Control of STN7 transcript abundance and transient STN7 dimerisation are involved in the regulation of STN7 activity.

Reversible phosphorylation of LHCII, the light-harvesting complex of photosystem II, controls its migration between the two photosystems (state transitions), and serves to adapt the photosynthetic machinery of plants and green algae to short-term changes in ambient light conditions. The thylakoid kinase STN7 is required for LHCII phosphorylation and state transitions in vascular plants. Regulation of STN7 levels occurs at the post-translational level, depends on the thylakoid redox state, and might involve reversible autophosphorylation. Here, we have analysed the effects of different light conditions and chemical inhibitors on the abundance of STN7 transcripts and their products. This analysis was performed in wild-type Arabidopsis thaliana plants, in several photosynthetic mutants, and in lines overexpressing STN7 (oeSTN7) or expressing mutant variants of STN7 carrying single or double cysteine-serine exchanges. It was found that accumulation of the STN7 protein is also controlled at the level of transcript abundance. Under certain conditions, exposure to high light or far-red light treatment, the relative decreases in LHCII phosphorylation can be attributed to decreases in STN7 abundance. Nevertheless, inhibitor experiments showed that redox control of LHCII kinase activity persists in oeSTN7 plants. STN7 dimers were found in oeSTN7 plants and in lines with single cysteine-serine exchanges, indicating that dimerisation involves disulphide bridges. We speculate that transient STN7 dimerisation is required for STN7 activity, and that the altered dimerisation behaviour of oeSTN7 plants might be responsible for the unusually high phosphorylation of LHCII in the dark found in this genotype.

The PsbW protein stabilizes the supramolecular organization of photosystem II in higher plants.

PsbW, a 6.1-kDa low-molecular-weight protein, is exclusive to photosynthetic eukaryotes, and associates with the photosystem II (PSII) protein complex. In vivo and in vitro comparison of Arabidopsis thaliana wild-type plants with T-DNA insertion knock-out mutants completely lacking the PsbW protein, or with antisense inhibition plants exhibiting decreased levels of PsbW, demonstrated that the loss of PsbW destabilizes the supramolecular organization of PSII. No PSII-LHCII supercomplexes could be detected or isolated in the absence of the PsbW protein. These changes in macro-organization were accompanied by a minor decrease in the chlorophyll fluorescence parameter F(V) /F(M) , a strongly decreased PSII core protein phosphorylation and a modification of the redox state of the plastoquinone (PQ) pool in dark-adapted leaves. In addition, the absence of PsbW protein led to faster redox changes in the PQ pool, i.e. transitions from state 1 to state 2, as measured by changes in stationary fluorescence (F(S) ) kinetics, compared with the wild type. Despite these dramatic effects on macromolecular structure, the transgenic plants exhibited no significant phenotype under normal growth conditions. We suggest that the PsbW protein is located close to the minor antenna of the PSII complex, and is important for the contact and stability between several PSII-LHCII supercomplexes.

New type of cells with multiple chromosome rearrangements.

A comparative analysis of the distribution and the frequency of multiaberrant cells (MAC) among lymphocytes in different categories of low dose (up to 0.5 Gy) irradiated people was carried out. The highest MAC frequency was observed in people exposed to alpha-radiation (Pu, Rn) and in cosmonauts. This fact allows MAC to be considered as an indicator of a high-energy local exposure. A new type of cells with multiple chromosome rearrangements was discovered in the course of analysis of stable aberrations by the fluorescence in situ hybridization (FISH) method. The biological consequences of MAC formation and possibility of revealing the whole diversity of cells with multiple aberrations by means of modern molecular-cytogenetic methods are discussed.

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.

A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis.

During photosynthesis, two photoreaction centers located in the thylakoid membranes of the chloroplast, photosystems I and II (PSI and PSII), use light energy to mobilize electrons to generate ATP and NADPH. Different modes of electron flow exist, of which the linear electron flow is driven by PSI and PSII, generating ATP and NADPH, whereas the cyclic electron flow (CEF) only generates ATP and is driven by the PSI alone. Different environmental and metabolic conditions require the adjustment of ATP/NADPH ratios and a switch of electron distribution between the two photosystems. With the exception of PGR5, other components facilitating CEF are unknown. Here, we report the identification of PGRL1, a transmembrane protein present in thylakoids of Arabidopsis thaliana. Plants lacking PGRL1 show perturbation of CEF, similar to PGR5-deficient plants. We find that PGRL1 and PGR5 interact physically and associate with PSI. We therefore propose that the PGRL1-PGR5 complex facilitates CEF in eukaryotes.

Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell.

Complex formation of Vipp1 depends on its alpha-helical PspA-like domain.

Vipp1 (vesicle-inducing protein in plastids 1) is found in Cyanobacteria and chloroplasts of photosynthetic eukaryotes where it is essential for the formation of the thylakoid membrane. Vipp1 is closely related to the phage shock protein A (PspA), a bacterial protein induced under diverse stress conditions. Vipp1 proteins differ from PspA by an additional C-terminal domain that is required for Vipp1 function in thylakoid biogenesis. We show here that in Cyanobacteria, green algae, and vascular plants, Vipp1 is part of a high molecular mass complex. The complex is formed by multiple copies of Vipp1, and complex formation involves interaction of the central alpha-helical domain that is common to Vipp1 as well as to PspA proteins. In chloroplasts of vascular plants, the Vipp1 complex can be visualized by green fluorescent protein fusion in discrete locations at the inner envelope. Green fluorescent protein fusion analysis furthermore revealed that complex formation is important for proper positioning of Vipp1 at the inner envelope of chloroplasts.