Many eukaryotic photosynthetic organisms enhance their carbon uptake by supplying concentrated CO2 to the CO2-fixing enzyme Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer this pyrenoid-based CO2-concentrating mechanism (PCCM) into crops to increase yields. Here we develop a computational model for a PCCM on the basis of the postulated mechanism in the green alga Chlamydomonas reinhardtii. Our model recapitulates all Chlamydomonas PCCM-deficient mutant phenotypes and yields general biophysical principles underlying the PCCM. We show that an effective and energetically efficient PCCM requires a physical barrier to reduce pyrenoid CO2 leakage, as well as proper enzyme localization to reduce futile cycling between CO2 and HCO3−. Importantly, our model demonstrates the feasibility of a purely passive CO2 uptake strategy at air-level CO2, while active HCO3− uptake proves advantageous at lower CO2 levels. We propose a four-step engineering path to increase the rate of CO2 fixation in the plant chloroplast up to threefold at a theoretical cost of only 1.3 ATP per CO2 fixed, thereby offering a framework to guide the engineering of a PCCM into land plants.
Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,032 candidate chloroplast proteins by using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of hundreds of poorly-characterized proteins, including identifying novel components of nucleoids, plastoglobules, and the pyrenoid. We discovered and further characterized novel organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and diverse unexpected spatial distributions of enzymes within the chloroplast. We observed widespread protein targeting to multiple organelles, identifying proteins that likely function in multiple compartments. We also used machine learning to predict the localizations of all Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to enable studies of chloroplast architecture and functions.
Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (FDR<0.11) 70 previously-uncharacterized genes required for photosynthesis. We then provide a resource of mutant proteomes that enables functional characterization of these novel genes by revealing their relationship to known genes. The data allow assignment of 34 novel genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Additional analysis uncovers at least seven novel critical regulatory proteins, including five Photosystem I mRNA maturation factors and two master regulators: MTF1, which impacts chloroplast gene expression directly; and PMR1, which impacts expression via nuclear-expressed factors. Our work provides a rich resource identifying novel regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis.
Many photosynthetic organisms enhance the performance of their CO2-fixing enzyme Rubisco by operating a CO2-concentrating mechanism (CCM). Most CCMs in eukaryotic algae supply concentrated CO2 to Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer an algal CCM into crops that lack a CCM to increase yields. To advance our basic understanding of the algal CCM, we develop a chloroplast-scale reaction-diffusion model to analyze the efficacy and the energy efficiency of the CCM in the green alga Chlamydomonas reinhardtii. We show that achieving an effective and energetically efficient CCM requires a physical barrier such as thylakoid stacks or a starch sheath to reduce CO2 leakage out of the pyrenoid matrix. Our model provides insights into the relative performance of two distinct inorganic carbon uptake strategies: at air-level CO2, a CCM can operate effectively by taking up passively diffusing external CO2 and catalyzing its conversion to HCO3-, which is then trapped in the chloroplast; however, at lower external CO2 levels, effective CO2 concentration requires active import of HCO3-. We also find that proper localization of carbonic anhydrases can reduce futile carbon cycling between CO2 and HCO3-, thus enhancing CCM performance. We propose a four-step engineering path that increases predicted CO2 saturation of Rubisco up to seven-fold at a theoretical cost of only 1.5 ATP per CO2 fixed. Our system-level analysis establishes biophysical principles underlying the CCM that are broadly applicable to other algae and provides a framework to guide efforts to engineer an algal CCM into land plants.
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