| Summary: | Despite accounting for approximately half of global primary productivity, photosynthesis in aquatic environments is often limited by the availability of dissolved carbon dioxide (CO$_{2(aq)}$). To overcome the slow diffusion of CO$_{2(aq)}$ as well as kinetic limitations of the primary photosynthetic carboxylase, Rubisco, carbon concentrating mechanisms (CCMs) have evolved in many aquatic photosynthetic organisms to improve photosynthetic efficiency and growth in CO2- limited environments. In the model eukaryotic green alga Chlamydomonas reinhardtii, the CCM is induced under low CO2 in the light and comprises: active inorganic carbon transport systems, carbonic anhydrases and the localisation of Rubisco to a central chloroplast microcompartment called the pyrenoid. In addition to changes in gene expression, acclimation to low CO2 is accompanied by alterations to metabolism, physiology and even cellular ultrastructure. However, mechanisms governing the regulation and interaction of these molecular components to increase CCM activity remain poorly understood. The overall aim of this study was to investigate regulation of the CCM in wild-type and mutant Chlamydomonas strains at both the whole cell and molecular level. Firstly, investigation of CCM induction in synchronised cultures of wild-type cells identified changes in CCM activity that were uncoupled from accumulation of CCM-related mRNA and protein, contrasting with the coordinated response to low CO2 observed in asynchronous control cultures. Pre-dawn induction of the CCM was coincident with preferential localisation of Rubisco and a thylakoid-lumenal carbonic anhydrase (CAH3) to the pyrenoid, highlighting the possible role of endogenous signals and post-translational modifications in modulating CCM activity. Secondly, in order to probe the relationship between pyrenoid formation and CCM induction and activity, CCM expression was investigated in pyrenoid-negative mutants with substituted Rubisco small subunits (RBCS). Low CO2-adapted pyrenoid-less RBCS mutants had impaired growth and low photosynthetic affinity for inorganic carbon (Ci). These pyrenoid-negative strains also showed a specific reduction in the accumulation of several CCM mRNAs, compared to pyrenoid- positive wild-type. Two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare the soluble proteome of one low CO2-adapted pyrenoid-less RBCS mutant compared to the pyrenoid-positive wild-type. This analysis identified only a few differentially expressed proteins, none of which were directly involved in CCM activity. Two primary metabolic enzymes were more abundant in the wild- type while eight proteins associated with protein synthesis and photosynthesis were more abundant in the pyrenoid-less mutant, suggesting that pyrenoid loss is accompanied by global metabolic, as well as CCM-specific, changes. A shotgun proteomics approach (LC-MS/MS) was used to extend the analysis of the pyrenoid-less RBCS mutant proteome to the whole genome level. Approximately 10% of the total proteins detected using this method were identified as differentially expressed between pyrenoid-negative and pyrenoid-positive strains. Increased abundance of photosynthetic proteins was found in the pyrenoid-less RBCS mutant, confirming the results of 2D-DIGE. In contrast, increased accumulation of CCM and primary metabolic enzymes was detected in the pyrenoid-positive wild-type. Overall, detailed investigation of the phenotype of pyrenoid-negative RBCS mutants indicates that pyrenoid loss leads to impaired induction of the CCM as well as altered metabolism under low CO2 conditions, perhaps as a result of decreased carbon fixation. The results of these studies are explored in the context of the identification of additional CCM components and regulatory mechanisms as well as possible connections between Rubisco aggregation and CCM activity.
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