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Article

Effects of pH, Temperature, and Light on the Inorganic Carbon Uptake Strategies in Early Life Stages of Macrocystis pyrifera (Ochrophyta, Laminariales)

by
Bárbara S. Labbé
1,2,
Pamela A. Fernández
3,*,
July Z. Florez
3,4 and
Alejandro H. Buschmann
3
1
Program of Magíster en Ciencias Mención Producción, Manejo y Conservación de Recursos Naturales, Universidad de Los Lagos, Puerto Montt 5400000, Chile
2
Institute of Marine and Antarctic Studies (IMAS), University of Tasmania, Hobart, TAS 7005, Australia
3
Centro i~mar, CeBiB & MASH, Universidad de Los Lagos, Puerto Montt 5400000, Chile
4
Departamento de Ciencias y Geografía, Facultad de Ciencias Naturales y Exactas, HUB Ambiental UPLA, Universidad de Playa Ancha, Valparaíso 2340000, Chile
*
Author to whom correspondence should be addressed.
Plants 2024, 13(23), 3267; https://doi.org/10.3390/plants13233267
Submission received: 8 October 2024 / Revised: 15 November 2024 / Accepted: 19 November 2024 / Published: 21 November 2024
(This article belongs to the Special Issue Advances in Algal Photosynthesis and Phytochemistry)
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Abstract

:
The responses of seaweed species to increased CO2 and lowered pH (Ocean Acidification: OA) depend on their carbon concentrating mechanisms (CCMs) and inorganic carbon (Ci) preferences. However, few studies have described these mechanisms in the early life stages of seaweeds or assessed the effects of OA and its interactions with other environmental drivers on their functionality and photophysiology. Our study evaluated the effects of pH, light (PAR), temperature, and their interactions on the Ci uptake strategies and photophysiology in the early stages of Macrocystis pyrifera. Gametophytes were cultivated under varying pH (7.80 and 8.20), light (20 and 50 µmol photons m−2s−1), and temperature (12 and 16 °C) conditions for 25 days. We assessed photophysiological responses and CCMs (in particular, the extracellular dehydration of HCO3− to CO2 mediated by the enzyme carbonic anhydrase (CA) and direct HCO3− uptake via an anion exchange port). This study is the first to describe the Ci uptake strategies in gametophytes of M. pyrifera, demonstrating that their primary CCM is the extracellular conversion of HCO3− to CO2 mediated by CA. Additionally, our results indicate that decreased pH can positively affect their photosynthetic efficiency and maximum quantum yield; however, this response is dependent on the light and temperature conditions.

1. Introduction

Anthropogenic greenhouse gas emissions are considered the primary driver of global change, causing increases in global sea surface temperatures (ocean warming: OW) and a global decrease in the pH of surface waters (ocean acidification: OA) [1]. Under the business-as-usual CO2 emissions scenario, sea surface temperatures are expected to increase globally by 2.0–4.3 °C, while the pH of surface waters may decline by 0.3 units by 2100 [1]. Decreasing surface water pH alters the carbonate chemistry of seawater (SW), causing changes in the concentrations of protons ([H+]) and the proportions of dissolved inorganic carbon (DIC) sources, including carbon dioxide (CO2), bicarbonate (HCO3−), and carbonate ions (CO3−2), each of which can differentially—as well as interactively—affect marine biota [2,3]. DIC and temperature are key factors influencing metabolic processes such as growth and photosynthesis in marine primary producers (i.e., phytoplankton and seaweeds); thus, the single and combined effects of OA and OW can substantially affect their physiological and ecological performance [4]. Along with OW and OA, light availability for benthic primary producers might also be reduced by anthropogenic activities (e.g., agriculture, forestry, pollution) [5,6], which may lead to decreased primary productivity in coastal ecosystems [7,8]. Thus, these global and local changes might directly affect the productivity rates, structure, and persistence of marine communities [9].
Seaweeds, including kelps (large brown algae of the Order Laminariales), have foundational functions in coastal ecosystems and are the base of the food web and major contributors to benthic primary production [9,10,11]. They provide food, habitat, and shelter to higher trophic levels (e.g., shellfish and fish) [2], as well as a range of ecosystem services, such as contributing to carbon and nitrogen cycling, climate regulation, and biodiversity [2,11,12]. In seaweed, as in all photosynthetic organisms, dissolved CO2 is the substrate for carbon fixation by the enzyme ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBisCO) [2,13]. However, at the current SW pH (7.9–8.1), CO2 represents a small proportion of the total DIC and, although it diffuses passively into the cell, this process is too slow to saturate photosynthesis in most species [11]. Therefore, most seaweeds (~70%) have developed specialized carbon concentrating mechanisms (CCMs) to increase the CO2 concentration close to RuBisCO and thus reduce Ci limitations [13,14]. It is assumed that most CCMs are based on the direct or indirect use of HCO3− from the bulk SW, representing about 91% of the total DIC [14]. These mechanisms include (1) HCO3− extracellular dehydration to CO2, catalyzed by the external enzyme carbonic anhydrase (CAext); (2) direct HCO3− uptake through an anion exchange (AE) protein located at the plasmalemma; and (3) active uptake of CO2 or HCO3−, which involves a proton motive force through an H+-ATPase pump [15,16,17]. Therefore, the increased CO2 and HCO3− concentrations associated with OA can strongly affect the photosynthetic physiology of seaweeds, depending on their CCMs and relative ability to use CO2 vs. HCO3− [16,17,18,19]. However, while high-affinity CCMs might be down-regulated by increased dissolved CO2 in some cases [20,21], such conditions might not cause physiological alterations in non-CCM species [18,22]. Additionally, regardless of whether they have a CCM or not, the physiological responses of seaweeds to OA may also be influenced by other environmental drivers, such as temperature and light.
The combined effect of OA with temperature and light will undoubtedly impact the Ci acquisition strategies of seaweeds and, consequently, their photosynthetic and growth rates [11]. In the case of kelp species, these responses are even more complex due to their intricate life cycle, which alternates between microscopic (haploid: meiospores and gametophytes and diploid: embryonic sporophytes) and macroscopic (diploid: adult sporophyte) stages [9]. Across this complex life cycle, micro- and macroscopic life stages are exposed to contrasting environmental conditions in terms of light, temperature, and CO2/pH along the water column. These conditions can modulate ontogenetic differences in their tolerances and sensitivities to environmental changes [23,24,25]; for example, previous studies in the giant kelp M. pyrifera have shown that, at pH 7.86, meiospore germination is unaffected, while gametophyte size is either positively or negatively affected [26]. At pH 7.61, meiospore germination is reduced, but gametophyte size remains unchanged. This may be due to protective mechanisms that minimize the effects of H+ ions on seaweed cell metabolism, which could operate at the expense, for example, of growth [18,26]. Similar results have been described for other Laminariales species, such as Undaria pinnatifida (Harvey) [27]. Juvenile sporophytes and gametophytes of U. pinnatifida exhibit similar photosynthetic responses at pH of 7.3 and 8.3; however, when the pH is increased to 9.3, photosynthetic rates of gametophytes are reduced, while those in sporophytes remain unchanged [27]. Similarly, Hollarsmith et al. [28] observed distinct responses to temperature across the early life stages of Chilean M. pyrifera populations. Meiospores showed higher tolerance to elevated and low temperatures (18 °C and 12 °C, respectively), and gametophytes from all populations grew and survived under all temperature treatments (18 °C and 12 °C). However, gametophytes of all Chilean populations tested failed to reach sexual maturity and produce eggs at elevated temperatures (≥18 °C). These distinct responses to temperature across the life stages of Chilean M. pyrifera may be explained by the local environmental variability experienced throughout their life cycle.
Previous studies have predicted that the early life stages of kelp might be more susceptible to abrupt changes in temperature [25,29,30] and highly vulnerable to OA [31,32]. Therefore, it is fundamental to understand how these micro and macroscopic stages will respond to changing climatic scenarios regarding the physiology of kelps. A recent study on the kelp Saccharina angustissima (Laminariales, Phaeophyceae) showed that each stage of its life cycle exhibits different responses to changes in temperature and light [25]. For example, meiospore germination occurred at temperatures ranging from 7 to 17 °C under light conditions between 20 and 80 μmol photon m−2s−1, except at 12 and 15 °C, where 80 μmol photon m−2s−1 considerably reduced the success of germination. Gametophytes grew best at temperatures between 8 and 13 °C and at the lowest irradiance (10 μmol photon m−2s−1), with light availability having a positive effect only at high temperatures (15 and 17 °C). For juvenile sporophytes, temperatures between 8 and 15 °C and light intensities between 10 and 100 μmol photon m−2s−1 are suitable for growth, while higher temperatures and light intensities considerably reduce growth. These results suggest that the combination of OW and the effects of high light intensity could put pressure on the early life cycle stages of S. angustissima. These differences are likely due to the close relationship between the functionality of CCMs and light. Recent studies have suggested that the presence or expression of CCMs in seaweeds is strongly associated with light availability [33,34,35], due to the varying physiological energy costs of each mechanism [17,19,35]. Thus, in seaweeds, the energy saved through the expression or downregulation of these mechanisms can be allocated to other physiological processes, such as growth, photosynthesis, or reproduction. However, the effects of light availability on CCMs in the early stages of kelps are still unknown.
The giant kelp M. pyrifera forms extensive forests widely distributed along the north and south Pacific coast, inhabiting latitudinal gradients of temperature, nutrients, salinity, and upwelling zones [9,36]. M. pyrifera is a major contributor to coastal productivity [10,37] and plays a key role in marine ecosystems [38]. M. pyrifera presents a biphasic life cycle with alternate generations between micro and macroscopic stages [9]. The development of microscopic early stages of M. pyrifera involves complex processes that are not yet fully understood [39,40], which may be essential to explain the abundance and distribution patterns of M. pyrifera populations [41]. This study evaluated the independent and interactive effects of pH/CO2, light, and temperature on the carbon metabolism and photophysiology of M. pyrifera in its early life stages. The presence of CCMs was assessed using specific inhibitors: acetazolamide (AZ), which inhibits CAext; etoxyzolamide (EZ), which inhibits both enzymes (CAext and CAint); and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), which inhibits the direct uptake of HCO3− via an AE protein [42]. M. pyrifera gametophytes were incubated under combined pH (pH 7.80 and 8.20), light PAR intensity (20 and 50 µmol photon m−2s−1), and temperature (12 °C and 16 °C) conditions for 25 days. At the end of the incubation, photosynthetic rates and photobiological aspects (i.e., Chlorophyll a fluorescence, and pigment concentrations) were measured in the gametophytes. We hypothesized that (1) the CCMs in the early stages of M. pyrifera differ from those described in adult individuals; and (2) the OA pH (7.80) positively affects the carbon metabolism and physiology of M. pyrifera gametophytes by increasing diffusive CO2 use, but this response is dependent on light availability and temperature.

2. Results

2.1. Experiment 1: pH × Light

2.1.1. The Effects of Specific Inhibitors on the Ci Acquisition Mechanisms

After the injection of specific CA inhibitors (AZ alone and AZ + EZ), the photosynthetic rate of M. pyrifera gametophytes, expressed as µmol O2 g−1 DW h−1, was significantly reduced across all treatment combinations, reaching values close to zero and even turning negative (Figure 1). Our results showed that, regardless of the pH and light conditions, the photosynthetic rate was completely inhibited by the presence of the inhibitors (p = 5.64 × 10−14; Table 1). The inhibitory effect on the photosynthetic rate was significantly affected by the pH × light interaction (p = 0.0048; Table 1), but not by pH (p = 0.3706) or light (p = 0.5369, Table 1) individually.
Following the injection of the direct HCO3− uptake inhibitor (DIDS), the photosynthetic rates of M. pyrifera gametophytes remained unchanged (p = 0.0748; Table 1; Figure 2). A slight decrease in the photosynthetic rate was observed under ambient pH and light conditions, compared to low light (Climate Global Change: CGC scenario), but this difference was not statistically significant (p = 0.0574; Table 1). Additionally, there were no significant interactions between pH, light, and the inhibitor (p = 0.9702; Table 1).

2.1.2. Photosynthetic Pigment Analysis

The Chl c/Chl a ratio in M. pyrifera gametophytes was significantly affected by light intensity (p = 0.0003, Table 1), but not by pH or its interaction with light (p = 0.0951; p = 0.7062, Table 1). The Chl c/Chl a ratio was higher at 50 µmol photon m−2s−1, compared to 20 µmol photon m−2s−1, regardless of pH treatment (Ambient pH: 8.20 and OA pH: 7.80; Table 2). The Fucox/Chl a ratio was significantly affected by light and pH, but not by their interaction (p = 0.4303; Table 1). The ratio was higher at 50 µmol photon m−2 s−1, compared to 20 µmol photon m−2s−1 (Table 2). Under ambient light conditions, the Fucox/Chl a ratio was considerably higher at pH 7.80, reaching values of 1.52 at 50 µmol photon m−2s−1; however, these differences were not observed under CGC light conditions at pH 8.20 (Table 2). Our results indicated a higher concentration of Chl a and proportion of photosynthetic pigments under lower light conditions, with a greater concentration of Chl a observed at 20 µmol photon m−2s−1 compared to 50 µmol photon m−2s−1 (p = 0.001, Table 1).

2.1.3. Photophysiology: Chlorophyll a Fluorescence

The photosynthetic efficiency (α) of M. pyrifera gametophytes was significantly affected by pH, but not by light or its interaction with pH (p = 0.6253, p = 0.7555; Table 1). At pH 7.80, α was significantly higher than that at pH 8.20, with values of 0.31 and 0.16 µmol photon m−2s−1, respectively (Table 1).
The maximum quantum yield (Fv/Fm) of M. pyrifera gametophytes was significantly affected by pH, but not by light or its interaction with pH (p = 0.275; p = 0.181; Table 1). At pH 7.80, Fv/Fm was higher compared to that at pH 8.20, with values of 0.53 and 0.44, respectively (Table 1; Figure 3). This suggests that, under future OA conditions (pH 7.80), Fv/Fm—which is known to be a parameter indicative of physiological stress—would not be negatively impacted in M. pyrifera gametophytes.
The ETRmax of M. pyrifera gametophytes was significantly affected by light and the interaction of pH × light, but not by pH alone (p = 0.0001; p = 0.0072; p = 0.3216; Table 1). At both pH 7.80 and pH 8.20, ETRmax was higher at 20 µmol photon m−2s−1 when compared to 50 µmol photon m−2s−1; notably, this difference was greater at ambient pH than at lowered pH (Table 1 and Table 3). Similarly, Ek values were significantly affected by pH, light, and their interaction (p = 0.0231; p = 0.0083; p = 0.0191; Table 1). At both pH 7.80 and pH 8.20, Ek values were higher at 20 µmol photon m−2s−1 compared to 50 µmol photon m−2s−1; however, this difference was greater at ambient pH than at lowered pH (Table 1 and Table 3).

2.2. Experiment 2: pH × Temperature

2.2.1. The Effects of Specific Inhibitors on the Ci Acquisition Mechanism

After the injection of AZ—a specific CAext inhibitor—the photosynthetic rate of M. pyrifera gametophytes was considerably reduced across all treatment combinations, reaching values close to zero and even negative values (Figure 4). The inhibitory effect was more pronounced under ambient pH conditions, compared to the OA treatment (p = 0.0024), regardless of the temperature treatment (p = 0.5086; Table 4; Figure 4).
After the simultaneous injection of both CA inhibitors (i.e., AZ and EZ), the photosynthetic rate of M. pyrifera gametophytes did not experience further inhibition in any pH treatment, compared to the photosynthetic rate after the injection of AZ alone, regardless of the temperature (Figure 4).
Following injection of the direct HCO3− uptake inhibitor (DIDS), the photosynthetic rate of M. pyrifera gametophytes increased slightly across all pH × temperature treatments (Figure 5). This increase was more pronounced under CGC temperature (p = 0.027; Table 4); however, this effect may be attributed to an experimental issue, such as DIDS dissolution, and it is not considered physiologically relevant for photosynthesis.

2.2.2. Photosynthetic Pigment Analysis

The Chl c/Chl a ratio of gametophytes of M. pyrifera was significantly affected by the pH × temperature interaction (p = 0.0449; Table 4). At the ambient temperature treatment, the Chl c/Chl a ratio showed greater values at pH 7.80 than at pH 8.20, reaching values of 0.88 and 0.69, respectively (Table 1). However, this increase in the Chl c/Chl a ratio was not observed under CGC temperature conditions. The Fucox/Chl a ratio of gametophytes of M. pyrifera was significantly affected by temperature, but not by pH or pH × temperature (p = 0.0032; p = 0.4655; Table 2), showing greater values at 16 °C than 12 °C (0.84 and 0.88, respectively), regardless of pH treatment (Table 4).

2.2.3. Photophysiology: Chlorophyll a Fluorescence

The photosynthetic efficiency (α) of M. pyrifera gametophytes was significantly affected by the interaction between pH and temperature (p = 1.415 × 10−06; Table 4). At pH 8.20, α was significantly higher at 12 °C, compared to all other treatments (Table 2).
The maximum quantum yield (Fv/Fm) of M. pyrifera gametophytes was significantly affected by the interaction between pH and temperature (p = 0.0003; Table 4). Fv/Fm was considerably reduced under CGC temperature scenarios (16 °C), when compared to ambient temperature (12 °C) under both pH treatments (Figure 6). At pH 8.2, Fv/Fm was significantly higher at 12 °C than at 16 °C, with values of 0.49 and 0.29, respectively (Figure 6). Furthermore, Fv/Fm at pH 8.20 and 12 °C was significantly higher, when compared to all temperature treatments at pH 7.80 (Table 3 and Table 4; Figure 6).
The ETRmax of M. pyrifera gametophytes was significantly affected by pH and temperature, but not by their interaction (p = 0.0262; p = 0.0193; p = 0.058; Table 4). ETRmax was higher at ambient pH than at lowered pH, reaching the highest value at 12 °C (Table 3). Contrary to ETRmax, Ek values were not significantly affected either by pH, light, or their interaction (p = 0.2139; p = 0.8207; p = 0.5629; Table 3 and Table 4).

3. Discussion

Our results support the hypothesis that CCMs in the early life stages of M. pyrifera differ from those described in adult individuals. We found that the photosynthetic rates of M. pyrifera gametophytes under all pH (7.80 and 8.20), light (20 and 50 µmol m−2s−1), and temperature (12 and 16 °C) treatments were 80–100% inhibited by the specific CA inhibitors (AZ + EZ). To the contrary, in the presence of the external HCO3− direct uptake inhibitor (DIDS), their photosynthetic rates mainly remained unaffected. These findings confirm that M. pyrifera gametophytes are able to use HCO3− from the external medium to support their photosynthesis via a CAext-mediated pathway, with the AE-type transmembrane transporter having a small contribution. These results are contrary to what has been described in adult individuals of M. pyrifera, where the use of HCO3− is primarily dependent on the direct HCO3− uptake via an AE port, with CAext having little contribution [17]. However, the photosynthetic rates of gametophytes under ambient pH (8.20) and light (50 µmol m−2s−1) conditions were partially inhibited by the presence of the direct HCO3− uptake inhibitor, suggesting that this mechanism might support photosynthesis in M. pyrifera gametophytes under certain environmental conditions.
The external dehydration of HCO3− to CO2 by the enzyme CAext enhances the CO2 concentration at the cell surface, facilitating the diffusion of CO2 into the cell [43,44]. This mechanism can be less energetically expensive than direct HCO3-uptake via the AE port and other CCMs such as direct CO2 uptake, implying that the energy saved might be directed to support other physiological processes such as growth [45]. Our results support the hypothesis previously raised by Leal et al. [32] who, when studying the effects of OA and elevated temperature on the early stages of this species, suggested that they may use CO2 either through passive diffusion or via the CAext, due to the enhanced growth rates observed at lowered pH (7.20). Similarly, the highest growth rates of M. pyrifera gametophytes were observed under lowered pH (7.86 and 7.61) by Roleda et al. [26], suggesting that increased CO2 or greater [H+] concentrations might favor the growth of M pyrifera in early life stages. The presence of CA enzymes has been successfully detected in other early life stages of large brown seaweeds; for example, both CAext and CAint have been characterized in gametophytes of S. japonica using molecular and cellular methods [46,47,48,49], with no HCO3− transporters detected [50]. Those results suggest that gametophytes of S. japonica might be unable to directly take up HCO3− from the external medium. Similarly, Zhang et al. [27] showed that gametophytes and sporophytes of U. pinnatifida use HCO3− via the CAext-mediated pathway to support their photosynthesis, with gametophytes having a lower affinity for HCO3− than juvenile sporophytes. In red seaweed species, which exhibit a different life cycle than kelps, it has also been demonstrated that Ci utilization varies between gametophytes and sporophytes. For example, Wang et al. [50] showed that, while the thallus (haploid) of Pyropia haitanensi mainly depends on CAext to acquire Ci, the conchocelis (diploid) uses both HCO3− utilization mechanisms; that is, AE transporters and CAext and CAint. These findings and our results strongly suggest that Ci uptake strategies can vary among the life stages (haploid vs. diploid) of seaweed species. These differences might be due to the differential energetic requirements across its life cycle; for example, early life stages might utilize less energetically costly Ci uptake strategies to support other physiological processes, such as growth and/or reproduction, or due to local environmental conditions.
In seaweeds, the existence or expression of different HCO3− utilization mechanisms can be influenced by external environmental conditions [51]; for example, species with moderate to high CCM capacity typically thrive in high-light environments with sufficient energy to support energetically expensive CCMs. Conversely, higher reliance on diffusive CO2 is commonly found in low-light environments [13,50,52]. These differences have even been observed within the same species; for example, in Ulva sp. growing in cold waters with low irradiance, the CA-mediated pathway predominates. In contrast, direct HCO3− uptake via the AE port predominates in Ulva sp. growing in warmer waters with higher irradiance [53]. The latter mechanism of HCO3− utilization is more efficient than the CA-mediated pathway. Hence, it can support high photosynthetic rates more efficiently, which typically occur under conditions of high temperature and high irradiance [53]. This advantage of alternating between Ci mechanisms could also occur in kelp stands, considering their global distribution and complex life cycles, which expose them to a wide range of temperatures, nutrient availability, hydrodynamic stresses, and light conditions [9,36].
Kelps inhabit subtidal habitats down to depths of 20–30 m, with settlement stages (gametophytes, embryonic sporophytes) growing at the upper limit zone, typically under the shade of the adult canopy with little exposure to high irradiances [9,54,55]. Thus, similar to the light-limited deep blades of M. pyrifera, which are well adapted to shade and exhibit lower photosynthetic rates than canopy blades [50,56,57], gametophytes and embryonic sporophytes also exhibit lower light requirements to support photosynthesis, when compared to the adult sporophytes (see [56] and the references therein). This could explain the predominance of the CA-mediated pathway for utilizing HCO3−. Given the environmental conditions to which these life stages are exposed—namely, low light and cold waters—the CA-mediated pathway is likely sufficient to supply CO2 to RuBisCO and support photosynthesis. Our findings, along with previous studies on the species [10,16,31], confirm its capacity to alternate its mode of HCO3− utilization depending on its environmental conditions and physiological requirements.
We also hypothesized that, under lowered pH (7.80), the carbon metabolism and photophysiology of M. pyrifera gametophytes will be positively affected by increased CO2 use; however, this response would depend on the availability of light and temperature. Contrary to our hypothesis, we did not observe a significant effect of lowered pH on the functioning of CCMs under any of the light or temperature treatments. For example, the impact of specific CA inhibitors on the photosynthetic rates of gametophytes was similar at both ambient and lowered pH, suggesting that CA activity was not downregulated by increased CO2 availability (i.e., enhanced CO2 use via passive diffusion). However, other physiological parameters showed differential responses to lowered pH and its interaction with light and temperature. We found that the gametophytes’ photosynthetic efficiency (α) and maximal quantum yield (Fv/Fm) were higher at lowered pH than at ambient pH, regardless of light availability. These results suggest that OA (lowered pH and increased CO2 availability) may increase photosynthesis in gametophytes of M. pyrifera. However, further metabolic studies are needed to elucidate the potential benefits associated with lowered pH and increased CO2 in the early life stages of kelps.
Conversely, our results indicated that a future decrease in light intensity is unlikely to impact the photosynthetic efficiency of M. pyrifera gametophytes negatively. This gametophytic resiliency may be attributed to their high acclimatization capacity to varying light intensities [58,59] and their relatively low light requirements to support photosynthesis (see [56] and the references therein). The low light treatment (20 µmol m−2 s−1) applied throughout our study may not have limited their photosynthesis. We found that Chl a concentrations were higher under the low light treatment, compared to 50 µmol m−2 s−1 treatment, reaching values up to 0.12 mg g−1 WW. Despite the lack of studies describing the pigment content in this life cycle stage of Macrocystis, a previous study in L. digitata gametophytes described similar values to our study, ranging from 0.042 to 0.139 mg g−1 WW [60]. Additionally, it has been shown that gametophytes can ameliorate the negative effects of photodamage through the upregulation of accessory pigments [60], which supports our findings. We observed that the Chlc/Chla and fucox/Chla ratios varied significantly between light treatments (20 and 50 µmol m−2 s−1). In particular, these ratios were higher under 50 µmol m−2 s−1 compared to 20 µmol m−2 s−1, suggesting that gametophytes were able to upregulate their accessory pigments while downregulating Chl a concentrations. This is a clear indicator of photoacclimation in algal species [58,61]. An increase in the concentrations of secondary pigments could enhance their ability to capture light and, thus, provide the energy to generate ATP and NADPH under conditions of greater CO2 availability [53]. Classical studies on M. pyrifera have shown how the composition of the pigments Chl a, Chl c, and fucoxanthin vary in juvenile sporophytes of M. pyrifera after they were transplanted at different depths [62]. These processes of variation and acclimatization have also been reported in other brown algae, such as Sargassum [63] or Ascoplhyllum nodosum [64]. A previous study on gametophytes of L. digitata showed that these stages exhibit great plasticity in their photosynthetic responses to light and temperature conditions [60]. Such physiological plasticity may enable gametophytes to thrive in diverse environments; this trait can be important for maintaining M. pyrifera populations in future oceans.
We also observed an interactive effect of OA and temperature on photosynthetic efficiency (α). Under OA pH conditions, α was similar under both temperature treatments. In contrast, at ambient pH, α was more than twice as high at 12 °C, when compared to the CGC temperature (16 °C). Similar to our study, Debelecq et al. [60] showed that the α value in gametophytes of L. digitata decreases with increasing temperature. However, increases in temperature also resulted in increases in the ETRmax and Ek values, which disagree with our results. The differences observed in our study regarding α values among temperature treatments were correlated with pigment content (Chl a and fucoxanthin), which decreased under the CGC temperature at ambient pH. This reduction in antennae pigments likely diminished light absorption at the higher temperature, contributing to a decrease in α [65]. Similarly, other photosynthetic parameters, such as Fv/Fm and ETRmax, were also reduced under elevated temperature at ambient pH. Interestingly, the negative impacts of elevated temperature on M. pyrifera gametophytes were observed only at ambient pH, and not under lowered pH. It has been shown that lower concentrations of pigments may limit energy transfer in the PS II [50], which could explain the low Fv/Fm values observed in gametophytes grown at 16 °C under ambient pH. Similar to those in our study, the Fv/Fm values in gametophytes of L. digitata decreased with increasing temperature. At the optimal temperature of 10 °C, Fv/Fm values ranged from 0.52 to 0.53 in gametophytes of L. digitata, showing a progressive decline with increasing temperature. In our study, the Fv/Fm values ranged from 0.29 at 16 °C to 0.49 at 12 °C, agreeing with the values described for L. digitata under optimal temperature conditions. However, additional information such as the tolerance and functionality of PSII—which is essential for the whole photochemistry process [60]—in the early life stages of kelps is still lacking.

4. Materials and Methods

4.1. Study Area and Sporophylls Collection

Fertile sporophylls were collected from ten sporophytes of M. pyrifera during low tide from the open coast site Carelmapu (Figure 7), Region de Los Lagos, South of Chile (41°44′45′′ S, 73°42′23′′ O) in February 2019. Sporophylls were transported to the laboratory using insulated containers filled with local seawater (SW). At the laboratory, eight to ten sporophylls (with mature sori) were dissected using a scalpel, and small disks (8–10 cm) were punched out from each sporophyll. These disks were gently rinsed, and any visible epibionts were brushed off with filtered ambient SW (0.22 μm pore, Merck Millipore, Burlington, MA, USA, at 12 °C). Later, the sporophyll disks were stored (wrapped in tissue and foil paper to induce dehydration) overnight at 4 °C before inducing meiospore release [66].

4.2. Experimental Design

Two independent experiments were conducted and, in each experiment, at least 5 to 10 disks with mature sori were immersed for 20 min in the respective pHT treatments: 7.80 and 8.20 (pH measured on the total scale) to obtain a stock meiospore suspension. An initial concentration of 25,000 cells mL−1 was separately inoculated into 6 small Petri dishes containing 20 mL SW with the corresponding pHT treatment (n = 6 independent replicates per pHT treatment). The initial meiospore density was calculated using a 0.1 mm depth hemocytometer (Neubauer improved bright-line, Marienfeld, Germany). Meiospores were allowed to settle for 12 h, and the culture medium was then renewed to eliminate unsettled meiospores and detritus.

4.2.1. Experiment 1: pH × Light

Meiospores of M. pyrifera were cultivated for 25 days in two identical, light-controlled growth chambers. One growth chamber was set up at 40 ± 5 µmol photon m−2s−1, representing the current light intensities (Ambient Light) described for the study site at a depth of 2 m. The other growth chamber was set up at 15 ± 5 µmol photon m−2s−1, simulating reduced light availability (Low Light), which can occur under the canopy in coastal marine ecosystems. Each growth chamber had a photoperiod of 16/8 h L:D (Light/Dark) PAR with a temperature of 12 °C, corresponding to the conditions at the study site. The light was provided using LED lamps (Philips, LED-EM-HO) and monitored with a PAR sensor (LI-COR, meter LI-250A, LI-COR, Lincon, NE, USA). For each light treatment, meiospore cultures were prepared at 25,000 cells mL−1 and the SW pH was adjusted to pH 7.80 (n = 6) or 8.20 (n = 6), simulating projected and current pH scenarios, respectively (see below for details of SW manipulation). Control cultures (SW without meiospores) corresponding to each pH × light treatment were prepared, and pH was monitored throughout the experiment. The culture medium was renewed every 3 days during the first 15 days of the experiment, and then every 1–2 days to prevent nutrient depletion. The pH of the culture medium was measured every 3–4 days, both after and before each medium renewal.

4.2.2. Experiment 2: pH × Temperature

Meiospores of M. pyrifera were cultivated for 25 days in two identical, temperature-controlled rooms. One room was set up at 12 °C, representing the current temperature (Ambient Temp) at the study site, and the other was set up at 16 °C (12 °C + ~4 °C), simulating future OW conditions [1]. Each controlled room had a photoperiod of 16/8 h L:D (Light/Dark) with a light intensity of 40 ± 5 µmol photon m−2s−1, representing the current light intensity described for the study site (Ambient Light) at a depth of 2 m. The light was provided using LED lamps (Philips, LED-EM-HO) and monitored with a PAR sensor (LI-COR, meter LI-250A, LI-COR, Lincon, NE, USA). For each temperature treatment, meiospore cultures were prepared at 25,000 cells mL−1, and the SW pH was adjusted either to pH 7.80 (n = 6) or 8.20 (n = 6), representing the OA and AMB pH scenarios, respectively (see below for details of SW manipulation). Control cultures (SW without meiospores) corresponding to each pH × temperature treatment were prepared, and their pH was monitored throughout the experiment. The culture medium was renewed every 3 days during the first 15 days of the experiment, and then every 1–2 days to prevent nutrient depletion. The pH of the culture medium was measured every 3–4 days, both after and before each medium renewal.
At the end of both experiments, we evaluated the effects of specific inhibitors—AZ, which inhibits CAext; EZ, which inhibits both enzymes (CAext and CAint); and DIDS, which inhibits the direct uptake of HCO3− via an AE protein—on the photosynthetic rates of M. pyrifera gametophytes. We also measured the concentrations of photosynthetic pigments (Chl a, Chl c, and Fucoxanthin) and Chlorophyll a fluorescence, as described below (see Section 4.5).

4.3. Seawater pH Manipulations

At the laboratory, the onsite SW was filtered at 0.2 µm using a vacuum filter system and stored overnight in sterilized 2 L Schott Duran® bottles at the respective experimental temperatures. After filtration and nutrient enrichment with Provasoli culture media [67], the ambient SW pH was 8.13. The two SW pHT (pH measured on the total scale) treatments, 7.80 and 8.20, were prepared daily at the respective pH and temperature before renewing the culture medium. The different SW pHT treatments were achieved using the acid/base method, mimicking the changes in SW carbonate chemistry due to OA [68]. To achieve the OA pH (7.80), equal volumes of 0.5 M HCl and 0.5 M NaHCO3 were used; meanwhile, for the SW ambient pH (8.20), equal volumes of 0.5 M NaOH and 0.5 M NaHCO3 were used.

4.4. Seawater pH Measurements

Seawater pHT was measured at 12 °C using a pH electrode (Thermo Scientific Orion ROSS Sure-Flow semi-micro, ORI8175BNWPW, Massachusetts, US) connected to a pH meter (Thermo Scientific Orion 720A pH/ION Meter, Walthma, MA, USA). Temperature-equilibrated pH buffers (pH 7.0 and pH 10.0, color-coded, NIST traceable, Thermo Scientific) were used to calibrate the electrode and determine its slope. A TRIS buffer, standardized against a seawater buffer, was then used to measure pH on the total scale [69]. Seawater samples representing both treatments—7.80 (OA scenario) and 8.20 (AMB scenario)—were collected and fixed with mercuric chloride for subsequent carbonate chemistry analysis. The total alkalinity (AT) was measured according to the potentiometric method with a controlled titration closed cell (Metrohm, 848 Titrino Plus, Herisau, Switzerland) [69]. The SW carbonate chemistry of each pH treatment was calculated from the measured AT, pHT, salinity, and temperature (Table 1) using the SWCO2 software (version 2.3) [70].

4.5. Physiological and Biochemical Parameters

4.5.1. Specific Inhibitors of Ci Acquisition Mechanism

We evaluated the Ci acquisition mechanisms of M. pyrifera gametophytes at the end of each experiment using specific inhibitors. The highly specific inhibitors used were AZ, which only inhibits CAext, as it is poorly permeable with respect to passage through the plasma membrane [42,71]; EZ, which inhibits both enzymes (CAext and CAint), as it passes quickly and easily across the cell membrane [17,72]; and DIDS, which inhibits the direct uptake of HCO3− via an AE port [17,73]. These inhibitors have been widely used in algae to study photosynthetic Ci acquisition mechanisms (e.g., [42,73]).
AZ and EZ were prepared in 0.02 M NaOH solutions [42], while DIDS was dissolved in MiliQ water. The final concentrations of the injected inhibitors were 100 µM for AZ and EZ, and 300 µM for DIDS. Each inhibitor was applied separately. The relative contribution of each Ci acquisition mechanism was estimated according to the effect of each inhibitor on the photosynthesis of the M. pyrifera gametophytes. Approximately 0.02 g of gametophytes from each Petri dish was transferred separately to Eppendorf tubes (1.5 mL) containing culture medium corresponding to each pH treatment. The tubes were incubated for 12 h before measurement, in order to minimize physiological stress. For each Petri dish (n = 5), O2 evolution was recorded using a fiber optic oxygen meter (Microx TX3, PreSens, Ragensburg, Germany, with temperature compensation and needle-type oxygen microsensors; PreSens, Ragensburg, Germany) for 20 min (initial photosynthesis time) under the light and temperature conditions of each treatment. After this period, the inhibitors were injected separately. For CA inhibitors, the order of injection was AZ first, followed by EZ. After the injection of each inhibitor, O2 evolution was measured for an additional 10 min (total measurement time per sample: 20 min initial + 10 min AZ + 10 min EZ). For DIDS, O2 evolution was measured continuously for 10 min post-injection. O2 evolution was also measured for control treatments for pH, as described above. The effect of each inhibitor was calculated through a linear regression during the last 5 min of incubation. The measurements were standardized according to the dry weight of the corresponding sample (g) and expressed as μmol O2 g−1 dried algae h−1 (modified from [42]).

4.5.2. Chlorophyll a Fluorescence

Chlorophyll a fluorescence of photosystem II (PSII) was measured on the last day of the experiment using a pulse amplitude modulated fluorometer Junior-PAM (Heinz Walz, Effeltrich, Germany), provided with an Emitter–Detector Unit (ED). Gametophytes were carefully detached from each Petri dish by hand, sieved through a 20 µm mesh, and then placed in a 96-well microplate (400 µL) (Costar-96-well model) with culture medium corresponding to each pHT treatment. Measurements were performed in each of the 5 experimental units (microplate well) by positioning the optical fiber (1.5 mm) directly inside each experimental unit, maintaining a constant distance of 10–15 mm between the optical fiber and the bottom of the microplate well. The potential maximum quantum yield (Fv/Fm) was determined according to Hanelt [74]. After a 15 min dark adaptation period, the minimal F0 was recorded with a pulsed measuring light of 650 nm followed by short pulses of completely saturating white light pulse (0.4–0.8 s, 1000–5000 μmol photons m−2 s−1), in order to record Fm (Fv = Fm − F0) (see [75] for reference).

4.5.3. Photosynthetic Pigment Analysis

Chlorophyll a, chlorophyll c, and fucoxanthin (Fucox) were analyzed on the final day of the experiment. Gametophytes were carefully detached from each Petri dish, filtered through a 20 µm mesh, and placed in 2 mL Eppendorf tubes. Gametophytes (0.05 g FW) were incubated in 200 µL Dimethylsulfoxide (DMSO) for 20 min in the dark. This extract was centrifuged at 10,000 rpm for 5 min at 4 °C, until a supernatant and a precipitate of gametophytes were obtained. Then, the supernatant was placed in a spectrophotometer microplate to read absorbances. To the obtained precipitate (gametophytes), an additional 20 min incubation was performed using 700 µL 90% (v/v) acetone. The acetone extracts were then centrifuged at 10,000 rpm for 5 min at 4 °C. The concentrations of all pigments were calculated according to the methodology of Seely et al. [76] for DMSO and acetone (90%) extraction. Absorbance readings were obtained using a microplate spectrophotometer (Thermo Scientific, TECAN, Infinite 200Pro) with 400 µL of samples. Chl a, Chl c, and Fucox contents (mg g−1 WW) were calculated based on absorbance measurements from DMSO (1) at 480.0 nm (A480), 582.0 nm (A582), 631.0 nm (A631), and 665.0 nm (A665) and from 90% (v/v) acetone (2) at 470.0 nm (A470), 581.0 nm (A581), 631.0 nm (A631), and 664.0 nm (A664), using the following equations:
(1)
DMSO extract:
Chl a (g L−1) = A665/72.8
Chl c (g L−1) = (A631 + A582 − 0.297A665)/61.8
Fucox (g L−1) = (A480 − 0.722 (A631 + A582 − 0.297A665) − 0.049A665)/130
(2)
Acetone extract:
Chl a (g L−1) = A664/73.6
Chl c (g L−1) = (A631 + A581 − 0.3A664)/62.2

4.6. Data Analysis

The effects of the experimental factors (pH, light, and temperature) on the physiological responses of M. pyrifera gametophytes (Chlorophyll a, fluorescence, and photosynthetic pigment concentrations) were analyzed via two-way analysis of variance (ANOVA). To analyze the data obtained from the experiments involving the specific photosynthesis inhibitors, a three-way ANOVA was applied, considering the factors of pH, light/temperature, and the type of inhibitor. In all of the datasets, outliers were detected through the Bonferroni outlier test [77], using the “Bonferroni outlier Test” function of the “car” package in R. Then, verification of the assumptions of normality and homoscedasticity was carried out using the Shapiro–Wilk and Levene tests, respectively. In cases where the normality assumption was not fulfilled, a logit transformation was carried out. A post hoc Tukey test (p < 0.05) was applied when a significant effect of independent variables was observed. All statistical analyses were performed using the R v4.2.2. software (www.r-project.org) (accessed on 19 January 2023).

5. Conclusions

The main conclusion of our study is that the Ci uptake mechanisms in gametophytes of the giant kelp M. pyrifera differ from those described in adult individuals. Specifically, gametophytes rely more on the CAext-mediated pathway for using bicarbonate (HCO3−) to support photosynthesis, while adults primarily use an AE-type transporter for taking up HCO3−. Our results suggest that gametophytes exhibit high photosynthetic plasticity, able to adapt to varying environmental conditions such as light, temperature, and pH. While the lowered pH (increased CO2 availability) did not significantly enhance photosynthesis, it did increase the photosynthetic efficiency and quantum yield of gametophytes, suggesting potential benefits of OA for early life stages. Additionally, temperature and light conditions influenced the photosynthetic efficiency, with gametophytes showing the capacity to acclimate to different light intensities. These findings highlight the importance of environmental drivers in shaping the Ci utilization strategies and physiological responses of M. pyrifera gametophytes.

Author Contributions

Conceptualization, B.S.L.; methodology, B.S.L. and J.Z.F.; writing—original draft preparation, B.S.L. and P.A.F.; writing—review and editing, A.H.B. and P.A.F.; supervision, A.H.B. and P.A.F.; funding acquisition, A.H.B. and P.A.F. All authors have read and agreed to the published version of the manuscript.

Funding

B.S.L. acknowledges the support of the Programa de Magíster en Ciencias mención Producción, Manejo y Conservación de Recursos Naturales, Universidad de Los Lagos, Puerto Montt, Chile. J.F. acknowledges the support of FONDECYT Postdoctoral 3220102. A.H.B. acknowledges the support of the following grants during the execution of this master thesis: FONDECYT 1110845, Programa Basal ANID: FB-0001 & AFB240001, and FONDECYT 1221161 during the preparation of this manuscript. P.A.F. acknowledges the support of the following grant during the execution of this master thesis: FONDECYT 11200474.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 13 03267 g001
Figure 1. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of CA inhibitors from Experiment 1: pH × light. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Light condition panels: AMB Light (50 µmol photon m−2s−1) and CGC Light (20 µmol photon m−2s−1). Control: gametophytes without the inhibitors AZ or EZ. Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Figure 1. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of CA inhibitors from Experiment 1: pH × light. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Light condition panels: AMB Light (50 µmol photon m−2s−1) and CGC Light (20 µmol photon m−2s−1). Control: gametophytes without the inhibitors AZ or EZ. Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Plants 13 03267 g001
Plants 13 03267 g002
Figure 2. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of the direct HCO3− uptake inhibitor from Experiment 1: pH × light. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Light condition panels: AMB Light (50 µmol photon m−2s−1) and CGC Light (20 µmol photon m−2s−1). Control: gametophytes without the inhibitor DIDS. Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Figure 2. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of the direct HCO3− uptake inhibitor from Experiment 1: pH × light. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Light condition panels: AMB Light (50 µmol photon m−2s−1) and CGC Light (20 µmol photon m−2s−1). Control: gametophytes without the inhibitor DIDS. Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Plants 13 03267 g002
Plants 13 03267 g003
Figure 3. Maximum quantum yield (Fv/Fm) in M. pyrifera gametophytes in Experiment 1 (corresponding to pH × light). pH conditions: Ambient (AMB) and Ocean Acidification (OA). Color of legends indicates temperature conditions: Ambient (Amb, 12 °C) and Climatic Global Changes (CGC, 16 °C). Values represent the mean ± SD (p < 0.005, two-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Figure 3. Maximum quantum yield (Fv/Fm) in M. pyrifera gametophytes in Experiment 1 (corresponding to pH × light). pH conditions: Ambient (AMB) and Ocean Acidification (OA). Color of legends indicates temperature conditions: Ambient (Amb, 12 °C) and Climatic Global Changes (CGC, 16 °C). Values represent the mean ± SD (p < 0.005, two-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Plants 13 03267 g003
Plants 13 03267 g004
Figure 4. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of CA inhibitors in Experiment 2: pH × temperature. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Temperature condition panels: AMB (12 °C) and CGC (16 °C). Control: gametophytes without inhibitors (AZ and EZ). Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Figure 4. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of CA inhibitors in Experiment 2: pH × temperature. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Temperature condition panels: AMB (12 °C) and CGC (16 °C). Control: gametophytes without inhibitors (AZ and EZ). Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Plants 13 03267 g004
Plants 13 03267 g005
Figure 5. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of the direct HCO3− uptake inhibitor in Experiment 2: pH × temperature. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Temperature condition panels: AMB (12 °C) and CGC (16 °C). Control: gametophytes without inhibitor (DIDS). Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Figure 5. Photosynthetic rate (µmoles O2 g−1 DW h−1) in M. pyrifera gametophytes after injection of the direct HCO3− uptake inhibitor in Experiment 2: pH × temperature. Color of legends indicates pH conditions: Ambient (Amb, red) and Ocean Acidification (OA, blue). Temperature condition panels: AMB (12 °C) and CGC (16 °C). Control: gametophytes without inhibitor (DIDS). Values represent the mean ± SD (p < 0.005, three-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Plants 13 03267 g005
Plants 13 03267 g006
Figure 6. Maximum quantum yield (Fv/Fm) in M. pyrifera gametophytes from Experiment 2 corresponding to pH × temperature. pH conditions: Ambient (AMB) and Ocean Acidification (OA). Color of legends indicates temperature conditions: Ambient (Amb, 12 °C) and Climatic Global Changes (CGC, 16 °C). Values represent the mean ± SD (p < 0.005, two-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Figure 6. Maximum quantum yield (Fv/Fm) in M. pyrifera gametophytes from Experiment 2 corresponding to pH × temperature. pH conditions: Ambient (AMB) and Ocean Acidification (OA). Color of legends indicates temperature conditions: Ambient (Amb, 12 °C) and Climatic Global Changes (CGC, 16 °C). Values represent the mean ± SD (p < 0.005, two-way ANOVA). Different letters indicate significantly different values (p < 0.05).
Plants 13 03267 g006
Plants 13 03267 g007
Figure 7. Sampling site of Macrocystis pyrifera sporophylls. (A) Map of Chile showing the study area. (B) Map of Southern Los Lagos Region with a zoom to Carelmapu locality (box). (C) Map of Carelmapu that indicates the specific study area (red point).
Figure 7. Sampling site of Macrocystis pyrifera sporophylls. (A) Map of Chile showing the study area. (B) Map of Southern Los Lagos Region with a zoom to Carelmapu locality (box). (C) Map of Carelmapu that indicates the specific study area (red point).
Plants 13 03267 g007
Table 1. Two-way and three-way ANOVA results and significance values for effects of seawater pHT treatments, light, and their interaction in Experiment 1: pH × light. Significant differences are indicated in bold.
Table 1. Two-way and three-way ANOVA results and significance values for effects of seawater pHT treatments, light, and their interaction in Experiment 1: pH × light. Significant differences are indicated in bold.
VariableSource of VariationDfSum of SquaresMean SquareFp
CA specific inhibitorspHT11361360.8190.37067
Light164640.3880.53696
Inhibitor223,31811,65970.2895.64 × 10−14
pHT × Light1147214728.8760.00484
pHT × Inhibitor29244622.7840.07348
Light × Inhibitor2181910.5460.58328
pHT × Light × Inhibitor21050.0300.97023
Residual416801166
Total52
Direct HCO3− uptake specific inhibitorpHT126262625.85.6700.0246
Light11010.30.0220.8827
Inhibitor115901590.53.4340.0748
pHT × Light118241824.23.9390.0574
pHT × Inhibitor1157156.70.3380.5656
Light × Inhibitor1124123.50.2670.6097
pHT × Light × inhibitor1915915.21.9760.1712
Residual2712,504463.1
Total34
Chl apHT10.000790.00076880.5770.4583
Light10.01934420.019344214.53360.001532
pHT × Light10.00392000.00392002.94520.105430
Residual160.02129600.00133
Total19
Chl cpHT10.0462720.04627276.62861.692 × 10−07
Light10.0025990.0025994.30440.0545066
pHT × Light10.0099460.00994616.47060.0009129
Residual160.009620.000604
Total19
FucoxanthinpHT10.01128130.01281370.35392.985 × 10−07
Light10.00010120.00010120.63140.438459
pHT × Light10.00257650.002576516.06770.001014
Residual160.00256560.0001603
Total19
Chl c/Chl a ratiopHT12.11252.11253.14670.0951083
Light113.568113.568120.21110.0003668
pHT × Light10.09880.09880.14720.7062785
Residual1610.74110.6713
Total19
Fucox/Chl a ratiopHT10.96220.96229.48160.007186
Light11.625301.6253016.01550.001028
pHT × Light10.066440.066440.65470.430320
Residual161.623720.10148
Total19
Alfa (α)pHT10.1269950.12699515.39190.001213
Light10.0020450.0020450.24790.625320
pHT × Light10.0007590.0007590.09200.765564
Residual160.1320120.008252
Total19
ETRmaxpHT1851.4851.41.04590.3216728
Light121,425.221,425.226.32080.0001007
pHT × Light17714.77714.79.47740.0071960
Residual1613,024.1814.0
Total19
EkpHT1410,680410,6806.30330.023173
Light1589,571589,5719.04900.008336
pHT × Light1442,039442,0396.78460.019156
Residual161,042,45065,153
Total19
Fv/FmpHT10.0836320.07089972.13324.09 × 10−07
Light10.0041490.0041491.28110.275
pHT × Light10.0022990.0022991.97320.181
Residual160.0173940.001164
Total19
Table 2. Photosynthetic pigment concentrations estimated from M. pyrifera gametophytes incubated under different pH, light, and temperature conditions (Experiment 1: pH × light; Experiment 2: pH × temperature). Chlorophyll a (Chl a): (mg g−1 WW); Chlorophyll c (Chl c): (mg g−1 WW); Fucoxanthin (Fucox): (mg g−1 WW); Chlorophyll c/Chlorophyll a ratio (Chl c/Chl a ratio); Fucoxanthin/Chlorophyll a ratio (Fucox/Chl c ratio). Values represent the mean of 5 replicates ± SD.
Table 2. Photosynthetic pigment concentrations estimated from M. pyrifera gametophytes incubated under different pH, light, and temperature conditions (Experiment 1: pH × light; Experiment 2: pH × temperature). Chlorophyll a (Chl a): (mg g−1 WW); Chlorophyll c (Chl c): (mg g−1 WW); Fucoxanthin (Fucox): (mg g−1 WW); Chlorophyll c/Chlorophyll a ratio (Chl c/Chl a ratio); Fucoxanthin/Chlorophyll a ratio (Fucox/Chl c ratio). Values represent the mean of 5 replicates ± SD.
Experiment 1pHLight
(µmol m−2s−1)		 Pigment Concentration
Chl a
(mg g−1 WW)		Chl c
(mg g−1 WW)		Fucox
(mg g−1 WW)		Chl c/Chl a RatioFucox/Chl a Ratio
7.8200.11 ± 0.030.15 ± 0.030.08 ± 0.011.45 ± 0.520.83 ± 0.32
500.07 ± 0.03 0.22 ± 0.030.10 ± 0.013.24 ± 1.17 1.52 ± 0.52
8.2200.12 ± 0.070.10 ± 0.020.06 ± 0.020.94 ± 0.520.51 ± 0.11
500.03 ± 0.010.08 ± 0.010.03 ± 0.012.45 ± 0.870.96 ± 0.15
Experiment 2pHTemperature
(°C)
7.8120.13 ± 0.030.12 ± 0.060.09 ± 0.010.88 ± 0.260.73 ± 0.16
160.13 ± 0.030.09 ± 0.020.11 ± 0.010.69 ± 0.080.84 ± 0.12
8.2120.18 ± 0.050.10 ± 0.060.11 ± 0.020.55 ± 0.180.62 ± 0.11
160.09 ± 0.010.06 ± 0.010.08 ± 0.010.69 ± 0.040.88 ± 0.07
Table 3. Photosynthetic parameters estimated from M. pyrifera gametophytes incubated under different pH, light, and temperature conditions (Experiment 1: pH × light; Experiment 2: pH × temperature). Photosynthetic efficiency (α): µmol m−2s−1. Maximal Electron Transport Rate (ETRmax): µmol e−1m−2s−1. Irradiance for the initial saturation of ETR (Ek): µmol m−2s−1. Maximal quantum yield (Fv/Fm): dimensionless. Values represent the mean of 5 replicates ± SD.
Table 3. Photosynthetic parameters estimated from M. pyrifera gametophytes incubated under different pH, light, and temperature conditions (Experiment 1: pH × light; Experiment 2: pH × temperature). Photosynthetic efficiency (α): µmol m−2s−1. Maximal Electron Transport Rate (ETRmax): µmol e−1m−2s−1. Irradiance for the initial saturation of ETR (Ek): µmol m−2s−1. Maximal quantum yield (Fv/Fm): dimensionless. Values represent the mean of 5 replicates ± SD.
Experiment 1pHLight
(µmol m−2s−1)		Photosynthetic Parameter
α
(µmol m−2s−1)		ETRmax
(µmol e−1m−2s−1)		Ek
(µmol m−2s−1)		Fv/Fm
7.8200.32 ± 0.0773.32 ± 29.62240.14 ± 102.610.53 ± 0.03
500.31 ± 0.1447.14 ± 19.16194.09 ± 150.140.53 ± 0.04
8.2200.17 ± 0.05125.65 ± 44.12824.07 ± 468.840.44 ± 0.05
500.14 ± 0.0820.91 ± 8.05183.35 ± 87.900.38 ± 0.04
Experiment 2pHTemperature
(°C)
7.8120.13 ± 0.0252.04 ± 8.62399.98 ± 113.280.41 ± 0.01
160.15 ± 0.0367.64 ± 42.96512.49 ± 463.860.33 ± 0.06
8.2120.29 ± 0.03154.52 ± 89.34558.14 ± 379.550.49 ± 0.01
160.13 ± 0,0276.95 ± 22.69610.89 ± 219.260.29 ± 0.07
Table 4. Two- and three-way ANOVA results and significance values for effects of seawater pHT treatments, temperature, and their interaction in Experiment 2: pH × temperature. Significant differences are indicated in bold.
Table 4. Two- and three-way ANOVA results and significance values for effects of seawater pHT treatments, temperature, and their interaction in Experiment 2: pH × temperature. Significant differences are indicated in bold.
VariableSource of VariationDfSum of SquaresMean SquaresFp
CA specific inhibitors pHT14813481310.2650.00244
Temperature12082080.4440.50865
Inhibitor223,04711,52324.5784.95 × 10−08
pHT × Temperature13283280.7010.40684
pHT × Inhibitor212346171.3160.27801
Temperature × Inhibitor248240.0510.95046
pHT × Temperature × Inhibitor28984490.9570.39124
Residual4722,036469
Total58
Chl apHT10.00012500.00012500.11440.739635
Temperature10.00907380.00907388.30120.010857
pHT × Temperature10.01200500.012005010.98280.004388
Residual160.01748920.0010931
Total19
Chl cpHT10.00210130.00210131.09020.319
Temperature10.00561120.00561122.91140.1073
pHT × Temperature10.0004410.0004410.21480.6492
Residual160.03083720.0019273
Total19
FucoxanthinpHT10.00020480.00020481.17900.2936401
Temperature10.00012500.00012500.71960.4087771
pHT × Temperature10.00307520.003075217.70410.0006678
Residual160.00277920.0001737
Total19
Direct HCO3− uptake specific inhibitorpHT10.00930.00930.02160.884258
Temperature12.32192.32195.37480.027682
Inhibitor13.86103.86108.93780.005643
pHT × Temperature10.88570.88572.05020.162876
pHT × Inhibitor10.68030.68031.57470.219540
Temperature × Inhibitor10.57810.57811.33830.256780
pHT × Temperature × Inhibitor10.03790.03790.08780.769070
Residual2912.52770.4320
Total36
Chl c/Chl a ratiopHT10.135580.1355844.97630.04036
Temperature10.002500.0025030.09190.76573
pHT × Temperature10.128900.1289034.73110.04496
Residual160.435940.027246
Total 19
Fucox/Chl a ratiopHT10.0079870.0079870.55880.465584
Temperature10.1701340.17013411.90380.003292
pHT × Temperature10.0292600.0292602.04730.171721
Residual160.2286800.014292
Total 19
Alfa (α)pHT10.0212580.02125830.3974.721 × 10−05
Temperature10.0238990.02389934.1732.483 × 10−05
pHT × Temperature10.0386600.03866055.2801.415 × 10−06
Residual160.0111890.000699
Total 19
ETRmaxpHT115,6191561.25.99810.02622
Temperature148014801.21.84380.01934
pHT × Temperature110,85310,853.54.16800.05804
Residual1641,6642604.0
Total19
EkpHT10.0096770.00967721.67530.2139
Temperature10.0003070.00030660.05310.8207
pHT × Temperature10.0020170.00201660.34910.5629
Residual160.0924220.0057764
Total19
Fv/FmpHT10.003370.003375.4660.03475
Temperature10.119130.11913193.0741.39 × 10−09
pHT × Temperature10.013930.0139322.5780.00031
Residual140.008640.00062
Total 17
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Labbé, B.S.; Fernández, P.A.; Florez, J.Z.; Buschmann, A.H. Effects of pH, Temperature, and Light on the Inorganic Carbon Uptake Strategies in Early Life Stages of Macrocystis pyrifera (Ochrophyta, Laminariales). Plants 2024, 13, 3267. https://doi.org/10.3390/plants13233267

AMA Style

Labbé BS, Fernández PA, Florez JZ, Buschmann AH. Effects of pH, Temperature, and Light on the Inorganic Carbon Uptake Strategies in Early Life Stages of Macrocystis pyrifera (Ochrophyta, Laminariales). Plants. 2024; 13(23):3267. https://doi.org/10.3390/plants13233267

Chicago/Turabian Style

Labbé, Bárbara S., Pamela A. Fernández, July Z. Florez, and Alejandro H. Buschmann. 2024. "Effects of pH, Temperature, and Light on the Inorganic Carbon Uptake Strategies in Early Life Stages of Macrocystis pyrifera (Ochrophyta, Laminariales)" Plants 13, no. 23: 3267. https://doi.org/10.3390/plants13233267

APA Style

Labbé, B. S., Fernández, P. A., Florez, J. Z., & Buschmann, A. H. (2024). Effects of pH, Temperature, and Light on the Inorganic Carbon Uptake Strategies in Early Life Stages of Macrocystis pyrifera (Ochrophyta, Laminariales). Plants, 13(23), 3267. https://doi.org/10.3390/plants13233267

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