Periplasmic carbonic anhydrase CAH1 contributes to high inorganic carbon affinity in Chlamydomonas reinhardtii.

Carbonic anhydrase (CA), an enzyme conserved across species, is pivotal in the interconversion of inorganic carbon (Ci; CO2 and HCO3-). Compared to the well-studied intracellular CA, the specific role of extracellular CA in photosynthetic organisms is still not well understood. In the green alga Chlamydomonas (Chlamydomonas reinhardtii), carbonic anhydrase 1 (CAH1), located at the periplasmic space, is strongly induced under CO2-limiting conditions by the Myb transcription factor LCR1. While the lcr1 mutant shows decreased Ci-affinity, the detailed mechanisms behind this phenomenon are yet to be elucidated. In this study, we aimed to unravel the LCR1-dependent genes essential for maintaining high Ci-affinity. To achieve this, we identified a total of 12 LCR1-dependent inducible genes under CO2-limiting conditions, focusing specifically on the most prominent ones - CAH1, LCI1, LCI6, and Cre10.g426800. We then created mutants of these genes using the CRISPR-Cas9 system, all from the same parental strain, and compared their Ci-affinity. Contrary to earlier findings that reported no reduction in Ci-affinity in the cah1 mutant, our cah1-1 mutant exhibited a decrease in Ci-affinity under high HCO3-/CO2-ratio conditions. Additionally, when we treated wild-type cells with a CA inhibitor with low membrane permeability, a similar reduction in Ci-affinity was observed. Moreover, the addition of exogenous CA to the cah1 mutant rescued the decreased Ci-affinity. These results, highlighting the crucial function of the periplasmic CAH1 in maintaining high Ci-affinity in Chlamydomonas cells, provide insights into the functions of periplasmic CA in algal carbon assimilation.

CO2-dependent migration and relocation of LCIB, a pyrenoid-peripheral protein in Chlamydomonas reinhardtii.

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO2-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO2-limiting conditions (low-CO2 [LC] and very low-CO2 [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO2-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3-15 µM CO2, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO2. Furthermore, LCIB relocated toward the pyrenoid at 2.6-3.4 µM CO2, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO2. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO2 concentration with ∼7 µM as the boundary.

A pyrenoid-localized protein SAGA1 is necessary for Ca2+-binding protein CAS-dependent expression of nuclear genes encoding inorganic carbon transporters in Chlamydomonas reinhardtii.

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic affinity for dissolved inorganic carbon (Ci) under CO2-limiting conditions. In the model alga Chlamydomonas reinhardtii, the pyrenoid-localized Ca2+-binding protein CAS is required to express genes encoding the Ci-transporters, high-light activated 3 (HLA3), and low-CO2-inducible protein A (LCIA). To identify new factors related to the regulation or components of the CCM, we isolated CO2-requiring mutants KO-60 and KO-62. These mutants had insertions of a hygromycin-resistant cartridge in the StArch Granules Abnormal 1 (SAGA1) gene, which is necessary to maintain the number of pyrenoids and the structure of pyrenoid tubules in the chloroplast. In both KO-60 and the previously identified saga1 mutant, expression levels of 532 genes were significantly reduced. Among them, 10 CAS-dependent genes, including HLA3 and LCIA, were not expressed in the saga1 mutants. While CAS was expressed normally at the protein levels, the localization of CAS was dispersed through the chloroplast rather than in the pyrenoid, even under CO2-limiting conditions. These results suggest that SAGA1 is necessary not only for maintenance of the pyrenoid structure but also for regulation of the nuclear genes encoding Ci-transporters through CAS-dependent retrograde signaling under CO2-limiting stress.