CGL160-mediated recruitment of the coupling factor CF1 is required for efficient thylakoid ATP synthase assembly, photosynthesis, and chloroplast development in Arabidopsis.

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CFO) and a soluble coupling factor (CF1). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF1-CFO assembly. In plants that lacked AtCGL160N, CF1-CFO content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF1 subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF1-ß subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF1 subcomplex, which is critical for the modulation of CF1-CFO activity and photosynthesis.

Introduction of the Carotenoid Biosynthesis α-Branch Into Synechocystis sp. PCC 6803 for Lutein Production.

Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous ß-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol-1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.