Calcium (Ca 2+)-dependent signalling plays a well-characterized role in the response to different environmental stimuli, both in plant and animal cells. In the model organism for green algae, Chlamydomonas reinhardtii, Ca 2+ signals were reported having a crucial role in different physiological processes, like stress responses, photosynthesis, and flagella functions. Recent reports identified the underlying components of the Ca 2+ signalling machinery at the level of specific subcellular compartments and reported in vivo imaging of cytosolic Ca 2+ concentration in response to environmental stimuli. The characterization of these Ca 2+-related mechanisms and proteins in C. reinhardtii is providing knowledge on how microalgae can perceive and respond to the environmental stimuli, but also on how this Ca 2+ signalling machinery has evolved. Here, we review the current knowledge on the cellular mechanisms underlying the generation, shaping, and decoding of Ca 2+ signals in C. reinhardtii, providing an overview of the known and possible molecular players involved in the Ca 2+ signalling of its different subcellular compartments. The advanced toolkits recently developed to measure time-resolved Ca 2+ signalling in living C. reinhardtii cells are also discussed, suggesting how they can improve the study of the role of Ca 2+ signals in microalgae cellular response to the environmental stimuli.
Microalgae are unicellular photosynthetic organisms that can be grown in artificial systems to capture CO2, release oxygen, use nitrogen- and phosphorus-rich wastes, and produce biomass and bioproducts of interest including edible biomass for space exploration. In the present study, we report a metabolic engineering strategy for the green alga Chlamydomonas reinhardtii to produce high-value proteins for nutritional purposes. Chlamydomonas reinhardtii is a species approved by the U.S. Food and Drug Administration (FDA) for human consumption, and its consumption has been reported to improve gastrointestinal health in both murine models and humans. By utilizing the biotechnological tools available for this green alga, we introduced a synthetic gene encoding a chimeric protein, zeolin, obtained by merging the γ-zein and phaseolin proteins, in the algal genome. Zein and phaseolin are major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris) that accumulate in the endoplasmic reticulum (ER) and storage vacuoles, respectively. Seed storage proteins have unbalanced amino acid content, and for this reason, need to be complemented with each other in the diet. The chimeric recombinant zeolin protein represents an amino acid storage strategy with a balanced amino acid profile. Zeolin protein was thus efficiently expressed in Chlamydomonas reinhardtii; thus, we obtained strains that accumulate this recombinant protein in the endoplasmic reticulum, reaching a concentration up to 5.5 fg cell-1, or secrete it in the growth medium, with a titer value up to 82 µg/L, enabling the production of microalga-based super-food.
Eukaryotic photosynthetic organisms have evolved an array of "antenna" complexes where both light harvesting and photoprotective mechanisms occur. Thermal dissipation of the excitation energy harvested in excess, named non-photochemical quenching (NPQ), is one of the main photoprotective mechanisms evolved in eukaryotic organisms to prevent photooxidative stress. Here, the role of the Photosystem II monomeric antenna CP26 was investigated in Chlamydomonas reinhardtii, model organism for green algae, using a genome editing approach to obtain cp26 knock-out mutant strains (named k6). The absence of CP26 caused a reduced growth at low or medium light but not at high irradiances. Photosystem II were partially affected by the absence of CP26 having reduced photochemical efficiency, light harvesting capacity and excitation energy transfer. However, the main phenotype observed in k6 was a strong reduction of NPQ, being reduced by more than 70% compared to wild type. The NPQ phenotype observed was rescued by genetic complementation of k6 mutant demonstrating that ~50% of CP26 content compared to wild type was able to restore the NPQ capacity. The comparison of k6 mutant with mutants deprived of the other Photosystem II monomeric antenna, CP29 or missing both CP26 and CP29, demonstrated that these monomeric antenna proteins have different specific functions in Chlamydomonas reinhardtii: CP26 plays a pivotal role in NPQ induction while the binding of CP29 to Photosystem II is crucial for its photochemical activity. The genetic engineering of these two proteins could be a promising strategy to regulate photosynthetic efficiency of microalgae under different light regimes.
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