Acclimation to the growth temperature and the high-temperature effects on photosystem II and plasma membranes in a mesophilic cyanobacterium, Synechocystis sp. PCC6803.

High-temperature effects on Photosystem II and plasma membranes, temperature dependence of growth, and acclimation to the growth temperature were studied in a mesophilic cyanobacterium, Synechocystis sp. PCC6803. The following results were obtained. (1) Small but distinct temperature acclimation of the PSII reaction center activity was shown for the first time when the activity was measured at inhibitory high temperatures. However, the reaction center activity showed no apparent acclimation when it was measured at growth temperatures after heat stress. (2) Oxygen-evolving activity and the permeability of plasma membranes showed higher resistance to heat when PCC6803 cells were grown at higher temperatures. (3) Acclimation of photosynthesis to the growth temperature seemed to occur so as to maintain photosynthesis activity not at a maximum level but in a certain range at the growth temperatures. (4) Neither sensitivity to high-temperature-induced dissociation of phycobilisomes from the PSII reaction center complexes nor degradation of phycocyanin were altered by changes in the environmental temperature. (5) A close relationship between the viability of cells and the structural changes of plasma membranes (but not the inactivation of photosynthesis) was observed. The denaturation process of PSII complexes and the relationship between the temperature dependence of the growth of Synechocystis PCC6803 cells and that of the photosynthetic activity are also discussed.

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a glaucocystophyte, Cyanophora paradoxa.

Conditions for preparing oxygen-evolving thylakoid membranes and PSII complexes, and those for observing the PSII activity were investigated in a glaucocystophyte, Cyanophora paradoxa. The active thylakoid membranes were isolated either with a medium containing glycerol or with that containing high concentrations of sucrose, phosphate, and citrate. Active PSII particles were solubilized by octyl-beta-D-glucoside from thylakoid membranes and were separated by sucrose density gradient centrifugation. The thylakoid membranes and PSII particles showed an oxygen-evolving activity only in high-ionic-strength media. The extrinsic 33 kDa protein (PsbO) and the cytochrome c(550) (PsbV) were found to be present in the PSII particles as in cyanobacteria or red algae, but no 12 kDa protein (PsbU) was detected. The PsbO protein was classified as a land-plant type by its N-terminal amino acid sequence.

An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes.

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. We have improved the traditional Tris/HCI buffer systems adopting a Tris/2-(N-morpholino)ethanesulfonic acid (MES) buffer system containing 6 M urea. This gel system was used with an 18-24% acrylamide gradient for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa. This buffer system can also be applied to the usual uniform concentration of acrylamide gel and also to minislab gels.

Effects of high-temperature treatments on a thermophilic cyanobacterium Synechococcus vulcanus.

Effects of high-temperature treatments on a thermophilic cyanobacterium, Synechococcus vulcanus, were studied, and the following results were obtained. (1) Oxygen evolution and the PSII photochemical reaction were the most sensitive sites and started to be inactivated at temperatures slightly higher than the cultivating temperature. (2) The decrease in the fluorescence Fv value reflected the inactivation of the charge separation reaction of PSII as well as that of the oxygen evolution reaction. (3) The dark fluorescence level, Fo, showed an increase at around 70 degrees C, which was partially reversed by further incubation at 50 degrees C. This increase reflected the inactivation of PSII reaction centers and probably dissociation of phycobilisomes from the PSII reaction center complexes. (4) At higher temperatures, phycobiliproteins disassembled and denatured in a pH-dependent manner, causing a large Fo decrease. (5) Cell membranes became leaky to low-molecular-weight substances at around 72 degrees C. (6) Inhibition of growth of the cells was recognized when the cells were pretreated at temperatures higher than 72 degrees C. Reversibility of the high-temperature effects and relationship between viability of the cells and the degradation of the cell membranes are discussed.

Identification of Photosystem I components from a glaucocystophyte, Cyanophora paradoxa: The PsaD protein has an N-terminal stretch homologous to higher plants.

Thylakoid membranes and Photosystem I (PS I) complexes were isolated from a glaucocystophyte, Cyanophora paradoxa, which is thought to have the most primitive 'plastids', and the proteins related to PS I were examined. The intrinsic light-harvesting chlorophyll protein complexes of PS I (LHC I) were not detected by an immunological method. The PS I complexes consisted of at least eight low-molecular-mass proteins in addition to PS I reaction center proteins. The N-terminal sequence of the PsaD protein has higher homology to that of Chlamydomonas reinhardtii and land plants, than to that of other algae or cyanobacteria. On the other hand, the PsaL sequence has the highest homology to those of cyanobacteria. Taking into account the other sequences of PS I components whose genes are encoded in the cyanelle genome, and the fact that LHC I is not detected, it is concluded that PS I of C. paradoxa has chimeric characteristics of both 'green' lineages and cyanobacteria.

Reduction of Q(A) in the dark: Another cause of fluorescence F(o) increases by high temperatures in higher plants.

Increases in the chlorophyll fluorescence F(o) (dark level fluorescence) during heat treatments were studied in various higher plants. Besides the dissociation of light-harvesting chlorophyll a/b protein complexes from the reaction center complex of PS II and inactivation of PS II, dark reduction of Q(A) via plastoquinone (PQ) seemed to be related to the F(o) increase at high temperatures. In potato leaves or green tobacco cultured cells, a part of the F(o) increase was quenched by light, reflecting light-induced oxidation of Q(A) (-) which had been reduced in the dark at high temperatures. Appearance of the F(o) increase due to Q(A) reduction depended on the plant species, and the mechanisms for this are proposed. The reductants seemed to be already present and formed by very brief illumination of the leaves at high temperatures. A ndhB-less mutant of tobacco showed that complex I type NAD(P)H dehydrogenase is not involved in the heat-induced reduction of Q(A). Quite strong inhibition of the Q(A) reduction by diphenyleneiodonium suggests that a flavoenzyme is one of the electron mediator to PQ from the reductant in the stroma. Reversibility of the heat-induced Q(A) reduction suggests that an enzyme(s) involved is activated at high temperatures and mostly returns to an inactive form at room temperature (25 degrees C).

Polypeptide composition of envelopes of spinach chloroplasts: two major proteins occupy 90% of outer envelope membranes.

Outer and inner envelope membranes of spinach chloroplasts were isolated using floatation centrifugation followed by sedimentation sucrose density gradient centrifugation after disruption of intact chloroplasts by freezing and thawing. Two major fractions with buoyant densities of 1.11 and 1.08 g cm-3 and a minor fraction with a density of 1.15 g cm-3 were obtained. They were identified as inner and outer envelope and thylakoid fractions, respectively by analyzing their polypeptide composition by high-resolution SDS-PAGE and the N-terminal sequences of their protein components. Due to the refinement of the isolation procedure, most of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which had always been observed as a contaminant, was eliminated from the outer envelope fraction. Application of high-resolution SDS-PAGE revealed that this fraction was rich in the low-molecular-mass outer envelope protein, E6.7 [Salomon et al. (1990) Proc. Natl. Acad. Sci. USA 87: 5778] and a protein with a molecular mass of 15 kDa which is homologous to the 16 kDa outer envelope protein of pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94: 9504]. The two proteins account for 90% of the total proteins present in outer envelope membranes. Proteins which are suggested to function in translocation of nuclear-encoded polypeptides were not identified in the envelopes from spinach in the present study. Differences in the protein composition of outer envelope membranes are discussed based on the developemental stages of chloroplasts.