Future CO₂-induced seawater acidification mediates the physiological performance of a green alga Ulva linza in different photoperiods

Introduction

Due to the activities of humans, the concentration of atmospheric CO₂ has increased to 400 ppm from 278 ppm during the pre-industrial revolution (Gattuso et al., 2015). It is estimated that the oceans, as a CO₂ sink, have taken up approximately 48% of the fossil-fuel and cement-manufacturing emissions (Sabine et al., 2004). The concentration of atmospheric CO₂ has been predicted to reach almost 1,000 ppm by the end of 21st century (Gattuso et al., 2015). This would result in the decline of surface ocean pH by 0.3–0.4 units, an increase of hydrogen ion concentration and bicarbonate concentration in seawater, which is termed ocean acidification (OA) (Orr et al., 2005; Koch et al., 2013; Albright, 2016). OA will be maintained for hundreds of years, and thus could cause a huge change in marine ecosystems (Kroeker et al., 2013; Wittmann & Pörtner, 2013).

Marine phytoplankton accounts for 50% of global primary production (Beardall, Stojkovic & Larsen, 2009) and macroalgae accounts for 10% of marine primary productivity (Smith, 1981; Charpy-Roubaud & Sournia, 1990; Heinrich et al., 2012). Macroalgae have evolved distinct strategies to capture inorganic carbon (Ci) for photosynthesis: (1) by the diffusive uptake of CO₂ (Giordano, Beardall & Raven, 2005; Raven et al., 2005), (2) the active transport of HCO${}_3^{-}$/CO₂ (termed carbon concentrating mechanisms (CCMs) (Maberly, Raven & Johnston, 1992; Raven, 2003; Giordano, Beardall & Raven, 2005). The elevated CO₂ might enhance the growth and photosynthesis of algae that depend on CO₂ diffusion or have weak CCMs (Kübler, Johnston & Raven, 1999; Gao et al., 2016a). Therefore, the physiological influences of OA on macroalgae might be species-specific. For example, OA has a positive influences on the growth of macroalgae, such as the red algae Pyropia yezoensis, Pyropia haitanensis and Gracilaria lemaneiformis (Gao et al., 1991; Xu & Gao, 2015; Chen et al., 2018), green algae Ulva prolifera, Ulva lactuca, Caulerpa taxifolia (Xu & Gao, 2012; Chen, Zou & Jiang, 2015; Roth-Schulze et al., 2018) and brown algae Hizikia fusiforme, Ecklonia radiata, Sargassum muticum (Zou, 2005; Britton et al., 2016; Xu et al., 2017). However, research has shown that high CO₂ levels did not have a detectable effect on the relative growth rate of several species, for example, Sargassum henslowianum, Ulva rigida, Ulva australis, the giant kelp Macrocystis pyrifera and Ulva australis (Chen & Zou, 2014; Rautenberger et al., 2015; Fernández, Roleda & Hurd, 2015; Reidenbach et al., 2017). Furthermore, increasing seawater CO₂ concentration reduced the growth of Gracilaria tenuistipitata, Porphyra leucosticte, Fucus vesiculosus, Halimeda opuntia and Ulva linza (García-Sánchez, Fernández & Niell, 1994; Mercado et al., 1999; Gutow et al., 2014; Johnson, Price & Smith, 2014; Gao et al., 2018). In addition, the various effects of ocean acidification may be also modulated by other environmental factors, such as temperature, nutrients and photoperiods (Gao et al., 2017a; Gao et al., 2019; Li et al., 2018).

Photoperiod is a key factor regulating seasonal responses of algae (Dring, 1988). Furthermore, daylength can influence algal affinity for dissolved inorganic carbon and cellular carbon demand, thus imposing an effect on CCMs regulation (Rost, Riebesell & Sültemeyer, 2006). Thus, the photoperiod may be critical for algae cultured under elevated CO₂ levels. Meanwhile, the interactive effects of OA and increased photoperiods could enhance the growth of U. prolifera, thus increase the opportunities for occurrence of green tides (Li et al., 2018). Elevated CO₂ levels enhanced the photosynthetic rate, but longer illumination periods reduced the photosynthetic efficiency in E. huxleyi (Nielsen, 1997; Rost, Riebesell & Sültemeyer, 2006). Therefore, at HC, the responses of algae to different photoperiods appear to be genera- and species-specific.

Green macroalgal bloom has occurred each year in the Yellow Sea (YS) since 2007, termed green tides (Xu, Zhang & Cheng, 2016). Green tides generate aesthetic problems and toxic gas when thalli decompose (Gao et al., 2016b). Ulva is the dominant genus contributing majority of green tides and U. linza is one of species causing green tides in the Yellow Sea (Fletcher, 1996; Kang et al., 2014; Gao et al., 2017a; Gao et al., 2017b). However, little is known about the interactive effects of ocean acidification and photoperiod on U. linza. Previous study showed that the effect of ocean acidification on diatoms was related to light intensity (Gao et al., 2012). In this study, based on previous studies, we hypothesized the effect of ocean acidification on U. linza may dependent on photoperiod. To test our hypothesis, the physiological responses of the green macroalga Ulva linza under two different CO₂ levels and three different photoperiods were examined.

Materials & Methods

Results

Both the elevated CO₂ levels and photoperiod altered carbonate parameters in seawater, and they both had an interactive effect (Tables 1 and 2). The elevated CO₂ decreased pH and CO${}_3^{2-}$, increased p CO₂, DIC, HCO${}_3^{-}$ and CO₂ in the seawater. Increased photoperiod did not affect carbonate parameters at LC but the longest photoperiod increased DIC, HCO${}_3^{-}$ and TA compared to shortest photoperiod.

The two-way ANOVA showed that elevated CO₂ and photoperiod had an interactive effect, and both elevated CO₂ levels and the photoperiods had a significant effect on the RGR of U. linza (Fig. 1 and Table 3). At LC, the RGR of adult U. linza increased with the extended light periods, and the highest RGR occurred at a L: D of 16:8. The effect of CO₂ also varied with photoperiod. Higher CO₂ levels enhanced RGR at L: D of 8:16, but had no effect at 12:12 and reduced it at 16:8.

The Yield and NPQ were measured under different CO₂ levels and photoperiod conditions (Fig. 2). Two-way ANOVA showed that elevated CO₂ and photoperiod had an interactive effect on Yield (Table 4). Higher CO₂ levels increased Yield when thalli were cultured under photoperiods of 8:16 and 12:12 but reduced it under 16:8. Photoperiod had the main effect on NPQ (Table 4). At LC, thalli cultured at L: D of 16:8 had higher NPQ compared to L: D of 8:16 while the difference between 8:16 and 12:12 was insignificant. At HC, NPQ increased with the increase in photoperiod although the increase was not statistically significant. The elevated CO₂ had neutral effect on NPQ of U. linza.

Maximum rETR (rETRmax), efficiency of electron transport (α), and saturating irradiance (I_k) were calculated from the rapid light curves (Fig. 3, Table 5). Photoperiod and elevated CO₂ levels had an interactive effect, and elevated CO₂ levels had a main effect on light-saturated electron transport rate (rETRmax) (Table 6). Higher CO₂ levels increased rETRmax during the 8:16 and 12:12 photoperiods, but did not affect it at 16:8. A similar pattern was also found for α. In contrast to rETRmax and α, CO₂ did not affect I_k while photoperiod had the main effect on it. At LC, I_k increased when L: D rose from 8:16 to 12:12 but did not change with the further increase in photoperiod. At HC, I_k did not change when L: D rose from 8:16 to 12:12 but was enhanced when L: D increased to 16:8 (Table 5).

In addition to photosynthetic parameters, the effects of p CO₂ and photoperiod on the respiration rate of adult U. linza were also investigated (Fig. 4). Photoperiod and elevated CO₂ levels had an interactive effect, and both photoperiod and elevated CO₂ levels had the primary effect on the respiration rate of U. linza (Table 7). Higher CO₂ levels did not affect the respiration rate at photoperiods of 8:16 or 12:12 but increased it by 56.39% at a photoperiod of 16:8.

Changes in photosynthetic pigments of U. linza grown under various conditions are shown in Fig. 5. Two-way ANOVA showed that CO₂ and photoperiod had an interactive effect, and both CO₂ levels and the photoperiods had the main effect on the Chl a content of U. linza (Table 8). Prolonged light periods reduced the synthesis of Chl a in thalli although the difference between the photoperiods of 8:16 and 12:12 at HC was not statistically significant. Higher CO₂ levels reduced Chl a at the photoperiods of 8:16 and 12:12 but did not affect it at a L: D of 12:12. The same trend was found for Chl b. The Chl a/b ratios were all greater than 1.0, suggesting a higher synthesis of Chl a than Chl b under all culture conditions. Photoperiod and elevated CO₂ levels had an interactive effect on the Chl a/b ratio (Table 8); the higher CO₂ levels increased the ratio at a photoperiod of 12:12 but not at the other photoperiods.

Discussion

In the present study, at LC, extended photoperiods had a positive effect on the relative growth rate of adult U. linza, similar to previous studies on Laminaria sacharina, Porphyra umbilicalis and Ulva prolifera (Fortes & Lüning, 1980; Green & Neefus, 2016; Li et al., 2018). Carbon isotope fractionation experiments suggested that extended photoperiods could enhance growth by influencing inorganic carbon capture and fixation rate in algae (Rost et al., 2003). This hypothesis is supported by the present study where extended photoperiods increased the maximum quantum yield in PS II. On the other hand, the highest growth rates of Compsopogon coeruleus were obtained in shorter light periods (L: D = 8:16) (Zucchi & Necchi, 2001), the highest growth rates of Porphyra umbilicalis was found under neutral (L: D = 12:12) and longer (L: D = 16:8) light periods using 110 µmol photons m⁻²s⁻¹ (Green & Neefus, 2016), and the relative growth rate of Chlorella vulgaris cultured at L: D = 20:4 was lower than the RGR of alga cultured at L: D = 16:8 (Kendirlioglu, Agirman & Cetin, 2015). Therefore, the effects of photoperiod on algae appear to be species-specific.

Although Ulva has efficient mechanisms for CO₂concentration, the growth of Ulva can be enhanced by elevated CO₂ (Young & Gobler, 2016; Gao et al., 2016a; Gao et al., 2017a; Gao et al., 2017b). However, in this study, we found that the effects of elevated CO₂ levels on the relative growth rate of U. linza depended on photoperiod. CO₂ enhanced the growth of adult U. linza under light/dark conditions of 8:16, had no effect on growth of U. linza under a L: D of 12:12, and reduced the relative growth rate under a L: D of 16:8. High CO₂ levels can down-regulate algal CO₂ concentration mechanisms (CCMs), meaning that energy would be saved, and thus enhancing the relative growth rate of algae (Gao et al., 2012; Gao et al., 2016a; Raven, Beardall & Sánchez-Baracaldo, 2017). This is supported by decreased pigment synthesis in thalli at higher CO₂ levels. However, higher CO₂ levels did not affect the growth rate of U. linza at medium photoperiods. The neutral effects of CO₂ on the growth of U. rigida (Rautenberger et al., 2015) and U. linza (Gao et al., 2018) were also reported. We speculate that this is a compromise between the positive effects of elevated CO₂ and negative effects of decreased pH. The negative effect of decreased pH on growth was documented for the brown alga Sargassum muticum (Xu et al., 2017). Algae might need to consume additional energy to act against acid–based perturbation caused by decreased pH, leading to reduced growth (Xu et al., 2017). This is supported by an enhanced respiration rate at the higher CO₂ levels in this study. The phenomenon of an increased respiration rate of algae under elevated CO₂ concentrations was found in Hizikia fusiformis (Zou, 2005), the microalgae Phaeodactylum tricornutum (Wu, Gao & Riebesell, 2010) and Emiliania huxleyi (Jin et al., 2015).

Furthermore, the higher CO₂ level reduced the growth rate of U. linza under the longest photoperiod in the present study. This may be due to the combination of down-regulated CCMs and excess light energy. The operation of CCMs is an energy-consuming process and the down-regulation of CCMs can result in additional energy (Raven, Beardall & Sánchez-Baracaldo, 2017). Higher light can usually reduce algal photosynthetic activity (Singh & Singh, 2015; Gao et al., 2016a). The energy saved due to down-regulation of CCMs at HC combined with high light intensity could synergistically damage the algal photosystem and photosynthetic rate (Gao et al., 2012; Gao et al., 2016a). Although light intensity did not change among different CO₂ treatments, the lengthened photoperiod may have similar effect to increased light intensity. This argument is supported by increased NPQ at longer photoperiods as NPQ is photo-protective process to dissipate excess light energy. Higher CO₂ levels also reduced maximum quantum yield and stimulated the respiration rate of U. linza during the longest photoperiod in this study, leading to the decrease in growth. Enhanced respiration rate is a signal that organisms are fighting against stress and damage (Xu et al., 2017).

In addition to growth, the interactive effect of CO₂ and photoperiod was also found in photosynthetic parameters. For instance, elevated CO₂ increased Yield, rETRmax and α at shorter photoperiods (L: D of 8:16 and 12:12) but did not affect rETRmax and reduced Yield at longest photoperiod (L: D of 16:8). These findings indicate the close connection between growth and photosynthesis in terms of responding to the combination of CO₂ and photoperiod. It is worth noting that the interaction of CO₂ and photoperiod on growth of U. linza in this study is different from the findings in U. prolifera reported by Li et al. (2018). Elevated CO₂ increased growth of U. prolifera at all three photoperiods (L: D of 12:12, 10:14 and 16:8). The different results may be due to differential physiological property between U. linza and U. prolifera. It has been documented that U. prolifera has a higher tolerance to high light intensity compared to U. linza (Cui et al., 2015). The strong capacity in dealing with high light intensity could contribute to the effect of elevated CO₂ and prolonged photoperiod on growth U. linza was still positive. Integrating our findings with Li et al. (2018) study, the interaction of CO₂ and photoperiod on Ulva species would be species dependent.

Conclusions

This work is the first attempt to clarify the interaction between light/dark and elevated CO₂ levels on the physiological responses of Ulva linza. We found that the effect of OA on U. linza depended on photoperiod. Outbreaks of green tides during spring and summer in China occur when the photoperiod is reaching its peak. Our findings indicate future OA may hinder the occurrence of green tides dominated by U. linza in combination with extended photoperiods. More environmental factors, such as temperature and nutrient levels, need to be investigated to obtain a more comprehensive understanding on development of green tides in future oceans.

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