Genetic and phenotypic analysis of Chlamydomonas pale green mutants

In this thesis it was performed the characterization of different Chlamydomonas reinhardtii pale green mutants, both to investigate some important photosynthetic mechanisms and to identify mutations which result in enhanced productivity, to be exploitable for industrial applications. The Chlamydomonas pale mutant as2.1 is characterized by an increased high light resistance and a faster growth in photoautotrophic conditions, compared to the WT. Sequencing data from this strain revealed a large deletion/insertion rearrangement including the gene CpFTSY, a soluble receptor belonging to the CpSRP pathway. Through a targeted mutagenesis by CRISPR-Cas9 genome editing approach, we aimed to understand if the growth advantage was related to the lack of CpFTSY . Unexpectedly, the CpFTSY-knock out (KO) mutants were unable to grow photoautotrophycally and showed a diminished abundance of LHCs and supercomplexes content than the control genotype; moreover, in mutant lines carotenoids biosynthesis was up-regulated, thus suggesting a higher susceptibility to excess light stress. To clarify these differences between as2.1 and the genome edited strains, all these mutants were complemented by the CDS of CpFTSY, demonstrating that this gene is responsible for the high-growth phenotype of as2.1; however, characterization of complemented lines revealed CpFTSY was not the only actor involved in growth phenotype, rather a side mutation likely contributes to this feature. The characterization of the first two cpftsy KO strains we obtained by genome editing revealed differences in LHC composition and in the relative abundance of monomeric vs. trimeric antennae, therefore additional KO lines were produced targeting this locus by a different sgRNA. The screening of several independent lines revealed a huge variation in the composition of the photosynthetic apparatus, that could tentatively be ascribed to an additional pathway or gene affecting the biogenesis of antenna systems, that might possibly compensate the missing CPFTSY during the strain propagation. cpftsy genome edited strains were also analyzed in comparison with Chlamydomonas cao mutants, to compare the effects of an impairment in LHCs insertion systems vs. chlorophyll biosynthesis, on the assembly of peripheral antenna system. Moreover, to understand which system is more important for light harvesting capacity, it was also produced a double mutant cpftsy-cao and the effects of the combined mutations were compared with those of the single ones. cao single mutants, despite a strong reduction in PSII functional antenna size and LHCII content, showed a remarkable resistance to excess light conditions, and a faster growth in photoautotrophic conditions compared to the wild type under saturating irradiances. Chlorophyll b biosynthetic pathway therefore appears crucial in the supramolecular organization of the photosystems, in the regulation of LHCII and LHCI relative abundance and in the structural stability of the LHCs, while its depletion does not significantly affect the core functionality. Instead, cpftsy single mutants were less affected in LHCII composition and in PSII functional antenna size than cao lines, however their photoautotrophic growth was impaired. Unexpectedly CpFTSY gene plays a minor role in regulating LHC system biogenesis, while the lower PsaA (PSI core-complex subunit) and CP47 (PSII core-complex subunit) contents suggested an involvement in the insertion of core proteins. The phenotype of cpftsy-cao double mutant is a cumulative result of the phenotypes of single mutants, thus it confirmed that CAO and CpFTSY regulate different steps of photosynthetic machinery biogenesis, and differently contributes to light-harvesting function. To obtain an efficient CRISPR-Cas9 procedure to be applied to Chlamydomonas, a long work of optimization of the available protocols was carried out. In the second part of the thesis, all the steps are illustrated.