Regulation Of Photosystem Research Articles

Photosystem stoichiometry adjustment is a photoreceptor-mediated process in Arabidopsis

Plant growth under spectrally-enriched low light conditions leads to adjustment in the relative abundance of the two photosystems in an acclimatory response known as photosystem stoichiometry adjustment. Adjustment of photosystem stoichiometry improves the quantum efficiency of photosynthesis but how this process perceives light quality changes and how photosystem amount is regulated remain largely unknown. By using a label-free quantitative mass spectrometry approach in Arabidopsis here we show that photosystem stoichiometry adjustment is primarily driven by the regulation of photosystem I content and that this forms the major thylakoid proteomic response under light quality. Using light and redox signaling mutants, we further show that the light quality-responsive accumulation of photosystem I gene transcripts and proteins requires phytochrome B photoreceptor but not plastoquinone redox signaling as previously suggested. In far-red light, the increased acceptor side limitation might deplete active photosystem I pool, further contributing to the adjustment of photosystem stoichiometry.

Yang cycle enzyme DEP1: its moonlighting functions in PSI and ROS production during leaf senescence

Ethylene-mediated leaf senescence and the compromise of photosynthesis are closely associated but the underlying molecular mechanism is a mystery. Here we reported that apple DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (MdDEP1), initially characterized to its enzymatic function in the recycling of the ethylene precursor SAM, plays a role in the regulation of photosystem I (PSI) activity, activating reactive oxygen species (ROS) homeostasis, and negatively regulating the leaf senescence. A series of Y2H, Pull-down, CO-IP and Cell-free degradation biochemical assays showed that MdDEP1 directly interacts with and dephosphorylates the nucleus-encoded thylakoid protein MdY3IP1, leading to the destabilization of MdY3IP1, reduction of the PSI activity, and the overproduction of ROS in plant cells. These findings elucidate a novel mechanism that the two pathways intersect at MdDEP1 due to its moonlighting role in destabilizing MdY3IP1, and synchronize ethylene-mediated leaf senescence and the compromise of photosynthesis.

Regulation of photosystem I-light-harvesting complex I from a red alga Cyanidioschyzon merolae in response to light intensities.

Photosynthetic organisms use different means to regulate their photosynthetic activity in respond to different light conditions under which they grow. In this study, we analyzed changes in the photosystem I (PSI) light-harvesting complex I (LHCI) supercomplex from a red alga Cyanidioschyzon merolae, upon growing under three different light intensities, low light (LL), medium light (ML), and high light (HL). The results showed that the red algal PSI-LHCI is separated into two bands on blue-native PAGE, which are designated PSI-LHCI-A and PSI-LHCI-B, respectively, from cells grown under LL and ML. The former has a higher molecular weight and binds more Lhcr subunits than the latter. They are considered to correspond to the two types of PSI-LHCI identified by cryo-electron microscopic analysis recently, namely, the former with five Lhcrs and the latter with three Lhcrs. The amount of PSI-LHCI-A is higher in the LL-grown cells than that in the ML-grown cells. In the HL-grown cells, PSI-LHCI-A completely disappeared and only PSI-LHCI-B was observed. Furthermore, PSI core complexes without Lhcr attached also appeared in the HL cells. Fluorescence decay kinetics measurement showed that Lhcrs are functionally connected with the PSI core in both PSI-LHCI-A and PSI-LHCI-B obtained from LL and ML cells; however, Lhcrs in the PSI-LHCI-B fraction from the HL cells are not coupled with the PSI core. These results indicate that the red algal PSI not only regulates its antenna size but also adjusts the functional connection of Lhcrs with the PSI core in response to different light intensities.

The role of water-water cycle in regulating the redox state of photosystem I under fluctuating light

The regulation of photosystem I (PSI) redox state under fluctuating light was investigated for four species using P700 measurement and electrochromic shift analysis. Species included the angiosperms Camellia japonica, Bletilla striata and Arabidopsis thaliana and the fern Cyrtomium fortunei. For the first seconds after transition from low to high light, all species showed relatively low levels of the proton gradient (ΔpH) across the thylakoid membranes. At this moment, PSI was highly oxidized in C. japonica and C. fortunei but was over-reduced in B. striata and A. thaliana. In B. striata and A. thaliana, the redox state of PSI was largely dependent on ΔpH. In contrast, the rapid oxidation of P700 in C. japonica was relatively independent of ΔpH, but was mainly dependent on the outflow of electrons to O2 via the water-water cycle. In the fern C. fortunei, PSI redox state was rapidly regulated by the fast photo-reduction of O2 rather than the ΔpH. These results indicate that mechanisms regulating PSI redox state under fluctuating light differ greatly between species. We propose that the water-water cycle is an important mechanism regulating the PSI redox state in angiosperms.

Regulation of photosystem I light harvesting by zeaxanthin.

In oxygenic photosynthetic eukaryotes, the hydroxylated carotenoid zeaxanthin is produced from preexisting violaxanthin upon exposure to excess light conditions. Zeaxanthin binding to components of the photosystem II (PSII) antenna system has been investigated thoroughly and shown to help in the dissipation of excess chlorophyll-excited states and scavenging of oxygen radicals. However, the functional consequences of the accumulation of the light-harvesting complex I (LHCI) proteins in the photosystem I (PSI) antenna have remained unclarified so far. In this work we investigated the effect of zeaxanthin binding on photoprotection of PSI-LHCI by comparing preparations isolated from wild-type Arabidopsis thaliana (i.e., with violaxanthin) and those isolated from the A. thaliana nonphotochemical quenching 2 mutant, in which violaxanthin is replaced by zeaxanthin. Time-resolved fluorescence measurements showed that zeaxanthin binding leads to a previously unrecognized quenching effect on PSI-LHCI fluorescence. The efficiency of energy transfer from the LHCI moiety of the complex to the PSI reaction center was down-regulated, and an enhanced PSI resistance to photoinhibition was observed both in vitro and in vivo. Thus, zeaxanthin was shown to be effective in inducing dissipative states in PSI, similar to its well-known effect on PSII. We propose that, upon acclimation to high light, PSI-LHCI changes its light-harvesting efficiency by a zeaxanthin-dependent quenching of the absorbed excitation energy, whereas in PSII the stoichiometry of LHC antenna proteins per reaction center is reduced directly.

Regulation of Photosystem I Reaction Center Genes in Synechocystis sp. Strain PCC 6803 during Light Acclimation

Cyanobacteria acclimate to changes in incident light by adjusting photosystem stoichiometry through regulation of PSI accumulation. To gain a deeper insight into this control mechanism in Synechocystis sp. strain PCC 6803, we studied the expression and regulation of the psaAB operon, encoding the reaction center proteins of PSI, during the initial stage of acclimation to changes in the intensity and quality of light. The psaAB operon was transcribed as a dicistronic transcript, which was processed into smaller, putatively monocistronic psaA and psaB transcript species. Dark treatment of the cells inhibited the psaAB transcription, whereas re-illumination of dark-adapted cells reactivated the transcription slowly in a process requiring de novo protein synthesis. Transfer of cells from white to orange light, favoring excitation of PSII, stimulated the psaAB transcription, whereas far-red light, primarily exciting PSI, down-regulated the transcription of the psaAB operon. These results, together with down-regulation of psaAB transcription upon the addition of electron transport inhibitors under constant white light illumination, suggested that the photosynthetic redox poise affects the psaAB transcription activity in the light. Pulse-labeling experiments demonstrated that light-induced modulations in the translation rate of the PsaA protein closely parallel the transcription rate of the psaAB operon, indicating that transcriptional regulation plays the major role in determining the content of PSI reaction center proteins and, thereby, PSI complexes, during light acclimation. The scantiness of PsaA translation in darkness despite the abundance of psaA transcripts demonstrated that the comprehensive regulation of PSI accumulation also involves regulation at the level of translation.

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level.

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.

Photosystem I Activity Is Increased in the Absence of the PSI-G Subunit

PSI-G is a subunit of photosystem I in eukaryotes. The function of PSI-G was characterized in Arabidopsis plants transformed with a psaG cDNA in antisense orientation. Several plants with significantly decreased PSI-G protein content were identified. Plants with reduced PSI-G content were indistinguishable from wild type when grown under optimal conditions, despite a 40% reduction of photosystem I. This decrease of photosystem I was correlated with a similar reduction in state transitions. Surprisingly, the reduced photosystem I content was compensated for by a more effective photosystem I because the light-dependent reduction of NADP(+) in vitro was 48% higher. Photosystem I antenna size determined from flash-induced P700 absorption changes did not reveal any significant effect on the size of the photosystem I antenna in the absence of PSI-G, whereas a 17% reduction was seen in the absence of PSI-K. However, nondenaturing green gels revealed that the interaction between photosystem I and the light-harvesting complex I was less stable in the absence of PSI-G. Thus, PSI-G plays a role in stabilizing the binding of the peripheral antenna. The increased activity in the absence of PSI-G suggests that PSI-G could have an important role in regulation of photosystem I.

Complex regulation of Photosystem II protein phosphorylation via redox state of chloroplast

Phosphorylation of photosystem II (PSII) core and light harvesting II complex (LHCII) proteins is regulated in vivo by complex redox systems involving reduction of plastoquinone and cytochrome b6f complex as well as thiol redox state in chloroplasts. This redox regulation is universal in all tested plant species including a model plant, Arabidopsis thaliana. CP43 protein was highly phosphorylated in darkness in several plant species, but some phosphorylated D1 and D2 proteins may also be detected in dark-adapted plants. The shift of leaves from darkness to very dim light best induced dephosphorylation of PSII core phosphoproteins, while further increase in irradiance up to the growth level significantly increased the number of PSII centres with phosphorylated core proteins. Both in vitro and in vivo experiments indicated that the kinase(s) for PSII core phosphoproteins is regulated via reduction of plastoquinone pool that strongly depends on metabolic state of plant leaf. Redox regulation of LHCII protein phosphorylation differs from that of PSII core proteins. The LHCII kinase is activated via reduction of plastoquinone and the cytochrome b6f complex. High redox state of chloroplast, on the other hand, inactivates the phosphorylation of LHCII, disclosing a second regulatory mechanism for LHCII phosphorylation. This novel regulation of LHCII phosphorylation is likely to be mediated by the ferredoxin-thioredoxin system. A cooperative model for the function of these two regulation mechanisms that determine the phosphorylation level of the LHCII proteins in vivo will be presented.

Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s).

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31-35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20-100 mumol photons m(-2) s(-1) as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness.

Structural Modifications of the Photosynthetic Apparatus in the Region of Photosystem I in Nicotiana tabacum as a Consequence of an Increased CO2-Content of the Atmosphere

Nicotiana tabacum was grown with a CO2-content in the atmosphereof 350 ppm and 700 ppm. After three weeksof growth in the respective atmospherewe were able to show that chloroplasts from plants grown under 700 ppm CO2 exhibited an 18% higher photosystem I-mediatedelectron transport activity. Furthermore, we were able to show that in plants grown under the higher CO2-concentration the peptide composition of photosystem I had been quantitatively changed. It appeared that the CPI-peptides were increased, whereasthe LHCPI peptides were decreased. Corresponding to this change in the peptide composition the ratio chlorophyll/carotenoids/protein changes from 1:0.16:3.6 in the control plants to 1:0.23:5 in the “CO2-plants”. The chlorophyll a/b ratio changes from 1.9 in the control plants to 2.3 in the “CO2 plants”. Furthermore, the carotenoid composition appears to be changed, β-carotene increases by 60% as expected from the increased CPI-content. The xanthophylls lutein, violaxanthin, neoxanthin and zeaxanthin increase also but to a lesser extent. Using specific antisera to lipids, we were able to show that photosystem I contains only the membrane lipids monogalactosyldiglyceride and phosphatidylglycerol. Both lipids are specifically bound in a restricted number onto peptides of the photosystem I complex. Both lipids differ with respect to their binding strength on the peptides of photosystem I. The changes in the peptide and lipid composition were established by chemical analyses and by immunological techniques (Western blot) using monospecific antisera to the photosystem I peptides, to the chloroplast lipids as well as to the chloroplast carotenoids. From the quantitative and qualitative analyses of the peptides, carotenoids and lipids, the molecular distribution of the carotenoids and lipids in the CPI and LHCPI complex of photosystem I was established.

Regulation of photosystem II by metabolic and environmental factors

The thylakoid membranes of higher plants possess several mechanisms that control both the distribution and rate of dissipation of absorbed light. These mechanisms, which allow regulation of photosynthetic electron transport in response to alteration in external and internal factors, can be observed as the various processes that quench chlorophyll fluorescence. By using the 'light-doubling techniques’, together with analysis of quenching relaxation, it is possible to assess quantitatively the extents of these regulatory processes and to allow their interrelations to be studied. These techniques can be applied toin vitrosystems or to leaves, and can be particularly useful when applied with electron-transport measurements and when models are used to aid interpretation. Results of quenching analysis at different light intensities in isolated thylakoids, intact chloroplasts, protoplasts, algae and leaves of a variety of species are presented.

Serological Investigations of the Function of Galactolipids in the Thylakoid Membrane

Summary We investigated the influence of monospecific antisera to monogalactosyl diglyceride and to digalactosyl diglyceride on the photosynthetic electron transport in chloroplasts of higher plants. Both antisera inhibit the photoreduction of dichlorophenolindophenol (DCPiP) with water as the native electron donor as well as the reduction of anthraquinone-2-sulfonate with the electron donor couple DCPiP/ascorbate. The degree of inhibition of the galactolipid antisera in the region of photosystem I depends on the pH and the temperature of the reaction assay. Treatment of the chloroplasts with sodium periodate or with lipase results in a complete loss of any inhibition by the galactolipid antisera. Treatment with β-galactosidase, however, had no influence on the reactions with galactolipid antisera. The sites of inhibition of the galactolipid antisera could be localized on the donor side of photosystem I as well as on the donor side of photosystem II. From this we conclude that the mono- and digalactosyl diglyceride molecules that are localized on the stroma side of the membrane are components of the photosystem I- and also of the photosystem II-protein-lipid-complex. We may conclude that there are obviously interactions between the galactolipid molecules and the photosynthetically active proteins, since the binding of antibodies leads to a partial blocking of electron transport.

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A 2 from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A 2 which destroyed all phospholipids in thylakoid membranes stimulated the NADP + reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A 2 may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP + photoreduction was inhibited whilst the methyl viologen-mediated O 2 uptake was stimulated. A subsequent addition of pancreatic phospholipase A 2 (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O 2 uptake and restored NADP + photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.

Measurements of cytochrome f and P-700 in intact leaves of Sinapis alba grown under high-light and low-light conditions

The oxidation and reduction of cytochrome f and P-700 is measured spectrophotometrically in leaves of low-light and high-light plants. After illumination with red light, an induction phenomenon for cytochrome f oxidation is observed which indicates a regulation of photosystem I activity through energy distribution between the pigment systems by the energy state of the membrane. After far-red excitation the reduction of cytochrome f in the dark is much slower in low-light leaves. This shows that cyclic electron transport is not improved in low-light plants under these conditions. P-700 is oxidized on excitation with far-red light. However, with high intensities of far-red light, P-700 is partially reduced again which is due to a low extent of photosystem II excitation with the far-red used in the experiments. The low-light leaves show greater sensitivity of photosystem II to this excitation. The initial rate of the cytochrome f oxidation-rate is the same in low-light and high-light leaves. This shows that several P-700 are connected with only one electron transport chain. The consequences of these results concerning the tripartite concept and the photosynthetic unit are discussed. In the high-light plants the experimental data can be well explained by the tripartite organization of the photosynthetic unit. In low-light plants, however, a multipartite organization has to be postulated. In the partition regions of the grana, several antennae systems I, antennae systems II, and light-harvesting complexes can communicate with one electron transport chain.

Antisera to the Coupling Factor of Photophosphorylation and Its Subunits

Abstract Stroma-freed chloroplasts were extracted with sucrose palmitate-stearate containing buffer. After the addition of dodecyl sulfate and mercaptoethanol to the extract a series of polypeptides was isolated from the mixture by gel filtration. These polypeptides were later used for immunization. Antisera to four polypeptides reacted in the Ouchterlony double diffusion test with authentic coupling factor yielding a precipitation band. According to the observed apparent molecular weights the polypeptides are the α, β , δ and ε subunits of the coupling factor. An antiserum to the γ subunit has been obtained already previously. All antisera inhibit photophosphorylation reactions and electron transport considerably. Addition of gramicidin inhibits photophosphorylation completely whereas gramicidin restores electron transport in the assays with the antisera to the α, β , γ and δ subunit. In the case of the antiserum to the ε subunit gramicidin does not regenerate electron transport. As in the presence of the serum to the ε subunit pH changes in the suspension medium are not observed, this serum seems to open a proton channel. Also, upon addition of dicyclohexyl carbodiimide (DCCD) pH changes in the suspension medium in the assay with antiserum do not reoccur. According to these unexpected results the identity of the antigen with the ε subunit of the coupling factor is not certain. ATP-ase reactions are only inhibited by the antisera to the α and γ subunit and what is thought to be the ε subunit. The antiserum to the α subunit uncouples electron transport as the only one when used in sufficient concentrations. The dosis-effect curves of the inhibition of the electron transport exhibits a maximum. The dosis-effect curves for the other components rise after a lag phase in an approximately hyperbolic manner. The inhibitory action on electron transport is exerted by all antisera in the region of the reaction center I or in its immediate vicinity. This is thought to be due to the fact that a protein of the reation center I is inhibited in its function by the increasing proton concentration inside the thylakoid. The inhibition of electron transport by the antiserum to the ε subunit is considered to be a direct serum effect. Besides the increase in fluorescence yield, due to the inhibition of electron transport in the region of photosystem I, decreases of the fluorescence yield are observed in the presence of DCMU, which do not depend on the redox state of Q but rather on the condition of the thylakoid mem­brane. Moreover, the antisera affect in a differing manner the energy spill-over of excitation from photosystem II to photosystem I.

Inhibition and uncoupling of photophosphorylation in isolated chloroplasts by organotin, organomercury and diphenyleneiodonium compounds.

1. Trialkyltin, triphenyltin and diphenyleneiodonium compounds inhibited ADP-stimulated O(2) evolution by isolated pea chloroplasts in the presence of phosphate or arsenate. Tributyltin and triphenyltin were the most effective inhibitors, which suggests a highly hydrophobic site of action. Phenylmercuric acetate was a poor inhibitor of photophosphorylation, which suggests that thiol groups are not involved. 2. Triethyltin was a potent uncoupler of photophosphorylation by isolated chloroplasts in media containing Cl(-), but had little uncoupling activity when Cl(-) was replaced by NO(3) (-) or SO(4) (2-), which are inactive in the anion-hydroxide exchange. It is suggested that uncoupling by triethyltin is a result of the Cl(-)-OH(-) exchange together with a natural uniport of Cl(-). Tributyltin, triphenyltin and phenylmercuric acetate had low uncoupling activity, probably because in these compounds the uncoupling activity is partially masked by inhibitory effects. 3. At high concentrations the organotin compounds caused inhibition of electron transport uncoupled by carbonyl cyanide m-chlorophenylhydrazone or NH(4)Cl. At these high concentrations the organotin compounds may be producing a detergent-like disorganization of the membrane structure. In contrast, diphenyleneiodonium sulphate inhibited uncoupled electron transport at low concentrations; however, this inhibition is less than the inhibition of photophosphorylation, which suggests that the compound also inhibits the phosphorylation reactions as well as electron transport. 4. The effects of these compounds on basal electron transport were complex and depended on the pH of the reaction media. However, they can be explained on the basis of three actions: inhibition of the phosphorylation reactions, uncoupling and direct inhibition of electron transport. 5. The inhibition of cyclic photophosphorylation in the presence of phenazine methosulphate by diphenyleneiodonium sulphate shows that it inhibits in the region of photosystem 1.

A chloroplast membrane conformational change activated by electron transport between the region of photosystem II and plastoquinone

Various partial redox reactions involved in photosynthetic electron transport were studied in relation to the electron transport dependent incorporation of the water soluble chemical modifier, diazonium benzene sulfonic acid (DABS)* into chloroplast membranes. This electron transport dependent diazonium incorporation reflects a conformational change (unspecified at this time) in membrane components. The redox reaction(s) responsible for this conformational change was shown to be localized after the site of DCMU inhibition but before plastoquinone by the following evidence: 1. Electron transport from water to lipophilic “Class III” electron acceptors such as dimethyl benzoquinone and high concentrations of dibromothymoquinone potentiate the extra DABS binding to the membranes. These compounds are reduced prior to or at the plastoquinone site. 2. Electron transfer from water to silicomolybdate plus ferricyanide, a DCMU insensitive reaction, does not result in the incremental diazonium binding. 3. Photosystem I cyclic electron flow mediated by menadione (anaerobic), which requires participation of plastoquinone does not give the extra diazonium binding.

Correlations between photophosphorylation and light-induced conformational changes of chloroplasts in whole cells of the halophilic green alga Dunaliella parva

Summary In whole cells of the extremely halophilic unicellular green alga Dunaliella parva slow changes in apparent absorbance, peaking at 535 nm, can be separated from the well-known fast 515 nm shift. It is concluded that this 535 nm change is caused by light scattering and indicates conformational changes (shrinkage) of the chloroplasts. Properties of the 535 nm change are compared with those of the light-induced increase of the endogenous ATP-level in vivo. From similar responses of both reactions to electron transport inhibitors and uncouplers it is concluded that the 535 nm change is correlated to light-induced ATP-formation and can be taken as a useful indicator for photophosphorylation in whole cells. In far-red light, exciting predominantly photosystem I, neither distinct conformational changes of chloroplasts nor an increase of the ATP-level could be observed. In red light, exciting both photosystem II and I, both the 535 nm change and ATP-formation are strongly suppressed by DCMU. These results suggest that in the presence of O2 cyclic photophosphorylation plays a minor role in Dunaliella. DBMIB, which inhibits photosynthetic electron transport between plastoquinone and cytochrome f, inhibits both ATP-formation and the 535 nm change. However, both reactions could be reactivated in vivo 1) by the addition of the Hill-reagent benzoquinone, which restored O2-evolution and 2) by the addition of the electron donor DAD, which caused O2-uptake. The former system was sensitive, the latter one insensitive to DCMU. FCCP inhibited ATP-formation and the 535 nm change in both systems. Photosynthetic control (stimulation of electron transport by FCCP) was observed only in the DBMIB/benzoquinone-system. These results seem to indicate two different ATP-sites in Dunaliella. One is located between the site of water oxidation and photosystem II. The second one is located after the DBMIB-inhibition site in the region of photosystem I.