Xiaozhuo Wang

The physiological role of chloroplast carbonic anhydrases (CAs) has long been debated, particularly in the context of photosynthesis. While early hypotheses proposed that CAs enhance CO2 assimilation by rapidly accessing the HCO3− pool, direct evidence has been lacking. In this study, we examined Arabidopsis (Arabidopsis thaliana) mutants lacking both chloroplast-localized βCA1 (AT3G01500) and βCA5 (AT4G33580) to assess their impact on plant growth and photosynthetic performance. Our results show that plants deficient in chloroplast CA activity are unable to grow under ambient CO2 conditions (400 μL L⁻1) but can complete their life cycle under elevated CO2 levels (≥12,000 μL L⁻1). However, CO2 assimilation rates and ΦII measurements in CA-deficient plants were comparable to those in wild-type plants under 0.04% (400 μL L⁻1), 0.4% (4,000 μL L⁻1), and 4% CO2 (40,000 μL L⁻1) concentrations, indicating that chloroplast CAs are not essential for photosynthetic CO2 fixation. Instead, our findings suggest that chloroplast CA activity is critical for supporting other metabolic pathways, namely amino acid, nucleic acid, and fatty acid biosynthesis. Expression of the Chlamydomonas (Chlamydomonas reinhardtii) bicarbonate transporter LCIA in chloroplast CA mutants partially rescued the growth phenotype under near-ambient CO2 conditions. These LCIA-complemented lines showed no difference in photosynthesis, further supporting the role of CAs in non-photosynthetic reactions. This work provides direct evidence that while chloroplast CAs are dispensable for photosynthesis, they are essential for plant growth and development under ambient CO2 due to their role in increasing the bicarbonate concentration for specific anaplerotic pathways.

Translation of psbA, the chloroplast gene that encodes the D1 subunit of PSII, is important for both PSII biogenesis and repair. The translation of psbA transcripts in the chloroplast is under the control of nuclear gene products. Using a forward genetic screen and whole-genome sequencing of the alga Chlamydomonas reinhardtii, we found a mutant defective in PSII activity and mapped the causative gene to be the homolog of Arabidopsis HIGH CHLOROPHYLL FLUORESCENCE 244 (HCF244), namely CrHCF244. We then demonstrated that CrHCF244 is required for psbA translation in the alga, consistent with the function of HCF244 in Arabidopsis, and found that AtHCF244 also partially complemented the algal mutant. These results experimentally support the functional conservation of the homologs in green algae and land plants. Intriguingly, the CrHCF244 mutant also exhibited a relatively high rate of suppressor mutants, pointing to the presence of alternative factor(s)/pathway(s) for D1 translational control. The establishment of CrHCF244 as a psbA translation factor in C. reinhardtii shows the similarities in psbA translation regulation in algae and plants. The future identification of the alternative factor(s) in this alga will provide insights on psbA translation in plants.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO₂-concentrating mechanism. BCT1 could enhance photosynthetic CO₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO₂-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO₂-concentrating mechanism components to improve plant photosynthesis.

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacteria CO ₂ concentrating mechanism, could enhance photosynthetic CO ₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO ₂ -dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana , but the transformed plants did not have higher carbon assimilation rates or decreased CO ₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO ₂ concentrating mechanism components to improve plant photosynthesis.