**Absorption and Transport of Inorganic Carbon in Kelps with Emphasis on** *Saccharina japonica*

Yanhui Bi and Zhigang Zhou

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62297

#### **Abstract**

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S0003701X12010161

110 Applied Photosynthesis - New Progress

Due to the low CO2 concentration in seawater, macroalgae including *Saccharina japonica* have developed mechanisms for using the abundant external pool of HCO3 − as an exogenous inorganic carbon (Ci ) source. Otherwise, the high photosynthetic efficiency of some macroalgae indicates that they might possess CO2 concentrating mechanisms (CCMs) to elevate CO2 concentration intracellularly around the active site of ribu‐ lose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo). As the photosynthetic modes of macroalgae are diverse (C3, C4 or a combination of C3 and C4 pathway), CCMs in different carbon fixation pathways should vary correspondingly. However, both in C3 and C4 pathways, carbonic anhydrase (CA) plays a key role by supplying either CO2 to RuBisCO or HCO3 − to PEPC. Over the past decade, although CA activities have been detected in a number of macroalgae, genes of CA family, expression levels of CA genes under different CO2 concentrations, as well as subcellular location of each CA have been rarely reported. Based on analysis the reported high-throughput sequencing data of *S. japonica*, 12 CAs of *S. japonica* (*SjCA*) genes were obtained. Neighbor-Joining (NJ) phylogenetic tree of SjCAs constructed using Mega6.0 and the subcellular location prediction of each CA by WoLFPSORT are also conducted in this article.

**Keywords:** Macroalgae, Inorganic carbon uptake, C3 and C4 metabolism, Carbonic anhydrase, *Saccharina japonica*

### **1. Introduction**

Kelps demonstrate high photosynthetic rates. According to the reports, productivity of large brown algae (e.g., *Macrocystis*, *Laminaria*, *Ecklonia*, *Sargassum*) ranges from 1000 to 3400 g m−2yr −1C or about 3300 to 11,300 g m−2yr−l dry weight, and red algae show a similar range of produc‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tion. Cultivated macroalgae can yield even higher values. The projected yield of cultivated *Laminaria japonica* on an annualized basis is equivalent to 1300 t ha−1 fresh weight or 6.5 times the maximum projected yield for sugarcane, the most productive of land plants under cultiva‐ tion. In general, 45% yield of the dry weight of plants is accounted by carbon, which is assimi‐ lated in plant through Calvin cycle. The high productivities of kelps indicate their higher photosynthetic efficiency than C4 terrestrial plants [1].

The enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo) is crucial in CO2 assimilation. This bifunctional enzyme could catalyse the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO2 and O2 with ribulose-1, 5-bisphosphate (RuBP) [2, 3]. RuBP carboxylation determines the net photosyn‐ thetic efficiency of photoautotrophs [4]. However, RuBisCo has a surprisingly low affinity for CO2 and the oxygenase activity is intrinsic to RuBisCo. For kelps, the enzymatic efficiency of RuBisCo is also limited by the low concentration and diffusion coefficient of CO2 in seawater [5]. At a natural pH of about 8, the major part of the dissolved inorganic carbon (DIC) is in the form of bicarbonate (HCO3 − ), and only about 12 μM is present as dissolved CO2 [6], which is much lower than the half-saturation constant (Ks) of RuBisCo for CO2 ranges from 30 μM to 60 μM in marine macroalgae [7, 8]. To support photosynthesis and growth, seaweeds require an exogenous inorganic carbon (Ci ), while only CO2 and HCO3 − can be used as a CO2 source for photosynthesis. Due to the low CO2 concentration in seawater, it is not surprising that most seaweed have developed mechanisms for using the abundant external pool of HCO3 − as an exogenous Ci source [9–11]. And it seems likely that those macrophytes that are able to use HCO3 <sup>−</sup> would possess advantages compared with that rely solely on diffusive CO2 entry. Here the question is how Ci is absorbed, transported to supply high CO2 concentration around RuBisCo in kelps since unlike CO2, HCO3 − cannot diffuse through the lipid bilayer of the plasma membrane [12] and the produced or absorbed CO2 are readily leaked out due to the high CO2 permeability of cytomembrane. Otherwise, different models of photosynthesis such as C3, C4 and CAM might employ different CCMs in kelps. Thus, this review mainly focuses on the mechanisms of Ci absorption, transportation and concentration mechanisms of multicel‐ lular marine algae, including representatives of Chlorophyceae, Rhodophyceae and Phaeo‐ phyceae with different photosynthetic types*.*

#### **2. Photosynthetic modes of macroalgae**

As with terrestrial angiosperms where a single family may possess species with divergent photosynthetic modes [13], the marine macroalgal divisions also exhibit diversity. The photosynthetic carbon fixation pathways of marine macrophytic algae generally follow that of C3 plants [14]. However, for certain genera, a number of studies have shown photosynthesis to possess C4-like photosynthetic characteristics, including the high phosphoenolpyruvate carboxykinase (PEPCK) activity with low phosphoenolpyruvate carboxylase (PEPC) activity, little photorespiration and the labelling of malate and aspartate as an early product of carbon fixation. Based on this, it has been suggested that these macroalgae are of the C4 type, or a combination of C3 and C4, type [15–17], although Kremer and Küppers [18] had contradicted the decision whether a species is a C4 plant or not based only on chromatographic and enzymatic analysis. In recent decades, our understanding of the possible metabolic pathways of macroalgae has been extended with using the available sequencing resources and molecular technologies and applying molecular approaches. Reiskind et al. [19] reported that a limited C4-like system in the green alga *Udotea* with the high PEPCK activity and low PEPC activity was a novel characteristic. Whereafter, Reiskind and Bowes [20] found that when PEPCK activity was inhibited *in vivo* with 3-mercaptopicolinic acid, thallus photosynthesis was decreased by 70% and the labelling of early photosynthetic products such as malate and aspartate was reduced by 66% and thus provided new evidences for the existence of C4 acid metabolism in this green alga. In contrast to *Udotea*, *Codium*, a macroalga closely related to *Udotea*, exhibits gas exchange characteristics that resemble terrestrial C3 plants, and neither C4 acids nor PEPCK plays a part in photosynthesis [19]. This demonstrates the diversity of photosynthetic mechanisms in the Chlorophyta. *Ulva*, a common green seaweed, was previ‐ ously reported as a typical C3 plant based on some biochemical evidences that 3-phosphogly‐ ceric acid (3-PGA) was the main primary product formed photosynthetically and a high RuBPcase/PEPcase ratio was found in it [21], while, recently, it was reported that *Ulva* possessed rather comprehensive carbon fixation pathways including C3, C4 and CAM mechanisms because key genes of enzymes involved in these photosynthetic modes were got from the expressed sequence tag (EST) using Kyoto encyclopedia of genes and genomes (KEGG) [22]. Recently, C4-like carbon fixation pathway was also found in representatives of Rhodophyceae and Phaeophyceae based on the analysis of ESTs or transcriptomes. In red algae, Fan et al. [23] speculated that the sporophyte of *Pyropia haitanensis* most likely possesses a C4-like carbon fixation pathway since genes of the key enzymes in the PCK-type C4 carbonfixation pathway were abundantly transcribed*.* Wang et al. [24] assumed that a C4-like carbonfixation pathway might play a special role in fixing inorganic CO2 in *Porphyra yezoensis* with the evidence that except pyruvate-phosphate dikinase all genes involved in C4-pathway were discovered from the transcriptome. Xu et al. [25] had reported that PEPCK, an important enzyme in carbon fixation in C4 plants, had very high activity in the sporophyte of *L. japoni‐ ca*. Besides, haploid gametophytes and diploid sporophytes of some marine macroalgae with dimorphic life cycles might even employ different photosynthetic mode. Wang et al. [24] found that both the RuBisCo content and the initial carboxylase activity were notably higher in gametophytes than in the sporophytes of four seaweed species — *P. yezoensis*, *P. haitanensis*, *Bangia fuscopurpurea* (Rhodophyte) and *L. japonica* (Phaeophyceae). They assumed that in the sporophyte of these algae, the major carbon fixation pathway may be a C4-like carbon fixation pathway, and thus a high abundance of RuBisCo would not be necessary for the sporophytes. And for *L. japonica*, the higher RuBisCo content and activity in gametophyte was corresponding to the lower photosynthetic rate, which implied there might be a greater difference between sporophytes and gametophytes of this alga in their photosynthetic mode. Conclusively, the existence of C4-like pathway in macroalgae has been verified using more evidence, while the distribution between C3 and C4 pathways was unknown during growth of macroalgae with comprehensive carbon fixation pathways including C3 and C4.

tion. Cultivated macroalgae can yield even higher values. The projected yield of cultivated *Laminaria japonica* on an annualized basis is equivalent to 1300 t ha−1 fresh weight or 6.5 times the maximum projected yield for sugarcane, the most productive of land plants under cultiva‐ tion. In general, 45% yield of the dry weight of plants is accounted by carbon, which is assimi‐ lated in plant through Calvin cycle. The high productivities of kelps indicate their higher

The enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo) is crucial in CO2 assimilation. This bifunctional enzyme could catalyse the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO2 and O2 with ribulose-1, 5-bisphosphate (RuBP) [2, 3]. RuBP carboxylation determines the net photosyn‐ thetic efficiency of photoautotrophs [4]. However, RuBisCo has a surprisingly low affinity for CO2 and the oxygenase activity is intrinsic to RuBisCo. For kelps, the enzymatic efficiency of RuBisCo is also limited by the low concentration and diffusion coefficient of CO2 in seawater [5]. At a natural pH of about 8, the major part of the dissolved inorganic carbon (DIC) is in the

much lower than the half-saturation constant (Ks) of RuBisCo for CO2 ranges from 30 μM to 60 μM in marine macroalgae [7, 8]. To support photosynthesis and growth, seaweeds require

photosynthesis. Due to the low CO2 concentration in seawater, it is not surprising that most seaweed have developed mechanisms for using the abundant external pool of HCO3

membrane [12] and the produced or absorbed CO2 are readily leaked out due to the high CO2 permeability of cytomembrane. Otherwise, different models of photosynthesis such as C3, C4 and CAM might employ different CCMs in kelps. Thus, this review mainly focuses on

lular marine algae, including representatives of Chlorophyceae, Rhodophyceae and Phaeo‐

As with terrestrial angiosperms where a single family may possess species with divergent photosynthetic modes [13], the marine macroalgal divisions also exhibit diversity. The photosynthetic carbon fixation pathways of marine macrophytic algae generally follow that of C3 plants [14]. However, for certain genera, a number of studies have shown photosynthesis to possess C4-like photosynthetic characteristics, including the high phosphoenolpyruvate carboxykinase (PEPCK) activity with low phosphoenolpyruvate carboxylase (PEPC) activity, little photorespiration and the labelling of malate and aspartate as an early product of carbon

−

), while only CO2 and HCO3

<sup>−</sup> would possess advantages compared with that rely solely on diffusive CO2 entry. Here

source [9–11]. And it seems likely that those macrophytes that are able to use

is absorbed, transported to supply high CO2 concentration around

absorption, transportation and concentration mechanisms of multicel‐

cannot diffuse through the lipid bilayer of the plasma

), and only about 12 μM is present as dissolved CO2 [6], which is

−

can be used as a CO2 source for

− as an

photosynthetic efficiency than C4 terrestrial plants [1].

−

form of bicarbonate (HCO3

112 Applied Photosynthesis - New Progress

exogenous Ci

the question is how Ci

the mechanisms of Ci

HCO3

an exogenous inorganic carbon (Ci

RuBisCo in kelps since unlike CO2, HCO3

phyceae with different photosynthetic types*.*

**2. Photosynthetic modes of macroalgae**

In C3 and C4 metabolisms, CO2 is the substrate of RuBisCo and assimilated through the Calvin cycle. In this cycle, CO2, catalysed with RuBisCo, combines with RuBP to form two molecules of 3-PGA. PGA is reduced to triose. RuBisCo, a bifunctional enzyme, may catalyse the combination of RuBP and CO2 for photosynthetic carbon reduction or may combine with O2 for C2 photorespiration [3]. The ratio of CO2 to O2 around RuBisCo is a major factor for the enzyme to choose the photosynthetic carbon reduction or C2 photorespiration carbon oxidation [26]. The low CO2 concentration around RuBisCo may not only impose restrictions on photosynthesis but also cause permanent light injuries to photosynthetic organelle [27–29]. The speciation of DIC (Ci ) is pH dependent. Above pH 4.5, the proportion occurring as CO2 (aq) decreases and HCO3 − increases, while above pH 8.3, the bicarbonate equivalence point, the equilibrium begins to shift towards carbonate (CO3 2− ). In the upper layer of the oceans, HCO3 − ions predominate, and the dissolved CO2 represents only about 1% of the total dissolved carbon with a concentration of about 21 μM [30]. The *K*m (CO2) value of RuBisCO is signifi‐ cantly higher than this, having been reported as being as high as 200 μM in some cyanobacteria [31]. To survive under the selective pressure of low CO2 concentration, high permeability of CO2 for plasma membrane and low affinity of CO2 for RuBisCo, many algae, including macroalgae living in the subtidal zone, have evolved with inorganic CCM that allows them to overcome this potentially limiting shortage of CO2 [9, 32–36]. So, the productivity of most macroalgae is not currently considered limited by DIC. Unlike terrestrial C4 plants possessing Kranz anatomy to prevent futile recycling of CO2 by segregating the initial carboxylation and decarboxylation reactions in different cells, macroalgae concentrate CO2 internally, which is mediated by Ci transporters at the plasma membrane or chloroplast envelope and CA. As for carboxylases are different between C3 and C4 metabolism, Ci acquisition, transportation and concentration mechanisms might be diverse.

Based on a series of reports on the presence of CCM in blue-green algae and *Chlamydomonas* (*Chlamydomonas reinhardtii*) and some other microalgae [37–40], Badger [41] reported that the CCM of algae possess at least three functional elements: (1) the transportation of the Ci dissolved in seawater into cells in the form of CO2 and/or HCO3 − ; (2) the accumulation of the Ci in cells in the form of HCO3 − , forming pools of the dissolved Ci and (3) the delivery of CO2 to the periphery of RuBisCo from such pools.

#### **3. Inorganic carbon absorption mechanisms of macroalgae**

The methods of CO2 and/or HCO3 − absorption of macroalgae cells (Figure 1) include the following: (1) non-CCM macroalgae (that do not possess or use CCM) rely exclusively on diffusive uptake of CO2, (2) CCM macroalgae uptake of Ci , as CO2 and/or HCO3 − via mechanisms of the external carbonic anhydrase (CAext) mechanism, the anion exchange (AE) transport mechanism, the plasma membrane associated with H+ -ATPase mechanism and passive transport of CO2 by diffusion. In the first mechanism, HCO3 − in the periplasmic space is converted to CO2 at the presence of CAext, an enzyme that is located in the cell wall in the

In C3 and C4 metabolisms, CO2 is the substrate of RuBisCo and assimilated through the Calvin cycle. In this cycle, CO2, catalysed with RuBisCo, combines with RuBP to form two molecules of 3-PGA. PGA is reduced to triose. RuBisCo, a bifunctional enzyme, may catalyse the combination of RuBP and CO2 for photosynthetic carbon reduction or may combine with O2 for C2 photorespiration [3]. The ratio of CO2 to O2 around RuBisCo is a major factor for the enzyme to choose the photosynthetic carbon reduction or C2 photorespiration carbon oxidation [26]. The low CO2 concentration around RuBisCo may not only impose restrictions on photosynthesis but also cause permanent light injuries to photosynthetic organelle [27–29].

) is pH dependent. Above pH 4.5, the proportion occurring as CO2

increases, while above pH 8.3, the bicarbonate equivalence point, the

). In the upper layer of the oceans, HCO3

acquisition, transportation and

; (2) the accumulation of the

−


in the periplasmic space is

via mechanisms

−

absorption of macroalgae cells (Figure 1) include the

−

, as CO2 and/or HCO3

, forming pools of the dissolved Ci and (3) the delivery of CO2

−

2−

ions predominate, and the dissolved CO2 represents only about 1% of the total dissolved carbon with a concentration of about 21 μM [30]. The *K*m (CO2) value of RuBisCO is signifi‐ cantly higher than this, having been reported as being as high as 200 μM in some cyanobacteria [31]. To survive under the selective pressure of low CO2 concentration, high permeability of CO2 for plasma membrane and low affinity of CO2 for RuBisCo, many algae, including macroalgae living in the subtidal zone, have evolved with inorganic CCM that allows them to overcome this potentially limiting shortage of CO2 [9, 32–36]. So, the productivity of most macroalgae is not currently considered limited by DIC. Unlike terrestrial C4 plants possessing Kranz anatomy to prevent futile recycling of CO2 by segregating the initial carboxylation and decarboxylation reactions in different cells, macroalgae concentrate CO2 internally, which is mediated by Ci transporters at the plasma membrane or chloroplast envelope and CA. As for

Based on a series of reports on the presence of CCM in blue-green algae and *Chlamydomonas* (*Chlamydomonas reinhardtii*) and some other microalgae [37–40], Badger [41] reported that the CCM of algae possess at least three functional elements: (1) the transportation of the Ci

following: (1) non-CCM macroalgae (that do not possess or use CCM) rely exclusively on

of the external carbonic anhydrase (CAext) mechanism, the anion exchange (AE) transport

converted to CO2 at the presence of CAext, an enzyme that is located in the cell wall in the

The speciation of DIC (Ci

114 Applied Photosynthesis - New Progress

−

equilibrium begins to shift towards carbonate (CO3

carboxylases are different between C3 and C4 metabolism, Ci

dissolved in seawater into cells in the form of CO2 and/or HCO3

**3. Inorganic carbon absorption mechanisms of macroalgae**

−

−

diffusive uptake of CO2, (2) CCM macroalgae uptake of Ci

mechanism, the plasma membrane associated with H+

transport of CO2 by diffusion. In the first mechanism, HCO3

concentration mechanisms might be diverse.

to the periphery of RuBisCo from such pools.

Ci in cells in the form of HCO3

The methods of CO2 and/or HCO3

(aq) decreases and HCO3

**Figure 1.** A schematic diagram on the photosynthetic carbon physiology of some macroalgae revised from [45].

majority of seaweeds and could be inhibited by the membrane impermeable acetazolamide (AZ), and then the resulting CO2 is readily taken into the cell by passive diffusion. This seems to be the most prevalent for HCO3 − utilization among seaweeds [42, 43], but it may be nonfunctional under high pH (>9.00) [44, 45]. The AE transport mechanism is HCO3 − direct uptake through the AE protein in plasma membrane [11, 43, 46–48], which is 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) sensitive. This operates equally well at pH 8.4 and 9.4 [44, 45]. H+ -ATPase mechanism refers to a plasma membrane associated H+ -ATPase pump that extrudes the excess cellular H+ to the outside of the plasma membrane facilitating a H<sup>+</sup> / HCO3 − co-transportation or enhancement of the external uncatalysed dehydration of HCO3 − to CO2 in the periplasmic space [49]. However, this has only been reported in some Laminariales such as *S. latissima* and *L. digitata*. Along with the uptake of CO2 and/or HCO3 − , the internal charge balance (OH<sup>−</sup> /H+ ) will be absolutely changed. To maintain intracellular ion balance, macroal‐ gae employ diverse strategies. In AE mechanism, the active transport of HCO3 − into the cell might result in an outward flux of OH<sup>−</sup> [50–53, 45] as this mechanism is involved in a one-forone exchange of anions across the plasma membrane. The OH<sup>−</sup> efflux can increase H+ in the cell [52]. To maintain the intracellular OH<sup>−</sup> /H+ balance, H+ extrusion might be required. In macroalgae possessing H+ -ATPase mechanism, their plasma membrane associated with H+ - ATPase pump might extrude excess cellular H+ to the outside of the plasma membrane, while in macroalgae that do not have H+ -ATPase pump in their plasma membrane, the regulation of intracellular ion balance might be related to a high activity of internal carbonic anhydrase (CAint), including the CA in cytoplasm, chloroplast stroma, thylakoid lumen and mitochondria [45].

The extent to which marine macroalgae are able to acquire HCO3 <sup>−</sup> for photosynthesis varies among taxa and/or species, and the special strategies by which the alga acquire Ci is closely related to habitat including pH and depth, conferring as adaptation advantage to the alga [9, 33, 36, 54–56]. Cornwall et al. [57] reported when light is low, CCM activity of macroalgae is reduced in favour of diffusive CO2 uptake and the proportion of non-CCM (diffusive uptake of CO2) species increased with depth. Otherwise, pH might also control Ci use by macroalgae. In *U. lactuca*, the CAext-mediated mechanism is the main method of HCO3 − utilization under normal pH conditions, whereas when they were grown at high pH, direct uptake of HCO3 − via a DIDS-sensitive mechanism can be induced [44]. Similar HCO3 <sup>−</sup> utilizing mechanisms were found in another green macroalgae *Enteromorpha intestinalis* [54]. For the red alga *Gracilaria gaditana*, the HCO3 − use is also carried out by the two DIC uptake mechanisms, in which the indirect use of HCO3 − by an external CA activity being the main pathway and the potential contribution to HCO3 − acquisition by the DIDS-sensitive AE mechanism was higher after culturing at a high pH [58]. However, these two mechanisms do not occur simultaneously, and the DIDS-sensitive mechanism is induced only under high pH. *Solieria filiformis*, another red marine macroalgae, in which the general form of Ci transported across the plasma membrane is CO2, but HCO3 − acquisition takes place simultaneously between CAext mechanism and direct uptake [59]. CAext mechanism is also the main pathway for DIC acquisition for the species of Phaeophyta. *S. latissima* mainly uses CAext mechanism for HCO3 − absorption, since when AZ is used to treat *S. latissima*, its photosynthetic efficiency drops by 80% [11]. Otherwise, *S. latissima* also has a H+ -ATPase mechanism, of which the proton pump may support the antiport of H<sup>+</sup> / HCO3 <sup>−</sup> or the discharge of H+ , creating an acid environment in the periplasmic space and causing the dehydration of HCO3 − into CO2 with CA to quickly diffuse into cells [49]. *S*imilar to *S. latissima*, *L. digitata* also has a CAext mechanism of absorbing HCO3 − and a P-H+ - ATPase mechanism [49]. Gametophytes of *Ectocarpus siliculosus* utilize the CAext mechanism and the HCO3 − transport protein [60] on the cell membrane to absorb HCO3 − . *Macrocystis pyrifera* utilizes the CAext mechanism and the AE protein mechanism to absorb HCO3 − , in which the main mechanism of HCO3 − uptake is via AE protein and CAext contributes little [45]. For *Sargassum henslowianum*, like most seaweed, the main Ci acquisition strategy is also CAext metabolism, since its photosynthetic O2 evolution could be drastically depressed by AZ at pH 8.1 (i.e., the normal seawater pH value) and at pH 9.0. And direct uptake for HCO3 − via DIDSsensitive AE protein mechanism was unlikely to be present in Ci acquisition of this kelp, because the photosynthesis in either blade or receptacle tissue of this alga was not affected by DIDS [61]. For *Hizikia fusiformis*, CAext+ diffusive uptake of CO2 could support its metabolic requirements sufficiently since there is no known other active Ci transport mechanisms [62]. For *S. japonica*, Yue et al. [63] found that the Ci absorption of the CAext mechanism in its juvenile sporophytes accounts for 75% of the total Ci absorption in algae cells, whereas free CO2 absorption accounts for 25% only.

Thus, the CAext mechanism plays an important role in the CCM macroalgae absorption and the utilization of the relatively abundant HCO3 − in seawater.

#### **4. Ci transition process in CCMs of macroalgae**

ATPase pump might extrude excess cellular H+ to the outside of the plasma membrane, while

intracellular ion balance might be related to a high activity of internal carbonic anhydrase (CAint), including the CA in cytoplasm, chloroplast stroma, thylakoid lumen and mitochondria

related to habitat including pH and depth, conferring as adaptation advantage to the alga [9, 33, 36, 54–56]. Cornwall et al. [57] reported when light is low, CCM activity of macroalgae is reduced in favour of diffusive CO2 uptake and the proportion of non-CCM (diffusive uptake of CO2) species increased with depth. Otherwise, pH might also control Ci use by macroalgae.

normal pH conditions, whereas when they were grown at high pH, direct uptake of HCO3

found in another green macroalgae *Enteromorpha intestinalis* [54]. For the red alga *Gracilaria*

culturing at a high pH [58]. However, these two mechanisms do not occur simultaneously, and the DIDS-sensitive mechanism is induced only under high pH. *Solieria filiformis*, another

and direct uptake [59]. CAext mechanism is also the main pathway for DIC acquisition for the

when AZ is used to treat *S. latissima*, its photosynthetic efficiency drops by 80% [11]. Otherwise,

ATPase mechanism [49]. Gametophytes of *Ectocarpus siliculosus* utilize the CAext mechanism

*Sargassum henslowianum*, like most seaweed, the main Ci acquisition strategy is also CAext metabolism, since its photosynthetic O2 evolution could be drastically depressed by AZ at pH

because the photosynthesis in either blade or receptacle tissue of this alga was not affected by DIDS [61]. For *Hizikia fusiformis*, CAext+ diffusive uptake of CO2 could support its metabolic

transport protein [60] on the cell membrane to absorb HCO3

−

*S*imilar to *S. latissima*, *L. digitata* also has a CAext mechanism of absorbing HCO3

*pyrifera* utilizes the CAext mechanism and the AE protein mechanism to absorb HCO3

8.1 (i.e., the normal seawater pH value) and at pH 9.0. And direct uptake for HCO3

use is also carried out by the two DIC uptake mechanisms, in which the

by an external CA activity being the main pathway and the potential

acquisition by the DIDS-sensitive AE mechanism was higher after

acquisition takes place simultaneously between CAext mechanism


uptake is via AE protein and CAext contributes little [45]. For

among taxa and/or species, and the special strategies by which the alga acquire Ci

In *U. lactuca*, the CAext-mediated mechanism is the main method of HCO3

species of Phaeophyta. *S. latissima* mainly uses CAext mechanism for HCO3

The extent to which marine macroalgae are able to acquire HCO3

a DIDS-sensitive mechanism can be induced [44]. Similar HCO3

red marine macroalgae, in which the general form of Ci

<sup>−</sup> or the discharge of H+

−

sensitive AE protein mechanism was unlikely to be present in Ci

−


<sup>−</sup> for photosynthesis varies

−

<sup>−</sup> utilizing mechanisms were

transported across the plasma

−

, creating an acid environment in the periplasmic

into CO2 with CA to quickly diffuse into cells [49].

is closely

− via

utilization under

absorption, since

and a P-H+

. *Macrocystis*

, in which

via DIDS-


−

−

−

−

acquisition of this kelp,

in macroalgae that do not have H+

116 Applied Photosynthesis - New Progress

[45].

*gaditana*, the HCO3

indirect use of HCO3

contribution to HCO3

membrane is CO2, but HCO3

*S. latissima* also has a H+

−

the main mechanism of HCO3

antiport of H<sup>+</sup> / HCO3

and the HCO3

−

−

−

space and causing the dehydration of HCO3

Ci acquisition mechanisms are extensively studied and well-known in microalgae [44, 38]. For instance, regardless of the Ci form (CO2 or HCO3 − ) taken up by the microalga *C. reinhardtii*, HCO3 − is the primary form accumulated into the cell to prevent CO2 leakage [38]. In macroalgae, most Ci use processes are speculated based on some biochemical evidence. For C3 photosynthesis, the CO2 that entered the cytoplasm is transformed into HCO3 <sup>−</sup> under the catalytic action of CA in the cytoplasm and stored in the cytoplasm [38] to maintain the equilibrium of different forms of Ci and to regulate the pH value of the cytoplasm [26, 38]. The HCO3 <sup>−</sup> in the cytoplasm enters the chloroplast stroma via the Ci transport protein on the chloroplast membrane, and the CO2 in the cytoplasm directly enters the stroma via the chloroplast membrane. In diatom *Phaeodactylum tricornutum*, genes with homology to bicarbonate transporters from SLC4 and SLC6 families, two HCO3 − transporters studied thoroughly in human, were got from its genome and one of these SLC4-type HCO3 − transporters has recently been confirmed to function as a Na+ -dependent HCO3 − transporter on the outer membrane [64, 65]. However, the molecular nature of HCO3 − transporters of macroalgae is unknown now, and their similarity to those found in diatoms is uncertain. The transportation of Ci from the cytoplasm to the chloroplast is the major Ci flux in the cell and the primary driving force for the CCM. This flux drives the accumulation of Ci in the chloroplast stroma and generates a CO2 deficit in the cytoplasm, inducing CO2 influx into the cell. Given that the pH value of the chloroplast stroma is closer to 8, the stroma Ci is mostly enriched in the form of HCO3 − , forming Ci pools [66]. In macroalgae, which have pyrenoids, HCO3 − is putatively carried into the thylakoid by the Ci transport protein on the thylakoid membrane, forming CO2 in the thylakoid space under the catalytic action of thylakoid CA [67, 68]. The thylakoid membrane partially sinks into the pyrenoids [69], where the diffused CO2 is quickly fixed by the RuBisCo in the pyrenoids. The diffused CO2 from the thylakoid space outside the pyrenoids or the unfixed CO2 leaked from the pyrenoids is transformed into HCO3 − under the action of CA in the starch sheath on the periphery of the pyrenoid, thus increasing the number of HCO3 − pools in the matrix [70]. For macroalgae without pyrenoids, such as *L. japonica*, HCO3 − entered the chloroplast stroma after being dehydrated under the action of chloroplast stroma CA and provided CO2 for the RuBisCo in the matrix (Figure 1).

For C4 photosynthesis, CA is required to convert CO2 to HCO3 <sup>−</sup> in the cytosol, and thus supply PEPC with substrate. HCO3 − will be fixed into malate. For non-PEPC algae with PEPCK, the CO2 entering the cytoplasm will be directly fixed in the form of four-carbon acid [71]. The produced four-carbon acid may be transported into the mitochondria, forming pyruvate after decarboxylation and CO2 release, which is fixed in the form of carbohydrate in the Calvin cycle. In fact, the presence of CA in C4 plants has been suggested to accelerate the rate of photosyn‐ thesis in C4 plants 104 -fold over what it would be if this enzyme were absent [72].

In conclusion, CA (CAext+CAint) is essential for the reversible HCO3 <sup>−</sup> –CO2 conversion both in the cell and in the periplasm. They participate in photosynthesis by supplying either CO2 to RuBisCO or HCO3 − to PEPC for C4 type.
