Yoshitaka Nishiyama

Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery

Absorption of excess light energy by the photosynthetic machinery results in the generation of reactive oxygen species (ROS), such as H2O2. We investigated the effects in vivo of ROS to clarify the nature of the damage caused by such excess light energy to the photosynthetic machinery in the cyanobacterium Synechocystis sp. PCC 6803. Treatments of cyanobacterial cells that supposedly increased intracellular concentrations of ROS apparently stimulated the photodamage to photosystem II by inhibiting the repair of the damage to photosystem II and not by accelerating the photodamage directly. This conclusion was confirmed by the effects of the mutation of genes for H2O2‐scavenging enzymes on the recovery of photosystem II. Pulse labeling experiments revealed that ROS inhibited the synthesis of proteins de novo. In particular, ROS inhibited synthesis of the D1 protein, a component of the reaction center of photosystem II. Northern and western blot analyses suggested that ROS might influence the outcome of photodamage primarily via inhibition of translation of the psbA gene, which encodes the precursor to D1 protein.

Glycinebetaine protects the D1/D2/Cytb559 complex of photosystem II against photo-induced and heat-induced inactivation

The presence of 1.0 mol/L glycinebetaine during isolation of D1/D2/Cytb559 reaction centre (RC) complexes from photosystem II (PSII) membrane fragments preserved the photochemical activity, monitored as the light-induced reduction of pheophytin and electron transport from diphenylcarbazide to 2.6-dichlorophenol-indophenol.-Glycinebetaine also protected the D1/D2/Cytb559 complexes against strong light-induced damage to the photochemical reactions and the irreversible bleaching of beta-carotene and chlorophyll. The presence of glycinebetaine also enhanced thermotolerance of the D1/D2/Cytb559 complexes isolated in the presence of 1.0 mol/L betaine with an increase in the temperature for 50% inactivation from 29 degrees C to 35 degrees C. The results indicate an increased supramolecular structural stability in the presence of glycinebetaine.

Molecular Characterization of the PEND Protein, a Novel bZIP Protein Present in the Envelope Membrane That Is the Site of Nucleoid Replication in Developing Plastids

Plastid nucleoids are known to bind to the envelope membrane in developing chloroplasts. Here, plastid DNA is extensively replicated. We previously detected a DNA binding protein in the inner envelope membranes of developing plastids in pea and named it PEND (for plastid envelope DNA binding) protein. In this study, we report on the structure and molecular characterization of a cDNA for the PEND protein. As a result of screening cDNA libraries in λgt11 with one of the target sequences of the PEND protein as a probe, we obtained a clone (PD2) for a novel DNA binding protein consisting of 633 amino acid residues. Analysis of the N-terminal sequence of the purified PEND protein indicated that the transit peptide is just 16 residues long. The PEND protein was detected specifically in the plastid envelope membrane of young unopened leaf buds by immunoblot analysis. The PEND protein consists of a basic region plus zipper region, an unprecedented sextuple repeat region, and a putative membrane-spanning region. The basic region with a zipper region seems to have diverged from that of other plant transcription factors. In addition, the PEND protein could be a distant homolog of the trans-Golgi network integral membrane proteins. The PEND protein is therefore a novel type of DNA binding protein that binds to the membrane as an intrinsic membrane protein.

No coordinated transcriptional regulation of the sod-kat antioxidative system in Synechocystis sp. PCC 6803

Summary The balanced expression of antioxidative enzymes is believed to be important for effective protection against oxidative stress. The cyanobacterium Synechocystis sp. PCC 6803 possesses the simplest antioxidative system, i.e., a single sod gene ( sodB ) for SOD and a single kat gene ( katG ) for catalase/peroxidase, so that it is an ideal model for analyzing the balance of expression. Here we show that sodB expression is induced by various stresses, i.e., O 2 − , H 2 O 2 , low and high temperatures and high salinity, whereas katG is constitutively expressed. These results suggest that coordinated expression is not essential for antioxidative protection at least in this cyanobacteria. The implications of these phenomena are discussed.

Membrane dynamics studied by FTIR spectroscopy in thylakoid and cytoplasmic membranes of Synechocystis PCC6803. Lipids and the effect of protein to lipid ratios

In Cyanobacteria the growth temperature determines the fatty acid composition of the cell membranes, having more unsaturated fatty acyl chains at lower growth temperatures. For proper functioning, membrane constituents require the liquid-crystalline state of the membrane lipids in which rotational transmembrane movements of lipid and protein molecules are possible. In photosynthetic membranes phase behaviour of glyc-erolipids is also regulated by the level of their unsaturation, a process mediated by the activity of fatty acid desaturases that introduce double bonds directly into the fatty acids of glycerolipids. The cyanobacterium strain Synechocystis PCC6803 is transformable and thus, desaturase deficient transformant strains can be obtained from it up to the point where oleic acids are the only unsaturated fatty acid species in the membranes of the mutant cells. We were interested what kind of membranes are constructed from the altered lipid choice, and by this to learn more about the role of lipids in membrane dynamics. For structural studies FTIR spectroscopy has been applied making use of our recent interpretation of the origin of the frequency upshift of vsymCH2 frequencies upon increasing lipid disorder as a consequence of increasing gauche and decreasing trans segment populations in the fatty acyl chains [1].

Genetically Engineered Modification of Plant Chilling Sensitivity and Characterization of Cyanobacterial Heat Shock Proteins

We conducted the following research in order to understand molecular mechanism of tolerance and adaptation in higher plants and microbiral plants to high and low temperature. (1) Glycerol-3-phosphate acyltransferase from Arabidopsis and squash was introduced into tobacco, resulting in a modified chilling tolerance in the leaves of the transgenic tobacco. (2) Cells of a cyanobacterium, Synechococcus PCC7002, were grown under high temperature, and aquired thermotolerance in the photosynthetic oxygen-evolving complex. The factors responsible for the enhanced thermal stability appeared to be associated with the thylakoid membrane. (3) Two groEL-homologous genes were cloned from Synechococcus PCC7002 and were then sequenced. Heat shock treatment markedly increased the mRNAs of both groEL-homologous genes. One gene was accompanied by an upstream groES gene, while the other was not.

Role of Psbu, an Extrinsic Protein of Photosystem II, In the Acquisition of Thermotolerance in Synechococcus sp. PCC 7002

During acclimation to high temperature, photosynthetic organisms enhance the thermal stability of their photosynthetic activity (1). This phenomenon has been observed in a number of species of plants (1) and cyanobacteria (2,3), whereas the physiological importance and the underlying mechanism of the acclimative response are unclear. The oxygen-evolving machinery of the photosystem II (PS II) complex is known to be the most susceptible to high temperature among various components of the photosynthetic machinery (1,4). We reported previously that in the PS II complex only the reaction of the evolution of oxygen was stabilized during acclimation to high temperature in the cyanobacterium Synechococcus sp. PCC 7002 (3). Therefore, it is likely that the stabilization of the oxygen-evolving machinery from heat-induced inactivation would be the mechanism that underlies the photosynthetic acclimation. We showed previously that thylakoid membranes isolated from cells of Synechococcus sp. PCC 7002, which had been grown at high temperatures, exhibited a greater thermal stability in the oxygen-evolving activity than those from cells grown at low temperatures (3). This finding suggested that factors for the thermal stability of the oxygen-evolving machinery were associated with thylakoid membranes. Biochemical investigations of the thylakoid membranes identified two proteins, cytochrome C550 and PsbU, as such factors (5,6). Cytochrome C550 and PsbU are the extrinsic proteins of the PS II complex that have been found in several species of cyanobacteria (7,8) and red algae (9), but not in higher plants (10). In the present study we inactivated the psbU gene in Synechococcus sp. PCC 7002 by targeted mutagenesis in order to examine the thermoprotective role of PsbU in vivo particularly, in terms of acclimation to high temperature.

Fatty Acids Unsaturation of Membrane Lipids is Involved in the Tolerance to Salt Stress

Cyanobacteria exhibit considerable tolerance to salt stress and are useful for studies of acclimation and tolerance to such stress. In a previous study we isolated a desA−/desD− mutant strain of Synechocystis sp. PCC 6803 in which the desA and desD genes for the Δl2 and Δ6 desaturases, respectively, had been inactivated by targeted mutagenesis (1). The desA−/desD− cells contain monounsaturated but not polyunsaturated fatty acids, whereas wild-type cells contain polyunsaturated fatty acids such as di-, tri-, and tetraunsaturated fatty acids (2,3). In the present study, we investigated the contribution of the unsaturation of fatty acids in membrane lipids to tolerance to salt stress by comparing desA − /desD − cells to wild-type cells of Synechocystis sp. PCC 6803. We demonstrated that the unsaturation of fatty acids is associated with the ability of the photosynthetic machinery to tolerate salt stress.