Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum.

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.

ACCUMULATION OF PHOTOSYSTEM ONE1, a member of a novel gene family, is required for accumulation of [4Fe-4S] cluster-containing chloroplast complexes and antenna proteins.

To investigate the nuclear-controlled mechanisms of [4Fe-4S] cluster assembly in chloroplasts, we selected Arabidopsis thaliana mutants with a decreased content of photosystem I (PSI) containing three [4Fe-4S] clusters. One identified gene, ACCUMULATION OF PHOTOSYSTEM ONE1 (APO1), belongs to a previously unknown gene family with four defined groups (APO1 to APO4) only found in nuclear genomes of vascular plants. All homologs contain two related motifs of approximately 100 amino acid residues that could potentially provide ligands for [4Fe-4S] clusters. APO1 is essentially required for photoautotrophic growth, and levels of PSI core subunits are below the limit of detection in the apo1 mutant. Unlike other Arabidopsis PSI mutants, apo1 fails to accumulate significant amounts of the outer antenna subunits of PSI and PSII and to form grana stacks. In particular, APO1 is essentially required for stable accumulation of other plastid-encoded and nuclear-encoded [4Fe-4S] cluster complexes within the chloroplast, whereas [2Fe-2S] cluster-containing complexes appear to be unaffected. In vivo labeling experiments and analyses of polysome association suggest that translational elongation of the PSI transcripts psaA and psaB is specifically arrested in the mutant. Taken together, our findings suggest that APO1 is involved in the stable assembly of several [4Fe-4S] cluster-containing complexes of chloroplasts and interferes with translational events probably in association with plastid nucleoids.

The universally conserved HCF101 protein is involved in assembly of [4Fe-4S]-cluster-containing complexes in Arabidopsis thaliana chloroplasts.

The seedling-lethal nuclear Arabidopsis hcf101 (high chlorophyll fluorescence) mutant is impaired in photosynthesis and complemented by the wild-type HCF101 cDNA. Photosystem I (PSI) activity is abolished, and PSI core complexes fail to accumulate in hcf101, whereas levels of other thylakoid membrane proteins are unaffected. Northern and in vivo labelling analyses as well as studies on polysome loading show that PSI transcript levels and translation rates of proteins, which belong to PSI, are normal in hcf101. PSI-specific fluorescence at 77 K is shifted from 735 to 728 nm in hcf101, indicating that exitons cannot efficiently be transferred to the PSI reaction centre, whereby the PSI antenna is almost unaffected. Mutant plants not only fail to accumulate mature PSI, which contains three [4Fe-4S]clusters (FSCs), but also are characterized by reduced levels of the soluble FSC-containing complex ferredoxin-thioredoxin reductase (FTR) in the stroma. Inhibited FTR maturation is not a secondary effect stemming from lack of PSI because the mutant hcf145, which also lacks PSI, accumulates FTR at normal levels. Levels of the [2Fe-2S] cluster-containing soluble and membrane proteins, ferredoxin and PetC, respectively, were unchanged in hcf101 plants. These data suggest a specific role of HCF101 in FSC biogenesis. HCF101 is plastid localized and belongs to an ancient and universally conserved family of P-loop ATPases previously designated as the 'MRP' (metGrelated protein) family. The function identified for HCF101 suggests a new designation, FSC, for this family.

A peptide chain release factor 2 affects the stability of UGA-containing transcripts in Arabidopsis chloroplasts.

Positional cloning of the hcf109 (high chlorophyll fluorescence) mutation in Arabidopsis has identified a nucleus-encoded, plastid-localized release factor 2-like protein, AtprfB, indicating that the processes of translational termination in chloroplasts resemble those of eubacteria. Control of atprfB expression by light and tissues is connected to chloroplast development. A point mutation at the last nucleotide of the second intron causes a new splice site farther downstream, resulting in a deletion of seven amino acid residues in the N-terminal region of the Hcf109 protein. The mutation causes decreased stability of UGA-containing mRNAs. Our data suggest that transcripts with UGA stop codons are terminated exclusively by AtprfB in chloroplasts and that AtprfB is involved in the regulation of both mRNA stability and protein synthesis. Furthermore, sequence data reveal a +1 frameshift at an internal in-frame TGA stop codon in the progenitor prfB gene of cyanobacteria. The expression pattern and functions of atprfB could reflect evolutionary driving forces toward the conservation of TGA stop codons exclusively in plastid genomes of land plants.