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Agricultural and Biological Sciences » "Applied Photosynthesis - New Progress", book edited by Mohammad Mahdi Najafpour, ISBN 978-953-51-2267-8, Published: March 30, 2016 under CC BY 3.0 license. © The Author(s).

Chapter 6

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

By Yanhui Bi and Zhigang Zhou
DOI: 10.5772/62297

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

Yanhui Bi and Zhigang Zhou
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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 ribulose-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 production. 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 cultivation. In general, 45% yield of the dry weight of plants is accounted by carbon, which is assimilated 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 photosynthetic 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 [911]. And it seems likely that those macrophytes that are able to use HCO3 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 multicellular marine algae, including representatives of Chlorophyceae, Rhodophyceae and Phaeophyceae 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 [1517], 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 previously reported as a typical C3 plant based on some biochemical evidences that 3-phosphoglyceric 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 carbon-fixation pathway were abundantly transcribed. Wang et al. [24] assumed that a C4-like carbon-fixation 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. japonica. 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.

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 [2729]. 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 (CO32−). 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 Km (CO2) value of RuBisCO is significantly 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, 3236]. 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 [3740], 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

media/Figure 1.png

Figure 1.

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

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 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 non-functional under high pH (>9.00) [44, 45]. The AE transport mechanism is HCO3 direct uptake through the AE protein in plasma membrane [11, 43, 4648], which is 4,4'-diisothiocyano-stilbene-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+/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/H+) will be absolutely changed. To maintain intracellular ion balance, macroalgae employ diverse strategies. In AE mechanism, the active transport of HCO3 into the cell might result in an outward flux of OH [5053, 45] as this mechanism is involved in a one-for-one exchange of anions across the plasma membrane. The OH efflux can increase H+ in the cell [52]. To maintain the intracellular OH/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 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, 5456]. 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 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+/HCO3 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]. Similar 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 DIDS-sensitive 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

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 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 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 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 photosynthesis 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 HCO3CO2 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.

5. Carbonic anhydrase

CAs are metalloenzymes that catalyse the reversible interconversion of CO2 and HCO3 [73]. They are encoded by six evolutionary divergent gene families and the corresponding enzymes are designated as α, β, γ, δ, ε and ζ-CA [39]. These six types of CAs share no sequence similarity in their primary amino acid sequences and seem to have evolved independently [26, 74]. In macroalgae, almost all known CAs belong to α, β and γ classes, with the β class predominating [26, 39]. The δ, ε and ζ classes of CA are found only in some diatoms [75], bacteria [76] and marine protists [77, 78]. The active site of CA contains a zinc ion (Zn2+), which plays a critical role in the catalytic activity of the enzyme. The ζ and γ classes of CAs represent exceptions to this rule since they can use cadmium (ζ), iron (γ) or cobalt (γ) as cofactors [7981]. CA plays an important role in photosynthesis by supplying either CO2 to RuBPCO or HCO3 to PEPC. They also participate in some other physiological reactions such as respiration, pH homeostasis, ion transport and catalysis of key steps in the pathways for the biosynthesis of physiologically important metabolites [41]. The CA synthesis in the cytoplasm [82] is located in the periplasmic space, mitochondria, chloroplast stroma and chloroplast thylakoid lumen, carboxysome and pyrenoid [66, 70, 83, 84]. Different subcellular localizations make different CA functions in CCM. Periplasmic CA (CAext) can catalyse the conversion of HCO3 into CO2 to promote the diffusion of CO2 at the cell surface across the plasma membrane [85, 86]. Therefore, CAext has been postulated to be part of the CCM in most macroalgae. The cytoplasm CA stores Ci in the form of HCO3 to avoid leakage of CO2 and to regulate the pH value of cytoplasm by maintaining the equilibrium of different forms of Ci, which is important for algal CCM [39]. CAs on the chloroplast membrane and in the stroma mainly provide CO2 for RuBisCo [26, 38, 87]. In cyanobacteria, CAs in the carboxysomal shell function to convert accumulated HCO3 into CO2 and pass it to RuBisCo inside the cytoxysome [88]. CA in the thylakoid lumen was proposed to function to create an efficient CO2 supply to RuBisCo by taking advantage of the acidity of the lumenal compartment [69]. Stromal CA is also thought to operate by converting leaking CO2 into HCO3 [70]. Recently, data provided by various genome sequencing studies have revealed the multiplicity of CA isoforms in algae. For example, in the model microalga C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three α, six β and three γ or γ-like CAs [39]. For marine diatom, nine and thirteen CA sequences were found in the genomes of P. tricornutum and Thalassiosira pseudonana, respectively [89]. P. tricornutum contains two β-CA genes, five α and two γ CA genes, whereas T. pseudonana has three α-, five γ-, four δ- and one ζ-CA genes [89]. As for macroalgae, CA genes have only been reported in few species. Six full-length CA of P. haitanensis (PhCA) genes were reported, which include two α-CAs, three β-CAs and one γ-CA [90]. Besides, one β-CA and one α-CA were reported in P. yezoensis [91] and S. japonica [92, 93]. Otherwise, although the activity of CAext and CAint has been detected in many macroalgae, the subcellular localization and functions of CAext and CAint remain unclear [71, 93].

Conclusively, CAs, including CAext and CAint (Figure 1), play an important role in the transportation or concentration process of the Ci. And as for C3 and C4 metablisms have different carboxylase, CAs might play different roles in CCMs of macroalgae with different photosynthetic mode. Thus, isolating of the CA genes, studies on their expression levels in different CO2 concentrations, in different life phase, and under different environmental stress, as well as studies on subcellular locations of CAs should be conducted in macroalgae to help reveal their Ci assimilation processes.

6. Studies of S. japonica CCM

S. japonica is an economically important brown seaweed. It has been cultivated extensively for food and industrial alginate in East Asia, such as in China, Japan and South Korea. China is by far the largest producer, and in 2009, its production in China rose sharply to 4.14×109 kg wet weight [94], accounting for approximately 80% of the global production, over several decades. This has been attributed to both its large-scale farming and high kelp yield per unit area. Production of this kelp in China under natural conditions is within the range of 3,300 to 11,300 g dry matter m−2·year−1, whereas that under artificial conditions is higher [1]. For example, its production during the 7-month cultivation is 15,000 g dry matter m−2 area (equivalent to 150 t per ha), which is 2.8 times higher than the maximum productivity of sugarcane in the United States (fresh weight about 95 t per ha·year) [1], which indicates that S. japonica has higher photosynthetic efficiency than sugarcane and other C4 plants. In fact, the photosynthetic efficiency of macroalgae (e.g., kelp) is 6%–8%, which is 1.8%–2.2% higher than that of land plants [95]. In seawater, the dominant species of Ci is HCO3 [11]. Since there is a fairly high photosynthetic rate in these kelps [34], a CCM involving an efficient HCO3 utilization mechanism is expected to exist. Indeed, 75% of the total Ci absorption in the juvenile sporophytes of this kelp is via the CAext mechanism [63], whereas CO2 diffusion accounts for 25% only. By analysis of genome annotation data of S. japonica [96], all the essential genes related to C3-pathway (23 unigenes) were discovered (Table 1), which provided the unequivocal molecular evidence that there existed C3-pathway in S. japonica. Otherwise, 16 enzyme-encoding unigenes involved in C4-pathway were found, covering almost all enzymes needed for C4-carbon fixation except the malic enzyme (Table 1). The results helped us to understand the carbon fixation process of this species.

Photosynthesis modesEnzyme namesUnigenes
Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (GAPDH)4
Phosphoglycerate kinase (PGK)5
Fructose-1,6-bisphosphatase (FBPase)1
Sedoheptulose-bisphosphatase (SBPase)3
Fructose-bisphosphate aldolase1
Ribulose-phosphate 3-epimerase2
Triose-phosphate isomerase (TIM)1
Ribose-5-phosphate isomerase1
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), small1
Ribulose-1,5-bisphosphate carboxylase/oxygenase(RuBisCo), large1
Malate dehydrogenase4
Aspartate aminotransferase (AST)4
Pyruvate kinase4
Phosphoenolpyruvate carboxylase (PEPC)1
Phosphoenolpyruvate carboxykinase (PEPCK)1
Pyruvate phosphate dikinase1
Arginine/alanine aminopeptidase1

Table 1.

Statistics of C3/C4-pathway related enzymes of S. japonica.

Considering CAs play key roles in CCMs of macroalgae, it is important to determine the numbers and characterizations of CA genes of S. japonica. Herein, based on unigene sequences [96], the high-throughput sequencing data of S. japonica [97, 98] and S. latissima [99], as well as combined with the preparatory work of our group [92, 93], 12 CAs of S. japonica (SjCA) genes were obtained. Among them, we have cloned the full-length complementary DNA (cDNA) sequences of SjαCA1, SjβCA1 and SjβCA2 using rapid amplification of cDNA ends, which are 2804 [94], 1291 and 1261 nucleotides, respectively. The encoded proteins were 290, 314 and 307 amino acids. For further analysis the gene subtypes of CAs, a phylogenetic tree was constructed by using the neighbour-joining algorithm of the MEGA6.0 software [100] with Poisson correction and pairwise deletion parameters. A total of 1000 bootstrap replicates were performed. On the basis of conserved motifs and phylogenetic tree analysis (Figure 2), the SjCAs were divided into three CA classes: from SjαCA1 to SjαCA7 are α-CA; SjβCA1 and SjβCA2 are β-CA; SjγCA1, SjγCA2 and SjγCA3 are γ-CA. Among them, only one α-CA (SjαCA1) has been localized in the chloroplast and thylakoid membrane of the gametocytes of S. japonica under immunogold electron microscopy [93]. To get a general idea of functions of each SjCA, herein, the subcellular localizations of SjCAs were predicted using WoLFPSORT ( Based on the predicted results (Table 2), SjαCA2 might be an external CA and exist in periplasmic space, SjαCA3; SjαCA4, SjαCA6, SjαCA7 and

media/Figure 2.png

Figure 2.

Phylogenetic tree constructed using SjCA amino acid sequences.

SjγCA1 might be cytoplasmic CA; SjαCA5, SjβCA2 and SjγCA2 might present in mitochondria; SjβCA1 and SjγCA3 might exist in chloroplasts. However, most of the SjCAs’ subcellular localizations are predicted, which need to be verified by further studies. Otherwise, sporophyte and gametophyte of this kelp might employ different carbon fixation process since the content and activity of RuBisCo enzyme in gametophyte are significantly higher than those in sporophyte implying they may have different types of photosynthetic metabolism [24]. As for CA might play different role in CCMs of C3 and C4 pathway, full-length cDNA as well as DNA sequences of each SjCA should be cloned from sporophytes and gametophytes of this kelp in the future studies. CA gene expression levels under different CO2 concentrations and the subcellular location of each CA should also be conducted to help reveal Ci assimilation process of S japonica.

EnzymeGene IDaAA no.Full length (Y/N)Subcellular location prediction
SjαCA1JF827608290YChloroplast and thylakoid membrane [93]
SjαCA5SJ13240294NMitochondrial inner membrane
SjβCA1SJ12311314YChloroplast thylakoid membrane

Table 2.

Prediction of subcellular locations of SjCAs.

Abbreviation: AA, amino acid.

a JF827608 is the NCBI gene accession number; ‘SJ’ in the table stands for the gene IDs for S. japonica.

The completion of the CCM modelling of sporophyte and gametophyte in S. japonica will give a solid foundation for further exploring its highly efficient photosynthetic mechanism. In addition, conducting studies on the inorganic carbon metabolism of macroalgae is of positive significance on developing the biomass energy from kelp and other algae and slowing down seawater acidification and global warming.


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