Chloroplast proteins without cleavable transit peptides: rare exceptions or a major constituent of the chloroplast proteome?

Most chloroplast proteins (cp proteins) are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence (cTP), and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs (non-canonical cp proteins) have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as approximately 30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein (RFP)-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein (PDII), and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others ('potential cp proteins') were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies.

Retrograde plastid redox signals in the expression of nuclear genes for chloroplast proteins of Arabidopsis thaliana.

Excitation imbalances between photosystem I and II generate redox signals in the thylakoid membrane of higher plants which induce acclimatory changes in the structure of the photosynthetic apparatus. They affect the accumulation of reaction center and light-harvesting proteins as well as chlorophylls a and b. In Arabidopsis thaliana the re-adjustment of photosystem stoichiometry is mainly mediated by changes in the number of photosystem I complexes, which are accompanied by corresponding changes in transcripts for plastid reaction center genes. Because chloroplast protein complexes contain also many nuclear encoded components we analyzed the impact of such photosynthetic redox signals on nuclear genes. Light shift experiments combined with application of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea have been performed to induce defined redox signals in the thylakoid membrane. Using DNA macroarrays we assessed the impact of such redox signals on the expression of nuclear genes for chloroplast proteins. In addition, studies on mutants with lesions in cytosolic photoreceptors or in chloroplast-to-nucleus communication indicate that the defective components in the mutants are not essential for the perception and/or transduction of light-induced redox signals. A stable redox state of glutathione suggest that neither glutathione itself nor reactive oxygen species are involved in the observed regulation events pointing to the thylakoid membrane as the main origin of the regulatory pathways. Our data indicate a distinct role of photosynthetic redox signals in the cellular network regulating plant gene expression. These redox signals appear to act independently and/or above of cytosolic photoreceptor or known chloroplast-to-nucleus communication avenues.

Knock-out of the genes coding for the Rieske protein and the ATP-synthase delta-subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression.

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b(6)/f (cyt b(6)/f) complex and the delta-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b(6)/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor Q(A) in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-delta destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b(6)/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.

Cytoplasmic N-terminal protein acetylation is required for efficient photosynthesis in Arabidopsis.

The Arabidopsis atmak3-1 mutant was identified on the basis of a decreased effective quantum yield of photosystem II. In atmak3-1, the synthesis of the plastome-encoded photosystem II core proteins D1 and CP47 is affected, resulting in a decrease in the abundance of thylakoid multiprotein complexes. DNA array-based mRNA analysis indicated that extraplastid functions also are altered. The mutation responsible was localized to AtMAK3, which encodes a homolog of the yeast protein Mak3p. In yeast, Mak3p, together with Mak10p and Mak31p, forms the N-terminal acetyltransferase complex C (NatC). The cytoplasmic AtMAK3 protein can functionally replace Mak3p, Mak10p, and Mak31p in acetylating N termini of endogenous proteins and the L-A virus Gag protein. This result, together with the finding that knockout of the Arabidopsis MAK10 homolog does not result in obvious physiological effects, indicates that AtMAK3 function does not require NatC complex formation, as it does in yeast. We suggest that N-acetylation of certain chloroplast precursor protein(s) is necessary for the efficient accumulation of the mature protein(s) in chloroplasts.

Covariations in the nuclear chloroplast transcriptome reveal a regulatory master-switch.

The evolution of the endosymbiotic progenitor into the chloroplast organelle was associated with the transfer of numerous chloroplast genes into the nucleus. Hence, inter-organellar signalling, and the co-ordinated expression of sets of nuclear genes, was set up to control the metabolic and developmental status of the chloroplast. Here, we show by the differential-expression analysis of 3,292 genes, that most of the 35 environmental and genetic conditions tested, including plastid signalling mutations, elicit only three main classes of response from the nuclear chloroplast transcriptome. Two classes, probably involving GUN (genomes uncoupled)-type plastid signalling, are characterized by alterations, in opposite directions, in the expression of largely overlapping sets of genes.