1. CO

1 CO

₂ Affinity of Photosynthesis versus Rubisco

affinity of photosynthesis versus Rubisco

Rubisco’s low affinity for external CO

drove the evolution of CCMs. Therefore, one of the most useful indicators of whether a photosynthetic organism has a CCM is to compare the affinity of photosynthesis for external CO

with the affinity of Rubisco for CO

. This approach was originally applied to higher plants, and by measuring the affinity K

(Rubisco CO

) of Rubisco extracted from photosynthetic organisms to substrate CO

, it was inferred that C

species might require a CCM [69]. However, due to the limitation of extraction technology, the affinity determination of photosynthetic organism Rubisco lacked accuracy. With the improvement of extraction and analysis techniques, this method has become widely used to evaluate algal CCMs. In general, the researchers measured the photosynthetic oxygen evolution rate under different inorganic carbon concentrations, and used the data analysis software to fit the inorganic carbon response curve (P-C curve) by using Michaelis-Menten equation, where K

is the substrate concentration (DIC concentration) when the photosynthetic rate is half of the maximum, from which we can infer the affinity of algae photosynthesis to CO

K

(photosynthetic CO

). Since marine algae may experience more persistent CO

limitations than fresh- and brackish-water autotrophs, it can be assumed that the type of water environment determines the effectiveness of CCM. Comparing the in vivo photosynthesis and the in vitro carboxylation of Rubisco under ambient oxygen levels can show its ability to concentrate CO

exceeds the level of environmental CO

[70]. For example, C. reinhardtii’s ratio of K

(photosynthetic CO

) to K

(Rubisco CO

) is approximately 1:30, and that of Ulva sp. is about 5–10:68 [1,71,72], both apparently rely on CCM to improve the efficiency of Rubisco, whereas other algae with close specific gravity may not use CCM.

2. Effects of CA Inhibitors

2 Effects of CA inhibitors

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

and CO

, but cannot affect the CO

equilibrium concentration and CO

mainly enters the cell through passive diffusion. This indicates that HCO

-utilization in this manner does not function well at high pH (pH > 9.4) because the equilibrium concentration of CO

is low. It is obvious that in addition to the extracellular CA catalytic utilization of HCO

, there must be another means of using HCO

in U. prolifera.

图Figure 3.CA抑制剂作用示意图。inCA 表示细胞内 CA，eCA 表示细胞外 CA，当 CA 图标为灰色时，CA 被抑制。

Schematic diagram of CA inhibitor actions. inCA means intracellular CA, eCA means extracellular CA, and when the CA icon is gray, CA is suppressed.

3. 使用HCO

3 Using HCO

₃⁻作为光合底物

as photosynthetic substrate

The function of HCO

in photosynthesis has been perceived as a property of algae [75]. The utilization of HCO

by algae depends on the involvement of external CA and plasma membrane transporters [76-78]. The use of HCO

is strongly correlated with the presence of a CCM. However, some macroalgae are not only capable of utilizing HCO

as a carbon source, some have demonstrated the use of CO

in specific situations. Through the detection of δ

C isotopes in Gracilaria and Ulva, it was found that when the concentration of environmental CO

was high, there was a physiological transition from using HCO

almost exclusively to predominantly using CO

. At the current seawater pCO

, many macroalgae use HCO

rather than dissolved CO

, and utilize CA to convert HCO

to CO

for use by Rubisco [79-82]. For example, Mercado et al. [83] found that under the current seawater CO

level, the chlorophyll plants U. rigida and U. compressa cannot obtain sufficient CO

through diffusion absorption alone; therefore, a CCM must be used to obtain HCO

. However, macroalgae may downregulate their CCM, reduce HCO

use, and become dependent on CO

as the main carbon source when CO

concentrations are high [1,84-86]. Consequently, when using these indicators to evaluate the CCM of Ulva sp., there may be a decrease in the use of HCO

in certain circumstances, where there is still CCM activity but with a certain degree of downregulation.

4. 与外部Ci的亲和力的变化取决于生长Ci条件

4 Changes in affinity to external Ci depends on growth Ci conditions

When there is Ci limitation in the external environment, the affinity of the Ci transporter for external CO

and HCO

^- increases, and the intracellular and extracellular CA activity also increases. The two work together to increase the affinity of microalgae for external Ci by more than 10 times [76]. This inducible change appears to be strong evidence that cellular infrastructure is involved in supplying CO

to Rubisco in chloroplasts These inducible CCM are not limited to microalgae but have been observed in many macroalgae as well, including Ulva [1], Gracilaria [87], Porphyra [88] and Fucus [89]. That is, some algae may have an inducible CCM to meet their environmental needs when facing external Ci periodic constraints, while other algae may not have such a flexible arrangement.