The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3-, the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed. © 2014 Springer Science+Business Media Dordrecht

Carbon fixation in diatoms

Diatoms are unicellular photoautotrophic algae and very successful primary producers in the oceans. Their high primary productivity is probably sustained by their high adaptability and a uniquely arranged metabolism. Diatom belongs to the Chromista, a large eukaryotic group, which has evolved by multiple endosymbiotic steps. As a result, diatoms possess a plastids with four membranes together with complicated translocation systems to transport proteins and metabolites including inorganic substances into and out of the plastids. In addition to the occurrence of potential plasma-membrane transporters, there are numerous carbonic anhydrases (CAs) within the matrix of the layered plastidic membranes, strongly suggesting large interconversion activity between CO2 and HCO3 − within the chloroplast envelope as a part of a CO2-concentrating mechanism (CCM). In diatoms also the Calvin cycle and its adjacent metabolism reveal unique characteristics as, for instance, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activase, the plastidic sedoheptulose-1,7-bisphosphatase (SBPase), and the plastidic oxidative pentose phosphate pathway (OPP) are absent. Furthermore, the Calvin cycle metabolism in diatoms is not under the strict redox control by the thioredoxin (Trx) system. Instead, a CO2-supplying system in the pyrenoid shows CA activities which are probably regulated by chloroplastic Trxs. Pyrenoidal CAs are also regulated on the transcriptional level by CO2 concentrations via cAMP as a second messenger, suggesting an intense control system of CO2 acquisition in response to CO2 availability. The photorespiratory carbon oxidation cycle (PCOC) is the major pathway to recycle phosphoglycolate in diatoms although this process might not be involved in recycling of 3-phosphoglycerate but instead produces glycine and serine. In this review we focus on recent experimental data together with supportive genome information of CO2 acquisition and fixation systems primarily in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana