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Review

Diversity of CO2 Concentrating Mechanisms in Macroalgae Photosynthesis: A Case Study of Ulva sp.

by
Jingyi Sun
1,†,
Chunyan Zhao
1,2,†,
Shuang Zhao
1,3,
Wei Dai
1,
Jinlin Liu
1,4,
Jianheng Zhang
1,5,*,
Juntian Xu
5,6,* and
Peimin He
1,5,7,*
1
College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China
2
Shanghai Primary School of Xuhui Districy, Shanghai 201306, China
3
Ocean College, Fujian Polytechnic Normal University, Fuzhou 350300, China
4
Ocean Institute, Northwestern Polytechnical University, Taicang 215400, China
5
Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Jiangsu Ocean University, Lianyungang 222005, China
6
Jiangsu Key Laboratory for Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang 222005, China
7
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Mar. Sci. Eng. 2023, 11(10), 1911; https://doi.org/10.3390/jmse11101911
Submission received: 1 September 2023 / Revised: 30 September 2023 / Accepted: 1 October 2023 / Published: 3 October 2023
(This article belongs to the Section Marine Biology)
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Abstract

:
Many algae respond to the CO2 limitation in seawater by inducing a CO2 concentrating mechanism (CCM) to obtain sufficient inorganic carbon to meet their photosynthetic needs, and Ulva sp. is a model population suitable for studying the ecological adaptability of macroalgae. As the dominant species of green tide disaster, Ulva sp. often faces strong inorganic carbon restriction due to its rapid growth and high population density and must have evolved a variety of carbon acquisition strategies, such as CCM, to overcome these limitations. This paper briefly summarizes the position and function of the important components of CCM (inorganic carbon transporters, carbonic anhydrase, Rubisco, and pyrenoid) and introduces several indexes suitable for evaluating the relative function of CCMs in macroalgae from the aspects of affinity between photosynthesis and Rubisco for CO2, and carbonic anhydrase inhibitor. The methods of judging the carbon sequestration pathway of Ulva sp., the CCM responses of diversity under different carbon sources, and the related genes that may be involved in the operation of CCMs were summarized. This work could provide a reference for revealing the CCMs of macroalgae and lay a foundation for further research on the inorganic carbon utilization strategy of the Ulva sp.

1. Introduction

Algae can obtain CO2, HCO3−, and CO32− types of inorganic carbon from the ocean, but only CO2 can easily pass through the cell membrane [1]. CO2 is the basic substrate of algae photosynthesis. Although seawater is rich in dissolved inorganic carbon (the concentration is as high as 2.2 mM, approximately 170 times that of the atmosphere, and HCO3− accounts for about 90% thereof), free CO2 accounts for less than 1% (approximately 13 μM) and only 1/10,000 as much CO2 diffuses in seawater as it does in the atmosphere [2]. The manner of obtaining free CO2 in seawater may be limited by low diffusion coefficient and surface boundary layer range [3]. Calvin cycle is the main way of carbon sequestration in seaweed, while ribulose-1,5-diphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of carbon fixation, which requires inorganic carbon in the form of CO2. However, the need for CO2 by seaweed photosynthesis is generally higher than the CO2 concentration in natural seawater [4]. For example, the half-saturation constant Km (DIC concentration when photosynthesis reaches half of the maximum rate) of Ulva prolifera to CO2 is about 250 μM [5], while the CO2 concentration in natural seawater is only 5–25 μM. The concentration of CO2 in the environment can not meet the needs of Rubisco, so it must rely on the CO2 concentrating mechanisms (CCMs) to increase the intracellular CO2 concentration to maintain normal photosynthesis. Therefore, CCMs evolved in most algae, including improved carbonic anhydrase (CA) activity, active absorption of HCO3−, and external acidification region [6,7]. In general, CO2 concentrating mechanisms refer to the sequentially acting of inorganic carbon transporters and CA in different cellular regions to change the availability of photosynthetic carbon so that CO2 accumulates in large quantities at the active site of Rubisco, which is beneficial for the enzyme to play the role of carboxylase and inhibit its oxidase activity.
Ulva sp. is a kind of seaweed with wide temperature, wide salt, strong stress resistance, and reproductive ability. Because of its ability to tolerate stress conditions, it has become a popular model organism for studying the biological resistance mechanism of photosynthesis [8]. Some seaweeds of Ulva sp., such as Enteromorpha (at present, it belongs to the genus Ulva, including the main population of green tide, U. prolifera, U. linza, U. compressa, U. flexuosa, etc., Figure 1), can rapidly accumulate biomass under suitable environmental conditions, causing macroalgal bloom disasters known as green tides, with serious economic and ecological consequences [9]. In the early stage of the green tide, the rapid proliferation of Enteromorpha can remove a large amount of DIC from the seawater, and the free-floating algae form an algal pad with a thickness of 0.5 m. It is difficult for the algae exposed to the atmosphere to obtain DIC from the sea to support photosynthesis, so using CO2 in the atmosphere as a carbon source leads to a decrease in the sea surface CO2 partial pressure (pCO2) [10,11]. It can be said that Ulva sp. has a high potential for adaptation to the lack of inorganic carbon. Scholars at home and abroad have done a lot of research on its origin, ecological habits, distribution characteristics, and stress resistance mechanism [8], but the research on its carbon utilization strategy is relatively scarce. Xu and Gao [12] measured the relationship between the photosynthetic oxygen evolution and the concentration of dissolved inorganic carbon (DIC) (P-C curve) in the green algae U. prolifera, which was used to reflect the relationship between photosynthesis and inorganic carbon affinity. P-C curve showed that U. prolifera grown under high CO2 condition had higher K1/2DIC or K1/2CO2. The decrease of photosynthetic affinity to DIC and/or CO2 indicated that CCM or CCM activity was decreased. U. prolifera grown under the condition of low CO2 showed higher DIC affinity. In other words, when the inorganic carbon is insufficient, U. prolifera will activate the CCM to obtain enough CO2 for photosynthesis. In addition, as technology advances, scientists are trying to record the overall pattern of photosynthetic carbon capture through the distribution of 13C/12C in plant tissues. Liu et al. [4] demonstrated that through the corresponding changes of δ13C tissue, the relationship between the activities of C3 enzyme (Rubisco), C4 enzymes (PEP-Case, PEPCKase, and PPDKase), and CA enzymes (extra- and intra-cellular CA) and photosynthate was analyzed, and the participation of C3 and C4 photosynthetic pathways and a CA mechanism (i.e., CCM) in U. prolifera was determined. The results show that C3 and C4 pathways and the HCO3− mechanism supported by CA coexist in U. prolifera, which is the first report on the existence of a CCM in Ulva sp. [4]. Understanding the CCMs of Ulva sp. is of great significance for studying the CCMs of macroalgae and predicting the development trend of green tide under long-term environmental CO2 increase. Therefore, this study summarized the organization, evaluation, and operational mechanism of CCM from Ulva sp. to provide a theoretical foundation for subsequent research.

2. Composition of CCMs

Equal concentrations of CO2 and O2 in surface waters around 500 million years ago may have led to the emergence of algal CCMs [13]. Eukaryotic algal CCMs play an important role in global productivity, molecular phylogeny, and diversity. More than four-fifths of oceans’ primary productivity is promoted by some form of CCM [14,15]. The CCM in Ulva sp. has three main components: (i) inorganic carbon transporters of plasma and chloroplast membranes; (ii) CAs located in key positions; and (iii) chloroplast microcompartments (pyrenoid) in which large amounts of Rubisco are accumulated [16]. Below, the roles and connections of inorganic carbon transporters, carbonic anhydrase, Rubisco, and pyrenoid in CCM are summarized.

2.1. Inorganic Carbon Transporters

In order to better understand CCM, it is very important to study related proteins, especially the possible existence of plasma membrane and chloroplast membrane inorganic carbon transporters. CO2 and HCO3− are the main sources of inorganic carbon in water. Active CCM leads to rapid uptake of inorganic carbon by plasma membrane [17]. HLA3 (high light activated 3) and LCI1 (low CO2 induced protein 1) HCO3− transporters have been confirmed to be involved in the active absorption of CO2 and HCO3− on the plasma membrane of Chlamydomonas reinhardtii [18]. HLA3 has the structural characteristics of hydrophilicity and transmembrane region of ABC transporter and can play a role in the form of HCO3− transporter on the plasma membrane under low CO2 conditions. LCI1 can not only enhance the absorption of HCO3−, but also proved to be necessary for active absorption of CO2 at low CO2 levels [19,20]. Wang and Spalding [21] reported that at very low CO2 concentrations, the active transport of HCO3− on the chloroplast envelope of C. reinhardtii requires LCIA, and LCIB may play a parallel role with HLA3 or LCIA in CO2 or HCO3− uptake pathways. So far, HCO3− transporters working on the plasma membrane and chloroplast envelope of C. reinhardtii have been identified, but not on the thylakoid membrane. Previous studies have found that three Bestrophin genes (BST1, BST2, and BST3) located on the thylakoid membrane controlled by the main regulator ‘CIA5’ are necessary to transport HCO3− accumulated in the chloroplast matrix to the thylakoid cavity [22]. CIA5 is essential for inducing the expression of several CCM genes encoding inorganic carbon transporters HLA3 and LCI1 [23]. At present, there is a lack of systematic and in-depth study on the inorganic carbon transporters of Ulva species. Gao [24] found the expression of low-CO2-inducible proteins, low-CO2-inducible membrane proteins, and CIA5 in some ESTs obtained from the transcriptome sequencing of U. prolifera. Rautenberger [25] found four hypothetical HLA3 ABC transporters in the ESTs of U. prolifera. Several low CO2 inducible proteins, a chloroplast Ci transporter (LCIB), several ABC transporters, and a nuclear transcriptional regulator (CIA5, LCR1) of the CCM element were also found in the U. linza transcription group [26].

2.2. Carbonic Anhydrase

CA is an enzyme that catalyzes the exchange of CO2 and HCO3− and other reactions (hydration of small CO2-like molecules, such as COS, and CS2 to form H2S, CO2, or some hydrolysis reactions) in solution [27]. Without CA, the transformation reaction between CO2 and HCO3− will occur; however, the mutual transformation is slow. In nature, CA has several families, denoted by the Greek letters α to θ. All CA active sites have a “bipolar” cavity, half of which are only aligned with hydrophobic amino acid residues, while the other half are aligned with hydrophilic amino acid residues. The hydrophobic part can capture CO2 molecules, while the hydrophilic part is involved in the release of ions (HCO3− and H+) produced by CO2 hydration from the cavity. CA is widespread in photosynthetic autotrophs and plays a crucial role in the CCM that promotes the transport of CO2 to Rubisco. In addition, CA has a similar effect in all types of algae, although the types of CA are different. For example, α-CA and θ-CA catalyze the conversion of bicarbonate to CO2 in green alga C. reinhardtii and diatom Phaeodactylum tricornutum, respectively [28]. Different subtypes of CA in plants and eukaryotic algae have also been described [27,29,30,31]. One of the most well-known functions of CA in algae is to catalyze the conversion between CO2 and HCO3−. The carboxysome CA in cyanobacteria has this effect, and the CA forms include β-CA [27,32], ε-CA [27,33], and γ-CA [27,34], etc. CA with the above functions also exists in the chloroplast thylakoid lumen of eukaryotic algae, including α-CA, CAH3 [35], CAH1, and θ-CA, etc. In addition, the protein complex LCIB/C, which is associated with the chloroplast pyrenoid, was found in the matrix, and the θ-CA-like protein may recapture CO2 that leaked from the pyrenoid [21]. In algal CCM, another function of CA is to promote the entry of external CO2 from the environment. These CA located in the cell wall or periplasmic space may be able to sense the level of CO2 in the environment, maintain the chemical balance among Ci types in the periplasm, and provide sufficient Ci for plasma membrane HCO3− transporters or active transport of CO2. Similarly, various CAs have been found on the cell walls of different algae, such as α-CA in green algae [30,36,37,38]. However, studies of CA in green macroalgae are few and far between. In Ulva sp., Zhang et al. [26] found a possible α-CA in U. linza, De Clerck et al. [39] annotated 9 CA homologous genes in the U. mutabilis genome. Wang et al. [40] cloned and identified a U. prolifera α-CA gene sensitive to environmental changes, and then Wang et al. [41] found two γ-CA genes highly expressed at low pH (pH7.5).

2.3. Rubisco and Pyrenoid

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in all oxygenic photosynthetic organisms, participates in the effect of CO2 on green plants and photoautotrophic bacteria, and is an important enzyme in the photosynthetic C3 reaction because it catalyzes the first step of photosynthesis by integrating CO2 in the air into ribulose-1-diphosphate (RuBP). It catalyzes the carboxylation of RuBP and then cleavage to form two PGA (3-phosphate-glyceric acid) molecules. Rubisco is thus the main gateway of inorganic carbon into the biosphere and has a profound impact on the global scale [42]. Although this enzyme is one of the most abundant proteins on earth, it is very inefficient [43]. Through the sequential action of inorganic carbon transporters and carbonic anhydrase in different cellular regions, CO2 is actively accumulated around Rubisco [44]. Compared to ordinary enzymes, Rubisco can catalyze both carboxylation and oxygenation. It has a bifunctional property and performs either carboxylase or oxygenase activity depending on the availability of the substrate, for which both CO2 and O2 can be used [43,45,46]. O2 and CO2 are competitive inhibitors of the carboxylase and oxygenase reactions, respectively. Rubisco, therefore, lies at the intersection of two opposite but interlocking cycles of photosynthetic carbon reduction (photosynthesis) and photosynthetic carbon oxidation (photorespiration) (Figure 2). When the concentration ratio of CO2:O2 is high, Rubisco catalyzes carboxylation, which catalyzes the first key carbon fixation reaction in the Calvin cycle in photosynthesis, converting free CO2 in the atmosphere into energy storage molecules in organisms. When the concentration ratio of CO2:O2 is low Rubisco promotes the catalyzation of oxygenation and participates in the photorespiratory cycle [45]. Since Rubisco tends to participate in oxygenase reactions and the catalytic conversion rate of active sites is low, organisms have evolved various strategies to optimize Rubisco’s performance when adapting to carbon limitations in a specific environment. There are mainly two strategies developed synergistically. On the one hand, Rubisco forms with different catalytic efficiencies have evolved. On the other hand, the underlying structure evolved to ensure that Rubisco received high concentrations of CO2 substrate, often referred to as the CCM [3], for example, C3 plants capture CO2 from the surrounding atmosphere by passive diffusion based on Rubisco, which has developed a high affinity for CO2; C4 plants that evolved from C3 ancestors added structural and biochemical specializations which allows them to concentrate CO2 around the Rubisco active site [47].
Pyrenoid is an evolutionary adaptation that enables algae to fix inorganic carbon from CO2-limited environments more efficiently. This contemporary definition was first reported in 1782, with an unannotated dot mark in a painting of Conferva jugalis (now Spirogyra) in the Flora Danica, one of the earliest records of pyrenoids [48]. The word pyrenoid comes from the Greek πυρην (pyren, kernel), created by Schmitz in 1882 in a monograph on algal chloroplasts [48]. Rubisco promotes about 1014 kg of carbon into the biosphere each year [49,50]. However, in many plants, the rate at which Rubisco fixes CO2 at atmospheric CO2 levels is less than 1/3 of its maximum rate, which restricts the growth of many crops. To overcome this limitation, numerous photosynthetic organisms comprising algae increased the CO2 fixation rate of Rubisco by providing it with concentrated CO2. In algae, this CCM occurs in a phase-separated organelle called the pyrenoid. Pyrenoid-based CO2 concentrating mechanisms (PCCMs) mediate approximately 1/3 of the global CO2 sequestration [50]. Nevertheless, not all algae have pyrenoid or CCMs. The appearance of CCM is not completely consistent with the existence of pyrenoids, which is not the only component of CCM. Kevekordes et al. [51] showed that the occurrence of CCM was not completely related to the presence of pyrenoids in Caulerpa.
Pyrenoids are permeable microchambers that contain tightly packed Rubisco that are present in the chloroplast stroma of many algae and operate CCM [47,52]. CCM increases the concentration of CO2 near Rubisco using an inorganic carbon pump, the synergy of one or more CA, and the packaging of Rubisco into pyrenoid [53]. Pyrenoids are found in almost all algae except Chrysophyta [53] and are relatively rare in algae with thallus but have been observed in Ulva, Monostroma, and Porphyra [54,55]. Some algae lacking pyrenoid increase the acquisition of CO2 by acidifying the extracellular environment, effluxing active protons, or exporting CA to periplasm [56]. Pyrenoids are not necessary for CCM. However, the CCM is more effective when pyrenoids are present [57]. For example, the accumulation of Ci in C. reinhardtii is 5–10 times higher than that of closely related species without pyrenoids, such as Chloromonas [58]. Therefore, evolutionary adaptation must take place in pyrenoids to improve the performance of the CCM. In many cases, pyrenoids and chloroplasts do not necessarily occur together. For green macroalgae, Ulvales are present in both, while in Caulerpales, some Codium and Caulerpa sp. have multiple chloroplasts but lack pyrenoid [51]. Most green algae contain pyrenoids, the thylakoid extends into the pyrenoid and starch grains are attached to the pyrenoid surface. The chloroplast of Ulvales is layered with thylakoid lumen and pyrenoid [47]. He et al. [59] reported that electron microscope photos of most U. clathrata showed that there were multiple pyrenoids in their chloroplasts, their morphological and structure were the same, and the pyrenoids were located in chloroplasts, oval- or bean-shaped. The pyrenoids were surrounded by starch sheaths and were connected to the thylakoid of the chloroplast through longitudinal channels extending to both ends. When cells move from a high CO2 concentration environment to a low CO2 concentration environment, the ultrastructure of the pyrenoid changes greatly, and the starch sheath surrounding the pyrenoid can be rapidly formed. However, Villarejo et al. [60] have shown that the starch sheath is closely related to CCM but is not involved in the process. He et al. [59] utilized metallographic immunomolecular localization and found that Rubisco gold-labeled granules of U. clathrata were mainly distributed in the pyrenoid and starch sheath chloroplasts and were rarely distributed in the chloroplast thylakoids and stroma. The molecular localization of Rubisco activase demonstrated that it was mainly distributed in the pyrenoid and starch sheath. These results indicated that the pyrenoid (and starch sheath) of U. clathrata was the same as that of unicellular green algae and had a photosynthetic function. In addition, Cai et al. [61] found that CO2 affects the distribution of Rubisco in U. clathrata. When CO2 concentration increased, Rubisco tended to diffuse into the chloroplast matrix, and when cultured with a low concentration of CO2 or without CO2, Rubisco was continuously concentrated in the pyrenoids.

3. Indicators to Measure CCMs in Macroalgae

According to the different initial photosynthetic products of carbon assimilation in photosynthesis, terrestrial plants were divided into three types: C3, C4, and CAM plants. The majority of the plants are C3 plants, and C4 and CAM plants developed from the C3 plants. The C3 pathway is the most common and primitive pathway among the three types of photosynthetic carbon metabolism pathways, and its initial photosynthetic product is 3-phosphoglycerate (PGA) [62]. Compared with C3 plants, C4 plants increase the CO2 concentration around Rubisco through a biochemical CCM [63,64]. Terrestrial C4 plants generally have Kranz anatomy, which is necessary for most terrestrial plants to perform the C4 photosynthetic pathway [65]. The Kranz anatomy is composed of mesophyll cells and their adjacent vascular bundle sheath cells, which can effectively prevent CO2 from escaping outward [66], thereby accumulating the CO2 concentration at the active site of Rubisco [67]. Algae are a diverse group of aquatic organisms and do not have the Kranz anatomy of terrestrial plants. In order to adapt to the low CO2 environment of seawater, most algae have evolved a CCM to ensure the supply of CO2 for photosynthesis. Although algae do not have Kranz anatomy like terrestrial C4 plants, they still possess the photosynthetic properties of the C4 pathway [68]. An example is the use of pyruvate orthophosphate dikinase (PPDK) to catalyze the formation of phosphoenolpyruvate (PEP), the initial molecule receptor for fixed CO2.
To assess the diversity of the CCM functions and activities in different algae, reliable metrics to measure and quantify the relative functions of CCM need to be established. As reported by Badger et al. [47], we screened out several indicators that may be suitable for measuring the CCM of macroalgae were demonstrated.

3.1. CO2 Affinity of Photosynthesis versus Rubisco

Rubisco’s low affinity for external CO2 drove the evolution of CCMs. Therefore, one of the most useful indicators of whether a photosynthetic organism has a CCM is to compare the affinity of photosynthesis for external CO2 with the affinity of Rubisco for CO2. This approach was originally applied to higher plants, and by measuring the affinity Km (Rubisco CO2) of Rubisco extracted from photosynthetic organisms to substrate CO2, it was inferred that C3 species might require a CCM [69]. However, due to the limitation of extraction technology, the affinity determination of the photosynthetic organism Rubisco lacked accuracy. With the improvement of extraction and analysis techniques, this method has become widely used to evaluate algal CCMs. In general, the researchers measured the photosynthetic oxygen evolution rate under different inorganic carbon concentrations and used the data analysis software to fit the inorganic carbon response curve (P-C curve) by using Michaelis–Menten equation, where Km is the substrate concentration (DIC concentration) when the photosynthetic rate is half of the maximum, from which we can infer the affinity of algae photosynthesis to CO2 Km (photosynthetic CO2). Since marine algae may experience more persistent CO2 limitations than fresh- and brackish-water autotrophs, it can be assumed that the type of water environment determines the effectiveness of CCM. Comparing the in vivo photosynthesis and the in vitro carboxylation of Rubisco under ambient oxygen levels can show its ability to concentrate CO2 exceeds the level of environmental CO2 [70]. For example, C. reinhardtii’s ratio of Km (photosynthetic CO2) to Km (Rubisco CO2) is approximately 1:30, and that of Ulva sp. is about 5–10:68 [1,71,72]. Both apparently rely on CCM to improve the efficiency of Rubisco, whereas other algae with close specific gravity may not use CCM.

3.2. Effects of CA Inhibitors

Because CA is central in all chloroplast CCM models, determining the effect of CA inhibitors is useful in studying the functional aspects of algal CCM. This is especially true for chloroplast-penetrating inhibitors, such as EZA (Ethoxzolamide), which are likely to inhibit most forms of internal CA. EZA is often used to obtain low inorganic carbon affinity for photosynthesis of algae. If the algae showed low inorganic carbon affinity under the action of EZA, it is proved that there is intracellular CA-mediated CCM in the algae, but no effect may not be conclusive evidence of CCM deletion. For CA inhibitors that cannot penetrate cell membranes, such as AZA (Acetazolamide), only apply to inhibit the activity of external CA [47], the effect on the function of chloroplast CCM is less easily explained. By comparing the relative effects of EZA and AZA, the relative contributions of internal and external CA forms to photosynthetic Ci absorption can be determined (Figure 3). For example, in the study of Gao et al. [73], after adding AZA the net photosynthetic rate of U. linza decreased, and the inhibition rate was noted to be 26.26%. Compared with AZA, EZA demonstrated a higher inhibition rate of photosynthesis (75.19%), which indicated that the internal CA contributed more to the absorption of photosynthesis. Xu et al. [74] found that U. linza and U. prolifera have obvious extracellular and intracellular CA activity by using different CA inhibitors. However, extracellular CA enzyme activity itself accelerates the mutual conversion between HCO3− and CO2 but cannot affect the CO2 equilibrium concentration, and CO2 mainly enters the cell through passive diffusion. This indicates that HCO3−-utilization in this manner does not function well at high pH (pH > 9.4) because the equilibrium concentration of CO2 is low. It is obvious that in addition to the extracellular CA catalytic utilization of HCO3−, there must be another means of using HCO3− in U. prolifera.

3.3. Using HCO3− as Photosynthetic Substrate

The function of HCO3− in photosynthesis has been perceived as a property of algae [75]. The utilization of HCO3− by algae depends on the involvement of external CA and plasma membrane transporters [76,77,78]. The use of HCO3− is strongly correlated with the presence of a CCM. However, some macroalgae are not only capable of utilizing HCO3− as a carbon source, some have demonstrated the use of CO2 in specific situations. Through the detection of δ13C isotopes in Gracilaria and Ulva, it was found that when the concentration of environmental CO2 was high, there was a physiological transition from using HCO3− almost exclusively to predominantly using CO2. At the current seawater pCO2, many macroalgae use HCO3− rather than dissolved CO2 and utilize CA to convert HCO3− to CO2 for use by Rubisco [79,80,81,82]. For example, Mercado et al. [83] found that under the current seawater CO2 level, the chlorophyll plants U. rigida and U. compressa cannot obtain sufficient CO2 through diffusion absorption alone; therefore, a CCM must be used to obtain HCO3−. However, macroalgae may downregulate their CCM, reduce HCO3− use, and become dependent on CO2 as the main carbon source when CO2 concentrations are high [1,84,85,86]. Consequently, when using these indicators to evaluate the CCM of Ulva sp., there may be a decrease in the use of HCO3− in certain circumstances, where there is still CCM activity but with a certain degree of downregulation.

3.4. Changes in Affinity to External Ci Depends on Growth Ci Conditions

When there is Ci limitation in the external environment, the affinity of the Ci transporter for external CO2 and HCO3− increases, and the intracellular and extracellular CA activity also increases. The two work together to increase the affinity of microalgae for external Ci by more than 10 times [76]. This inducible change appears to be strong evidence that cellular infrastructure is involved in supplying CO2 to Rubisco in chloroplasts. These inducible CCM are not limited to microalgae but have been observed in many macroalgae as well, including Ulva [1], Gracilaria [87], Porphyra [88] and Fucus [89]. That is, some algae may have an inducible CCM to meet their environmental needs when facing external Ci periodic constraints, while other algae may not have such a flexible arrangement.

4. Operation of Ulva sp. CCM

To determine the type of photosynthetic carbon metabolism of seaweed, it is usually investigated from two aspects: a. The flow direction of C element in photosynthetic metabolites during the carbon assimilation process; b. The activity of photosynthetic enzymes in the carbon assimilation pathway. The former uses 14C isotope tracing technology to focus on the radioactivity intensity of key metabolites (3-phosphoglyceric acid, malic acid, oxaloacetic acid, aspartic acid, etc.) after supplying 14C to seaweed. If the initial fixed product is 3-phosphoglyceric acid (3-PGA), it may be the C3 pathway. If the initial fixed products are malic acid, oxaloacetate, and aspartic acid, it may be the C4 pathway. The latter can focus on the activity differences of Rubisco carboxylase and PEPC enzymes, the key enzymes of the C3 and C4 pathways, from an enzymatic perspective.
Enteromorpha is the dominant species of green tide, which is a large-scale algal bloom disaster. Its thick floating pad is bound to be strongly restricted by inorganic carbon (Figure 4). Thus, it must have evolved a variety of carbon acquisition strategies, such as CCM, to overcome these limitations. Karekar and Joshi [90] found in U. lactuca and U. tubulosa that 14C appeared not only in 3-PGA but also in aspartic acid after feeding for 10 s, while considerable 14C labeled malic acid appeared in U. lactuca after 30 s. Therefore, it is considered that there are both C3 and C4 pathways in Ulva sp. Ulva sp. has weak photorespiration and a low CO2 compensation point, demonstrating characteristics of C4 plants in gas exchange [91]. However, Beer et al. [92] examined an Ulva sp. alga, the 14C isotope tracer results showed that approximately 90% of the radioactive 14C appeared in 3-phosphoglyceric acid (3-PGA), and the Rubisco carboxylase activity was about 10-times that of the PEPC activity. At the same time, due to the efficient uptake of HCO3−, algae may transport high concentrations of CO2 to chloroplasts, which inhibits the activity of Rubisco oxygenase. This hypothetical inorganic carbon enrichment mechanism was confirmed in the green algae U. compressa and U. fasciata. However, after a few seconds, about 12% of the radioactive 14C was present in aspartic acid, indicating that Ulva sp. could use other metabolic pathways besides C3 pathway [93]. Xu et al. [94] and Liu et al. [4] reported that the C3 and C4 pathways and CCM coexist in U. prolifera. Based on the above research, we listed all Ulva sp. known to have CCMs (Table 1) and attempted to summarize the CCMs of Ulva sp.
Ulva mainly uses HCO3− as a carbon source in normal seawater. The CCMs have three pathways to utilize HCO3− (Figure 5): (i) Extracellular CA bound to the cell wall promotes the dehydration of HCO3−, forming CO2 in the cell membrane. (ii) When the pH of the environment is high, the low concentration of CO2 in the cell membrane induces the DIDS-sensitive membrane anion exchanger to transport HCO3− into the cell and, under the action of intracellular CA, dehydrated to form CO2. (iii) The proton pump located in the cell membrane utilizes the energy generated by the hydrolysis of ATP to pump H+ out of the cell membrane, forming an acidic region in the cell wall, and promoting the dehydration of HCO3− to form CO2, which enters the cell membrane.
In the work of Björk et al. [105], the existence of pathway (i) in the green algae (U. rigida C. Ag.) was demonstrated. They believe that for U. rigida C. Ag., the main form of inorganic carbon entering the cell through the plasma membrane is CO2, while HCO3− is dehydrated on the cell wall surface or in the cell wall under the catalysis of external CA to form CO2, and then enters the cell. External CA-mediated dehydration preceded Ci absorption. CA-mediated HCO3− uptake is a conventional form of CCM in macroalgae, and when the environment changes, macroalgae appear to have inducible CCMs similar to microalgae.
Drechsler and Beer [106] and Drechsler et al. [107] reported the mechanism of direct absorption of HCO3− in Ulva sp. (pathway (ii)). Its function is similar to the anion transport in red blood cells mediated by the anion exchange protein AE1. In subsequent studies, it was found that growing algae in high pH conditions induces an AE1-like system. In addition, AE1 plays a crucial role in the electron neutralization exchange of HCO3− and inorganic carbon in red blood cells, which is a basic process of clearing respiratory CO2 through blood flow to the lungs [108]. Due to the functional similarity between the putative HCO3−-transporter of Ulva sp. and the anion exchange of erythrocytes, we found that there are structural similarities between them. This functional similarity, combined with the current findings at the structural level, suggests that similar HCO3−-transport systems exist in the cells of two different organisms: mammalian and green macroalgae [109]. That is, the uptake of HCO3− by Ulva sp. is mediated by a protein that has a function similar to that of an anion exchanger on mammalian cell membranes, especially erythrocyte cell membranes. The evidence for the absorption of HCO3− by Ulva sp. (without an external CA) is that the photosynthetic rate observed under pH 8.2 was higher than that predicted by the extracellular CO2 supply, which indicates that Ci must be absorbed in the form of ions. Once inside the cell, the lower internal pH and presence of an internal CA promote the dehydration of HCO3−, thus providing Rubisco with sufficient CO2 to meet the measured high photosynthetic rate [89].
Gao et al. [73] treated Ulva sp. with Tris buffer and found that it had an inhibitory effect on photosynthesis, demonstrating and reporting for the first time that the Ulva cell wall has an acidic region as one of the CCM pathways (pathway (iii)). Acidic regions have previously been found in other macrophytes, such as Ruppia cirrhosa [110], Zostera marina, Z. noltii [111,112], and Laminaria saccharina [113]. Acidic regions are created by expelling protons out of the plasma membrane, making the CO2 concentration in these regions higher than its level in the medium, which can facilitate the rapid diffusion of CO2 into the cell through the membrane itself or the protein pores [113,114]. The acidic region located in the cell wall of Ulva sp. promotes the dehydration of external HCO3−, and the increase of extracellular CO2 concentration is conducive to the entry of CO2 into the cell membrane to participate in follow-up reaction, that is, CCM of Ulva sp. can absorb exogenous Ci under the cooperation of acidic region and external CA.
Pathway (i) seems to be ubiquitous in Ulva sp., in this mechanism HCO3− is dehydrated outside the cell with the external CA, and then the formed CO2 is absorbed into the cell. Photosynthesis that is dependent on this mechanism is completely and selectively inhibited by CA inhibitors that do not pass through the plasma membrane (e.g., dextran-bound sulfonamide) or pass very slowly through the plasma membrane (e.g., acetazolamide), but is not sensitive to DIDS. Due to the efficient utilization of CA enzymes, this method of using HCO3− has a high proportion in normal seawater at pH 8.1. With the increased pH, the pathway (i) is gradually suppressed. Correspondingly, algae have evolved an inducible CCM similar to those described by several green microalgae to utilize HCO3− under high pH conditions. For a few green macroalgae, the photosynthesis induced by high pH could be inhibited by anion exchange protein inhibitors (DIDS). The basic mechanism is insensitive to AZA. With the support of this mechanism, photosynthesis under high pH conditions is faster than spontaneous dehydration of HCO3−. Therefore, the direct uptake of HCO3− through the plasma membrane is mediated by the anion exchangers process [115].
In addition, some studies have found that although Ulva sp. has high expression of key enzymes in the C4 pathway under stress [68], Ulva sp.’s PEPCK may be involved in other biological processes (such as gluconeogenesis or providing PEP for antibacterial intermediate metabolites) or indirectly affect CCM (by affecting amino acid metabolism and transport, indirectly affect the carbon sequestration process), and it is more likely to use NAD-ME C4 type CCM to tolerate stress caused by CO2 deficiency [8,116].

5. CCM Gene of Ulva sp.

Many aquatic organisms, especially green algae and cyanobacteria, have evolved CCMs to increase the concentration of intracellular CO2 to compensate for the low affinity of Rubisco to CO2. For example, Synechocystis PCC6803, Synechococcus 7942, and C. reinhardtii are model organisms for studying CCMs. The proteins involved in the algae CCM mainly include various CA, transcriptional regulators, low CO2-inducible membrane proteins, low CO2-inducible proteins, various carrier proteins, chloroplast proton extrusion proteins, plasma membrane-type H+-ATPase (proton pump) and various key enzymes involved in the carbon fixation process (Table 2). We used SequenceServer 2.0.0.rc8 [117] to perform BLASTP alignment of the collected predicted CCM proteins using the U. prolifera genome data. When e < 10−5, the aligned sequences were considered to have high homology (Table 2).

6. Conclusions

The study of the mechanism of photosynthetic carbon sequestration is the basis for understanding the photosynthetic physiology, ecological adaptation, and biomass accumulation of macroalgae, and Ulva sp. is a model population suitable for the study of inorganic carbon utilization of macroalgae. The green tide formed by the outbreak of Ulva sp. can occur all over the world, which shows the great potential of Ulva sp. to adapt to the limitation of inorganic carbon. Ulva sp. may form an effective adaptation strategy to survive and develop under the inorganic carbon limitation caused by high population density, which may contain diverse and flexible CCMs. So far, the CCMs of Ulva sp. have been reported to include: under normal seawater pH, extracellular CA, and acidic region act synergistically; when the green tide breaks out, a large number of algae cover the sea surface, resulting in a sharp increase in pH, which will induce the anion exchange protein AE to transport HCO3− into the cell. In the future ocean acidification environment, higher CO2 concentration in the environment will inhibit the extracellular CA and anion exchange protein AE pathways and mainly rely on the acidic region and CO2 diffusion or transport pathways; under high light stress, extracellular CA pathway and C4 pathway cooperate to supplement C3 cycle; in addition, Ulva sp. also uses NAD- ME C4 type CCM to tolerate stress caused by lack of CO2.
The study of Ulva sp. CCM is helpful in revealing the adaptation strategies of macroalgae to inorganic carbon limitation, but there are still some unsolved problems. The function and expression of specific genes of CCMs, the physiological function of various transporter proteins of CCMs, and the changes in cell level, expression level, and metabolite level in the response process of CCMs are all urgent problems to be solved. With the continuous advancement of science and technology, the method of multiomics has been applied to the analysis of various metabolic mechanisms of algae. By comparing the differences of Ulva sp. in CCM using physiology and multiomics, it may be possible to make some progress in elucidating how U. prolifera gains a competitive advantage and explaining the internal reasons for its success in the Yellow Sea green tide outbreak.

Author Contributions

Conceptualization, J.S.; methodology, J.S., C.Z., S.Z. and W.D.; writing—original draft preparation, J.S., J.L. and S.Z.; writing—review and editing, J.S., J.L. and S.Z.; supervision, P.H., J.X. and J.Z.; project administration, P.H., J.X. and J.Z.; funding acquisition, P.H. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (2022YFC3106001, 2022YFC3106004), Project of Prevention Strategies for Green Tides of Yellow Sea, M.N.R., Natural Science Foundation of Shanghai (21ZR1427400).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are available on request.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

CCM: CO2 concentrating mechanism; DIC, dissolved inorganic carbon; CA, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; ME, malic enzyme; NAD, nicotinamide adenine dinucleotide.

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Figure 1. U. prolifera, the dominant species of the Yellow Sea green tide.
Figure 1. U. prolifera, the dominant species of the Yellow Sea green tide.
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Figure 2. Schematic diagram of Rubisco functions. RuBP is ribulose 1, 5-diphosphate, and PGA is 3-phosphoglyceric acid.
Figure 2. Schematic diagram of Rubisco functions. RuBP is ribulose 1, 5-diphosphate, and PGA is 3-phosphoglyceric acid.
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Figure 3. Schematic diagram of CA inhibitor actions. inCA means intracellular CA, eCA means extracellular CA, and when the CA icon is gray, CA is suppressed.
Figure 3. Schematic diagram of CA inhibitor actions. inCA means intracellular CA, eCA means extracellular CA, and when the CA icon is gray, CA is suppressed.
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Figure 4. Thick floating pad in 2018 Qingdao green tide.
Figure 4. Thick floating pad in 2018 Qingdao green tide.
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Figure 5. Model diagrams of different methods of CO2 concentrating mechanism (By Figdraw). Different colors are used to represent different molecules/ions: purple: HCO3−, blue: H2O, orange: CO2, green: H+. ① Extracellular CA bound to the cell wall promotes the dehydration of HCO3−, forming CO2 in the cell membrane. ② When the pH of the environment is high, the low concentration of CO2 in the cell membrane induces the DIDS-sensitive membrane anion exchanger to transport HCO3− into the cell and, under the action of intracellular CA, dehydrated to form CO2. ③ The proton pump located in the cell membrane utilizes the energy generated by the hydrolysis of ATP to pump H+ out of the cell membrane, forming an acidic region in the cell wall, and promoting the dehydration of HCO3− to form CO2, which enters the cell membrane. When the environmental pH and the content of HCO3− were low, but the concentration of CO2 was high, Ulva sp. preferred to use CO2 as a carbon source, and CO2 was transported into the cells through ④ passive diffusion or ⑤ possible CO2 transporters. ⑥ NAD-ME catalyzes the oxidative decarboxylation of malic acid to produce CO2, which is added to the Calvin cycle.
Figure 5. Model diagrams of different methods of CO2 concentrating mechanism (By Figdraw). Different colors are used to represent different molecules/ions: purple: HCO3−, blue: H2O, orange: CO2, green: H+. ① Extracellular CA bound to the cell wall promotes the dehydration of HCO3−, forming CO2 in the cell membrane. ② When the pH of the environment is high, the low concentration of CO2 in the cell membrane induces the DIDS-sensitive membrane anion exchanger to transport HCO3− into the cell and, under the action of intracellular CA, dehydrated to form CO2. ③ The proton pump located in the cell membrane utilizes the energy generated by the hydrolysis of ATP to pump H+ out of the cell membrane, forming an acidic region in the cell wall, and promoting the dehydration of HCO3− to form CO2, which enters the cell membrane. When the environmental pH and the content of HCO3− were low, but the concentration of CO2 was high, Ulva sp. preferred to use CO2 as a carbon source, and CO2 was transported into the cells through ④ passive diffusion or ⑤ possible CO2 transporters. ⑥ NAD-ME catalyzes the oxidative decarboxylation of malic acid to produce CO2, which is added to the Calvin cycle.
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Table 1. Species of Ulva known to have CO2 concentrating mechanisms (CCMs).
Table 1. Species of Ulva known to have CO2 concentrating mechanisms (CCMs).
SpeciesReferences
Ulva australis[95]
U. pertusa[96,97]
U. lactuca[98]
U. linza[68,99,100]
U. prolifera[4,74,100,101,102]
U. rigida[1,103,104]
U. compressa[100]
U. pulchra[1]
U. reticulata[1]
Table 2. Copy number of proteins that may be involved in algal CCM found in the U. prolifera genome database.
Table 2. Copy number of proteins that may be involved in algal CCM found in the U. prolifera genome database.
ProteinNCBI Accession NumberCopy Number in U. prolifera genomeReferences
alpha carbonic anhydrasesCAH1 [BAA14232]5[24,25,118]
CAH2 [CAA38360.1]5
CAH3 [EDP00852.1]5
beta carbonic anhydrasesCAH6 [AAR82947.1]0
CAH8 [ABS87675.1]0
gamma carbonic anhydraseCAG2 [XP_001701594]3
carboxysomal-located carbonic anhydraseccaA/icfA [P27134.1]0[119]
phosphoribosyl aminoimidazole carboxylasepurK [AAB05791]1
NADH dehydrogenasendhB [CAA46161.1]0
nuclear transcriptional regulators of CCM elementsCIA5 [AAG37909.1]2[24,25,118,120]
CIA5 [AF317732_1]2
LCR1 [BAD13492.1]1
low-CO2-inducible proteinsLCIA [BAD16681.1]1
LCIB [BAD16682.1]3
LCIB [EDP04243.1]3
LCIC [BAD16683.1]3
Lci2 [AAC31958.1]1
low-CO2-inducible membrane protein[KAF5834422.1]1
LCIA [XP_001703387.1]0
chloroplast carrier protein 1CCP1 [EDP04147.1]27[25,118]
chloroplast proton extrusion proteinCemA [XP_001696592]1
pyruvate orthophosphate dikinasePPDK [JN222388.1]4[68]
PPDK [JN936854.1]2
ribulose-1, 5-biphosphate carboxylaseRuBPCase [AAR19268.1]2
high and medium affinity HCO3− transportersSbtA [UOW71290.1]0[24,121]
BicA [Q14SY0.1]1
putative ABC transporter/high light-activated 3MRP1/HLA3 [AAL35383.1]26[25,118,122]
HLA3 [XP_001700040.1]26
plasma membrane-type H+-ATPase[AQM50087.1]12[73,123,124]
[P19456.2]7
bestrophin-like proteinBSTs [NP_191691.2]7[22]
proton gradient regulation 5PGR5 [OAP09444.1]1[44]
proton gradient regulation like proteinPGRL1 [XP_001692513.1]1
flavodiiron protein BFlvB [AMJ52190.1]2
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Sun, J.; Zhao, C.; Zhao, S.; Dai, W.; Liu, J.; Zhang, J.; Xu, J.; He, P. Diversity of CO2 Concentrating Mechanisms in Macroalgae Photosynthesis: A Case Study of Ulva sp. J. Mar. Sci. Eng. 2023, 11, 1911. https://doi.org/10.3390/jmse11101911

AMA Style

Sun J, Zhao C, Zhao S, Dai W, Liu J, Zhang J, Xu J, He P. Diversity of CO2 Concentrating Mechanisms in Macroalgae Photosynthesis: A Case Study of Ulva sp. Journal of Marine Science and Engineering. 2023; 11(10):1911. https://doi.org/10.3390/jmse11101911

Chicago/Turabian Style

Sun, Jingyi, Chunyan Zhao, Shuang Zhao, Wei Dai, Jinlin Liu, Jianheng Zhang, Juntian Xu, and Peimin He. 2023. "Diversity of CO2 Concentrating Mechanisms in Macroalgae Photosynthesis: A Case Study of Ulva sp." Journal of Marine Science and Engineering 11, no. 10: 1911. https://doi.org/10.3390/jmse11101911

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