the MCu and HCu treatments at a lower pCO2 level. High copper concentrations have been shown to reduce growth in Gracilariopsis longissima [1], Gracilaria lemaneiformis [40], Hizikia fusiformis [41], P. haitanensis [10], Chondrus crispus, and Palmaria palmata [42]. Excess copper in algae may trigger the synthesis of intracellular ROS [41,43] and impair nutrient assimilation [10,41,44], resulting in decreased growth. On the other hand, the harmful effects of copper on algal growth can be attributed to photosynthetic inhibition. However, in the present study, we found a decoupling between reduced growth and increased photosynthetic efficiency, which is consistent with the reports for Porphyra haitanesis [45], Ascophyllum nodosum, and Fucus vesiculosus [46]. The reason may be that under stress conditions, algae reallocate energy from growth to the maintenance of cellular integrity and the accumulation of compounds such as phytochelatins [47], enzymes [48], pigments [12], and other antioxidant molecules [49], thus resisting the adverse effects of copper [1,50]. This result was evidenced by the enhanced pigment contents in HCu and lower-pCO2 conditions (Figure 3). Thus, when juvenile sporophytes of S. japonica are exposed to higher copper concentrations, the redistribution of energy and disruption in physiological processes may lead to a reduction in algal growth.
In terms of the characteristics relating to chlorophyll fluorescence, excessive copper exposure increased the algal Fv/Fm and rETR but decreased NPQ at the lower pCO2 level. A similar response has been shown in Fucus serratus under copper exposure [51]. The chlorophyll fluorescence parameter Fv/Fm is an indicator of photoinhibition and a measure of the efficiency of the photosynthetic apparatus of PSII in macroalgae [42]. Given that the toxicity of heavy metals is typically thought to be dose-dependent, exposure to higher copper concentrations or for a longer duration may cause greater damage [42,52]. Hence, the increased Fv/Fm and rETR in this investigation may suggest that the addition of 1 μmol/L copper to the culture may be not adequate to impair the photosynthesis of S. japonica. This is supported by research on the brown alga A. nodosum [46], which showed that its Fv/Fm and rETR values were not significantly affected at lower concentrations of Cu2+ (up to 1 mg/L) but were significantly inhibited at 5 mg/L. In addition, the growth rate of A. nodosum was significantly reduced at a 0.1 mg/L copper concentration. As a result, this study’s findings that copper exposure enhanced photosynthesis (Fv/Fm and rETR) and reduced the growth rate reveal that photosynthesis in juvenile S. japonica has a higher tolerance for copper toxicity than growth. The higher electron transfer rate in the excess copper condition in this study may also be due to the production of functional Cu-containing proteins involved in photosynthetic electron transport, such as plastocyanin [53,54], as copper participates in important biological processes as an electron carrier in photosynthesis at certain concentrations [55]. The photosynthetic efficiency would also benefit from the increased pigment concentration. At HCu under the lower-pCO2 condition, NPQ was decreased, in contrast to the elevated Fv/Fm and rETR. In macroalgae, non-photochemical quenching is a form of photoprotective dissipation of entrapped excitation energy through the activation of the xanthophyll cycle, mostly as heat, preventing damage to the photosynthetic apparatus [56]. The decrease in NPQ under copper exposure may indicate that copper affected the photoprotection system of this alga and caused the xanthophyll pool to shrink or become less robust. Hence, the lower NPQ found in this study may indicate that NPQ, rather than Fv/Fm or rETR, would be the first target of copper toxicity in juvenile sporophytes of S. japonica.
Chl a, Chl c, carotenoid, and soluble carbohydrate contents in juvenile S. japonica were increased with rising copper concentrations at the lower pCO2 level. Previously, it was noted that H. fusiformis [41], Sargassum cymosum [4], U. prolifera [12], and Cystoseira tamariscifolia [57] all had higher levels of photosynthetic pigments. Increased pigments may be crucial in reducing the toxicity of copper, even if photosynthetic pigments are responsible for light-energy absorption in the photosynthesis process of algae [58]. First, increased Chl a and Chl c at higher copper levels could be an adaptive strategy, completing the pool of chlorophyll to counteract copper toxicity, since magnesium (Mg2+) in chlorophyll molecules could replace intercellular Cu2+ to decrease the copper toxicity in algae [59]. Secondly, because carotenoids have antioxidative properties [43], and toxic compounds increase the amount of ROS [60], the enhancement of carotenoid content under excess copper conditions in this work may indicate that the alga is subjected to oxidative stress, and carotenoids appear to be an antioxidant substrate used to defend against ROS. Costa et al. [4] and Celis-Plá et al. [57] reported similar responses of photosynthetic pigments, but in these studies, a decoupling phenomenon between the increase in pigments and the decrease in photosynthesis was observed. This contradicts the finding of this study, which found that S. japonica was more resistant to excess copper.
In terms of soluble carbohydrates as photosynthetic products, active photosynthesis may lead to their synthesis by increasing Fv/Fm, rETR, and pigment contents. Furthermore, according to some studies, the carbohydrates excreted by algae can regulate the concentration of copper in the environment and restrict metal uptake by forming complexes [8,61,62]. Consequently, in order to release more carbohydrates into seawater, the alga may tend to stimulate the production of photosynthetic organic compounds, establishing an important barrier to protect cells from the harmful effects of excess copper.

3.2. The Effects of Elevated pCO2 Level on Growth and Photosynthetic Physiology in Ambient Copper Condition

In contrast to the effects of copper, the elevated pCO2 level did not significantly change the growth rate, chlorophyll fluorescence parameters, or biochemical composition in the absence of copper. Kang and Chung [36] also reported that higher pCO2 concentrations had no effect on the growth or chlorophyll fluorescence parameters of S. japonica. Similar phenomena have been observed in other brown algae, including S. fusiforme [7] and Sargassum horneri [63]. To overcome the carbon limitation in seawater, most macroalgae can use gaseous CO2 and the ionic form of HCO3− to drive photosynthesis via carbon-concentrating mechanisms (CCMs). Some macroalgae whose photosynthesis is carbon-limited at current pCO2 levels would benefit from OA because CO2 and HCO3− concentrations in seawater increased markedly with OA. The lack of changes in the photosynthetic apparatus under the elevated-pCO2 condition in this study may indicate that algal photosynthesis is nearing saturation at the ambient inorganic carbon level [6], and thus, no more carbon was fixed by photosynthesis or contributed to growth.

3.3. The Effects of Higher Copper on Growth and Photosynthetic Physiology at Elevated pCO2 Level

When exposed to higher copper concentrations (0.2~1 µM), no significant differences in any of the parameters were observed at the elevated pCO2 level. This indicates that OA weakened the sensitivity of the growth and photosynthetic physiology of S. japonica to excess copper under interactive conditions. In this study, when the copper concentration was increased to 0.5 µM, elevated pCO2 offset the negative effect of copper on growth at the lower pCO2 level. Furthermore, at 1 µM copper concentration, the alleviated effect was more noticeable. These findings indicate that the effects of excess copper on this alga at the ambient pCO2 level could be mitigated by OA, and the combined effects of elevated pCO2 and copper levels could be found when the copper concentration was increased to 0.5 µM.
Previous studies suggest that the lower sensitivity of the growth and photosynthetic physiology of juvenile S. japonica to excess copper at higher pCO2 levels may be related to the competition between H+ and Cu2+ [12,64,65]. When H+ in seawater increased, Cu2+ would be competitively excluded from binding to ligands at the cell surface [64,65], reducing the toxicity of copper. This phenomenon was also found by Gao et al. [12], who noted that the growth of U. prolifera was less negatively affected by 0.5 μmol/L copper at 1000 ppmv compared to 390 ppmv. Moreover, increased concentrations of other free trace metal ions (Me2+) at low pH may be another reason. At low pH, free trace metals became more soluble and less likely to bind to organic matter due to proton competition; thus, the concentrations of the other free Me2+ were increased in water [66]. However, low-toxicity free Me2+, such as Mn2+, Zn2+, and Fe3+, may be able to outcompete the highly toxic Cu2+ for binding ligands at the cell surface, lowering copper uptake and toxicity in algae [66]. Because of the poorer absorption of copper in algae and the competition between Cu2+ and H+ and/or other free Me2+ for binding biological ligands at the cell surface when pH decreased, we hypothesized that OA protected the thalli of S. japonica against copper toxicity. However, further studies are required to assess the accumulated volume of copper in the alga under different pCO2 and copper treatments.
In conclusion, this work evaluated the interaction between pCO2 and the copper concentration on the growth and physiological characteristics of the juvenile sporophyte of S. japonica. At an ambient pCO2 level, the growth of S. japonica was inhibited by higher copper concentrations (0.5~1 µM), but its photosynthesis (Fv/Fm and rETR), pigment contents (Chl a, Chl c, and Car), and SC contents were all improved. This may suggest that when this alga responds to copper stress, its growth and photosynthesis become decoupled. It is interesting to note that high pCO2 reduced the toxic effects of copper on the growth of the kelp. Likewise, OA also reduced the beneficial impacts of excess copper on the photosynthetic physiology and biochemical compositions at low pCO2. As a result, our findings show that S. japonica was more resistant to excess copper at elevated pCO2 levels, suggesting that it would be well adapted to future oceanic conditions and have the potential to be used in the treatment of wastewater. This finding may also indicate that, throughout the artificial seedling process, higher pCO2 levels in seawater will help juvenile S. japonica resist the harmful effects of heavy metals. However, in order to obtain a more comprehensive view of the combined effects of OA and copper pollution on the growth and physiology of S. japonica, further research on the response of different developmental stages of this alga is needed.

4. Materials and Methods

4.1. Sample Collection and Maintenance

Juvenile sporophytes of S. japonica were collected from cultivated populations in Lianjiang, Fujian, China (26°07′ N, 120°17′ E), in December 2021. These samples were put into a cooling box filled with seawater and transported quickly to the laboratory within 2 h. Selected healthy individuals (approximately 5 cm in length) were rinsed with filtered natural seawater to remove epiphytic organisms and then pre-cultured in an intelligent illumination incubator (GXZ-380B, Jiangnan Instrument Factory, Ningbo, China). In order to prevent nutrient depletion, filtered sterile seawater enriched with 25% PESI [67] was used to culture the samples. Natural seawater was obtained from the coastal region of Pingtan, Fuzhou. A preliminary analysis of seawater using an atomic absorption spectrophotometer (AA6300C, SHIMADZU, Japan) showed that the copper concentration was 0.01 μmol/L. The temperature was set to 9 °C with a 12 h:12 h (light/dark) photoperiod of 60 μmol photons m−2 s−1.
After 3 days, they were cultured in side-arm flasks containing 500 mL of 25% PESI-enriched seawater. Cultures were maintained under four different copper regimes (control, C; 0.2 μM, LCu; 0.5 μM, MCu; 1 μM, HCu) and two pCO2 levels (400 and 1000 ppmv) to investigate the combined effects of OA and copper. The choice of copper concentration was based on the report by Li et al. [68]. Natural seawater without the addition of CuSO4·5H2O (Sigma Aldrich, Steinhelm, Germany) was regarded as the control, and the copper content in the natural seawater was 0.01 μmol/L. The two pCO2 levels were maintained in two separate CO2 incubators and automatically regulated by controlling the flows of ambient air and pure CO2 gas. The algal stocking density was about 0.3 g fresh weight. The cultures were conducted in three replicates for each experiment and lasted for 9 days and bubbled with ambient or CO2-enriched air at a rate of 0.4 L min−1 to make the thalli roll up and down. The culture medium was changed every 3 days. The temperature and light conditions were consistent with the preincubation conditions described above. The TA and pH of the culture medium were determined after 24 h aeration before the algae were cultured. TA samples were immediately collected in acid-washed glass bottles without empty space, preserved with a few drops of saturated HgCl2 solution, and stored at room temperature until analysis. The pH (NBS scale) was measured using a pH meter (Orion STAR A211; Thermo Scientific), and the TA was calculated by titration. The seawater carbonate system parameters were calculated using the CO2SYS software [65,69], according to known parameters, such as temperature, salinity, TA, and pH.

4.2. Measurement of Growth Rates

Before and after the experiment, the fresh weights of all juvenile sporophytes were measured. The samples were dried on paper towels until no changes in biomass were detectable. The RGR (% day−1) of each replicate was estimated as follows:
RGR = 100 × lnW t − lnW 0 / t    
where W0 is the initial fresh weight, and Wt is the final fresh weight after t days of culture.

4.3. Measurement of Chlorophyll Fluorescence

After the culture experiment, the chlorophyll fluorescence parameters of juvenile sporophytes were measured using a pulse modulation fluorometer (Imaging-PAM, Walz, Germany). Before determination, the samples were kept in the dark for 15 min. The basal fluorescence (F0) was obtained at low light, and the maximum fluorescence (Fm) was measured by the saturation pulse method with an intensity of 4000 µmol m−2 s−1. Fv/Fm was calculated using the equation Fv/Fm = (Fm − F0)/Fm. The rETR and NPQ were detected under actinic light in our experiments after 15 min of dark adaptation. The rETR = ΔF/Fm × PAR × 0.5, where ΔF/Fm represents the effective PSII quantum yield, PAR is the light irradiance, and 0.5 refers to the fraction of incident PAR absorbed by chlorophyll associated with PSII. NPQ was calculated as follows: NPQ = (Fm − Fm’)/Fm’, where Fm represents the maximum fluorescence after 15 min of darkness adaptation, and Fm’ represents the maximum fluorescence yield under actinic light.

4.4. Measurements of Pigments

Approximately 0.1 g (fresh weight) of samples from each culture condition was used for the extraction of Chl a and Car. These samples were placed in 10 mL of methanol for 24 h in the dark, and the absorption of the supernatant was measured at 750, 665, 652, and 510 nm with an ultraviolet absorption spectrophotometer (U-2900, HITACHI, Tokyo, Japan). The contents of Chl a and Car were estimated according to [70,71].
Approximately 0.2 g (fresh weight) of samples in each treatment was used for the extraction of Chl c. These samples were placed in 2 mL of dimethyl sulfoxide for 5 min, and the absorption of the supernatant was detected at 665, 631, and 582 nm using an ultraviolet absorption spectrophotometer. Next, the same samples were placed in 3 mL of acetone for 2 h. The supernatant was transferred into a 10 mL tube, and 1 mL of methanol and 1 mL of distilled water were then added. The absorbance of the supernatant was read at 664, 631, and 581 nm. The concentrations of Chl c were estimated according to [72]. Pigment contents were determined as mg g−1 FW.

4.5. Measurement of Soluble Carbohydrates

About 0.1 g of the fresh alga was homogenized in 2 mL of distilled water and diluted to 10 mL. The homogenate was centrifuged at 1400× g for 5 min at 4 °C. After centrifugation, 1 mL of the supernatant was transferred to a glass tube and diluted to 2 mL with distilled water, and then 8 mL of anthrone reagent was added. The mixture was immersed in boiling water for 10 min. After cooling at room temperature, the absorbance value of the sample at 620 nm was measured. The contents of soluble carbohydrates were calculated according to [73] and expressed as mg g−1 FW.

4.6. Statistical Analysis

All data are expressed as the mean ± SD (n = 3). Prior to the analysis, tests for the normal distribution (Shapiro–Wilk, p > 0.05) and homogeneity (Levene’s test, p > 0.05) of variance were conducted. A two-way analysis of variance (ANOVA) was used to test the combined effects of the pCO2 level and copper on the seawater carbonate parameters, RGR, Fv/Fm, rETR, NPQ, Chl a, Chl c, Car, and soluble carbohydrates. A Duncan test was used to analyze the significance level of the factors (p < 0.05). All data analyses were performed using SPSS 22.0 software.

Supplementary Materials

The following supporting information can be downloaded at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/plants12051140/s1: Table S1: Analysis of variance (two-way ANOVA) examining the effects of pCO2 level and copper condition on RGR, Fv/Fm, rETR, NPQ, Chl a, Chl c, Car, and soluble carbohydrates of juvenile sporophytes of Saccharina japonica.

Author Contributions

Conceptualization, W.Z., X.C. and Y.C.; methodology, L.H., X.C. and S.L.; investigation, J.P., Y.Z., R.G. and Y.S.; resources, L.H., X.C. and Y.C.; supervision, L.H., X.C., Y.C., J.P., Y.Z. and R.G.; writing—original draft preparation, W.Z. and Y.C.; visualization, W.Z., X.C. and Y.C.; funding acquisition, L.H., X.C. and Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Educational Research Project for Young and Middle-aged Teachers of Fujian Provincial Department of Education, grant number KLa21019A, and the National College Students Innovation and Entrepreneurship Training Program: China, grant number X202210389161.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We sincerely thank Xu Gao of the Ocean University of China for helpful comments on the design of the experiment.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Brown, M.T.; Newman, J.E. Physiological responses of Gracilariopsis longissima (S.G. Gmelin) Steentoft, L.M. Irvine and Farnham (Rhodophyceae) to sub-lethal copper concentrations. Aquatic. Toxicol. 2003, 64, 201–213. [Google Scholar] [CrossRef]
  2. Contreras, L.; Moenne, A.; Correa, J.A. Antioxidant responses in Scytosiphon Lomentaria (Phaeophyceae) inhabiting copper-enriched coastal environments. J. Phycol. 2005, 41, 1184–1195. [Google Scholar] [CrossRef]
  3. Correa, J.A.; González, P.; Sánchez, P.; Muoz, J.; Orellana, M.C. Copper-algae interactions: Inheritance or adaptation? Environ. Monit Assess 1996, 40, 41–54. [Google Scholar] [CrossRef]
  4. Costa, G.B.; de Felix, M.R.L.; Simioni, C.; Ramlov, F.; Oliveira, E.R.; Pereira, D.T.; Maraschi, M.; Chow, F.; Horta, P.A.; Lalau, C.M.; et al. Effects of copper and lead exposure on the ecophysiology of the brown seaweed Sargassum cymosum. Protoplasma 2016, 253, 111–125. [Google Scholar] [CrossRef]
  5. González, A.; Vera, J.; Castro, J.; Dennett, G.; Mellado, M.; Morales, B.; Correa, J.A.; Moenne, A. Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess. Plant Cell Environ. 2010, 33, 1627–1640. [Google Scholar] [CrossRef]
  6. Wen, J.; Zou, D. Interactive effects of increasing atmospheric CO2 and copper exposure on the growth and photosynthesis in the young sporophytes of Sargassum fusiforme (Phaeophyta). Chemosphere 2021, 269, 129397. [Google Scholar] [CrossRef]
  7. Huovinen, P.; Leal, P.; Gomez, I. Interacting effects of copper, nitrogen and ultraviolet radiation on the physiology of three south Pacific kelps. Mar. Freshw. Res. 2010, 61, 330–341. [Google Scholar] [CrossRef]
  8. Yruela, I. Copper in plants: Acquisition, transport and interactions. Funct. Plant Biol. 2009, 36, 409–430. [Google Scholar] [CrossRef] [Green Version]
  9. Mamboya, F.; Lyimo, T.J.; Landberg, T.; Bjoerk, M. Influence of combined changes in salinity and copper modulation on growth and copper uptake in the tropical green macroalga Ulva reticulata. Estuar. Coast Shelf Sci. 2009, 84, 326–330. [Google Scholar] [CrossRef]
  10. Zhu, X.; Zou, D.; Huang, Y.; Cao, J.; Sun, Y.; Chen, B.; Chen, X. Physiological responses of Porphyra haitanensis (Rhodophyta) to copper and cadmium exposure. Bot. Mar. 2017, 60, 27–37. [Google Scholar] [CrossRef]
  11. Gouveia, C.; Kreusch, M.; Schmidt, É.C.; Felix, M.R.D.; Osorio, L.K.P.; Pereira, D.T.; dos Santos, R.; Ouriques, L.C.; Martins, R.D.; Latini, A.; et al. The effects of lead and copper on the cellular architecture and metabolism of the red alga Gracilaria domingensis. Microsc. Microanal. 2013, 19, 513–524. [Google Scholar] [CrossRef]
  12. Gao, G.; Liu, Y.; Li, X.; Feng, Z.; Xu, Z.; Wu, H.; Xu, J. Expected CO2-induced ocean acidification modulates copper toxicity in the green tide alga Ulva prolifera. Environ. Exp. Bot. 2017, 135, 63–72. [Google Scholar] [CrossRef]
  13. Kumar, K.S.; Han, Y.S.; Choo, K.S.; Kong, J.A.; Han, T. Chlorophyll fluorescence based copper toxicity assessment of two algal species. Toxicol. Environ. Health Sci. 2009, 1, 17–23. [Google Scholar] [CrossRef]
  14. Raven, J.; Caldeira, K.; Elderfifield, H.; Hoegh-Guldberg, H.; Liss, P.; Riebesell, U.; Shepherd, J.; Turley, C.; Watson, A. Ocean Acidifification Due to Increasing Atmospheric Carbon Dioxide; The Royal Society: London, UK, 2005; ISBN 978-0-85403-617-2. [Google Scholar]
  15. Feely, R.A.; Sabine, C.L.; Lee, K.; Berelson, W.; Kleypas, J.A.; Fabry, V.J.; Millero, F.J. Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 2004, 305, 362–366. [Google Scholar] [CrossRef] [Green Version]
  16. Israel, A.; Hophy, M. Growth, photosynthetic properties and Rubisco activities and amounts of marine macroalgae grown under current and elevated seawater CO2 concentrations. Glob. Change Biol. 2002, 8, 831–840. [Google Scholar] [CrossRef]
  17. Punchai, P.; Ishimatsu, A.; Nishihara, G.N. The effect of elevated CO2 on the production and respiration of a Sargassum thunbergii community: A mesocosm study. Phycol. Res. 2020, 68, 169–177. [Google Scholar] [CrossRef]
  18. Wei, Z.; Zhang, Y.; Yang, F.; Liang, J.; Long, L. Increased light availability modulates carbon and nitrogen accumulation in the macroalga Gracilariopsis lemaneiformis (Rhodophyta) in response to ocean acidification. Environ. Exp. Bot. 2021, 187, 104492. [Google Scholar] [CrossRef]
  19. Chu, Y.; Liu, Y.; Li, J.; Gong, Q. Effects of Elevated PCO2 and Nutrient Enrichment on the Growth, Photosynthesis, and Biochemical Compositions of the Brown Alga Saccharina Japonica (Laminariaceae, Phaeophyta). PeerJ 2019, 7, e8040. [Google Scholar] [CrossRef] [Green Version]
  20. Ober, G.T.; Thornber, C.S. Divergent responses in growth and nutritional quality of coastal macroalgae to the combination of increased pCO(2) and nutrients. Mar. Environ. Res. 2017, 131, 69–79. [Google Scholar] [CrossRef] [Green Version]
  21. Hepburn, C.D.; Pritchard, D.W.; Cornwall, C.E.; McLeod, R.J.; Beardall, J.; Raven, J.A.; Hurd, C.L. Diversity of carbon use strategies in a kelp forest community: Implications for a high CO2 ocean. Glob. Change Biol. 2011, 17, 2488–2497. [Google Scholar] [CrossRef]
  22. Giordano, M.; Beardall, J.; Raven, J.A. CO2 concentrating mechanisms in algae: Mechanisms, environmental modulation, and evolution. Annu. Rev. Plant Biol. 2005, 56, 99–131. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Raven, J.A.; Beardall, J. Carbon acquisition mechanisms of algae: Carbon dioxide diffusion and carbon dioxide concentrating mechanisms. In Photosynthesis in Algae; Advances in Photosynthesis and Respiration; Larkum, A.W.D., Douglas, S.E., Raven, J.A., Eds.; Springer: Dordrecht, The Netherlands, 2003; Volume 14, pp. 225–244. ISBN 978-94-010-3772-3. [Google Scholar]
  24. Raven, J.A. Inorganic Carbon Acquisition by Marine Autotrophs. Adv. Bot. Res. 1997, 27, 85–209. [Google Scholar] [CrossRef]
  25. Todgham, A.E.; Stillman, J.H. Physiological Responses to Shifts in Multiple Environmental Stressors: Relevance in a Changing World. Integr. Comp. Biol. 2013, 53, 539–544. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Gunderson, A.R.; Armstrong, E.J.; Stillman, J.H. Multiple stressors in a changing world: The need for an improved perspective on physiological responses to the dynamic marine environment. Annu. Rev. Mar. Sci. 2016, 8, 357–378. [Google Scholar] [CrossRef] [PubMed]
  27. Ma, J.; Wang, W.; Liu, X.; Wang, Z.; Xu, J. Zinc toxicity alters the photosynthetic response of red alga Pyropia yezoensis to ocean acidification. Environ. Sci. Pollut. R 2020, 27, 3202–3212. [Google Scholar] [CrossRef]
  28. Millero, F.J.; Woosley, R.; Ditrolio, B.; Waters, J. Effect of ocean acidification on the speciation of metals in seawater. Oceanography 2009, 22, 72–85. [Google Scholar] [CrossRef]
  29. Xu, T.; Cao, J.; Qian, R.; Song, Y.; Wang, W.; Ma, J.; Gao, K.; Xu, J. Ocean acidification exacerbates copper toxicity in both juvenile and adult stages of the green tide alga Ulva linza. Mar. Environ. Res. 2021, 170, 105447. [Google Scholar] [CrossRef]
  30. Zhong, Z.H.; Qin, S.; Wang, Y.; Ma, C.; Liu, Z.Y. Low pH in simulated acid rain promotes the toxicity of copper on the physiological performance in Sargassum horneri. Photosynthetica 2020, 58, 853–861. [Google Scholar] [CrossRef]
  31. Koh, C.H.; Shin, H.C. Growth and size distribution of some large brown algae in Ohori, east coast of Korea. Hydrobiologia 1990, 204–205, 225–231. [Google Scholar] [CrossRef]
  32. Liu, F.L.; Wang, X.L.; Liu, J.D.; Fu, W.D.; Duan, D.L.; Yang, Y.X. Genetic mapping of the Laminaria japonica (Laminariales, Phaeophyta) using amplifified fragment length polymorphism markers. J. Phycol. 2009, 45, 1228–1233. [Google Scholar] [CrossRef]
  33. Graham, M.H. Effects of local deforestation on the diversity and structure of Southern California giant kelp forest food webs. Ecosystems 2004, 7, 341–357. [Google Scholar] [CrossRef]
  34. Liu, F.; Sun, X.; Wang, F.; Wang, W.; Liang, Z.; Lin, Z.; Dong, Z. Breeding, economic traits evaluation, and commercial cultivation of a new Saccharina variety “Huangguan No. 1”. Aquac. Int. 2014, 5, 1665–1675. [Google Scholar] [CrossRef]
  35. Liu, F.; Yao, J.; Wang, X.; Repnikova, A.; Galanin, D.A.; Duan, D. Genetic diversity and structure within and between wild and cultivated Saccharina japonica (Laminariales, Phaeophyta) revealed by SSR markers. Aquaculture 2012, 358–359, 139–145. [Google Scholar] [CrossRef]
  36. Kang, J.W.; Chung, I.K. The Interactive Effects of Elevated CO2 and Ammonium Enrichment on the Physiological Performances of Saccharina Japonica (Laminariales, Phaeophyta). Ocean Sci. J. 2018, 53, 487–497. [Google Scholar] [CrossRef]
  37. Xu, D.; Wang, D.; Li, B.; Fan, X.; Zhang, X.W.; Ye, N.H.; Wang, Y.; Mou, S.; Zhuang, Z. Effects of CO2 and Seawater Acidification on the Early Stages of Saccharina Japonica Development. Environ. Sci. Technol. 2015, 49, 3548–3556. [Google Scholar] [CrossRef]
  38. Gao, X.; Kim, J.H.; Park, S.K.; Yu, O.H.; Kim, Y.S.; Choi, H.G. Diverse responses of sporophytic photochemical efficiency and gametophytic growth for two edible kelps, Saccharina japonica and Undaria pinnatifida to ocean acidification and warming. Mar. Pollut. Bull 2019, 142, 315–320. [Google Scholar] [CrossRef]
  39. Wang, Q.; Fang, T. Effects of copper on the growth and development of female gametophytes and young sporophyte of Laminaria japonica. J. Shandong Coll. Oceanol. 1981, 11, 53–60. (In Chinese) [Google Scholar]
  40. Xia, J.R.; Li, Y.J.; Lu, J.; Chen, B. Effects of copper and cadmium on growth, photosynthesis, and pigment content in Gracilaria lemaneiformis. Bull. Environ. Contam. Toxicol. 2004, 73, 979–986. [Google Scholar] [CrossRef]
  41. Zhu, X.; Zou, D.; Hong, D. Physiological responses of Hizikia fusiformis to copper and cadmium exposure. Bot. Mar. 2011, 54, 431–439. [Google Scholar] [CrossRef]
  42. Baumann, H.A.; Morrison, L.; Stengel, D.B. Metal accumulation and toxicity measured by PAM-Chlorophyll fluorescence in seven species of marine macroalgae. Ecotoxicol. Environ. Saf. 2009, 72, 1063–1075. [Google Scholar] [CrossRef]
  43. Collén, J.; Pinto, E.; Pedersén, M.; Colepicolo, P. Induction of oxidative stress in the red macroalga Gracilaria tenuistipitata by pollutant metals. Arch. Environ. Con. Tox. 2003, 45, 337–342. [Google Scholar] [CrossRef] [PubMed]
  44. Tripathi, B.N.; Mehta, S.K.; Gaur, J.P. Recovery of uptake and assimilation of nitrate in Scenedesmus sp previously exposed to elevated levels of Cu2+ and Zn2+. J. Plant Physiol. 2004, 161, 543–549. [Google Scholar] [CrossRef] [PubMed]
  45. Li, Y.; Zhou, S.; Zhao, F.; Liu, Y.; Fan, P.; Wang, G. Physiological responses of Porphyra haitanesis to different copper and zinc concentrations. Braz. J. Oceanogr. 2010, 58, 261–267. [Google Scholar] [CrossRef]
  46. Connan, S.; Stengel, D.B. Impacts of ambient salinity and copper on brown algae: 1. Interactive effects on photosynthesis, growth, and copper accumulation. Aquat. Toxicol. 2011, 104, 94–107. [Google Scholar] [CrossRef]
  47. Pawlik-Skowronska, B.; Pirszel, J.; Brown, M.T. Concentrations of phytochelatins and glutathione found in natural assemblages of seaweeds depend on species and metal concentrations of the habitat. Aquat. Toxicol. 2007, 83, 190–199. [Google Scholar] [CrossRef] [PubMed]
  48. Contreras, L.; Moenne, A.; Gaillard, F.; Potin, P.; Correa, J.A. Proteomic analysis and identification of copper stress-regulated proteins in the marine alga Scytosiphon gracilis (Phaeophyceae). Aquat. Toxicol. 2010, 96, 85–89. [Google Scholar] [CrossRef]
  49. Contreras, L.; Mella, D.; Moenne, A.; Correa, J.A. Differential responses to copper-induced oxidative stress in the marine macroalgae Lessonia nigrescens and Scytosiphon lomentaria (Phaeophyceae). Aquat. Toxicol. 2009, 94, 94–102. [Google Scholar] [CrossRef]
  50. Cid, A.; Herrero, C.; Torres, E.; Abalde, J. Copper toxicity on the marine microalga Phaeodactylum tricornutum: Effects on photosynthesis and related parameters. Aquat. Toxicol. 1995, 31, 165–174. [Google Scholar] [CrossRef] [Green Version]
  51. Nielsen, H.D.; Nielsen, S.L. Photosynthetic responses to Cu2+ exposure are independent of light acclimation and uncoupled from growth inhibition in Fucus serratus (Phaeophyceae). Mar. Pollut. Bull. 2005, 51, 715–721. [Google Scholar] [CrossRef]
  52. Xiao, X.; Li, W.; Jin, M.; Zhang, L.; Qin, L.; Geng, W. Responses and tolerance mechanisms of microalgae to heavy metal stress: A review. Mar. Environ. Res. 2023, 183, 105805. [Google Scholar] [CrossRef]
  53. Maksymiec, W. Effect of copper on cellular processes in higher plants. Photosynthetica 1997, 34, 321–342. [Google Scholar] [CrossRef]
  54. Yamasaki, H.; Pilon, M.; Shikanai, T. How do plants respond to copper deficiency? Plant Signal Behav. 2008, 3, 231–232. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Andrade, L.R.; Farina, M.; Amado, G.M. Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro. Ecotoxicol. Environ. Saf. 2004, 58, 117–125. [Google Scholar] [CrossRef] [PubMed]
  56. Nielsen, H.D.; Brownlee, C.; Coelho, S.M.; Brown, M. Inter-population differences in inherited copper tolerance involve photosynthetic adaptation and exclusion mechanisms in Fucus serratus. New Phytol. 2003, 160, 157–165. [Google Scholar] [CrossRef] [PubMed]
  57. Celis-Plá, P.S.M.; Brown, M.T.; Santillán-Sarmiento, A.; Korbee, N.; Sáez, C.A.; Figueroa, F.L. Ecophysiological and metabolic responses to interactive exposure to nutrients and copper excess in the brown macroalga Cystoseira tamariscifolia. Mar. Pollut. Bull. 2018, 128, 214–222. [Google Scholar] [CrossRef]
  58. Caron, L.; Douady, D.; Martino, A.D.; Quinet, M. Light harvesting in brown algae. Cah. Biol. Mar. 2001, 42, 109–124. [Google Scholar]
  59. Chojnacka, K.; Chojnacki, A.; Górecka, H. Biosorption of Cr3+, Cd2+ and Cu2+ ions by blue–green algae Spirulina sp.: Kinetics, equilibrium and the mechanism of the process. Chemosphere 2005, 59, 75–84. [Google Scholar] [CrossRef]
  60. Dummermuth, A.L.; Karsten, U.; Fisch, K.M.; König, G.M.; Wiencke, C. Responses of marine macroalgae to hydrogen-peroxide stress. J. Exp. Mar. Biol. Ecol. 2003, 289, 103–121. [Google Scholar] [CrossRef] [Green Version]
  61. Romeo, M.; Gnassia-Barelli, M. Organic ligands and their role in complexation and transfer of trace metals (micronutrients) in marine algae. In Macroalgae, Eutrophication and Trace Metal Cycling in Estuaries and Lagoons: Proceedings of the Coast-48 Symposium of Sub Group III, Thessaloniki, 1993; Rijstenbil, J.W., Haritonidis, S., Eds.; Delta Instituut voor Hydrobiologisch Onderzoek (DIHO): Wageningen, The Netherlands, 1993; pp. 121–135. [Google Scholar]
  62. Van den Berg, C.M.G.; Wong, P.; Chau, Y. Measurement of Complexing Materials Excreted from Algae and Their Ability to Ameliorate Copper Toxicity. J. Fish Res. Board Can. 1979, 36, 901–905. [Google Scholar] [CrossRef]
  63. Liu, Y.; Cao, J.; Chu, Y.; Liu, Y.; Li, J. The brown algae Saccharina japonica and Sargassum horneri exhibit species-specific responses to synergistic stress of ocean acidification and eutrophication. J. Ocean Univ. China 2021, 20, 1253–1262. [Google Scholar] [CrossRef]
  64. Franklin, N.M.; Stauber, J.L.; Markich, S.J.; Lim, R.P. pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.). Aquat. Toxicol. 2000, 48, 275–289. [Google Scholar] [CrossRef]
  65. Peterson, H.G.; Healey, F.P.; Wagemann, R. Metal Toxicity to Algae: A Highly pH Dependent Phenomenon. Can. J. Fish Aquat. Sci. 1984, 41, 974–979. [Google Scholar] [CrossRef]
  66. Kochoni, E.; Fortin, C. Iron Modulation of Copper Uptake and Toxicity in a Green Alga (Chlamydomonas reinhardtii). Environ. Sci. Technol. 2019, 53, 6539–6545. [Google Scholar] [CrossRef]
  67. Tatewaki, M. Formation of a Crustose Sporophyte with Unilocular Sporangia in Scitosiphon L. Phycologia 1966, 6, 62–66. [Google Scholar] [CrossRef]
  68. Li, T.; Sun, G.; Yang, C.; Liang, K.; Ma, S.; Huang, L. Using self-organizing map for coastal water quality classification: Towards a better understanding of patterns and processes. Sci. Total Environ. 2018, 628–629, 1446–1459. [Google Scholar] [CrossRef]
  69. Pierrot, D.; Lewis, E.; Wallace, D.W.R. MS Excel Program Developed For CO2 System Calculations; Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, US Department of Energy: Oak Ridge, TN, USA, 2006. [Google Scholar]
  70. Porra, R.J. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth. Res. 2002, 73, 633–640. [Google Scholar] [CrossRef]
  71. Parsons, T.R.; Strickland, J.D. Discussion of spectrophotometric determination of marine plant pigments with revised equations for ascertaining chlorophylls. J. Mar. Res. 1963, 21, 155–163. [Google Scholar]
  72. Seely, G.R.; Duncan, M.J.; Vidaver, W.E. Preparative and analytical extraction of pigments from brown algae with dimethyl sulfoxide. Mar. Biol. 1972, 12, 184–188. [Google Scholar] [CrossRef]
  73. Yemm, E.W.; Willis, A.J. The Estimation of Carbohydrates in Plant Extracts by Anthrone. Biochem. J. 1954, 57, 508–514. [Google Scholar] [CrossRef] [Green Version]
Plants 12 01140 g001 550
Figure 1. Relative growth rates of S. japonica grown under different pCO2 levels and copper concentrations. C (control, natural seawater); Lcu (low copper concentration, 0.2 μM); Mcu (medium copper concentration, 0.5 μM); Hcu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Figure 1. Relative growth rates of S. japonica grown under different pCO2 levels and copper concentrations. C (control, natural seawater); Lcu (low copper concentration, 0.2 μM); Mcu (medium copper concentration, 0.5 μM); Hcu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Plants 12 01140 g001
Plants 12 01140 g002a 550Plants 12 01140 g002b 550
Figure 2. Photosynthetic parameters ((A), Fv/Fm; (B), rETR; (C), NPQ) of S. japonica grown at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Figure 2. Photosynthetic parameters ((A), Fv/Fm; (B), rETR; (C), NPQ) of S. japonica grown at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Plants 12 01140 g002aPlants 12 01140 g002b
Plants 12 01140 g003 550
Figure 3. Chlorophyll a (A), chlorophyll c (B), and carotenoid content (C) of S. japonica grown at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Figure 3. Chlorophyll a (A), chlorophyll c (B), and carotenoid content (C) of S. japonica grown at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
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Plants 12 01140 g004 550
Figure 4. Soluble carbohydrate contents of S. japonica grown at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Figure 4. Soluble carbohydrate contents of S. japonica grown at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). All results are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Plants 12 01140 g004
Table
Table 1. Carbonate parameters of seawater at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). Data are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
Table 1. Carbonate parameters of seawater at different pCO2 levels and copper concentrations. C (control, natural seawater); LCu (low copper concentration, 0.2 μM); MCu (medium copper concentration, 0.5 μM); HCu (high copper concentration, 0.2 μM). Data are shown as mean value ± SD (n = 3). Different letters indicate statistical differences (p < 0.05) among experimental treatments.
CopperpCO2
(ppmv)		pHTA
(μmol L−1)		DIC
(μmol kg−1)		CO32−
(μmol kg−1)		HCO3−
(μmol kg−1)		CO2
(μmol kg−1)
Control4007.90 ± 0.02 a2230 ± 18 a2132 ± 24 a84.8 ± 3.1 a2020.9 ± 25.6 a26.6 ± 1.6 a
10007.53 ± 0.04 c2223 ± 8 a2232 ± 7 b38.5 ± 3.7 c2128.2 ± 4.8 b65.4 ± 6.8 c
LCu4007.89 ± 0.02 a2234 ± 15 a2137 ± 21 a84.4 ± 3.1 a2026.0 ± 22.3 a26.9 ± 1.6 a
10007.58 ± 0.01 b2228 ± 18 a2223 ± 20 b42.9 ± 0.7 bc2122.3 ± 18.7 b57.9 ± 1.8 b
Mcu4007.87 ± 0.03 a2227 ± 21 a2138 ± 16 a80.1 ± 5.0 a2029.0 ± 14.3 a28.4 ± 1.7 a
10007.58 ± 0.03 b2232 ± 4 a2228 ± 6 b42.8 ± 3.0 bc2126.5 ± 4.7 b58.6 ± 4.2 b
Hcu4007.88 ± 0.02 a2224 ± 7 a2131 ± 8 a81.1 ± 2.6 a2023.9 ± 8.9 a27.9 ± 1.1 a
10007.60 ± 0.03 b2216 ± 13 a2205 ± 17 b44.7 ± 2.9 b2105.6 ± 16.0 b55.0 ± 4.1 b
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Zhang, W.; He, L.; Pan, J.; Zhou, Y.; Ge, R.; Li, S.; Shi, Y.; Chen, X.; Chu, Y. Response of Juvenile Saccharina japonica to the Combined Stressors of Elevated pCO2 and Excess Copper. Plants 2023, 12, 1140. https://0-doi-org.brum.beds.ac.uk/10.3390/plants12051140

AMA Style

Zhang W, He L, Pan J, Zhou Y, Ge R, Li S, Shi Y, Chen X, Chu Y. Response of Juvenile Saccharina japonica to the Combined Stressors of Elevated pCO2 and Excess Copper. Plants. 2023; 12(5):1140. https://0-doi-org.brum.beds.ac.uk/10.3390/plants12051140

Chicago/Turabian Style

Zhang, Wenze, Lianghua He, Jiangqi Pan, Yuhong Zhou, Ruxiang Ge, Sufang Li, Yunyun Shi, Xinhua Chen, and Yaoyao Chu. 2023. "Response of Juvenile Saccharina japonica to the Combined Stressors of Elevated pCO2 and Excess Copper" Plants 12, no. 5: 1140. https://0-doi-org.brum.beds.ac.uk/10.3390/plants12051140

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