Identification of STN7/STN8 kinase targets reveals connections between electron transport, metabolism and gene expression.

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.

The Peptide Microarray ChloroPhos1.0: A Screening Tool for the Identification of Arabidopsis thaliana Chloroplast Protein Kinase Substrates.

We designed the peptide microarray ChloroPhos1.0 to screen for substrates of chloroplast protein kinases. The peptides represented on the microarray were selected from phosphoproteomics data, and the identified chloroplast phosphopeptides were spotted as 15-mers on a glass slide with the phosphorylation site centered. Altogether, 905 distinct peptides from chloroplast proteins are present on the array. Here we describe how the array can be used to identify the target protein spectrum of chloroplast kinases. We present the method and discuss limitations and challenges associated with the determination of phosphorylation activity on peptide substrates in vitro.

The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.

We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.

Signal integration by chloroplast phosphorylation networks: an update.

Forty years after the initial discovery of light-dependent protein phosphorylation at the thylakoid membrane system, we are now beginning to understand the roles of chloroplast phosphorylation networks in their function to decode and mediate information on the metabolic status of the organelle to long-term adaptations in plastid and nuclear gene expression. With the help of genetics and functional genomics tools, chloroplast kinases and several hundred phosphoproteins were identified that now await detailed functional characterization. The regulation and the target protein spectrum of some kinases are understood, but this information is fragmentary with respect to kinase and target protein crosstalk in a changing environment. In this review, we will highlight the most recent advances in the field and discuss approaches that might lead to a comprehensive understanding of plastid signal integration by protein phosphorylation.

High-throughput sequencing of complete human mtDNA genomes from the Caucasus and West Asia: high diversity and demographic inferences.

To investigate the demographic history of human populations from the Caucasus and surrounding regions, we used high-throughput sequencing to generate 147 complete mtDNA genome sequences from random samples of individuals from three groups from the Caucasus (Armenians, Azeri and Georgians), and one group each from Iran and Turkey. Overall diversity is very high, with 144 different sequences that fall into 97 different haplogroups found among the 147 individuals. Bayesian skyline plots (BSPs) of population size change through time show a population expansion around 40-50 kya, followed by a constant population size, and then another expansion around 15-18 kya for the groups from the Caucasus and Iran. The BSP for Turkey differs the most from the others, with an increase from 35 to 50 kya followed by a prolonged period of constant population size, and no indication of a second period of growth. An approximate Bayesian computation approach was used to estimate divergence times between each pair of populations; the oldest divergence times were between Turkey and the other four groups from the South Caucasus and Iran (~400-600 generations), while the divergence time of the three Caucasus groups from each other was comparable to their divergence time from Iran (average of ~360 generations). These results illustrate the value of random sampling of complete mtDNA genome sequences that can be obtained with high-throughput sequencing platforms.

Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes.

Heteroplasmy, the existence of multiple mtDNA types within an individual, has been previously detected by using mostly indirect methods and focusing largely on just the hypervariable segments of the control region. Next-generation sequencing technologies should enable studies of heteroplasmy across the entire mtDNA genome at much higher resolution, because many independent reads are generated for each position. However, the higher error rate associated with these technologies must be taken into consideration to avoid false detection of heteroplasmy. We used simulations and phiX174 sequence data to design criteria for accurate detection of heteroplasmy with the Illumina Genome Analyzer platform, and we used artificial mixtures and replicate data to test and refine the criteria. We then applied these criteria to mtDNA sequence reads for 131 individuals from five Eurasian populations that had been generated via a parallel tagged approach. We identified 37 heteroplasmies at 10% frequency or higher at 34 sites in 32 individuals. The mutational spectrum does not differ between heteroplasmic mutations and polymorphisms in the same individuals, but the relative mutation rate at heteroplasmic mutations is significantly higher than that estimated for all mutable sites in the human mtDNA genome. Moreover, there is also a significant excess of nonsynonymous mutations observed among heteroplasmies, compared to polymorphism data from the same individuals. Both mutation-drift and negative selection influence the fate of heteroplasmies to determine the polymorphism spectrum in humans. With appropriate criteria for avoiding false positives due to sequencing errors, next-generation technologies can provide novel insights into genome-wide aspects of mtDNA heteroplasmy.