A Novel Prokaryote-Type ECF/ABC Transporter Module in Chloroplast Metal Homeostasis.

During evolution, chloroplasts, which originated by endosymbiosis of a prokaryotic ancestor of today's cyanobacteria with a eukaryotic host cell, were established as the site for photosynthesis. Therefore, chloroplast organelles are loaded with transition metals including iron, copper, and manganese, which are essential for photosynthetic electron transport due to their redox capacity. Although transport, storage, and cofactor-assembly of metal ions in chloroplasts are tightly controlled and crucial throughout plant growth and development, knowledge on the molecular nature of chloroplast metal-transport proteins is still fragmentary. Here, we characterized the soluble, ATP-binding ABC-transporter subunits ABCI10 and ABCI11 in Arabidopsis thaliana, which show similarities to components of prokaryotic, multisubunit ABC transporters. Both ABCI10 and ABCI11 proteins appear to be strongly attached to chloroplast-intrinsic membranes, most likely inner envelopes for ABCI10 and possibly plastoglobuli for ABCI11. Loss of ABCI10 and ABCI11 gene products in Arabidopsis leads to extremely dwarfed, albino plants showing impaired chloroplast biogenesis and deregulated metal homeostasis. Further, we identified the membrane-intrinsic protein ABCI12 as potential interaction partner for ABCI10 in the inner envelope. Our results suggest that ABCI12 inserts into the chloroplast inner envelope membrane most likely with five predicted α-helical transmembrane domains and represents the membrane-intrinsic subunit of a prokaryotic-type, energy-coupling factor (ECF) ABC-transporter complex. In bacteria, these multisubunit ECF importers are widely distributed for the uptake of nickel and cobalt metal ions as well as for import of vitamins and several other metabolites. Therefore, we propose that ABCI10 (as the ATPase A-subunit) and ABCI12 (as the membrane-intrinsic, energy-coupling T-subunit) are part of a novel, chloroplast envelope-localized, AAT energy-coupling module of a prokaryotic-type ECF transporter, most likely involved in metal ion uptake.

Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis.

Metallochaperones traffic copper (Cu(+)) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu(+) chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu(+) chaperone delivers Cu(+) with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu(+) import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu(+)-delivery mechanism and provides a unique snapshot into the evolution of a Cu(+) distribution pathway. We also provide evidence for an interaction between PAA2, the Cu(+)-ATPase in thylakoids, and the Cu(+)-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu(+) network that has evolved to fine-tune Cu(+) distribution.

RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron.

The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previously described to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1 knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid development and plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function in protection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered from oxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants were characterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 protein increase in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of other transition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency, suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading. In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated in PIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. In consequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particular fruit development.