The chloroplast F 1 F o -ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b9, and c) and a peripheral domain (subunits a, b, g, d, and «). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism.

Biogenesis of plastid ribosomes is facilitated by auxiliary factors that process and modify ribosomal RNAs (rRNAs) or are involved in ribosome assembly. In comparison with their bacterial and mitochondrial counterparts, the biogenesis of plastid ribosomes is less well understood, and few auxiliary factors have been described so far. In this study, we report the functional characterization of CONSERVED ONLY IN THE GREEN LINEAGE20 (CGL20) in Arabidopsis (Arabidopsis thaliana; AtCGL20), which is a Pro-rich, ;10-kD protein that is targeted to mitochondria and chloroplasts. In Arabidopsis, CGL20 is encoded by segmentally duplicated genes of high sequence similarity (AtCGL20A and AtCGL20B). Inactivation of these genes in the atcgl20ab mutant led to a visible virescent phenotype and growth arrest at low temperature. The chloroplast proteome, pigment composition, and photosynthetic performance were significantly affected in atcgl20ab mutants. Loss of AtCGL20 impaired plastid translation, perturbing the formation of a hidden break in the 23S rRNA and causing abnormal accumulation of 50S ribosomal subunits in the high-molecular-mass fraction of chloroplast stromal extracts. Moreover, AtCGL20A-eGFP fusion proteins comigrated with 50S ribosomal subunits in Suc density gradients, even after RNase treatment of stromal extracts. Therefore, we propose that AtCGL20 participates in the late stages of the biogenesis of 50S ribosomal subunits in plastids, a role that presumably evolved in the green lineage as a consequence of structural divergence of plastid ribosomes.

ATP synthases in chloroplasts (cpATPase) and mitochondria (mtATPase) are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multisubunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 (CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11) is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLD11 function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the β subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential.

Proteins belonging to the enhancer of RNA interference-1 subfamily of 3'-5' exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE-1-LIKE-1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3'-5' exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus-encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.

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 β-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.