Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - PPi inorganic pyrophosphate - PGA 3-phosphoglycerate - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(aminoethyl ether) - N,N tetraacetic acid     

Characterisation of the effects of Antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts

High energy state quenching of chlorophyll fluorescence (qE) is inhibited by low concentrations of the inhibitor antimycin A in intact and osmotically shocked chloroplasts isolated from spinach and pea plants. This inhibition is independent of any effect upon pH (as measured by 9-aminoacridine fluorescence quenching). A dual control of qE formation, by pH and the redox state of an unidentified chloroplast component, is implied. Results are discussed in terms of a role for qE in the dissipation of excess excitation energy within photosystem II.Abbreviations 9-AA_max = Maximum yield of 9-aminoacridine fluorescence - DCMU = 3(3,4-dichlorophenyl)-1,1-dimethylurea; F_max ± Maximum yield of chlorophyll fluorescence - hr = hour - PAR = Photosynthetically Active Radiation - Q_A = Primary stable electron acceptor within photosystem II - qE = High energy state quenching of chlorophyll fluorescence - qI = quenching of chlorophyll fluorescence related to photoinhibition - qP = Quenching of chlorophyll fluorescence by oxidised plastoquinone - qQ = photochemical quenching of chlorophyll fluorescence - qR = (F_max—maximum level of chlorophyll fluorescence induced by the addition of saturating DCMU) - qT = Quenching of chlorophyll fluorescence attributable to state transitions     

The role of monovalent cations for photosynthesis of isolated intact chloroplasts

The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l^-1), CO₂-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K⁺, Rb⁺, or Na⁺ (50–100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in low-salt medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K⁺>Rb⁺>Na⁺). Of the divalent cations tested, Mg²⁺ also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO₂-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO₂-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg²⁺ but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg²⁺ in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50–100 mM) or Mg²⁺ (20–50 mM).The relevance of a light-triggered K⁺/H⁺ exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg²⁺ concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes.     

Fractional control of photosynthesis by the QB protein,the cytochrome f/b 6 complex and other components of the photosynthesic apparatus

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the Q_B protein of the electron-transport chain. Fractional control by the cytochrome f/b ₆ complex was insignificant under these conditions. Control by the cytochrome f/b ₆ complex dominated at high energy fluence rates where the contribution to control of the Q_B protein was very small. Uncoupling shifted control from the cytochrome f/b ₆ complex to the Q_B protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b ₆ complex and of the Q_B protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO₂ saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b ₆ complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the Q_B protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2,4-dinitro phenylether of 2-iodo-4-nitrothymol - pCMBS p-chloromercuribenzosulfonate     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO₂ at 20°C at 100, 300 and 750 mol·m^–2·s^–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m^–2·s^–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C _infRubisco ^supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C _infRubisco ^supA increases as incident irradiance rises, (iii) When low-light (100 mol·m^–2·s^–1)-grown plants are exposed to high (750–1000 mol·m^–2·s^–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m^–2·s^–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m^–2·s^–1), C _infRubisco ^supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C _infRubisco ^supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A rate of photosynthesis - C _infRubisco ^supA flux control coefficient of Rubisco for photosynthesis - c_i internal CO₂ concentration - qE energy-dependent quenching of chlorophyll fluorescense - qQ photochemical quenching of chlorophyll fluorescence - NADP-MDH NADP-dependent malate dehydrogenase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose-1,5-bisphosphate This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Potential mechanisms of low-temperature tolerance of C<Subscript>4</Subscript> photosynthesis in<Emphasis Type="Italic"> Miscanthus</Emphasis> ×<Emphasis Type="Italic"> giganteus</Emphasis>: an in vivo analysis

Miscanthus × giganteus (Greef & Deuter ex Hodkinson & Renvoize) is unique among C₄ species in its remarkable ability to maintain high photosynthetic productivity at low temperature, by contrast to the related C₄ NADP-malic enzyme-type species Zea mays L. In order to determine the in vivo physiological basis of this difference in photosynthesis, water vapor and CO₂ exchange and modulated chlorophyll fluorescence were simultaneously monitored on attached leaf segments from plants grown and measured at 25/20°C or 14/11°C (day/night temperature). Analysis of the response of photosynthesis to internal CO₂ concentration suggested that ribulose bisphosphate carboxylase/oxygenase (Rubisco) and/or pyruvate orthophosphate dikinase (PPDK) play a more important role in determining the response to low temperature than does phosphoenolpyruvate carboxylase (PEPc). For both species at both temperatures, the linear relationship between operating efficiency of whole-chain electron transport through photosystem II (_PSII) and the efficiency of CO₂ assimilation (_CO2) was unchanged and had a zero intercept, suggesting the absence of non-photosynthetic electron sinks. The major limitation at low temperature could not be solely at Rubisco or at any other point in the Calvin cycle, since this would have increased leakage of CO₂ to the mesophyll and increased _PSII/_CO2. This in vivo analysis suggested that maintenance of high photosynthetic rates in M. × giganteus at low temperature, in contrast to Z. mays, is most likely the result of different properties of Rubisco and/or PPDK, reduced susceptibility to photoinhibition, and the ability to maintain high levels of leaf absorptance during growth at low temperature.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with ‘antisense’ rbcS

Transgenic tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to produce a series of plants with a progressive decrease in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been used to investigate the contribution of Rubsico to the control of photosynthesis at different irradiance, CO₂ concentrations and vapour-pressure deficits. Assimilation rates, transpiration, the internal CO₂ concentration and chlorophyll fluorescence were measured in each plant. (i) The flux-control coefficient of Rubisco was estimated from the slope of the plot of Rubisco content versus assimilation rate. The flux-control coefficient had a value of 0.8 or more in high irradiance, (1050 mol·m^–2·s^–1), low-vapour pressure deficit (4 mbar) and ambient CO₂ (350 bar). Control was marginal in enhanced CO₂ (450 bar) or low light (310 mol·m^–2·s^–1) and was also decreased at high vapour-pressure deficit (17 mbar). No control was exerted in 5% CO₂. (ii) The flux-control coefficients of Rubisco were compared with the fractional demand placed on the calculated available Rubisco capacity. Only a marginal control on photosynthetic flux is exerted by Rubisco until over 50% of the available capacity is being used. Control increases as utilisation rises to 80%, and approaches unity (i.e. strict limitation) when more than 80% of the available capacity is being used. (iii) In low light, plants with reduced Rubisco have very high energy-dependent quenching of chlorophyll fluorescence (qE) and a decreased apparent quantum yield. It is argued that Rubisco still exerts marginal control in these conditions because decreased Rubisco leads to increased thylakoid energisation and high-energy dependent dissipation of light energy, and lower light-harvesting efficiency. (iv) The flux-control coefficient of stomata for photosynthesis was calculated from the flux-control coefficient of Rubisco and the internal CO₂ concentration, by applying the connectivity theorem. Control by the stomata varies between zero and about 0.25. It is increased by increased irradiance, decreased CO₂ or decreased vapour-pressure deficit. (v) Photosynthetic oscillations in saturating irradiance and CO₂ are suppressed in decreased-activity transformants before the steady-state rate of photosynthesis is affected. This provides direct evidence that these oscillations reveal the presence of excess Rubisco. (vi) Comparison of the flux-control coefficients of Rubisco with mechanistic models of photosynthesis provides direct support for the reliability of these models in conditions where Rubisco has a flux-control coefficient approach unity (i.e. limits photosynthesis), but also indicates that these models are less useful in conditions where control is shared between Rubisco and other components of the photosynthetic apparatus.Abbreviations A assimilation rate - C_i intercellular CO₂ concentration in the leaf - C_R flux-control coefficient of Rubisco for photosynthesis - qE high-energy-state-dependent quenching of chlorophyll fluorescence - Q_A primary acceptor of PSII - rbc S gene for the nuclear-encoded small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Ru1,5bisP ribulose-1,5-bisphosphate - VPD vapour-pressure deficit     

Photoinactivation of photosystem II during photoinhibition in the cyanobacterium Microcystis aeruginosa

Sites of photoinhibition and photo-oxidative damage to the photosynthetic electrontransport system of the unicellular cyanobacterium Microcystis aeruginosa were identified by studies of the kinetics of chlorophyll fluorescence induction by whole cells at room temperature and from partial photosynthetic electron-transport reactions in vitro in thylakoid preparations. Chlorophyll fluorescence intensity decreased following photoinhibitory light treatment. This was attributed to decreases both in the activity of photosystem II and in electron flow through the primary electron acceptor, Q. This inhibition was only partially reversed over a 50-min dark recovery period. Partial photosynthetic electron-transport experiments in vitro demonstrated that photosystem I was not affected by the photoinhibitory treatment. Light damage was associated exclusively with the light reactions, of photosystem II, at a site close to the reaction centre, between the site where diphenylcarbazide can donate electrons and the site where silicomolybdate can accept electrons. This damage presumably reduced production of ATP by noncyclic photophosphorylation and production of NADPH by photosystem I, decreasing the availability of these co-factors for reducing CO₂ in the dark reactions of photosynthesis. The importance of these findings is discussed.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenylcarbazide - PSI photosystem I - PSH photosystem II     

Predicting CO2 gain and photosynthetic light acclimation from fluorescence yield and quenching in cyano-lichens

Modulated chlorophylla fluorescence is useful for eco-physiological studies of lichens as it is sensitive, non-invasive and specific to the photobiont. We assessed the validity of using fluorescence yield to predict CO₂ gain in cyano-lichens, by simultaneous measurements of CO₂ gas exchange and chlorophylla fluorescence in five species withNostoc-photobionts. For comparison, O₂ evolution and fluorescence were measured in isolated cells ofNostoc, derived fromPeltigera canina (Nostoc PC). At irradiances up to the growth light level, predictions from fluorescence yield underestimated true photosynthesis, to various extents depending on species. This reflected the combined effect of a state transition in darkness, which was not fully relaxed until the growth light level was reached, and a phycobilin contribution to the minimum fluorescence yield (F_o). Above the growth light level, the model progressively overestimated assimilation, reflecting increased electron flow to oxygen under excess irradiance. In cyanobacteria, this flow maintains photosystem II centres open even up to photoinhibitory light levels without contributing to CO₂ fixation. Despite this we show that gross CO₂ gain may be predicted from fluorescence yield also in cyanolichens when the analysis is made near the acclimated growth light level. This level can be obtained even when measurements are performed in the field, since it coincides with a minimum in non-photochemical fluorescence quenching (NPQ). However, the absolute relation between fluorescence yield and gross CO₂ gain varies between species. It may therefore be necessary to standardise the fluorescence prediction for each species with CO₂ gas exchange.Abbreviations CCM CO₂-Concentrating mechanism - Chl chlorophyll - C_i inorganic carbon - 0 convexity (curvature of the light response curve) - ETR electron transport rate - F_o minimum fluorescence yield - F_m maximal fluorescence yield - F_s fluorescence yield at steady-state photosynthesis - F_v variable fluorescence yield - F_v/F_m dark ratio of variable to maximal fluorescence yield after dark adaptation - F_vF_mmax ratio of variable to maximal fluorescence yield in the absence of quenching - _CO2 maximum quantum yield of CO₂ assimilation - _PS quantum yield of photosystem II photochemistry - GP gross photosynthesis - I irradiance (mol quanta·m^–2·s^–1) - NPQ non photochemical fluorescence quenching - qp photochemical fluorescence quenching     

The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Photosynthetic activity of isolated chloroplasts from Euglena gracilis

Functional chloroplasts from photoheterotrophic Euglena gracilis can be isolated in isoosmotic gradients of 10–80% Percoll. The chloroplasts display rates of CO₂ dependent O₂ evolution and CO₂ fixation of 30–50 mol mg^-1 chlorophyll h^-1 or 25–35% of the net O₂ evolution by the whole cells and appear to be strikingly different from spinach chloroplasts in several respects: 1. tolerance to high concentration of orthophosphate in the assay medium; 2. inability to support oxaloacetate-dependent O₂ evolution; 3. ability to support only low to moderate rates of 3-phosphoglycerate-dependent O₂ evolution; 4. an apparent absence of a phosphate translocator in the terms described by Heldt and Rapley ([1970] FEBS Lett. 10, 143–148).University of California, Dept. of Plant and Soil Biology, 108 Hilgard Hall, Berkeley, CA 94720 USA     

The Influence of Leaf Windows on the Utilization and Absorption of Radiant Energy in Seven Desert Succulents

Four fluorescence parameters [F_v/F_m = the intrinsic efficiency of energy conversion via photosystem 2 (PS2); F_v/F_m= the efficiency of energy conversion via PS2 in the light; P = fraction of absorbed radiant energy utilized for photosynthesis; and D = fraction of absorbed radiant energy dissipated as heat] were measured on leaves of seven species of succulents having epidermal windows. While the function of leaf windows has reportedly been to increase absorption of radiant energy and, hence, the rate of photosynthesis in these species, recent evidence indicates that this translucent portion of epidermal tissue, lacking chlorophyll, may also result in photoinhibition in these species, especially for those with growth habits aboveground. Species with aboveground and belowground growth habits were compared with their leaf windows covered with reflective tape and with windows unobstructed. Results showed no increase in photoinhibition for these species resulting from the radiant energy penetrating the window tissue. Although the efficiency of the photosynthetic mechanism was not significantly influenced by the additional radiant energy provided by the window for individual species, there were significant differences in the efficiencies of radiant energy capture (F_v/F_m) and utilization (P) between the two growth habits. Species with an aboveground growth habit were less efficient in radiant energy utilization compared with the species having a belowground growth habit.     

Temperature-sensitive coupling and uncoupling of ATPase-mediated,nonradiative energy dissipation: Similarities between chloroplasts and leaves

The effects of temperature on the dark relaxation kinetics of nonradiative energy dissipation in photosystem II were compared in lettuce (Lactuca sativa L.) chloroplasts and leaves of Aegialitis annulata R. Br. After high levels of violaxanthin de-epoxidation in the light, Aegialitis leaves showed a marked delay in the dark relaxation of nonradiative dissipation, measured as non-photochemical quenching (NPQ) of photosystem II chlorophyll a fluorescence. Aegialitis leaves also maintained a moderately high adenylate energy charge at low temperatures during and after high-light exposure, presumably because of their limited carbon-fixation capacity. Similarly, dark-sustained NPQ could be induced in lettuce chloroplasts after de-epoxidizing violaxanthin and light-activating the ATP synthase. The duration and extent of dark-sustained NPQ were strongly enhanced by low temperatures in both chloroplasts and leaves. Further, the NPQ sustained at low temperatures was rapidly reversed upon warming. In lettuce chloroplasts, low temperatures sharply decreased the ATP-hydrolysis rate while increasing the duration and extent of the resultant trans-thylakoid proton gradient that elicits the NPQ. This was consistent with a higher degree of energy-coupling, presumably due to reduced proton diffusion through the thylakoid membrane at the lower temperatures. The chloroplast adenylate pool was in equilibrium with the adenylate kinase and therefore both ATP and ADP contributed to reverse coupling. The low-temperature-enhanced NPQ quenched the yields of the dark level (F_o) and the maximal (F_m) fluorescence proportionally in both chloroplasts and leaves. The extent of NPQ in the dark was inversely related to the efficiency of photosystem II, and very similar linear relationships were obtained over a wide temperature range in both chloroplasts and leaves. Likewise, the dark-sustained absorbance changes, caused by violaxanthin de-epoxidation (A_508nm) and energy-dependent light scattering (A_536nm) were strikingly similar in chloroplasts and leaves. Therefore, we conclude that the dark-sustained, low-temperature-stimulated NPQ in chloroplasts and leaves is apparently directly dependent on lumen acidification and chloroplastic ATP hydrolysis. In leaves, the ATP required for sustained NPQ is evidently provided by oxidative phosphorylation in the mitochondria. The functional significance of this quenching process and implications for measurements of photo-protection versus photodamage in leaves are discussed.Abbreviations and Symbols A antheraxanthin - Chl chlorophyll - DPS de-epoxidation state of the xanthophyll cycle, ([Z+A]/[V+A+Z]) - F, F steady-state fluorescence in the absence, presence of thylakoid energization - F_o, F_o dark fluorescence level in the absence, presence of thylakoid energization - F_m, F_m maximal fluorescence in absence, presence of thylakoid energization - NPQ nonphotochemical quenching (F_m/F_m)–1 - V violaxanthin - Z zeaxanthin - NRD nonradiative dissipation - PFD photon flux density - [2ATP+ADP] - pH trans-thylakoid proton gradient - S pH-dependent light scattering - _PSII (F_m–F)/F_m, photon yield of PSII photochemistry at the actual reduction state in the light or dark - [ATP+ADP+AMP] We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. Support from an NSF/USDA/DOE postdoctoral training grant to A.G. is gratefully acknowledged. A.G. also wishes to thank Prof. Govindjee for valuable discussions. C.I.W.-D.P.B. Publication No. 1197.     

Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator

Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix ¹⁴C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations P_i orthophosphate - PLP pyridoxal 5-phosphate - TP triosephosphate     

Regulation of adenylate levels in intact spinach chloroplasts

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.Abbreviations G1P Glucose 1-phosphate - P_i orthophosphate - Tris Tris (hydroxymethyl)aminomethane - MES 2(N-morpholino)ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Light-dependent modification of Photosystem II in spinach leaves

In dark-adapted spinach leaves approximately one third of the Photosystem II (PS II) reaction centers are impaired in their ability to transfer electrons to Photosystem I. Although these inactive PS II centers are capable of reducing the primary quinone acceptor, Q_A, oxidation of Q_A ^– occurs approximately 1000 times more slowly than at active centers. Previous studies based on dark-adapted leaves show that minimal energy transfer occurs from inactive centers to active centers, indicating that the quantum yield of photosynthesis could be significantly impaired by the presence of inactive centers. The objective of the work described here was to determine the performance of inactive PS II centers in light-adapted leaves. Measurements of PS II activity within leaves did not indicate any increase in the concentration of active PS II centers during light treatments between 10 s and 5 min, showing that inactive centers are not converted to active centers during light treatment. Light-induced modification of inactive PS II centers did occur, however, such that 75% of these centers were unable to sustain stable charge separation. In addition, the maximum yield of chlorophyll fluorescence associated with inactive PS II centers decreased substantially, despite the lack of any overall quenching of the maximum fluorescence yield. The effect of light treatment on inactive centers was reversed in the dark within 10–20 mins. These results indicate that illumination changes inactive PS II centers into a form that quenches fluorescence, but does not allow stable charge separation across the photosynthetic membrane. One possibility is that inactive centers are converted into centers that quench fluorescence by formation of a radical, such as reduced pheophytin or oxidized P680. Alternatively, it is possible that inactive PS II centers are modified such that absorbed excitation energy is dissipated thermally, through electron cycling at the reaction center.Abbreviations A518 absorbance change at 518 nm, reflecting the formation of an electric field across the thylakoid membrane - A_FL1 amplitude of the fast (<100 ms) phase of A518 induced by the first of two saturating, single-turnover flashes spaced 30 ms apart - A_FL2 amplitude of the fast (<100 ms) phase of A518 induced by the second of two saturating, single-turnover flashes spaced 50 ms apart - DCBQ 2,6-dichloro-p-benzoquinone - Fo yield of chlorophyll fluorescence when Q_A is fully oxidized - Fm yield of chlorophyll fluorescence when Q_A is fully reduced - Fx yield of chlorophyll fluorescence when Q_A is fully reduced at inactive PS II centers, but fully oxidized at active PS II centers - Pheo pheophytin - P680 the primary donor of Photosystem II - PPFD photosynthetic photon flux density - Q_A Primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Photosynthetic characteristics of mesophyll cells isolated from cladophylls ofAsparagus officinalis L.

Intact mesophyll cells can be rapidly isolated from the cladophylls ofAsparagus officinalis by gentle scraping with a plastic card, the yield being higher than 80% on a chlorophyll basis. The cells can be stored for at least 24h without loss of photosynthetic capacity and were found to be stable under a variety of conditions. In contrast to cell preparations from other plant species, photosynthetic activity was little affected by the presence of sorbitol as an osmoticum up to a concentration of 1.5 M. Similarly, the pH value of the medium influenced photosynthesis to only a small extent at a constant [CO₂] of 200 M. The response of the cells' photosynthetic capacity to light, temperature and CO₂ concentration was similar to those reported for isolated cells from other plant species. Isolated cells ofA. officinalis can be used under a large range of conditions which gives them a measure of flexibility not possible with most plant cells which have sharply defined optimal conditions for photosynthesis. The isolated cells have a photosynthetic capacity of 40–60% of that of the intact cladophyll. The loss of photosynthetic activity observed upon isolation could not be accounted for by breakage of the cells. Virtually all of the cells were shown to be intact on the basis of Evans Blue exclusion and more than 80% of the cells contained intact chloroplasts and vacuoles. The entire loss of photosynthetic activity could be accounted for by a decrease in sucrose synthesis rather than by an equal decrease in the synthesis in all products. A six- to seven fold increase in the level of¹⁴C in hexose phosphates in the isolated cells supports the notion of inhibition of the sucrose-synthesis pathway.