Control of the Quantum Efficiencies of Photosystems I and II, Electron Flow, and Enzyme Activation following Dark-to-Light Transitions in Pea Leaves: Relationship between NADP/NADPH Ratios and NADP-Malate Dehydrogenase Activation State

The quantum efficiencies of photosystems I and II (PSI and PSII), [NADP]/[NADPH] ratios, and the activities of chloroplastic fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were measured in intact pea (Pisum sativum L.) leaves in air following the transition from darkness to 750 microeinsteins per square meter per second irradiance. PSII efficiency declined from a low value to a minimum within the first 10 to 15 seconds of irradiance, after which it increased progressively to the steady-state value. The resistance of electron flow between the photosystems was high at this time, but it was not the principal factor limiting electron flow. Oxidation of P700 was restricted by acceptor side processes for approximately the first 60 seconds of illumination. Once the acceptor side limitation was relieved, the oxidation state of P700 was used to estimate the quantum efficiency of electron transport by PSI. This was observed to increase progressively with time. The quantum efficiencies of both photosystems increased in parallel, consistent with a predominant role for noncyclic electron transport. Fructose-1,6-bisphosphatase activity increased in an approximately sigmoidal fashion with time of irradiance, paralleling the changes in the quantum efficiencies of the photosystems. In contrast, the activation of NADP-malate dehydrogenase did not show a lag period but increased with time, reaching a maximum value at about 50 seconds of illumination, after which it declined. The NADP pool was not extensively reduced during the first 10 seconds of illumination, but became so subsequently. It remained in the reduced state until about 60 seconds of illumination and then became relatively oxidized. The empirical relationship between NADP-malate dehydrogenase activity and the reduction state of the NADP pool supports the suggestion that NADP-malate dehydrogenase activity is a useful estimate of the reduction state of the stroma.     

Time of Appearance of Photosystems I and II in Chloroplasts of Greening Jack Bean Leaves

The time of appearance of photochemical activities togetherwith the chlorophyll-protein complexes associated with photosystemsI and II was followed in greening primary leaves of jack bean(Canavalia ensiformis (L.) DC.). When greening of the etiolatedleaves occurred under high relative humidity conditions, nolag phase in chlorophyll-accumulation was observed. These environmentalconditions also promoted rapid and uniform development of thechloroplast lamellar system. Chlorophyll-protein complexes ofthe lamellae were separated by means of sodium dodecylsulphate-polyacrylamidegel electrophoresis and by hydroxylapatite chromatography. Thephotosystem II complex, containing chlorophyll a/b-protein,was detected after 2 h of greening. Its appearance was correlatedwith a sharp decrease in the chlorophyll a/b ratio and withthe onset of oxygen evolution. Subsequently, the photosystemI complex, containing a chlorophyll a-protein, was detected—after6 h of illumination. Its appearance coincided with the detectionof light-induced bleaching of P700 and the beginning of a risingchlorophyll a/b ratio that plateaued some time later.     

Action Spectra of Photosystems I and II in State 1 and State 2 in Intact Sugar Maple Leaves

Photochemical activity, measured as energy storage of photosystems I (PSI) and II (PSII) together and individually, is studied in sugar maple (Acer saccharum Marsh.) leaves in the spectral range between 400 and 700 nm in state 1 and state 2. Total photochemical activity remains the same in both state 1 and state 2 between 580 and 700 nm, but it is lower in state 2 between 400 and 580 nm. Both PSI and PSII activities change significantly during the state transition due to the migration of light-harvesting chlorophyll a/b protein complex of PSII (LHCII). In the action spectra of PSI and PSII, peak positions vary depending on the association or dissociation of LHCII, except for the peak at 470 nm in the PSII spectrum. PSII activity is about 3 times higher than or equal to PSI in state 1 or state 2, respectively, over most of the spectrum except in the blue and far-red regions. At 470 nm, PSII activity is 8 or 1.6 times higher than PSI in state 1 or state 2, respectively. The amplitude of LHCII coupling-induced change is the same in both PSI and PSII between 580 and 700 nm, but it is less in PSI than in PSII between 400 and 580 nm, which explains the lower photochemical activity of the leaf in state 2 than in state 1. This may be due to a decrease in energy transfer efficiency of carotenoids to chlorophylls in LHCII when it is associated with PSI.     

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO(2)-Free Air : Evidence for Cyclic Electron Flow in Vivo

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO₂ compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO₂ had been removed. P700 was more oxidized at any measured irradiance in CO₂-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO₂-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO₂-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO₂-free air, with an activation state 50% of maximum. We conclude that, at the CO₂ compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane.     

Photoproduction and Detoxification of Hydroxyl radicals in Chloroplasts and Leaves and Relation to Photoinactivation of Photosystems I and II

The light-dependent production of hydroxyl radicals (HO{dot})by thylakoids, chloroplasts and leaves of Spinacia oleraceawas investigated using dimethylsulfoxide as HO{dot} trappingagent. Maximum rates of HO{dot} production by thylakoids asindicated by the formation of methane sulfinic acid were observedunder aerobic conditions in the absence of added electron acceptors.They were higher than 2 µmol (mg Chl h)^–1. Saturationof HO{dot} production occurred at the low photon flux densityof 100 µmol m^–2 s^–1. Trapping of HO{dot} bydimethylsulfoxide suppressed, but did not eliminate light-dependentinactivation of PSI and II suggesting that HO{dot} formationcontributed to the photosensitivity of isolated thylakoids.DCMU inhibited HO{dot} formation. Importantly, methylviologendecreased HO{dot} formation in the absence, but stimulated itin the presence of Fe³⁺. In intact chloroplasts, HO{dot} formation became appreciableonly after KCN had been added to inhibit effective H₂O₂ scavengingby ascorbate peroxidase. It was stimulated by ferrisulfate,but not by ferricyanide which does not penetrate the chloroplastenvelope. Infiltrated spinach leaves behaved similar in principleto intact chloroplasts in regard to HO{dot} formation but HO{dot}production was very slow if detectable at all by the formationof methylsulfinic acid indicating effective radical detoxification. HO{dot} formation is interpreted to be the result of a Fenton-typereaction which produces HO{dot} in chloroplasts from H₂O₂ andreduced ferredoxin, when O₂ is electron acceptor in the Mehlerreaction and radical detoxification reactions are inhibited. (Received November 13, 1996; Accepted April 23, 1996)     

Immunocytochemical and Cytochemical Localization of Photosystems I and II

Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P₇₀₀ chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P₇₀₀ chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae.     

Changing concepts about the distribution of Photosystems I and II between grana-appressed and stroma-exposed thylakoid membranes

Thylakoid membranes of higher plants and some green algae, which house the light-harvesting and energy transducing functions of the chloroplast, are structurally unique. The concept of the photosynthetic unit of the 1930s (Robert Emerson, William Arnold and Hans Gaffron), needing one reaction center per hundreds of antenna molecules, was modified by the discovery of the Enhancement effect in oxygen evolution in two different wavelengths of light (Robert Emerson and his coworkers) in the late 1950s, followed by the 1960 Z scheme of Robin Hill and Fay Bendall. It was realized that two light reactions and two pigment systems were needed for oxygenic photosynthesis. Changing ideas about the distribution of Photosystem II (PS II) and PS I between the green-appressed and stroma-exposed thylakoid membrane domains, which led to the concept of lateral heterogeneity, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Action Spectra for Photosystems I and II in Formaldehyde Fixed Algae

Action spectra were obtained for photosystems I and II in chemically fixed algal cells and for photosystem I in unfixed lysozyme treated cells. Untreated algal cells yielded neither of the 2 light reactions with the reaction mixtures used. The action spectra for photosystem I in the blue-green alga Anacystis nidulans and red alga Porphyridium cruentum follow the absorption spectrum of chlorophyll a with a small peak in the region of the accessory pigments. In the green alga Chlorella pyrenoidosa the photosystem I action spectrum follows the absorption spectrum of chlorophyll a. Photosystem II action spectra in A. nidulans and P. cruentum follow the absorption spectra of the accessory pigments while that in C. pyrenoidosa is shifted slightly toward the blue spectral region. These results provide additional evidence that formaldehyde fixed cells are valid models for studying the light reactions of photosynthesis.     

The Quantum Yield of Photoinactivation of Photosystem II in Pea Leaves is Greater at Low Than High Photon Exposure

The quantum yield of PSII photoinactivation in pea leaves atlow photon exposure was 3 x 10^-7 mol PSII per mol photons absorbedby PSII and 2.5 x 10^-8 at high photon exposures (mol photonsm^-2), regardless of photomodulation of chloroplasts. We postulatethat the doseresponse behaviour of PSII photoinactivation, whichdepends on the number of photons absorbed, results from thebalance of light consumption with supply. (Received July 18, 1995; Accepted August 8, 1995)     

Fluorescent kinetics of chlorophyll in Photosystems I and II enriched fractions of spinach

The fluorescent emission kinetics of spinach subchloroplast Photosystems I and II particles have been studied on a picosecond time scale. Using picosecond laser pulses and an optical Kerr gate, the fluorescent decay times are measured to be 60±10 ps, and 200±20 ps for Photosystems I and II, respectively. The quantum yields are calculated to be 0.004 for Photosystem I and 0.013 for Photosystem II. Theory of exciton energy transfer and trapping is applied for the determination of intermolecular potential energy in the photosystems.     

Antenna Size Dependency of Photoinactivation of Photosystem II in Light-Acclimated Pea Leaves

Utilization of absorbed light energy by photosystem (PS) II for O2 evolution depends on the light-harvesting antenna size, but the role of antenna size in the photoinactivation of PSII seems controversial. To address this controversy, pea (Pisum sativum L.) plants were grown in low (50 [mu]mol m-2 s-1) or high (650 [mu]mol m-2 s-1) light. The doubled functional antenna size of PSII in low light allows each PSII to utilize twice as many photons at given flash light energies for O2 evolution. The application of a target theory to depict the photon dose dependency of PSII photoinactivation measured by repetitive-flash O2 yield and the ratio of variable to maximal chlorophyll fluorescence indicates that photoinactivation of PSII is probably a single-hit process in which repair or photoprotective mechanisms are only slightly involved. Furthermore, the exacerbation of photoinactivation of PSII with greater antenna size under anaerobic conditions strongly indicates that photoinactivation of PSII depends on antenna size.     

Effects of O(2) and CO(2) Concentrations on Quantum Yields of Photosystems I and II in Tobacco Leaf Tissue

The interactive effects of irradiance and O₂ and CO₂ levels on the quantum yields of photosystems I and II have been studied under steady-state conditions at 25°C in leaf tissue of tobacco (Nicotiana tabacum). Assessment of radiant energy utilization in photosystem II was based on changes in chlorophyll fluorescence yield excited by a weak measuring beam of modulated red light. Independent estimates of photosystem I quantum yield were based on the light-dark in vivo absorbance change at 830 nanometers, the absorption band of P700⁺. Normal (i.e. 20.5%, v/v) levels of O₂ generally enhanced photosystem II quantum yield relative to that measured under 1.6% O₂ as the irradiance approached saturation. Photorespiration is suspected to mediate such positive effects of O₂ through increases in the availability of CO₂ and recycling of orthophosphate. Conversely, at low intercellular CO₂ concentrations, 41.2% O₂ was associated with lower photosystem II quantum yield compared with that observed at 20.5% O₂. Inhibitory effects of 41.2% O₂ may occur in response to negative feedback on photosystem II arising from a build-up in the thylakoid proton gradient during electron transport to O₂. Covariation between quantum yields of photosystems I and II was not affected by concentrations of either O₂ or CO₂. The dependence of quantum yield of electron transport to CO₂ measured by gas exchange upon photosystem II quantum yield as determined by fluorescence was unaffected by CO₂ concentration.     

The Involvement of the Complexes of Photosystems in the Organization of Chloroplast Ultrastructure in Pea Mutants

We studied the involvement of pigment-protein complexes of photosystems (PS) in the development and spatial arrangement of thylakoids in chloroplasts of pea (Pisum sativum L.) leaves. The initial line (cv. Torsdag) and its mutants, chlorotica 2004 displaying primary disturbances in the PSI reaction centers and chlorotica 2014 containing only 50% of chlorophyll and, as a sequence, the reduced amount of all pigment-protein complexes. A proportional decrease in the content of PSI and PSII complexes in the chlorotica 2014 mutant resulted in a partial reduction of the whole chloroplast membrane system, whereas grana and stroma thylakoid regions were well developed. In contrast, a loss of only 20% of chlorophyll and destruction of PSI complexes in the chlorotica 2004 mutant by 50% resulted in the destruction of stroma thylakoid regions and disturbed longitudinal thylakoid and grana orientation. It was concluded that protein-protein interactions in pigment-protein complexes played a key role in the structure of thylakoid membranes and their longitudinal orientation.     

Differential Effects of Nitrogen Limitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae

The effects of nitrogen starvation on photosynthetic efficiency were examined in three unicellular algae by measuring changes in the quantum yield of fluorescence with a pump-and-probe method and thermal efficiency (i.e. the percentage of trapped energy stored photochemically) with a pulsed photoacoustic method together with the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea to distinguish photosystems I and II (PSI and PSII). Measured at 620 nm, maximum thermal efficiency for both photosystems was 32% for the diatom Thalassiosira weissflogii (PSII:PSI ratio of 2:1), 39% for the green alga Dunaliella tertiolecta (PSII:PSI ratio of 1:1), and 29% for the cyanobacterium Synechococcus sp. PCC 7002 (PSII:PSI ratio of 1:2). Nitrogen starvation decreased total thermal efficiency by 56% for T. weissflogii and by 26% for D. tertiolecta but caused no change in Synechococcus. Decreases in the number of active PSII reaction centers (inferred from changes in variable fluorescence) were larger: 86% (T. weissflogii), 65% (D. tertiolecta), and 65% (Synechococcus). The selective inactivation of PSII under nitrogen starvation was confirmed by independent measurements of active PSII using oxygen flash yields and active PSI using P700 reduction. Relatively high thermal efficiencies were measured in all three species in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting the potential for significant cyclic electron flow around PSI. Fluorescence or photoacoustic data agreed well; in T. weissflogii, the functional cross-sectional area of PSII at 620 nm was estimated to be the same using both methods (approximately 1.8 x 102 A2). The effects of nitrogen starvation occur mainly in PSII and are well represented by variable fluorescence measurements.     

Expressing an RbcS Antisense Gene in Transgenic Flaveria bidentis Leads to an Increased Quantum Requirement for CO2 Fixed in Photosystems I and II

It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469-477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.     

Relationship between Photosynthetic Electron Transport and Stromal Enzyme Activity in Pea Leaves : Toward an Understanding of the Nature of Photosynthetic Control

The responses of the quantum efficiencies of photosystem (PS) II and PSI measured in vivo simultaneously with estimations of the activities and activation states of NADP-malate dehydrogenase, chloroplast fructose-1,6-bisphosphatase, and ribulose-1,5-bisphosphate carboxylase were used to study the relationship between electron transport and carbon metabolism. The effects of varying irradiance and CO₂ partial pressure on the relationship between the quantum efficiencies of PSI and II, and the activity of these enzymes shows that the interrelationships vary according to the limitations placed on the system. The relationship between the quantum efficiencies of PSII and PSI was linear in most situations. In response to increasing irradiance, the activity of all three enzymes increased. In the case of NADP-malate dehydrogenase this increase was well correlated with the estimated flux of electrons through PSI and PSII. The other two enzymes showed a more complex relationship with the estimated flux of electrons through both photosystems. These relationships are consistent with the known interactions between these stromal enzymes and the thylakoids. The response to varying CO₂ partial pressure is more complex. The efficiencies of PSI and II declined with decreasing CO₂ partial pressure and the activity of each enzyme varied uniquely. However, there are clear correlations between the activities of the enzymes and the flux of electrons through the photosystems. In contrast to the data obtained under conditions of varying irradiance, there is clear evidence of photosynthetic control of electron transport when the CO₂ concentration is varied.     

Photoinhibition of Reaction Centers of Photosystems I and II in Intact Bryopsis Chloroplasts under Anaerobic Conditions

Illumination of intact Bryopsis corticulans chloroplasts under anaerobic conditions induced a decline of chlorophyll fluorescence and photoinhibition of Photosystems I and II. The time course of the light-induced decline of chlorophyll fluorescence and the decreases of activities of reactions sensitized by Photosystems I and II were compared. Photosystem I activity decreased in parallel with the disappearance of active P700. The time course of the destruction of the reaction center of Photosystem II was similar to that of photoinhibition of 2,6-dichlorophenolindophenol-Hill reaction.

It appears that the initial events in photoinhibition are the destruction of the reaction centers of Photosystems I and II and that the reaction centers that are inhibited become quenchers of chlorophyll fluorescence.

Effects of inhibitors of electron transfer and of an electron donor to Photosystem I showed that photoinhibition was related to Photosystem I activity.