Loss of CpFTSY Reduces Photosynthetic Performance and Affects Insertion of PsaC of PSI in Diatoms

Abstract

CpFTSY is a component of the chloroplast signal recognition particle (CpSRP) pathway that post-translationally targets light-harvesting complex proteins (LHCPs) to the thylakoid membranes in plants and green algae containing chloroplasts derived from primary endosymbiosis. In plants, CpFTSY also plays a major role in co-translational incorporation of chloroplast encoded subunits of photosynthetic complexes to the thylakoids. This role has not been demonstrated in green algae. So far, its function in organisms with chloroplasts derived from secondary endosymbiotic events has not been elucidated. Here, we report the generation and characterization of mutants lacking CpFTSY in the diatom Phaeodactylum tricornutum. We found that this protein is not involved in inserting LHCPs into thylakoid membranes, indicating that the post-translational part of the CpSRP pathway is not active in this group of microalgae. Lack of CpFTSY caused an increased level of photoprotection, low electron transport rates, inefficient repair of PSII, reduced growth, a strong decline in the PSI subunit PsaC and upregulation of proteins that might compensate for a non-functional co-translational CpSRP pathway during light stress conditions. The phenotype was highly similar to the one described for diatoms lacking another component of the co-translational CpSRP pathway, the CpSRP54 protein. However, in contrast to cpsrp54 mutants, only one thylakoid membrane protein, PetD of the Cytb6f complex, was downregulated in cpftsy. Our results point to a minor role for CpFTSY in the co-translational CpSRP pathway, suggesting that other mechanism may partially compensate for the effect of a disrupted CpSRP pathway.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin

“…Amplification of the two ZEP genes by PCR and Sanger sequencing of the PCR products revealed a lack of polymorphism in the sequence for three out of four mutants, indicating that only one allele had been amplified for these mutant lines. This phenomenon is typically caused by large indels or mitotic gene conversion affecting the target site, as observed previously [35,36]. No background signal indicating the presence of a non-mutated WT sequence was observed in any of the mutants where only one allele was amplified by PCR.…”

<em>Zeaxanthin Epoxidase</em> 3 Knockout Mutants of the Model Diatom <em>Phaeodactylum tricornutum</em> Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin

“…Amplification of the two ZEP genes by PCR and Sanger sequencing of the PCR products revealed a lack of polymorphism in the sequence for three out of four mutants, indicating that only one allele had been amplified for these mutant lines. This phenomenon is typically caused by large indels or mitotic gene conversion affecting the target site as observed previously [35,36]. No background signal indicating the presence of a nonmutated WT sequence was observed in any of the mutants where only one allele was amplified by PCR.…”

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects

“…Dual-gene ( CpFTSY and ZEP ) knockout led to greater photosynthetic activity and zeaxanthin production in C. reinhardtii [ 158 ], implying the wide prospect of CRISPR/Cas9-induced mutation in environmentally friendly biotechnology [ 176 ]. The functions of genes involved in the CpSRP pathway were also investigated in diatoms using CRISPR/Cas9 technology [ 178 , 179 ]. The functional loss of one member of the CpSRP pathway, CpSRP 54 kDa ( CpSRP54 ), in P. tricornutum led to the decreased accumulation of chloroplast-encoded photosynthetic complex subunits, indicating that CpSRP54 acts in the co-translational part of the CpSRP pathway in P. tricornutum [ 178 ].…”

“…However, the LHC and pigment contents did not decrease in CpSRP54 mutant lines as plants and green algae do, emphasizing the different pathways for the integration of thylakoid membrane proteins between plants, green algae, and diatoms [ 178 ]. Correspondingly, the phenotype of CpFTSY mutants created by using the CRISPR/Cas9 system also indicates that CpSRP54 and CpFTSY of the CpSRP pathway have not yet evolved post-translational functions in diatoms [ 179 ]. In addition, Sharma et al attempted to simultaneously introduce indels in multiple Lhcf genes in P. tricornutum , and the visible color changes of mutant lines demonstrated the successful modification [ 180 ].…”

“…Light-harvesting complex (LHC); chloroplast signal recognition particle 54 kDa (CpSRP54); CpFTSY; cryptochrome; violaxanthin de-epoxidase (VDE); pyruvate orthophosphate dikinase (PPDK); Chl c synthase (CHLC) [178][179][180][181][182][183][184][185]188] Porphyridium sp. Chlorophyll synthase (CHS) [187] Lipid production and fatty acid metabolism…”