Photosynthetic responses of Halimeda scabra (Chlorophyta, Bryopsidales) to interactive effects of temperature, pH, and nutrients and its carbon pathways

Introduction

Photosynthetic parameters respond faster to environmental changes than algae C and N content, hence their usefulness in short-term studies (Figueroa et al., 2009). Photochemical and biochemical reactions of photosynthesis continually respond to environmental conditions. Irradiance, temperature, and nutrient concentration including CO₂ levels are among the main environmental factors limiting photosynthesis (Raven & Hurd, 2012; Zweng, Koch & Bowes, 2018). Algal ecophysiology studies have traditionally quantified temperature dependence using the metabolic quotient Q ₁₀, which describes the metabolic increase accompanied by an increase of 10 °C in an optimal temperature range (Bruno, Carr & O’Connor, 2015; Vásquez-Elizondo & Enríquez, 2016). This quotient Q₁₀ has also been used as a proxy to analyze the effect of temperature on nutrient absorption where it was found that by doubling the temperature the rate of nutrient absorption is doubled (Harrison & Hurd, 2001).

For aquatic plants, another limiting factor for photosynthesis is CO₂, since it is the only source of carbon that can be assimilated by the Ribulose 1,5 bisphosphate carboxylase oxygenase enzyme (RuBisCO) (Falkowski & Raven, 2007). At seawater pH (8.1–8.3) CO₂ is only between 0.5–1% of all dissolved inorganic carbon, while more than 91% is in the form of HCO₃⁻ and the remaining 8% is in the form of CO₃²⁻ (Hurd et al., 2009; Diaz-Pulido et al., 2016). Moreover, since the diffusion of CO₂through the cell membrane is slower in water than in air; many algae and higher plants have acquired mechanisms that promote intracellular CO₂ accumulation, allowing photosynthetic organisms to reduce carbon limitation by increasing the concentration of CO₂ in the vicinity of RuBisCO (CO₂ Concentration Mechanisms, CCM). Parallel to this, CCM’s contribute to decreasing photorespiration due to the oxygenase activity of RuBisCO (Ogren, 1984; Enríquez & Rodríguez-Román, 2006; Cornwall, Revill & Hurd, 2015). In general, most algae can acquire inorganic carbon (C_i) for RuBisCO through diffusion and active absorption of both, CO₂ and HCO₃⁻ (Badger & Price, 1994; Giordano, Beardall & Raven, 2005; Hurd et al., 2009). In many cases, the activity of CCMs has been associated with the direct or indirect use of HCO₃⁻ (Reiskind, Seamon & Bowes, 1988; Invers et al., 2001; Enríquez & Rodríguez-Román, 2006). Some macroalgae convert bicarbonate (HCO₃⁻) into CO₂ extracellularly using carbonic anhydrase (CA) thus CO₂ enters the cell by active transport or diffusion. Other algae incorporate HCO₃⁻ actively through the cell membrane and, intracellularly, an internal CA converts HCO₃⁻ into CO₂ (Badger & Price, 1994). The activity of carbonic anhydrases has been widely documented in algae (Reiskind, Seamon & Bowes, 1988; Invers, Perez & Romero, 1999; Enríquez & Rodríguez-Román, 2006) and they play a significant role in CCMs.

Many studies have examined the combined effects of environmental variables on algae photosynthetic responses: CO₂ and temperature (Campbell et al., 2016; Kram et al., 2016; Vásquez-Elizondo & Enríquez, 2016); CO₂ and light (Vogel et al., 2015); light and nutrients (Zubia, Freile-Pelegrín & Robledo, 2014); CO₂ and nutrients (Hofmann et al., 2014; Hofmann et al., 2015; Bender-Champ, Diaz-Pulido & Dove, 2017); CO₂, nutrients, and temperature (Stengel et al., 2014), and CO₂, nutrients and light (Celis-Plá et al., 2015). Multiple stressors could have an interactive influence causing complex responses at the physiological and ecological level (Hofmann et al., 2014), which makes them difficult to interpret. Therefore, studies that combine ocean acidification scenarios with other factors such as temperature, light, and nutrient availability are particularly necessary since changes in these parameters are co-occurring with changes in carbonate chemistry in the seawater (Harley et al. , 2012; Hofmann et al., 2014).

Halimeda is a calcifying genus of siphonous green algae (Bryopsidales, Chlorophyta) which are important components of tropical and subtropical reefs and lagoons. Some species of this genus often appear dominating Caribbean coral reefs (Beach et al., 2003; Hofmann et al., 2014) where they contribute as primary producers, food source and habitat, sand production, and coral-reef formation. Halimeda scabra Howe is particularly abundant in the front reef and shallow rocky areas of the Caribbean Reefs (Alcolado et al., 2003). Despite the ecological studies above-mentioned, to our knowledge, no previous physiological studies have been reported for this species.

Photosynthetic responses to the combined effect of environmental variables have been studied in some Halimeda species, for example in H. opuntia, the effect of nutrients and pH (Hofmann et al., 2014; Hofmann et al., 2015), in H. incrassata and H. simulans the effect of pH and temperature (Campbell et al., 2016), and in H. opuntia the effect of pH and light (Vogel et al., 2015). These studies have suggested that an increase in both, CO₂ (low pH) and high temperature could have a positive synergistic effect on photosynthetic rates (Kram et al., 2016). However, Halimeda responses to high CO₂ have been diverse; in some species a decrease in photosynthesis with the reduction of pH has been observed (Price et al., 2011; Sinutok et al., 2012; Meyer et al., 2016) others have shown the opposite effect (Peach, Koch & Blackwelder, 2016) or a lack of a significant response (Price et al., 2011; Campbell et al., 2016). In general, there are still insufficient studies on the physiology of the genus Halimeda that allow us to understand the diversity of physiological responses to the interactive effects of environmental variables and the mechanisms involved in those responses.

In this study, we hypothesized that a synergistic increase in environmental factors (temperature, pH, and nutrients) would enhance H. scabra photosynthesis, which absorbs bicarbonate supported by a CCM. We evaluated the interactive effect of temperature, pH, and nutrient levels on photosynthetic responses of H. scabra. Additionally, we determined the C_i uptake mechanisms by measuring the effect of CA inhibitors on P_max, analyzing δ¹³C values, and evaluating the incorporation of stable isotope ¹³C into resulting products of photosynthesis.

Materials and Methods

Results

Photosynthetic responses to the interactive effect of temperature, pH, and nutrient levels

The three-way ANOVA showed a significant interactive effect of temperature, pH, and nutrients on H. scabra gross photosynthesis, P_gross (F_12;216 = 4.57, p ≤ 0.001) (Fig. 1; Table S2). The highest P_gross values (1.83 mg O₂ g DW h⁻¹) were obtained at the highest nutrient concentration (10:1.0 µM) under elevated temperature (33 °C) at a pH of 8.6 and in the control treatment (no added nutrients) at 33 °C but, at the lowest pH (7.5) (P_gross = 1.78 mg O₂ g DW h⁻¹). In contrast, low and medium nutrient level treatments had lowest P_gross at intermediate temperatures. In general, H. scabra photosynthetic rates were higher at the highest temperature tested regardless of the nutrient or pH levels, except for the low nutrient treatment, which had higher P_gross at the lowest temperature for all pH treatments.

It is noteworthy how P_gross responds to temperature and pH changes in relation to nutrient concentrations in an opposite pattern between the highest nutrient concentration (10:1.0) and the control treatment (no added nutrients) at the highest temperature tested.

Individual analysis of factors showed that pH alone had no effect on P_gross (F = 2.84, p >0.05), whereas the individual effect of nutrients (F = 4.77, p ≤ 0.05) and temperature (F = 45.30, p ≤ 0.001) were significant (Table S2). All interactions involving two factors were significant: temperature-pH (F = 2.70, p ≤ 0.05); nutrient-temperature ( F = 10.32, p ≤ 0.001); and pH-nutrients (F = 9.23, p ≤ 0.001).

HCO₃⁻ Uptake

The δ¹³C value in H. scabra was −23.9‰ suggesting uptake of both HCO₃⁻ and CO₂, and the presence of a CCM. The carbonic anhydrase assays corroborate the latter since the addition of EZ caused a significant inhibition (22.2%) of maximum photosynthesis rates P_max (F_3,24 = 18.674, p ≤ 0.001) whereas the combination of both inhibitors produced a similar effect to that found with EZ (Fig. 2). AZ inhibitor showed no effect on P_max implying a lack of external CA and direct uptake of HCO₃⁻ with a CCM depending on internal CA.

Incorporation of ¹³C into products of photosynthesis

¹³C isotope labeling in H. scabra observed by signal multiplicity (coupling) also showed bicarbonate uptake, since ¹³C isotope was incorporated into an amino acid akin to aspartate in the three photoperiod treatments analyzed. The incorporation in darkness indicates non-photosynthetic β-carboxylation. Aspartate also appears in the three control treatments (simple decoupled signal) highlighting its abundance in the species (Fig. 3).

Discussion

The results of our work support the hypothesis that a synergistic increase in pH, temperature, and nutrients enhances H. scabra photosynthesis. An increase in temperature could enhance P_gross at high pH if there are sufficient nutrients. Environmental conditions of high seawater temperature (Robledo & Freile-Pelegrín, 2005; Rodríguez-Martínez et al., 2010), alkaline pH seawater, likely because of the karstic origins of the Yucatán Peninsula (Cejudo et al., 2020), and pulsatile nutrient enrichment due to the submarine groundwater discharge (Hernández-Terrones et al., 2015), are common in Quintana Roo coastal areas where H. scabra and other Halimeda species colonize shallow environments. Interestingly, in the control treatment (no added nutrients) at low pH and high temperature high P_gross was also observed, most probably because at low pH CO₂ availability is higher (Falkowski & Raven, 2007) and an increase in temperature facilitates photosynthesis (Bruno, Carr & O’Connor, 2015; Vásquez-Elizondo & Enríquez, 2016). Our results also emphasize that interactive effects are more reliable indicators than those observed under individual analyses since pH alone had no effect on P_gross while all interactions were significant. Variation of only one factor can modify the photosynthetic response to other factors highlighting the importance of interactive studies.

Conversely, the interactive effect of decreasing pH (low and medium) with increases in temperature and nutrient enrichment kept P_gross below its potential capacity. Thus, potential deleterious effects on H. scabra performance are expected to occur under future scenarios of ocean acidification, global warming, and their complex interactions with nutrient enrichment due to the continuous coastal development in the area.

In agreement with our results with H. scabra, significant reductions in gross photosynthetic rates have been reported for H. macroloba and H. cylindracea when exposed to elevated CO₂ combined with elevated temperature, showing an additive negative effect (Sinutok et al., 2012). In contrast, for H. incrassata, H. simulans, and H. opuntia no significant effects in net photosynthesis were reported for the interactions among species, pH, and temperature (Campbell et al., 2016). While, in H. opuntia no interactive effect of CO₂ and nutrient enrichment on net photosynthesis was found (Hofmann et al., 2015).

These contrasting results among congeners indicate that the photosynthetic responses to the interactive effects of several environmental variables are complex since, in addition to the factors being evaluated, the physiological mechanisms could be responding to other interrelated processes that were not assessed during assays. For example, Campbell et al. (2016) found in three Halimeda species that photosynthesis was positively correlated to calcification rates and, an increase in temperature increased activity of both processes. In this context, processes with high carbon requirements such as calcification could indirectly stimulate photosynthesis (Carvalho & Eyre, 2017), generating protons that are used to facilitate the absorption of nutrients and bicarbonate (McConnaughey & Whelan, 1997). The reduction of NO₃⁻ to NH₄⁺ is another process with high energy requirements (Ale, Mikkelsen & Meyer, 2011), and it is related to carbon fixation (Cabello-Pasini & Figueroa, 2005) so it is likely to be more plausible to affect photosynthesis rather than calcification since the latter appears to be more dependent on photosynthetic activity of many calcifying primary producers. Nutrient enrichment supported a rapid increase in the physiological performance of H. opuntia (Teichberg, Fricke & Bischof, 2013). Therefore, the photosynthetic increase found with the nutrient addition (KNO₃:K₃PO₄) in our experiments could be the result of its effect on processes related to nutrient uptake and these, to photosynthesis. Moreover, it is also known that nutrient uptake rates increase with temperature increases (Harrison & Hurd, 2001), consequently, our results not only are a response to the interactive effect of environmental factors but also, the result of the direct and indirect response of other metabolic processes on photosynthesis.

Temperature is a significant factor controlling metabolic rates, including photosynthesis; increasing temperature increases photosynthetic rates linearly up to an optimum rate, beyond this thermal threshold rates, tend to decline (Bruno, Carr & O’Connor, 2015; Vásquez-Elizondo & Enríquez, 2016). It is generally accepted that Q ₁₀ values greater than 2 characterize an active nutrient absorption process across cell membranes, while Q ₁₀ ∼1 values describe passive processes that are not greatly affected by temperature (Lobban & Harrison, 1994). According to this, our calculated Q ₁₀ P_gross for the medium and high nutrient treatments are in the range of active nutrient absorption, expected by organisms living in highly illuminated habitats and with high elevated metabolic activity (Vásquez-Elizondo & Enríquez, 2016).

Mechanisms of photosynthetic carbon uptake can influence the isotopic composition of organic matter. Values of δ¹³C between -30 and -10‰ indicate active uptake of both HCO₃⁻ and CO₂ and species whose fixation fall within this range are classified as species with active CCM (Maberly, Raven & Johnston, 1992; Raven et al., 2002; Diaz-Pulido et al., 2016; Bender-Champ, Diaz-Pulido & Dove, 2017). Species with δ¹³C signatures between −32‰ and −22‰ are considered as C3 plants while δ¹³C between −16‰ and –10‰ are typical for C4 plants (Valiela et al., 2018). Considering these ranges and the results obtained in this work, H. scabra could be classified as a C3 plant with a CCM that uses both, HCO₃⁻ and CO₂ as a resource of C_i for photosynthesis. The δ¹³C values of H. scabra found within this study are in the range of those reported in other Halimeda species, such as H. opuntia, ∼−21‰ (Zweng, Koch & Bowes, 2018) and H. tuna, ∼−21‰ (Duarte et al., 2018).

The extracellular CA inhibitor (AZ), did not show any adverse effect on photosynthesis, indicating a direct uptake of HCO₃⁻ while a significant reduction in P_max with EZ, confirmed HCO₃⁻uptake and the presence of a CCM (Badger et al., 1998) along with the role of internal CA (Badger & Price, 1994). The reduction of photosynthesis under the activity of EZ was only about 22.2% relative to control samples, likely because there was still enough CO₂ in the proximity of RuBisCO to maintain a reduced level of photosynthesis. The available CO₂ may come from the following alternatives: (1) as the result of a CCM, mainly related to efficient HCO₃⁻ utilization (Raven, 1997); (2) respiratory CO_2, (Borowitzka & Larkum, 1976); (3) CO₂ supplied from interutricular spaces, although this source is not sufficient to sustain photosynthesis (De Beer & Larkum, 2001); and (4) by CO₂ diffusion from both, the external medium and the intercellular space (ICS) (Borowitzka & Larkum, 1976). All previous explanations could maintain RuBisCO CO₂ saturated and minimize photosynthetic losses after inhibiting intracellular CA. This suggests that inorganic carbon supply for photosynthesis in H. scabra does not depend entirely on the activity of the CA’s, and might be maintained by several mechanisms which may be advantageous during adverse conditions. Bicarbonate (HCO₃⁻) uptake has also been found in H. discoidea, H. macroloba, and H. tuna (Borowitzka & Larkum, 1976), while the lack of extracellular CA has been found in H. discoidea (De Beer & Larkum, 2001) and H. cuneata f. digitate (Hofmann et al., 2015a).

Some Halimeda species possess CCM and use bicarbonate as an alternate source of inorganic carbon (Borowitzka & Larkum, 1976; Price et al., 2011) keeping their photosynthesis carbon saturated under the current seawater C_i concentrations (Beer, 1994; Beardall, Beer & Raven, 1998; Fernández, Hurd & Roleda, 2014). Therefore, this ability may also be responsible for P_gross enhancement under elevated temperature observed in this study in H. scabra through a decrease in photorespiration (Ogren, 1984). According to Giordano, Beardall & Raven (2005), the number of resources that a cell invests in acquiring carbon through a CCM is likely to be coupled with the availability of nutrients. This could also explain the increase in P_gross observed at the highest nutrient level since most CCM’s require the de novo synthesis of specific proteins, which represents a demand for cellular nitrogen (Giordano, Beardall & Raven, 2005). The low oxygen production observed at low pH (under high nutrient concentrations) in H. scabra could be a delay in the induction of the CCM relying on passive diffusion of CO₂ alone, thus leading to reduced efficiency of carbon assimilation (Price et al., 2011; Cornwall et al., 2012; Meyer et al., 2016).

In this study, H. scabra incorporated ¹³C isotope into aspartate in the three photoperiod treatments demonstrating photosynthetic and non-photosynthetic NaH¹³CO ₃assimilation. Moreover, the incorporation of ¹³C isotope in aspartate in 24-hour darkness treatment indicates β-carboxylation which facilitates a metabolic alternative to inorganic carbon carboxylation resulting in an important contribution to CCMs (Raven & Osmond, 1992; Enríquez & Rodríguez-Román, 2006). β-carboxylation has multiple functions for algal metabolism such as providing essential compounds for growth that cannot be produced photosynthetically (Falkowski & Raven, 2007). Carbon fixation of these compounds can be done in both light and darkness (Axelsson, 1988), and it is generally less than 5% of maximum photosynthesis (Cabello-Pasini & Alberte, 1997). In marine algae, the end products of this carbon fixation independent of light are typically organic compounds and amino acids rather than triose sugars generated during photosynthesis (Cabello-Pasini & Alberte, 1997). Although the δ¹³C values in H. scabra suggest a C3 pathway, the abundance of aspartate in control and experimental treatments also suggest the existence of a C4 pathway. In C4 plants, the C4 acids malate and aspartate are the major initial photosynthetic products, these products are rapidly decarboxylated releasing CO₂ for its refixation by RuBisCO functioning as photosynthetic intermediates (Holaday & Bowes, 1980). In this sense, a C4 mechanism could explain the increase in P_gross under all of our high-end treatments (temperature, pH, and nutrient concentration), as well as the insensitivity of the photosynthetic response of the alga to AZ and, its low inhibition in the presence of EZ. C4 plants have an active CCM, which is mainly related to the efficient use of HCO₃⁻ through an initial carboxylation reaction by a Phosphoenolpyruvate enzyme (Badger & Price, 1994; Raven, 1997). C4 mechanisms have been reported in some Chlorophyta, including the semi-calcified Udotea flabellum, which shows an initial carboxylation by phosphoenolpyruvate carboxykinase enzyme (Reiskind, Seamon & Bowes, 1988), whereas in Ulva prolifera evidence of both C3 and C4 pathway has been found (Xu et al., 2012).

Conclusions

H. scabra uses both CO₂ and HCO₃⁻ for photosynthesis and it seems to have different mechanisms for C_i acquisition incorporating bicarbonate through photosynthetic and non-photosynthetic pathways. Our results suggest the presence of both C3 and C4 pathways, with the latter relying on β-carboxylation. These strategies give H. scabra physiological plasticity to acclimate to possible environmental changes in the short term. Our study strongly suggests that H. scabra acclimatizes better to environmental conditions of high pH and high temperature with enough nutrient enrichment. Although these conditions could exacerbate the presence of epiphytes and opportunistic algae, the availability of pulsatile nutrients likely plays a role in maintaining Halimeda populations by enhancing algal photosynthetic performance. Such conditions are typical in the Yucatan peninsula coast where Halimeda species grow in abundance. Opposite interactive conditions of decreasing pH in combination with increases in temperature and nutrient availability, could keep photosynthesis at a sub-optimal level which has strong ecological implications due to the potential for decline in Halimeda abundance and the resulting consequences to sediment production and carbon balance in coral reefs where these algae thrive.

Supplemental Information