Arabidopsis thaliana contains three genes with high homology to potato p24 which was described as a member of the Whirly family of nuclear transcriptional activators. Computer-based analysis revealed that all Arabidopsis Whirly (Why) proteins contain targeting sequences for either plastids or mitochondria. The functionality of these sequences was demonstrated by in vitro import assays into isolated organelles. Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. The possession of organellar targeting sequences seems to be conserved among Why proteins of higher plant species, including potato p24.

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The thylakoid-associated kinases STN7 and STN8 are involved in short- and long-term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA-binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin-NADP reductase as targets for the thylakoid-associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7-dependent phosphorylation site are similar to those of phosphorylated light-harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the -1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long-term adaptation processes affecting metabolite accumulation and gene expression.

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Research interest in proteomics is increasingly shifting toward the reverse genetic characterization of gene function at the proteome level. In plants, several distinct gene defects perturb photosynthetic capacity, resulting in the loss of chlorophyll and an albino or pale-green phenotype. Because photosynthesis is interconnected with the entire plant metabolism and its regulation, all albino plants share common characteristics that are determined by the switch from autotrophic to heterotrophic growth. Reverse genetic characterizations of such plants often cannot distinguish between specific consequences of a gene defect from generic effects in response to perturbations in photosynthetic capacity. Here, we set out to define common and specific features of protein accumulation in three different albino/pale-green plant lines. Using quantitative proteomics, we report a common molecular phenotype that connects the loss of photosynthetic capacity with other chloroplast and cellular functions, such as protein folding and stability, plastid protein import, and the expression of stress-related genes. Surprisingly, we do not find significant differences in the expression of key transcriptional regulators, suggesting that substantial regulation occurs at the posttranscriptional level. We examine the influence of different normalization schemes on the quantitative proteomics data and report all identified proteins along with their fold changes and P values in albino plants in comparison with the wild type. Our analysis provides initial guidance for the distinction between general and specific adaptations of the proteome in photosynthesisimpaired plants.

As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts is encoded in the nucleus and synthesized in the cytosol as precursor molecules carrying N-terminal transit peptides for the transport into the respective target organelle. In most instances, transport takes place into either mitochondria or chloroplasts, although a few examples of dual targeting into both organelles have been described. Here, we show by a combination of three different experimental strategies that also cytochrome c(1) of potato, a component of the respiratory electron transport chain, is imported not only into mitochondria, but also into plastids. In organello import experiments with isolated mitochondria and chloroplasts, which were analyzed in both single and mixed organelle assays, demonstrate that the processing products accumulating after import within the two endosymbiotic organelles are different in size. Dual targeting of cytochrome c(1) is observed also in vivo, after biolistic transformation of leaf epidermal cells with suitable reporter constructions. Finally, Western analyses employing cytochrome c(1)-specific antiserum provide evidence that the protein accumulates in significant amounts in mitochondria and chloroplasts of both pea and spinach. The possible consequences of our findings on the relevance of the dual targeting phenomenon are discussed.

Reanalysis of published mass spectrometry data on Tyr-phosphorylated chloroplast proteins indicates that the majority of peptide spectrum matches reporting Tyr phosphorylation are ambiguous.

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution.