Presentation Information
[P04-580]The function of Tpθ-CA2 in the thylakoid lumen of the marine diatom Thalassiosira pseudonana
○Yuzu Sakaura1, Hermanus Nawaly1, Yusuke Matsuda1 (1. Grad. Sch. Tech., Kwansei Gakuin Univ. (Japan))
Keywords:
Diatom,CCM,Pyrenoid,Thylakoid
[Purpose]
In seawater under present atmosphere, the dissolved CO2 concentration is low and its diffusion is extremely slow, making it far insufficient to saturate Rubisco carboxylation. However, seawater contains abundant HCO3- because of its high salinity and alkalinity. Marine diatoms possess a CO2-concentrating mechanism (CCM) that actively transports dissolved inorganic carbon (DIC) into the chloroplast. There is a Rubisco condensate body called pyrenoid at the center of the diatom chloroplast and the Pyrenoid-penetrating thylakoid (PPT) membranes traverses across the axis of the pyrenoid. The PPT lumen is a machinery to evolve high concentration of CO2 from accumulated HCO3-, and the marine diatom, Thalassiosira pseudonana, possesses θ-type carbonic anhydrase 2 (Tpθ-CA2) in the PPT lumen. Tpθ-CA2 is supposed to be a critical component of the CO2-evolving machinery of T. pseudonana but it is still wait to be proven. We generated Tpθ-CA2 knockout strains and, the present study aims to characterize the phenotype of the ΔTpθ-CA2 strain and to generate a complementation strain to observe the phenotype recovery.
[Method]
Growth rates of diatom cells were measured by the optical density at 730 nm, and photosynthetic parameters were determined using an oxygen electrode. Relative accumulations of CCM- and photosynthesis-related proteins were quantified by western blotting with β-actin as a loading control. For complementation, a Tpθ-CA2::GFP expression cassette driven by the nitrate reductase promoter was introduced into the knockout strain by bacterial conjugation, and the GFP fluorescence was observed by confocal microscopy.
[Results]
Under atmospheric CO2 (LC), the knockout strain showed 40% reduced growth compared with the wild type, while no difference appeared 1% CO2 (HC). Pmax decreased to 82% of WT and K1/2[DIC] increased 502 times in the LC-grown KO strain, indicating a severe impairment of DIC affinity. Under HC, Pmax was unchanged, but K1/2[DIC] increased 87 times. PyShell1/2 accumulated slightly higher (1.2-1.5 times) relative to that of WT, whereas RbcL remained stable. Cryo ET observation clearly showed the mislocalization of PPT and PyShell structure in the pyrenoid, indicating the critical function of Tpθ-CA2 not only in the CCM but also in the pyrenoid formation. This intense phenotype of KO strain was restored by complementation of Tpθ-CA2::GFP.
[Discussion and Conclusion]
The strong increase in K1/2[DIC] demonstrates that Tpθ-CA2 is essential for maintaining high local CO2 concentrations around Rubisco, which is essential to maintain CO2 supply to Rubisco both under LC and HC. Structural defects of the pyrenoid by the Tpθ-CA2 KO suggest the importance of Tpθ-CA2 in proper pyrenoid assembly and PPT formation.
In seawater under present atmosphere, the dissolved CO2 concentration is low and its diffusion is extremely slow, making it far insufficient to saturate Rubisco carboxylation. However, seawater contains abundant HCO3- because of its high salinity and alkalinity. Marine diatoms possess a CO2-concentrating mechanism (CCM) that actively transports dissolved inorganic carbon (DIC) into the chloroplast. There is a Rubisco condensate body called pyrenoid at the center of the diatom chloroplast and the Pyrenoid-penetrating thylakoid (PPT) membranes traverses across the axis of the pyrenoid. The PPT lumen is a machinery to evolve high concentration of CO2 from accumulated HCO3-, and the marine diatom, Thalassiosira pseudonana, possesses θ-type carbonic anhydrase 2 (Tpθ-CA2) in the PPT lumen. Tpθ-CA2 is supposed to be a critical component of the CO2-evolving machinery of T. pseudonana but it is still wait to be proven. We generated Tpθ-CA2 knockout strains and, the present study aims to characterize the phenotype of the ΔTpθ-CA2 strain and to generate a complementation strain to observe the phenotype recovery.
[Method]
Growth rates of diatom cells were measured by the optical density at 730 nm, and photosynthetic parameters were determined using an oxygen electrode. Relative accumulations of CCM- and photosynthesis-related proteins were quantified by western blotting with β-actin as a loading control. For complementation, a Tpθ-CA2::GFP expression cassette driven by the nitrate reductase promoter was introduced into the knockout strain by bacterial conjugation, and the GFP fluorescence was observed by confocal microscopy.
[Results]
Under atmospheric CO2 (LC), the knockout strain showed 40% reduced growth compared with the wild type, while no difference appeared 1% CO2 (HC). Pmax decreased to 82% of WT and K1/2[DIC] increased 502 times in the LC-grown KO strain, indicating a severe impairment of DIC affinity. Under HC, Pmax was unchanged, but K1/2[DIC] increased 87 times. PyShell1/2 accumulated slightly higher (1.2-1.5 times) relative to that of WT, whereas RbcL remained stable. Cryo ET observation clearly showed the mislocalization of PPT and PyShell structure in the pyrenoid, indicating the critical function of Tpθ-CA2 not only in the CCM but also in the pyrenoid formation. This intense phenotype of KO strain was restored by complementation of Tpθ-CA2::GFP.
[Discussion and Conclusion]
The strong increase in K1/2[DIC] demonstrates that Tpθ-CA2 is essential for maintaining high local CO2 concentrations around Rubisco, which is essential to maintain CO2 supply to Rubisco both under LC and HC. Structural defects of the pyrenoid by the Tpθ-CA2 KO suggest the importance of Tpθ-CA2 in proper pyrenoid assembly and PPT formation.
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