The molecular basis of Chlamydomonas reinhardtii responses to the environment: the role of intracellular Ca2+ signalling and minor antenna proteins

Photosynthetic organisms are constantly exposed to climate fluctuations. In some cases, these environmental changes can exceed the physiologically favourable range, negatively affecting their growth and performance. As a consequence, crop yield and microalgal biomass productivity are regularly influenced by unstable environmental conditions. It is thus becoming of uttermost importance to gain a deep understanding on the molecular mechanisms underlying the sensing and response of these organisms to stressful conditions. This will strongly help the identification of entry points for genetic engineering and will clarify the evolution of eukaryotic fundamental cellular processes. Calcium (Ca2+)-dependent signalling plays a well-characterized role in the perception and response mechanisms to environmental stimuli in plant and algal cells. In the model organism for green algae, Chlamydomonas reinhardtii, Ca2+ signals have a crucial role in different physiological processes, such as stress responses, photosynthesis, and flagella functions. In the first part of this work, we reviewed the current knowledge on the cellular mechanisms underlying the generation, shaping, and decoding of Ca2+ signals in C. reinhardtii, providing an overview of the known and possible molecular players involved in the Ca2+ signalling of its different subcellular compartments. Thereafter, to investigate in C. reinhardtii the role of compartment-specific Ca2+ signalling in the perception and response mechanisms to different environmental stressors, the genetically encoded ratiometric Ca2+ indicator Yellow Cameleon (YC3.6) was expressed and targeted to cytosol, chloroplast, and mitochondria. As a major factor influencing algal growth, light exposure was firstly investigated. Light dependent Ca2+ response was detected in C. reinhardtii cells only in the chloroplast as an organelle-autonomous response, influenced by light intensity and photosynthetic electron transport. The absence of blue and red-light photoreceptor aCRY strongly reduced the light dependent chloroplast Ca2+ response. In parallel, Chlamydomonas Ca2+ signalling was investigated in responses to other environmental stressors: nutrient availability, osmotic stress, temperature fluctuations and carbon sensing. Obtained data report cytosolic and plastidial compartment-specific Ca2+ transients, characterized by stimulus-specific kinetic parameters. A relevant role of the chloroplast Ca2+ signalling was also identified in response to hyperosmotic shocks, heat stress and different exogenous carbon sources. Our findings demonstrate the role of intracellular Ca2+ signalling in the perception of the environment in green algae, suggesting the presence of conserved mechanisms among Viridiplantae, but also the existence of uncharacterized responses. In the second part of this work, to deepen our understandings on how microalgae can sense and respond to light, we investigated the functional role of the monomeric Photosystem II antenna CP26 in photoprotection and light harvesting processes. The absence of CP26 partially affected Photosystem II activity causing a reduced growth at low or medium light but not at high irradiances. CP26 knockout mutants displayed more than 70% reduction of the non-photochemical quenching (NPQ) mechanism compared to wild-type, demonstrating a pivotal role for CP26 in NPQ induction, while a crucial function of CP29 for Photosystem II activity. Taken together, our data will provide new understanding of the molecular mechanisms that green algae exploit to sense and respond to the environment, besides suggesting novel strategies to improve their growth and biomass accumulation in challenging and unstable conditions.