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Repair of photosystem II (PSII) during photoinhibition involves replacement of photodamaged D1 protein by newly synthesized D1 protein. In this review, we summarize evidence for the indispensability of ATP in the degradation and synthesis of D1 during the repair of PSII. Synthesis of one molecule of the D1 protein consumes more than 1,300 molecules of ATP equivalents. The degradation of photodamaged D1 by FtsH protease also consumes approximately 240 molecules of ATP. In addition, ATP is required for several other aspects of the repair of PSII, such as transcription of psbA genes. These requirements for ATP during the repair of PSII have been demonstrated by experiments showing that the synthesis of D1 and the repair of PSII are interrupted by inhibitors of ATP synthase and uncouplers of ATP synthesis, as well as by mutation of components of ATP synthase. We discuss the contribution of cyclic electron transport around photosystem I to the repair of PSII. Furthermore, we introduce new terms relevant to the regulation of the PSII repair, namely, "ATP-dependent regulation" and "redox-dependent regulation," and we discuss the possible contribution of the ATP-dependent regulation of PSII repair under environmental stress.

Tomato (Lycopersicon esculentum cv. 'Moneymaker') was transformed with a codA gene, from Arthrobacter globiformis, for choline oxidase that had been modified to allow targeting to both chloroplasts and the cytosol. Glycinebetaine (GB) accumulated in seeds of transformed plants up to 1 µmol g(-1) dry weight (DW), while no detectable GB was found in wild-type (WT) seeds. The codA-transgenic seeds germinated faster and at higher frequency than WT seeds with high temperature treatment. After heat stress, levels of expression of a mitochondrial small heat-shock protein (MT-sHSP), heat-shock protein 70 (HSP70) and heat-shock cognate 70 (HSC70) were higher in transgenic seeds than in WT seeds during heat stress, and the accumulation of HSP70 was more prominent in codA-transgenic seeds than in WT seeds. Addition of GB to the germination medium or imbibition of seeds in a solution of GB enhanced the tolerance of WT seeds to high temperatures. WT seeds treated with exogenous GB also expressed heat-shock genes at elevated levels and accumulated more HSP70 than controls. Our results suggest that GB, either applied exogenously or accumulated in vivo in codA-transgenic seeds, enhanced the expression of heat-shock genes in and improved the tolerance to high temperature of tomato seeds during germination.

Various compatible solutes enable plants to tolerate abiotic stress, and glycinebetaine (GB) is one of the most-studied among such solutes. Early research on GB focused on the maintenance of cellular osmotic potential in plant cells. Subsequent genetically engineered synthesis of GB-biosynthetic enzymes and studies of transgenic plants demonstrated that accumulation of GB increases tolerance of plants to various abiotic stresses at all stages of their life cycle. Such GB-accumulating plants exhibit various advantageous traits, such as enlarged fruits and flowers and/or increased seed number under non-stress conditions. However, levels of GB in transgenic GB-accumulating plants are relatively low being, generally, in the millimolar range. Nonetheless, these low levels of GB confer considerable tolerance to various stresses, without necessarily contributing significantly to cellular osmotic potential. Moreover, low levels of GB, applied exogenously or generated by transgenes for GB biosynthesis, can induce the expression of certain stress-responsive genes, including those for enzymes that scavenge reactive oxygen species. Thus, transgenic approaches that increase tolerance to abiotic stress have enhanced our understanding of mechanisms that protect plants against such stress.

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants were transformed with a gene for choline oxidase (codA) from Arthrobacter globiformis. The gene product (CODA) was targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). These three transgenic plant types accumulated different amounts and proportions of glycinebetaine (GB) in their chloroplasts and cytosol. Targeting CODA to either the cytosol or both compartments simultaneously increased total GB content by five- to sixfold over that measured from the chloroplast targeted lines. Accumulation of GB in codA transgenic plants was tissue dependent, with the highest levels being recorded in reproductive organs. Despite accumulating, the lowest amounts of GB, Chl-codA plants exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses. This suggests that chloroplastic GB is more effective than cytosolic GB in protecting plant cells against chilling, high salt and oxidative stresses. Chloroplastic GB levels were positively correlated with the degree of oxidative stress tolerance conferred, whereas cytosolic GB showed no such a correlation. Thus, an increase in total GB content does not necessarily lead to enhanced stress tolerance, but additional accumulation of chloroplastic GB is likely to further raise the level of stress tolerance beyond what we have observed.

The tolerance of various species of plant to abiotic stress has been enhanced by genetic engineering with certain genes. However, the use of such transgenes is often associated with negative effects on growth and productivity under non-stress conditions. The codA gene from Arthrobacter globiformis is of particular interest with respect to the engineering of desirable productive traits in crop plants. The expression of this gene in tomato plants resulted in significantly enlarged flowers and fruits under non-stress conditions. The enlargement of flowers and fruits was associated with high levels of glycinebetaine that accumulated in reproductive organs, such as flower buds and fruits. The enlargement of flowers was related to an increase in the size and number of cells, and reflected the pleiotropic effect of the codA transgene on the expression of genes involved in the regulation of cell division.

Oxidative stress caused by reactive oxygen species and, in particular, to hydrogen peroxide (H(2)O(2)) has a major impact on all biological systems, including plants and microorganisms. We investigated the H(2)O(2)-inducible expression of genes in the cyanobacterium Synechocystis sp. PCC 6803 using genome-wide DNA microarrays. Our systematic screening of a library of mutant lines with defects in histidine kinases (Hiks) by RNA slot-blot hybridization and DNA-microarray analysis suggested that four Hiks, namely, Hik33, Hik34, Hik16 and Hik41, are involved in the perception and transduction of H(2)O(2) signals that regulate the gene expression of 26 of the 77 H(2)O(2)-inducible genes with induction factors higher than 4.0. Among the four Hiks, Hik33 was the main contributor and was responsible for 22 of the 26 H(2)O(2)-inducible genes under the control of the Hiks. By contrast to Hik33, PerR encoding putative peroxide-sensing protein is involved in the regulation of only nine H(2)O(2)-inducible genes.

Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.

Acclimation of living organisms to cold stress begins with the perception and transduction of the cold signal. However, traditional methods failed to identify the sensors and transducers of cold stress. Therefore, we combined systematic mutagenesis of potential sensors and transducers with DNA microarray analysis in an attempt to identify these components in the cyanobacterium Synechocystis sp. PCC 6803. We identified histidine kinase Hik33 as a potential cold sensor and found that Hik33 participates in the regulation of the expression of more than 60% of the cold-inducible genes. Further study revealed that Hik33 is also involved in the perception of hyperosmotic stress and salt stress and transduction of the signals. Complexity of responses to cold and other environmental stresses is discussed.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

The cis-unsaturated molecular species of phosphatidylglycerol (PG) in chloroplasts have been implicated in the chilling sensitivity of plants. Homozygous lines of transgenic tobacco (Nicotiana tabacum) that overexpressed the cDNA for glycerol-3-phosphate acyltransferase, a key enzyme in the determination of the extent of cis-unsaturation of PG, were established from a chilling-sensitive squash (Cucurbita moschata). In transgenic plants, the proportion of saturated plus trans-monounsaturated molecular species of PG increased from 24 to 65%. However, this change did not affect the architecture of the chloroplasts. Chilling stress decreased the growth and biomass production of young seedlings of transgenic plants more severely than those of wild-type plants, and this observation suggests that the changes in the proportion of cis-unsaturated PG affected not only leaves but also developing plants. Chilling stress also damaged inflorescences. In particular, the abscission of flower buds and inflorescence meristems from transgenic plants occurred more frequently than that from wild-type plants. Thus, it is likely that decreases in the proportion of cis-unsaturated PG enhanced the sensitivity to chilling of reproductive organs.

Previously, we showed that transformation with the codA gene for choline oxidase allows plants to synthesize glycine betaine (GB) and enhances their ability to tolerate various kinds of stress during germination and vegetative growth. In this study, we examined the tolerance of transformed plants to salt stress at the reproductive stage, which is the stage at which plants are most sensitive to environmental stress. Salt-shock treatment of wild-type plants for 3 days resulted in the abortion of flower buds and decreased the number of seeds per silique. These deleterious effects were clearly visible 6 days after the termination of salt-shock treatment. Microscopic examination of floral structures revealed that salt stress inhibited the development of anthers, pistils, and petals. In particular, the production of pollen grains and ovules was dramatically inhibited. These effects of salt stress were significantly reduced by transformation with the codA gene, and our observations suggested that the enhanced tolerance of the transgenic plants was a result of the accumulation of GB in the reproductive organs. Indeed, levels of GB in flowers, siliques, and inflorescence apices were about five times higher than in leaves.

Thylakoid membranes are crucial to photosynthesis in cyanobacteria and plants. In cyanobacteria, genetic modification of membrane lipid composition strongly influences cold tolerance and susceptibility to photoinhibition. We have used fluorescence recovery after photobleaching to measure the diffusion of a lipid-soluble fluorescent marker in cells of the cyanobacterium Synechococcus sp. PCC 7942. We have compared the wild-type strain with a transformant with an increased level of fatty acid unsaturation. The transformant showed a six-fold increase in the diffusion coefficient for the fluorescent marker at growth temperature. This is the first direct measurement of lipid diffusion in a photosynthetic membrane.

The unicellular cyanobacteria Synechocystis sp. (PCC6803) has become a model organism for a range of biochemical and molecular biology studies aimed at investigating environmental stress responses. In this study the soluble proteins of Synechocystis were analysed using narrow pH range (pH 4.5-5.5) zoom gels, automated matrix-assisted laser desorption/ionization mass spectrometry acquisition, spectral processing and database searching. The work sets the foundation for investigations of proteomic changes following stress treatment. One hundred and ninety-two protein spots were analysed and 105 proteins identified, of these 37 were novel proteins not previously seen on two-dimensional gels. Proteins involved in amino acid biosynthesis, energy metabolism and protein modification were identified using this fully automated procedure demonstrating that automated acquisition and processing will be a useful tool for proteomic analyses on this organism.

The stress imposed on living organisms by hyperosmotic conditions and low temperature appears to be perceived via changes in the physical state of membrane lipids. We compared genome-wide patterns of transcription between wild-type Synechocystis sp. PCC 6803 and cells with a mutation in the histidine kinase Hik33 using a DNA microarray. Our results indicated that Hik33 regulated the expression of both osmostress-inducible and cold-inducible genes. The respective genes that were regulated by Hik33 under hyperosmotic and low-temperature conditions were, for the most part, different from one another. However, Hik33 also regulated the expression of a set of genes whose expression was induced both by osmotic stress and by cold stress. These results indicate that Hik33 is involved in responses to osmotic stress and low-temperature stress but that the mechanisms of the responses differ.

The acclimation of a plant to a constantly changing environment involves the accumulation of certain organic compounds of low molecular mass, known collectively as compatible solutes, in the cytoplasm. The evidence from numerous investigations of the physiology, genetics, biophysics and biochemistry of plants strongly suggests that glycine betaine (GB), an amphoteric quaternary amine, plays an important role as a compatible solute in plants under various types of environmental stress, such as high levels of salts and low temperature. Plant species vary in their capacity to synthesize GB and some plants, such as spinach and barley, accumulate relatively high levels of GB in their chloroplasts while others, such as Arabidopsis and tobacco, do not synthesize this compound. Genetic engineering has allowed the introduction into GB-deficient species of biosynthetic pathways to GB from both micro-organisms and higher plants; this approach has facilitated investigations of the importance of GB in stress protection. In this review, we summarize recent progress in the genetic manipulation of the synthesis of GB, with special emphasis on the relationship between the protective effects of GB in vivo and those documented in vitro.

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold-inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a ΔHik33 mutant using the DNA microarray technique. In wild-type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold-repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold-regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.

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.

Summary The expression of the cytM gene, which encodes cytochrome cM in Synechocystis sp. PCC 6803, is induced under stress conditions such as low temperature. Results of spectrophotometric studies revealed that cytochrome cM was oxidized by light absorbed by photosystem I in cells that had acclimatized to a low temperature. However, no similar light-induced oxidation of cytochrome cM was observed in D cytMmutant cells. The kinetics of the oxidation and reductionofP700beforeandafteracclimationofSynechocystiscells to low temperature suggested that cytochrome cM might donate electrons to photooxidized P700. Our observations indicate that cytochrome cM is synthesized under low-temperature stress and that it might carry electrons directly to P700 + in photosystem I. IUBMBLife, 51: 93‐97, 2001

To examine the effect of abscisic acid (ABA) on the photoinduced inactivation of the photosystem II (PSII) complex, a suspension culture of Chlamydomonas reinhardtii was treated with ABA for 24 h in darkness and then, after removal of ABA, the cells were exposed to strong light at a photon flux density of 2000 micromol m(-2) s(-1) at various temperatures. The activity of PSII, as estimated in terms of chlorophyll fluorescence and the evolution of oxygen, decreased significantly during the exposure of cells to the strong fight, and the extent of the photo-induced decrease in PSII activity was much greater at lower temperatures. Irrespective of temperature, the decrease in PSII activity in ABA-treated cells was significantly smaller than that in control cells. Moreover, the recovery of PSII activity from the photo-inactivated state in ABA-treated cells was significantly faster than that in control cells. The recovery of PSII activity in both ABA-treated and control cells was almost entirely prevented by the presence of chloramphenicol. These results indicate that ABA protects the PSII complex in C. reinhardtii against photo-induced inactivation by accelerating the recovery of this complex.

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis, which encodes choline oxidase, the enzyme that synthesizes glycinebetaine from choline. The transformation enabled the plants to accumulate glycinebetaine in chloroplasts, and significantly enhanced the freezing tolerance of plants. Furthermore, the photosynthetic machinery of transformed plants was more tolerant to freezing stress than that of wild-type plants. Exogenous application of glycinebetaine also increased the freezing tolerance of wild-type plants, suggesting that the presence of glycinebetaine in transformed plants had enhanced their ability to tolerate freezing stress. Northern blotting analysis revealed that the enhancement of freezing tolerance was not related to the expression of four cold-regulated genes. These results suggest that engineering of the biosynthesis of glycinebetaine by transformation with the codA gene might be an effective method for enhancing the freezing tolerance of plants.

Low temperature is an important environmental factor that has effects on all living organisms. Various low-temperature-inducible genes encode products that are essential for acclimation to low temperature, but low-temperature sensors and signal transducers have not been identified. However, systematic disruption of putative genes for histidine kinases and random mutagenesis of almost all the genes in the genome of the cyanobacterium Synechocystis sp. PCC 6803 have allowed us to identify two histidine kinases and a response regulator as components of the pathway for perception and transduction of low-temperature signals. Inactivation, by targeted mutagenesis, of the gene for each of the two histidine kinases and inactivation of the gene for the response regulator depressed the transcription of several lowtemperature-inducible genes.

Summary Arabidopsis thaliana was transformed with the codA gene for choline oxidase from Arthrobacter globiformis under control of the 35S RNA promoter of cauliflower mosaic virus. As a result, high levels of glycinebetaine accumulated in the seeds of transformed plants. Transformation with the codA gene significantly enhanced the tolerance to high temperatures during the imbibition and germination of seeds, as well as during growth of young seedlings. The extent of enhancement of the tolerance to high temperature was correlated with levels of choline oxidase expressed and of glycinebetine accumulated in the transformed plants. The induction of homologues of heat shock protein 70 at high temperature was less conspicuous in the transformed plants than in the wild-type plants, suggesting that the transformation alleviated the hightemperature stress.

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light-acclimated and of dark-acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki-Zorba. 1997. Biochim. Biophys. Acta 1335: 1-4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na + and K + salts caused rapid (t 1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCI, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCI and water.

We transformed Arabidopsis thaliana with the codA gene from Arthrobacter globiformis. This gene encodes choline oxidase, the enzyme that converts choline to glycinebetaine. The presence of choline oxidase and glycinebetaine in seeds of transformed lines was confirmed by Western blotting and nuclear magnetic resonance (NMR) spectrometry, respectively. The transformation with the codA gene significantly enhanced the tolerance of seeds to low temperatures, such as 0 °C, during imbibition. The transformation accelerated the germination and growth of seedlings at 10 and 15 °C. It appears that the presence of glycinebetaine in transformed plants enhances their ability to tolerate low-temperature stress during the imbibition and germination of seeds and the growth of seedlings.

We examined the temperature-dependent regulation of the expression of the desA gene, which encodes delta 12 desaturase of Synechocystis PCC6803. The level of desA transcript increased 10-fold within 1 h upon a decrease in temperature from 36 degrees C to 22 degrees C. This suggests that the low-temperature-induced desaturation of membrane lipid fatty acids is regulated at the level of the expression of the desaturase genes. The accumulation of the desA transcript depended on the extent of temperature change over a certain threshold level, but not on the absolute temperature.

Glycinebetaine (betaine), an osmoregulant in halophilic plants, stabilized the evolution of oxygen and the synthesis of ATP by thylakoid membranes from the cyanobacterium Synechocystis PCC6803 when it was present during the preparation and incubation of the thylakoid membranes. Moreover, betaine enhanced the evolution of oxygen and the synthesis of ATP when present during assays. When betaine at 1.0 M was present during the preparation of thylakoid membranes and during the measurement of activity, the rate of evolution of oxygen was equivalent to that of intact cells.

Low-molecular-mass polypeptides in spinach photosystem II membranes were separated by SDS-polyacrylamide gel electrophoresis. The partial amino acid sequence of one of the polypeptides was determined. Comparison of this sequence with the entire nucleotide sequence of the tobacco chloroplast genome revealed that this polypeptide is encoded in the chloroplast genome. The gene for the polypeptide, designated as psbK, is located between the genes for tRNASer and tRNAGln in the large single-copy region and oriented in the direction opposite to the tRNA genes. The amino acid sequence deduced from the gene indicates that the translation product consists of 98 amino acid residues and its 62nd residue corresponds to the amino-terminus of the mature polypeptide. The putative polypeptide consists of 37 amino acid residues with a molecular mass of 4285 Da and has a single membrane-spanning segment. Northern blot hybridization analysis revealed that psbK was transcribed in the chloroplast.

The interaction of Cl− with the extrinsic proteins of 18 kDa, 24 kDa and 33 kDa in the photosynthetic oxygen-evolution complex was studied by comparing spinach photosystem II particles of different protein compositions. The 33-kDa protein decreased the Cl− concentration optimum for oxygen evolution from 150 to 30 mM, and the 24-kDa protein decreased it from 30 to 10 mM. The 18-kDa protein did not change the optimum Cl− concentration, but sustained oxygen evolution at Cl− concentrations lower than 3 mM. The presence of the 24-kDa and 18-kDa proteins, but not each protein alone, markedly suppressed inactivation of oxygen evolution at a very low Cl− concentration and its restoration by readdition of Cl−.

Treatment of Photosystem II particles from spinach chloroplasts with Triton X-100 with 2.6 M urea in the presence of 200 mM NaCl removed 3 polypeptides of 33 kDa, 24 kDa and 18 kDa, but left Mn bound to the particles. The (urea + NaCl)-treated particles could evolve oxygen in 200 mM, but not in 10 mM NaCl. Mn was gradually released with concomitant loss of oxygen-evolution activity in 10 mM NaCl but not in 200 mM Cl−. The NaCl-treated particles, which contained Mn and the 33-kDa polypeptide but not the 24-kDa and 18-kDa polypeptides, did not lose Mn or oxygen-evolution activity in 10 mM NaCl. These observations suggest that the 33-kDa polypeptide maintains the binding of Mn to the oxygen-evolution system and can be functionally replaced by 200 mM Cl−.

Spinach chloroplasts and their various fractions enriched in either photochemical reaction centre 1 (P700) or reaction centre 2 (P680) have been analyzed for pigment composition by high-performance liquid chromatography (HPLC). The results clearly establish a stoichiometry of chlorophyll (Chl) a'/P700 = 2, where Chl a' represents the C 10 epimer of Chl a. This is the first demonstration of the association of Chl a' with any functional site in vivo. It is further shown that a Chl a' dimer prepared in vitro reproduces fairly well the redox and spectroscopic behaviour of P700. These findings provide strong evidence that a Chl a dimer constitutes P700.

The amino acid sequence of the 33 kDa protein isolated as an extrinsic protein from spinach photosystem II particles was determined by using solid-phase sequencing and conventional procedures. The 33 kDa protein was found to be composed of 248 amino acid residues, to lack histidine and to have a molecular mass of 26 663 Da, which was considerably smaller than the value deduced from SDS-polycrylamide gel electrophoresis. The sequence of the 33 kDa protein was compared with those of the bacterial Superoxide dismutases (SOD) with Mn atoms at an active site. A part of the sequence of the 33 kDa protein was similar to a region in Mn-SODs from Bacillus stearothermophilus and Escherichia coli, which was expected to be the Mn-binding site.

— The induction transient of delayed light of chlorophyll a, excited by repetitive flashes (0.5 ms in duration) and emitted 0.1 - 1.2 ms after the flashes, was measured in system II particles derived from spinach chloroplasts. An uncoupler, gramicidin S, was always added to the particles in order to eliminate the influence of the phosphorylation system on the delayed light and to isolate a direct relationship between the delayed light emission and the primary photochemical reaction, except for the experiments described in the next paragraph. The yield of delayed light emission from the system II particles was found to be about three–times higher than that of chloroplasts on a chlorophyll content basis. System I particles, on the other hand, emitted much weaker delayed light than chloroplasts. Upon intermittent illumination, induction of delayed light in system II particles showed a decrease from the initial rise level to the steady-state level. The initial rise level was the maximum. The fluorescence induction, on the other hand, exhibited an increase from the initial rise level to the maximum steady-state level. The induction of both delayed light emission and fluorescence arrived at their final steady-state levels after the same period of illumination. Induction of delayed light emission was measured under various conditions that changed the oxidation-reduction state of the primary electron acceptor, X, of photoreaction II: by adding an electron acceptor and an inhibitor of electron transport, and by changing the light intensity. The state of A'was monitored by measuring the fluorescence yield. The yield of delayed light emission excited by each flash was found to depend on the amount of oxidized form of X present before the flash. To examine the role of the primary electron donor Y of photoreaction II in delayed light emission, effects of electron donors of photoreaction II such as Mn2+, hydroquinone and p-phenylenediamine were investigated. These agents were found to markedly decrease the yield of delayed light emission without altering the pattern of its induction. They had little effect on the induction of fluorescence. These findings are interpreted by a mechanism in which transformation of the reaction center from the form (X-Y+) into (X Y) produces a singlet excitation of chlorophyll a that is the source of millisecond delayed light emission. This reaction is probably non–physiological and must be very slow if compared to the transformation of (X-Y+) into (X-Y). Since the form (X-Y+) is produced when the excitation is transferred to the reaction center in the form (XY), it is expected in this scheme that the yield of delayed light emission should depend on the amount of the form (X Y) present before the excitation flashes. Electron donors stimulate transformation of the reaction center from (X-Y+) into (X-Y). Since this reaction competes with the process of delayed light emission, electron donors are expected to suppress delayed light emission.

— –Fraction-1 particles were prepared by passing spinach chloroplasts three times through the French pressure cell and centrifuging in a sucrose gradient. With the electron donor DAD (diaminodurol or 2,3,5,6-tetramethyl-p-phenylenediamine) and ascorbate, a light-induced difference spectrum revealed the oxidation of both cytochrome f and P700 upon illumination of these particles. The oxidation of cytochrome f was completed in less than 0.5 msec. P700 and cytochrome f thus seem to be tightly bound to each other in these particles. Addition of Triton X-100 abolished the fast oxidation of cytochrome f but not that of P700. Artificial electron donors such as DAD, DCIP (2,6-dichlorophenol indophenol), and PMS (N-methylphenazonium methosulfate) were good electron donors for photoreaction 1 in these particles, while neither plastocyanin, Porphyra cytochrome 553, nor Euglena cytochrome-552 reduced P700 efficiently. However, after treatment of fraction 1 particles with Triton X-100 reduced DAD, DCIP and PMS were no longer efficient electron donors, while plastocyanin and the algal cytochromes were highly active in reducing P700. Mammalian cytochrome c was not a good electron donor either before or after Triton treatment. Measurements of the effectiveness of P700 reduction as a function of concentration in Triton-treated particles showed plastocyanin to be about four times more active than Porphyra or Euglena cytochromes which in turn were about fourteen times more active than mammalian cytochrome c. Recent studies by Murata and Brown have shown that plastocyanin is not required for the reduction of NADP in these particles with DCIP and isoascorbate as electron donors. The present investigation and that of Murata and Brown indicate that disintegration of chloroplasts with the French pressure cell and centrifugation in a sucrose gradient is the best method to separate system-1 particles having an electron-transport system in almost the native state as in chloroplasts.

— Highly purified Chi a and b were prepared from spinach leaves in a short time by a combined use of the column chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B. The former chromatography eliminated carotenoids, phaeophytin and chlorophyllide, and the latter chromatography efficiently separated Chi a and b.