In vivo and in silico investigation of green microalgae adaptation to environmental conditions

Microalgae are unicellular photosynthetic organisms increasingly recognized as sustainable and valuable biorefineries, but the domestication process to favorably utilize them in industrial processes started only in recent years. One of the major obstacles in large-scale applications is a lacking knowledge about their response to diverse environmental conditions. This shortfall implies both difficulties in precisely controlling their growth, and sub-optimal induction conditions for the biosynthesis of high-value secondary products, itself often linked to the exposure to abiotic stressors. It is thus becoming of paramount importance to acquire a deep understanding on the mechanisms that regulate the interaction between algal cells and the surrounding environment in order to tackle present limitations and fully exploit their biotechnological potential. A major factor, strongly influencing algal growth, is light exposure. In the first part of this work we examined different aspects of photoprotection mechanisms in two green microalgae, the model species Chlamydomonas reinhardtii and the important industrial species Chlorella vulgaris. In the light of recent structural discoveries on C. reinhardtii photosystem II (PSII) supercomplex, we decided to investigate the role in this species of the two monomeric PSII antennae CP26 and CP29. By means of DNA-free CRISPR/Cas9 technology, we were able to obtain in C. reinhardtii single and double knockout mutants. Several measurements, including photospectrometric analyses and phenotypical characterization with PAM fluorimetry, highlighted in mutants an impairment in photoprotection and photosynthetic efficiency. In particular, we observed a strong increase in LHCII antennae and a marked decrease in chlorophyll a/b ratio along with all major photosynthetic efficiency parameters. Moreover, state transitions were severely inhibited and NPQ was strongly reduced or absent. We thus demonstrated that CP26 and CP29 are crucial for the correct docking of LHCII on PSII and PSI, for state transitions, and for NPQ, and that they are unsuitable targets for future domestication attempts on this species. Thereafter we looked instead into xanthophyll cycle, being a fundamental component of photoprotection. Since C. reinhardtii utilizes an atypical VDE enzyme, we investigated if this was the case also for C. vulgaris. We identified instead a VDE gene closely related to higher plant homologs, that we expressed in E. coli and characterized. We then validated this result by examining in vivo and in vitro (thylakoid) de-epoxidation. Furthermore, to better understand zeaxanthin role in this species, we evaluated several parameters including NPQ response and time-resolved fluorescence decay, demonstrating that C. vulgaris, in contrast with C. reinhardtii, has a zeaxanthin-dependent NPQ mechanism which, evolution-wise, has also proved successful in higher plants. Another promising perspective to better understand how microalgae respond to environmental conditions could be directly examining cellular signalling proteins linked with abiotic stress response, and for the last part of this work we then decided to center our inquiries around the role of G-proteins in both C. reinhardtii and C. vulgaris. By thorough in silico examinations we putatively identified and classified numerous G-proteins in both species, highlighting novel aspects on GTPase distribution in green algae. Briefly, both species encode a small subset of plant GTPases, with representatives for all subfamilies except for monomeric ROP and heterotrimeric G-proteins. Conversely, C. reinhardtii also encodes for several flagellar-related G-proteins, absent in higher plants. Moreover, DGE analysis of GTPases in multiple conditions suggested a connection with environmental response also in this species. Finally, we presented preliminary results on the generation and characterization of C. reinhardtii knockout mutants for RJL1, an atypical flagellar GTPase.