Functional studies of CpSRP54 in diatoms show that the mechanism of thylakoid protein insertion differs from that in plants and green algae

Abstract

SUMMARY The chloroplast signal recognition particle 54 kDa (CpSRP54) protein is a member of the CpSRP pathway known to target proteins to thylakoid membranes in plants and green algae. Loss of CpSRP54 in the marine diatom Phaeodactylum tricornutum lowers the accumulation of a selection of chloroplast‐encoded subunits of photosynthetic complexes, indicating a role in the co‐translational part of the CpSRP pathway. In contrast to plants and green algae, absence of CpSRP54 does not have a negative effect on the content of light‐harvesting antenna complex proteins and pigments in P. tricornutum, indicating that the diatom CpSRP54 protein has not evolved to function in the post‐translational part of the CpSRP pathway. Cpsrp54 KO mutants display altered photophysiological responses, with a stronger induction of photoprotective mechanisms and lower growth rates compared to wild type when exposed to increased light intensities. Nonetheless, their phenotype is relatively mild, thanks to the activation of mechanisms alleviating the loss of CpSRP54, involving upregulation of chaperones. We conclude that plants, green algae, and diatoms have evolved differences in the pathways for co‐translational and post‐translational insertion of proteins into the thylakoid membranes.

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 ].…”

“…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 ].…”

“…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 ].…”

“…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…”

Both major xanthophyll cycles present in nature can provide Non-Photochemical Quenching in the model diatomPhaeodactylum tricornutum

“…KO mutants were maintained for several months at 16 °C and 25 µmol photons m -2 s -1 of white light with 16:8 h light:dark photoperiod on 16 g/L salinity f/2 agar plates without antibiotic selection, to facilitate the expulsion of the CRISPR/Cas9 episome. In addition, prolonged cultivation of transgenic lines was necessary to achieve a stable phenotype before performing experiments, as it is known that in P. tricornutum the effects of mutations can be alleviated via compensatory mechanisms such as upregulation of other genes (87). All KO strains generated for this study were later cryopreserved according to (88).…”