The role of two putative Ca2+ transporters in the Chlamydomonas reinhardtii CO2-concentrating mechanism

There is an urgent need to double global crop production by 2050 to meet the demands of population growth, diet changes and biofuel production. A promising solution is to increase crop yields by enhancing plant photosynthesis. One approach is to engineer a CO2-concentrating mechanism (CCM) into C3 crops to increase photosynthetic carbon fixation efficiency. The CCM of the green alga Chlamydomonas reinhardtii is a good candidate as it shares close phylogeny and similar photosynthetic traits with higher plants. The CCM is only induced under low CO2 conditions, but it is unknown how the alga senses CO2 changes to regulate this process. To dissect the CO2-sensing pathway in the CCM, this study set out to identify candidate CO2 sensors in Chlamydomonas by adopting a target reverse genetic screen. A list of candidate genes were identified as homologues of characterised CO2-sensors in other species and their Chlamydomonas mutants were screened in an autotrophic growth assay. The mutants of two genes, CGLD1 and CPLD63, were found to have a disturbed CCM and photosynthesis respectively, and failed to correctly transcriptionally regulate known CCM genes like the HCO3- transporter LCIA. Bioinformatics study showed that they belong to the UPF0016 family and contain two highly conserved motifs that are important for Ca2+ transport in their yeast homologue Gdt1p. Fluorescence protein tagging also revealed that CGLD1 localises to the thylakoid membrane while CPLD63 concentrates at the chloroplast envelope. Meanwhile, growth assays in different Ca2+ concentrations showed that CGLD1 is important for the limiting-CO2 growth of Chlamydomonas in low Ca2+. As Ca2+ elevation in the algal pyrenoid is important for the CCM, data in this study indicates that CGLD1 and CPLD63 are putative Ca2+ transporters with potential roles in regulating the Chlamydomonas CCM via a Ca2+-induced retrograde signal from the chloroplast to the nucleus.