**6. Studies of** *S. japonica* **CCM**

For C4 photosynthesis, CA is required to convert CO2 to HCO3

In conclusion, CA (CAext+CAint) is essential for the reversible HCO3

to PEPC for C4 type.

−

PEPC with substrate. HCO3

118 Applied Photosynthesis - New Progress

thesis in C4 plants 104

RuBisCO or HCO3

−

CA stores Ci in the form of HCO3

−

to operate by converting leaking CO2 into HCO3

accumulated HCO3

**5. Carbonic anhydrase**

<sup>−</sup> in the cytosol, and thus supply

<sup>−</sup> –CO2 conversion both in

<sup>−</sup> [73].

−

−

to PEPC.

into CO2

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‐

the cell and in the periplasm. They participate in photosynthesis by supplying either CO2 to

CAs are metalloenzymes that catalyse the reversible interconversion of CO2 and HCO3

an important role in photosynthesis by supplying either CO2 to RuBPCO or HCO3

CA functions in CCM. Periplasmic CA (CAext) can catalyse the conversion of HCO3

−

They also participate in some other physiological reactions such as respiration, pH homeosta‐ sis, ion transport and catalysis of key steps in the pathways for the biosynthesis of physiolog‐ ically 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

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

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

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

genome sequencing studies have revealed the multiplicity of CA isoforms in algae. For

to avoid leakage of CO2 and to regulate the pH value of

<sup>−</sup> [70]. Recently, data provided by various

into CO2 and pass it to RuBisCo inside the cytoxysome [88]. CA in the

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 [79–81]. CA plays


*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 HCO<sup>3</sup> − [11]. Since there is a fairly high photosynthetic rate in these kelps [34], a CCM involving an efficient HCO<sup>3</sup> − utilization mechanism is expected to exist. Indeed, 75% of the total C<sup>i</sup> 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 unequiv‐ ocal molecular evidence that there existed C3-pathway in *S. japonica*. Otherwise, 16 enzymeencoding 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.


**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 Sj*α*CA1, Sj*β*CA1 and Sj*β*CA2 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

**Figure 2.** Phylogenetic tree constructed using SjCA amino acid sequences.

for C4-carbon fixation except the malic enzyme (Table 1). The results helped us to understand

**Photosynthesis modes Enzyme names Unigenes** C3-pathway 23

C4-pathway 16

Total 39

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 Sj*α*CA1, Sj*β*CA1 and Sj*β*CA2 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

**Table 1.** Statistics of C3/C4-pathway related enzymes of *S. japonica*.

Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (GAPDH) 4 Transketolase 1 Phosphoribulokinase 2 Phosphoglycerate kinase (PGK) 5 Fructose-1,6-bisphosphatase (FBPase) 1 Sedoheptulose-bisphosphatase (SBPase) 3 Fructose-bisphosphate aldolase 1 Ribulose-phosphate 3-epimerase 2 Triose-phosphate isomerase (TIM) 1 Ribose-5-phosphate isomerase 1 Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), small 1 Ribulose-1,5-bisphosphate carboxylase/oxygenase(RuBisCo), large 1

Malate dehydrogenase 4 Aspartate aminotransferase (AST) 4 Pyruvate kinase 4 Phosphoenolpyruvate carboxylase (PEPC) 1 Phosphoenolpyruvate carboxykinase (PEPCK) 1 Pyruvate phosphate dikinase 1 Arginine/alanine aminopeptidase 1

the carbon fixation process of this species.

120 Applied Photosynthesis - New Progress

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 *SjCA*s were divided into three CA classes: from Sj*α*CA1 to Sj*α*CA7 are *α-*CA; Sj*β*CA1 and Sj*β*CA2 are β-CA; Sj*γ*CA1, Sj*γ*CA2 and Sj*γ*CA3 are γ-CA. Among them, only one α-CA (Sj*α*CA1) 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 (http://www.genscript.com/wolf-psort.html). Based on the predicted results (Table 2), Sj*α*CA2 might be an external CA and exist in periplasmic space, Sj*α*CA3; Sj*α*CA4, Sj*α*CA6, Sj*α*CA7 and Sj*γ*CA1 might be cytoplasmic CA; Sj*α*CA5, Sj*β*CA2 and Sj*γ*CA2 might present in mito‐ chondria; Sj*β*CA1 and Sj*γ*CA3 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*.


Abbreviation: *AA,* amino acid.

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

**Table 2.** Prediction of subcellular locations of SjCAs.

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.

#### **Author details**

Yanhui Bi and Zhigang Zhou\*

\*Address all correspondence to: zgzhou@shou.edu.cn

College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, People's,

Republic of China

chondria; Sj*β*CA1 and Sj*γ*CA3 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

**Enzyme Gene IDa AA no. Full length (Y/N) Subcellular location prediction**

Sj*α*CA5 SJ13240 294 N Mitochondrial inner membrane

Sj*β*CA1 SJ12311 314 Y Chloroplast thylakoid membrane

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

Sj*α*CA2 SJ07762 205 N Secreted Sj*α*CA3 SJ07765 160 N Cytoplasmic Sj*α*CA4 SJ13238 151 N Cytoplasmic

Sj*α*CA6 SJ18135 257 N Cytoplasmic Sj*α*CA7 SJ18141 189 N Cytoplasmic

Sj*β*CA2 SJ17783 307 Y Mitochondrial Sj*γ*CA1 SJ07587 305 N Cytoplasmic Sj*γ*CA2 SJ22175 161 N Mitochondrial Sj*γ*CA3 SJ21158 246 N Chloroplast

Sj*α*CA1 JF827608 290 Y Chloroplast and thylakoid membrane [93]

process of *S japonica*.

122 Applied Photosynthesis - New Progress

Abbreviation: *AA,* amino acid.

**Author details**

Yanhui Bi and Zhigang Zhou\*

**Table 2.** Prediction of subcellular locations of SjCAs.

seawater acidification and global warming.

a

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**Section 2**

**Section two**
