Yn-Hui Bi, Cui-Ling Ling, Ji-Li Li, Ho Yin, Ruo-Tong Tin, Zhi-Gng Zhou,b,＊

aKey Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Ministry of Education, Shanghai, 201306, China

bInternational Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, 201306, China

cNational Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China

Keywords:

Saccharina japonica

Gametophytes

Carbonic anhydrase

Inorganic carbon

pH

ABSTRACT

Carbonic anhydrase (CA) was considered to be an important component of carbon concentrating mechanism(CCM) of algae. It was an inducible enzyme. Environmental factors, especially dissolved inorganic carbon and pH, were known to affect CA activity. Effects of inorganic carbon (CO2 and) and pH on CA activity of gametophytes of Saccharina japonica were evaluated in this study. Under high-CO2 condition (3% CO2), the activity of external CA (CAext) was significantly decreased (P ＜0.05) from 33.92 REA/g FW to 27.69 REA/g FW.In contrast, the internal CA (CAint) and total CA activities were elevated significantly (p ＜0.01) from 36.83 REA/g FW to 48.80 REA/g FW, and from 70.75 REA/g FW to 76.49 REA/g FW, respectively. Addition of an appropriate concentration of HCO−3 to the medium, CAint and total CA activities were promoted significantly, although the activity of CAext was inhibited significantly (P ＜ 0.05). In higher HCO−3 concentration (up to 1680 mg/L), the activity of CAint was promoted to 60.81REA/g FW, which was twice that of the control. Higher activities of CAint and total CA were induced at low pH. At pH 6.0, the highest activities of CAint (110.85 REA/g FW) and total CA(128.17 REA/g FW) were induced. However, at pH 9.0, the activities of CAint and total CA were reduced to the lowest of 23.31REA/g FW and 42.19 REA/g FW, respectively. This is the first report about the successful detection of CAext activity of gametophytes of S. japonica. The results would provide data for the analysis of Ci acquisition and transport mechanism in S.japonica gametophytes.

1.Introduction

Saccharina japonica

(Areschoug) C. E. Lane, C. Mayes, Druehl et G. W.Saunders or

Saccharina japonica

Aresch., belonging to Phaeophyta and Laminariales, is one of the most important economic algae in China. Its total production was the largest in the world, and was about 1.5 million tons in 2017 (China Fishery Statistical Yearbook, 2018). This kelp has been cultured on a large scale in China, not only because of its important nutritional, medical and industrial value, but also due to its huge biomass brought out by the high photosynthetic efficiency (6%—8%). For a 7-month growing season, the biomass of

S. japonica

could reached more than 15,000 g mdry weight (

i

.

e

.150 t ha) (Gao & McKinley,1994).In today's oceans, the concentration of dissolved total inorganic carbon (DIC) in seawater is about 2.1 mmol/L (Fernández, Roleda, &Hurd, 2015). The main form of DIC is bicarbonate (HCO, accounting for 91% of the total concentration of DIC), followed by carbonate (CO,accounting for 8%). The proportion of dissolved COis the lowest, only 1%, with a concentration of about 21 μmol/L (Fernández et al., 2015),which is far lower than the Km value of COat RuBisCo in macroalgae(30—60 μmol/L) (Johnston, 1991). To support high photosynthesis rates,most macroalgae including

S.japonica

, have developed abilities to utilize HCOin addition to COas carbon source (Gao & McKinley, 1994).In macroalgae, three mechanisms of HCOacquisition have been described. The most common mechanism of HCOuptake involves the dehydration of HCOto COoutside the plasmalemma under the catalization of external carbonic anhydrase (CA) (Gao & McKinley, 1994;Raven, 1997). In addition, HCOcould also be absorbed directly through an anion exchange protein (AE) in the plasmalemma in macroalgae such as

Ectocarpus siliculosus

,

Laminaria digitata

and

S. latissima

et al. (Klenell, Snoeijs, & Pedersén, 2004; Schmid, 1998), or with the help of acid zones produced by a P-type H-ATPase pump in

L

.

digitata

(Klenell, Snoeijs, & Pedersén, 2002) and

S

.

latissima

(Axelsson, Mercado,& Figueroa, 2000). Other than in the AE mechanism, the produced high concentration of COat the cell surface facilitated the diffusion of COinto cell to RuBisCo. In

S. japonica

sporophytes, 75% of the DIC acquisition was depended on CAmechanism (Yue, Wang, Wang, Zhou, &Zeng, 2001).

In addition to CAin periplasmic space, internal CAs (CA) also played an important role in the storage and transportation of inorganic carbon (Ci) in many algae. They have been localized at the mitochondria, chloroplast thylakoid, and cytoplasm in some algae (Hanson,Franklin, Samuelsson, & Badger, 2003; Moroney et al., 2011; Ye, Yu,Shi, Gao, Bi, & Zhou, 2014; Gee & Niyogi, 2017) and involved in several metabolic functions including regulation of pH, influence lipid biosynthesis, and participating photosynthesis and respiration (Ashok, Susanna, & Seppo, 2018). In addition, the physiological functions of CAin Ci transportation and concentration have also been studied extensively in photosynthetic organisms. They were proved to participate in concentrating COin plastids, and reducing the leakage of COfrom plastids (Hoang & Chapman, 2002). Thus, it has been well accepted that CAand CAwere crucial components of COconcentrating mechanism (CCM) in most algae.

In many large brown algae including

L.digitata

,

L. hyperborea

, and

L. saccharina

, CAand CAactivities had been detected successfully(Surif & Raven, 1989), and their activities were reported to be affected by several environmental factors (Ci, pH, blue light, N/P, and UV),especially by COand pH (Bi et al., 2019). In the sporophytes of

S. japonica

, there was also CAactivity outside the plasmalemma (Yue et al., 2001). In contrast, it was reported that there might be no CAin the gametophytes of this kelp (Yue, Ji, Wang, & Zhou, 2000). Recently, a series of CA genes including 4 CAand 7 CAgenes of

S. japonica

gametophytes were documented by Bi et al. (2019). These reports reminded us to clarify whether there was CAactivity in the gametophytes of

S. japonica

. In this paper, CAand CAactivities in gametophytes of

S. japonica

were assayed, and the effects of different Ci concentrations as well as pH values on the CA activity were studied. The results would be helpful for the further analysis of the mechanism of Ci acquisition and transportation in

S. japonica

gametophytes.

2.Materials and methods

2.1.Gametophytes of Saccharina japonica

The gametophytes of

S. japonica

were kept in our laboratory and cultured in PES medium according to the reported method (Zhou & Wu,1998). The culture conditions were as follows: temperature 17 ± 1 °C,light intensity 40 μmol photons/(m⋅s), and light time 16 h/d. White cold light source was provided by Philips straight fluorescent lamp. The medium was replaced every two weeks.

2.2.Experimental treatment and culture conditions

The gametophytes of 1.0 g (FW) were placed in 25 ml sterilized seawater and treated for 4 h with 1000 r/min magnetic stirrer to make the gametophytes be similar to unicellular algae in shape. The gametophytes were cultured for 24 h and then treated for 2 h under different culture conditions respectively. To access the effects of COon CA activity, the gametophytes were grown in PES medium gassed with the filtered air and 3% CO, respectively. NaHCOwas added to PES medium in a series of concentrations, i.e., 0, 84, 420, 840 and 1680 mg/L,in which the 0 group was the normal PES medium and used as control to access the effects of HCO. For pH treatment, PES medium regulated by HCl or NaOH solution were set at 6.0, 7.0 and 9.0, respectively. The normal PES medium (pH =8.50) was used as control. Each experimental condition was set in three parallel. Then, the activities of CAand CAwere assayed.

Total organic carbon analyzer (TOC-L) and pH electrode were used to determine the concentration of DIC and pH value in culture medium under different experimental conditions.

2.3.Assay of CAext activity in S. japonica gametophytes

The gametophytes of

S. japonica

were collected by using filtration method for CAactivity determination. The activity of CA was detected by Wilbur and Anderson (1948) methods. The collected gametophytes were suspended immediately in 5 ml barbiturate buffer solution with pH 8.4, and added 3 ml CO-saturated distilled water at 4 °C. The changes of pH in the reaction system were measured using a pH electrode. The time required for the decrease of pH by 0.4 units was recorded using a timer.The whole process was operated on ice. The equation for calculating the relative enzyme activity (REA) of CA is:

REA

=10 ×(

T

/

T

-1).

T

was the time required in the reaction system without algae cells, and

T

was that in the reaction system added sample. CA activity was standardized using the equation: REA/g FW =10 ×(

T

/

T

-1)/m, and m is the fresh weight of algae (g).

2.4.Assay of CAint activity in S. japonica gametophytes

After the activity of CAwas determined, each material was ground into powder in liquid nitrogen to break up the cells and used to assay the activity of total CA in gametophytes. The determination method is the same as 2.3. REA/g FW of CAwas determined using the following equations: REA=(REA- REA)/m, m is the weight of algae powder (g).

2.5.Statistical analysis

Three parallel samples were set up in the experiment, and the experimental data were reported as means ±S.D. analyzed using SPSS 20.0 software (Chicago, IL, USA). The data were analyzed by variance analysis using one-way ANOVA and multiple comparisons containing Tukey's test, with P

＜

0.05 as the significant difference.

3.Results and analysis

3.1.Effects of CO2 on CA activity of S. japonica gametophytes

The activities of CA, CAand total CA of

S. japonica

gametophytes under different COconcentrations were shown in Fig. 1. Under high-COcondition (3% CO), the CAactivity decreased significantly from 33.92 REA/g FW in the control group to 27.69 REA/g FW (P

＜

0.01), but the CAactivity and the total CA activity increased significantly from 36.83 REA/g FW to 48.80 REA/g FW (P

＜

0.05) and from 70.75 REA/g FW to 76.49 REA/g FW (P

＜

0.01).

Fig. 1.Effect of CO2 concentration on the activity of CAs in S. japonica gametophytes and the DIC concentration in each condition.

After being bubbled with 3% CO, the concentration of DIC in the medium was elevated to 47.31 mg/L, which was nearly twice that in the control group (24.38 mg/L) (Fig. 1), and the pH value decreased from 8.5 in the control group to 6.18 (Fig. 2).

Fig. 2.pH values in two CO2 treatments.

3.2.Effects of HCO−3 on CA activity of S. japonica gametophytes

NaHCOwas added in PES medium in a series of concentrations of 0,84, 420, 840 and 1680 mg/L. The CA, CAand total CA activities of each sample were shown in Fig. 3. With increased HCOconcentration,the CAactivity decreased significantly. At 84 mg/L HCO, the CAactivity was 25.03 REA/g FW, which was significantly lower than the control value of 33.92 REA/g FW (P

＜

0.05). At 420, 840, and 1680 mg/L HCO, the CAactivity decreased, resulting in a minimum CAvalue at 1680 mg/L HCO. This value was 6.96 REA/g FW, which was significantly lower than the control value, but not significantly different from the value at 840 mg/L HCO(P

＞

0.05).

Fig. 3.Effect of HCO−3 on the activity of CAs in S. japonica gametophytes and the DIC concentrations in five HCO−3 treatments.

The activities of the CAand total CA decreased first and then increased with the elevation of HCOin medium. Addition of 84 mg/L of HCO, the activities of the CAand total CA significantly decreased from 36.83 REA/g FW and 70.75 REA/g FW in control group to 26.17 REA/g FW and 51.20 REA/g FW (P

＜

0.05). At 420 mg/L of HCO, the two values were decreased to 32.85 REA/g FW and 44.30 REA/g FW,respectively. But, when the concentration of HCOwas elevated to 1680 mg/L, the CAactivity increased to 60.81 REA/g FW, nearly twice as much as that of the control group. Furthermore, the total CA activity increased to 67.77 REA/g FW, which was not significantly different from that of the control (P

＞

0.05).

When the medium was treated with the four concentrations of NaHCO, DIC concentration gradually increased from 24.38 mg/L in the control group to 202.93 mg/L at 1680 mg/L HCO(Fig. 3). And pH value decreased gradually from 8.50 in the control group to 7.66 (1680 mg/L of HCO) (Fig. 4).

Fig. 4.pH values of PES medium in five HCO−3 treatments.

3.3.Effects of pH on CA activity of S. japonica gametophytes

The activities of the CA, CAand total CA of

S. japonica

gametophytes under different pH conditions were shown in Fig. 5. The CAactivity of

S. japonica

gametophytes in the control group (pH =8.5) was the highest (33.92 REA/g FW). Both increasing and decreasing the pH value of the medium resulted in a significant decrease in CAactivity(P

＜

0.05).

Fig. 5.Effect of pH on the activity of CAs in S. japonica gametophytes and the DIC concentrations in four pH treatments.

In contrast, the CAand total CA activities increased with the decrease of pH value (P

＜

0.05), and resulting the maximum CA values at pH 6.0. These values were 110.85 REA/g FW and 128.17 REA/g FW respectively, both of which were significantly higher than the control values (P

＜

0.05). When pH was elevated to 9.0, both CAactivity and total CA activity were decreased to the lowest values, which were 23.31 REA/g FW and 42.19 REA/g FW, respectively.

The concentration of DIC in different pH conditions are shown in Fig. 5. In the control medium at pH 8.5, the concentration of DIC reached to the highest value of 24.38 mg/L. Decreasing or increasing the pH value of the medium resulted in the decrease of the concentration of DIC. At pH 6.0, the DIC concentration decreased to a minimum value of 10.93 mg/L.

4.Discussion

4.1.The activity of CAext in gametophytes of S. japonica and its regulation

In this study, the activity of CAwas detected successfully in gametophytes of

S. japonica,

and it was found that the CAactivity was regulated by the concentrations of external DIC, including COand HCO, as well as pH value. This is consistent with the report that four CAs of the gametophytes of

S. japonica

were predicted to be located in the secreted pathway (Bi et al., 2019). Considering that

S. japonica

gametophytes could not absorb HCOas carbon source for photosynthesis,CAhere might mainly play a role in regulation of pH on the surface of gametophytes.Unlike in some other brown algae, CAof the gametophytes of

S. japonica

might promote a transient acidification on the surface of the cell, which was documented to occur during photosynthesis in the Phaeophyceae (Raven & Giordano, 2017). The formed acid zones on the surface would facilitate the diffusion of COinto cell (Price & Badger,1985; Raven & Giordano, 2017).CA is an inducible enzyme. When COconcentration in culture medium could support photosynthesis, the CAactivity was often been inhibited (Xia & Gao, 2005). Therefore, in this study, the activity of CAdecreased significantly under high CO, high HCO(lower pH)and low pH conditions. In

C. reinhardtii,

the decrease of CAactivity under high COconcentration might be caused by the down-regulation of the expression of CAgene, such as

cah

1 (Fukuzawa, Fujiwara,Yamamoto, Dionisio-Sese, & Miyachi, 1990). Whether the regulation of CAactivity in gametophytes of

S. japonica

also occurs on the molecular level needs to be furtherly verified.

4.2.The activity of CAint in gametophytes of S. japonica and its regulation

Based on the available reports, CAwas mainly located in cytosol,chloroplast, mitochondria and endoplasmic reticulum in algae. For example, CAH3 of

C. rheinensis

was localized in thylakoid lumen(Karlsson et al., 1998; Mitra et al., 2005), CAH4 and CAH5 were localized in mitochondria (Eriksson, Karlsson, Ramazanov, Gardeström, &Samuelsson, 1996), CAH6 was in flagella (Mackinder et al., 2017), and CAH9 was in cytoplasm (Mackinder et al., 2017). In macroalgae, activities of CAs in plasma membrane and chloroplast membrane were detected in

Gracilaria tenuistipitata

(Haglund, Björk, Ramazanov, García-Reina, & Pedersén, 1992),

Porphyra leucosticte

(Mercado, Viñegla,Figueroa, & Niell, 1999),

Ulva intestinalis

(Andría, Vergara, &Pérez-Lloréns, 2000) and

Soliera filiformis

(Gómez-Pinchetti, Ramazanov, & Garcia-Reina, 1992). In

S. japonica

, one CA had been localized in chloroplast (Ye et al., 2014) and one other CA was predicted to be localized in the chloroplast, 4 might be in the mitochondria, and 1 might be in the stroma (Bi et al., 2019). These reports were consistent with the result that CAactivity was successfully detected in the gametophytes of

S. japonica

in this study.On the basis of the subcellular localization and the expression characteristics of the coding genes of CA, it was believed that they played an important role in the process of providing enough substrate of COfor photosynthetic carbon reduction (PRC) of RuBisCo. The increase of CAand total CA activity implied the increase of photosynthetic rate,as well as the biomass of algae (Zhou et al., 2016). Many reports proved the positive correlation between CAactivity and biomass of algae. For example, in COsaturated media, both the activity of CAand biomass of

Platymonas subcordiformis

increased significantly (Liu, Mo, & Yang,2017). Under COenrichment, the activities of CA, photosynthetic rate,and photosynthetic carbon sequestration rate of three marine microalgae,

Skeletonema costatum

,

Heterosigam akashiwo

and

Chlorella autotropica

, increased significantly. And it was speculated that the enriched COpromoted the activities of CA of the three marine microalgae,thereby increasing their photosynthetic rate and photosynthetic carbon sequestration rate (Xu et al., 2010). Zhou et al. (2016) found that proper addition of bicarbonate could improve CA activity, photosynthetic pigments synthesis and photosynthesis of macroalgae, thereby promoting their growth. After adding bicarbonate to medium for 3 days, both CA activity and relative growth rate of

Cladophora oligoclora

Kütz increased significantly (Ma et al., 2018). In this study, the results that CAactivity was enhanced under conditions of increased COand HCO, as well as decreased pH value suggested that photosynthetic rate, and the biomass of

S. japonica

gametophytes would be promoted under these conditions.

5.Conclusion

In this study, CA activities were successfully detected both outside and inside the plasmalemma of

S. japonica

gametophytes. Under conditions of increasing the dissolved CO, HCOand decreasing the pH value resulted in the reduction of CAactivity, but the enhancement of the CAactivity in gametophytes of

S. japonica

. Based on the results above and the report that

S. japonica

could not absorb HCOfrom the seawater, it was speculated that CAmight play a role in making acid zones on the surface of gametophytes to facilitate the diffusion of COinto cell. Generally, the increase of CA activity implied the elevation of photosynthetic rate, as well as the biomass of algae. At pH 6.0, CA activity of gametophytes reached to a maximum of 128.17 REA/g FW,suggesting that compared with the addition of DIC, lowering pH value of medium were more beneficial to photosynthesis of

S. japonica

gametophytes. Simultaneously, it also implied that 10.93 mg/L of DIC in the medium at pH 6.0 might be enough to meet the Ci demand for the growth of gametophytes of

S. japonica

.

CRediT authorship contribution statement

Yan-Hui Bi: Conceptualization, Methodology, Writing - review &editing. Cui-Ling Liang: Investigation, Writing - original draft. Jia-Li Li:Data curation. Hao Yin: Investigation. Ruo-Tong Tian: Investigation.Zhi-Gang Zhou: Supervision.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgments

This research was supported by the National Key R & D Program of China (Grant No. 2018YFD0901500), the National Natural Science Foundation of China (Grant No. 41376136) and the Double First-Class Discipline of Fisheries Science of China.